The applications are: 1. RDT in Helping Us to Stay Healthy 2. RDT in Making our Agriculture more Productive 3. RDT in the Generation of Transgenic Animals 4. RDT in Forensics 5. RDT in Keeping Our Environment Pollution Free.
Application # 1.
RDT in Helping Us to Stay Healthy:
(a) Human Therapeutics from RDT:
One of the greatest benefit of the recombinant DNA technology has been the production of human therapeutics such as hormones, growth factors and antibodies which are not only scarcely available but also are very costly for human use.
Recombinant therapeutics include proteins that help the body to fight infection or to carry out specific functions such as blood factors, hormones, growth factors, interferon’s and interleukins. Starting with simple protein such as insulin (the first recombinant therapeutic product) and then growth hormone, recombinant biopharmaceuticals has increased considerably in recent years.
Therapeutic proteins are preferred over conventional drugs because of their higher specificity and absence of side effects. Therapeutic proteins are less toxic than chemical drugs and are neither carcinogenic nor teratogenic (able to disturb the growth and development of an embryo or fetus).
Further, once the biologically active form of a protein is identified for medical application, its further development into a medicinal product involves fewer risks than chemical drugs.
Notable diseases for which recombinant therapeutics have been produced include diabetes, hemophilia, hepatitis, myocardial infarction and various cancers. Till today, around 165 biopharmaceuticals (recombinant proteins, antibodies, and nucleic acid based drugs) have been approved. (Table 2.1) lists the type of biomolecules that have been produced by the recombinant DNA technology.
Today for the first time we have been successful in isolating a naturally occurring protein and using it for the therapeutic purposes. This has become possible only due to RDT. The development of one such product, tissue plasminogen activator, illustrates the techniques that have been used.
In most people, when a wound begins bleeding, a blood clot soon forms to stop the flow. Later, as the wound heals, the clot dissolves. How does the blood perform these conflicting functions at the right times? Mammalian blood contains an enzyme called plasmin that catalyses the dissolution of the clotting proteins.
But plasmin is not always active; if it were, a blood clot would dissolve as soon as it formed! Instead, plasmin is “stored” in the blood in an inactive form called plasminogen.
The conversion of plasminogen to plasmin is activated by an enzyme, appropriately called tissue plasminogen activator (TPA), which is produced by cells lining the blood vessels:
Heart attacks and many strokes are caused by blood clots that form in major blood vessels leading to the heart or the brain, respectively. During the 1970s, a bacterial enzyme, called streptokinase, was found to stimulate the dissolution of clots in some patients.
Treating these persons with this enzyme saved lives, but its use had side effects. Streptokinase was a protein foreign to the body, so patients’ immune systems reacted against it. More importantly, the drug sometimes prevents clotting throughout the entire circulatory system, leading to an almost hemophilia-like condition in some patients.
The discovery of TPA and its isolation from human tissues led to the hope that this enzyme would bind specifically to clots, and that it would not provoke an immune reaction. But the amounts of TPA available from human tissues were tiny, certainly not enough to inject at the site of a clot in the emergency room.
Recombinant DNA technology solved this problem. TPA mRNA was isolated and used to make a cDNA copy, which was then inserted into an expression vector and transfected into E. coli (Fig. 2.3). The transgenic bacteria made the protein in quantity, and it soon became available commercially. This drug has had considerable success in dissolving blood clots in people undergoing heart attacks and, especially, strokes.
(b) Antisense Therapeutics:
The base-pairing rules can be used not only to make artificial genes, but they can also be employed to stop the translation of mRNA. As is often the case, this technique is an example of scientists imitating nature. In normal cells, a rare mechanism for controlling gene expression is the production of an RNA molecule that is complementary to mRNA.
This complementary molecule is called antisense RNA because it binds by base pairing to the “sense” bases on the mRNA that codes for a protein. The formation of a double-stranded RNA hybrid inhibits translation of the mRNA, and the hybrid tends to be broken down rapidly in the cytoplasm.
Although the gene continues to be transcribed, translation does not take place. After determining the sequence of a gene and its mRNA in the laboratory, scientists can make and add specific antisense RNA to a cell to prevent translation of that gene’s mRNA (Fig. 2.4).
The antisense RNA can be added as itself—RNA can be inserted into cells in the same way that DNA is—or it can be made in the cell by transcription from a DNA molecule introduced as a part of a vector. A related technique takes advantage of interference RNA (RNAi), a rare way of naturally inhibiting mRNA translation, that occurs in the inactivation of the X chromosome.
In this case, a short (about 20 nucleotides) double- stranded RNA is unwound to single strands by a protein complex that guides this RNA to a complementary region on mRNA. The protein complex catalyses the breakdown of the targeted mRNA.
Armed with this knowledge, scientists can custom-synthesize a small interfering RNA (siRNA) to inhibit the translation of any known gene. Since these double stranded siRNAs are more stable than antisense RNAs, RNAi is a much easier technique to use than antisense RNA.
Antisense RNA and RNAi have been widely used to test cause-and-effect relationships. For example, when antisense RNA was used to block the synthesis of a protein essential for the growth of cancer cells, the cells reverted to a normal phenotype. Gene silencing offers great potential for the development of drugs to treat diseases that are the result of the inappropriate expression of specific genes.
(c) Genetically Engineered Proteins in Therapeutics:
Genetically engineered proteins are the proteins which are obtained by the process of RDT. They are specially structured proteins which are tactfully developed to fight against the host either by preventing it or by cheating it.
Genetically engineered proteins are known to block or mimic surface receptors present on the cell membranes. For example, we can design a protein which can mimic a receptor protein that HIV binds to in entering WBC. The HIV binds to this genetically engineered protein molecule instead and fails to enter the WBC.
(d) Gene Therapy:
Genetic engineering has also allowed biologists to try to treat genetic disorders in different ways. One method is a technique called gene therapy. In gene therapy a genetic disorder is treated by introducing a gene into the patient’s cells. Gene therapy works best for disorders that result from the loss of a single protein. For example, the lung disease cystic fibrosis results from the lack of a functional gene called the CFTR gene.
When functional, the gene encodes a protein that helps transport ions into and out of cells in the breathing passages. Without that gene, poor ion exchange causes the symptoms of cystic fibrosis, including the build-up of sticky mucus that blocks the airways. (Fig. 2.5) summarizes the steps involved in gene therapy.
In steps, researchers first isolate the functional gene (e.g., CFTR gene). Then they insert the healthy gene into a viral vector. In the next step, they introduce the recombinant virus to the patient by infecting the patient’s airway by means of a nasal spray.
The healthy copy of the CFTR gene temporarily produces the missing protein and improves ion exchange. The traditional treatment for cystic fibrosis involves thumping sessions— clapping on the back and chest for half-hour periods several times a day to dislodge mucus. Cystic fibrosis research has accelerated since the discovery of the CFTR gene in 1989.
In the laboratory, researchers were able to add a healthy copy of CFTR into the DNA of cystic fibrosis cells. The result was an immediate return to a normal ion transport mechanism. However, trials in the laboratory are different from trials on living humans.
Apparently, the cells that express the highest levels of CFTR are deeper in the lungs than the surface cells that current forms of gene therapy can reach, because the cells that line the airway slough periodically, the treatment must be repeated.
In addition, patients may suffer immune reactions to the treatment. Researchers hope to overcome these obstacles one day and to provide a permanent cure. People with certain kinds of haemophilia, acquired immunodeficiency syndrome (AIDS), or some cancers are future candidates for gene therapy.
Until recombinant DNAs can be inserted into the correct cells, however, and immune reactions can be prevented, gene therapy may continue to be a short-term solution.
A vaccine is a substance containing all or part of a harmless version of a pathogen that physicians introduce into the body to produce immunity to disease. The immune system recognizes the pathogen’s surface proteins and responds by making defensive proteins called antibodies. A DNA vaccine is a vaccine made from the DNA of a pathogen but does not have disease-causing capability.
The DNA vaccine is injected into a patient where it directs the synthesis of a protein. The immune system mounts a defense against the protein. If the vaccinated person contacts the disease agent in the future, his or her new immunity should provide protection. Researchers are currently working on developing DNA vaccines to prevent AIDS, malaria, and certain cancers.
A plantibody is an antibody produced by genetically modified crops. Antibodies are part of animal immune systems, and are produced in plants by transforming them with antibody genes from animals. This was first done in 1989, with a mouse antibody made by tobacco plants. Although plants do not naturally make antibodies, plantibodies have been shown to function in the same way as normal antibodies do.
The plantibodies include the followings:
1. Hepatitis B vaccine
2. Antibody to fight cavity causing bacteria
3. Antibodies to prevent sexually transmitted diseases
4. Antibody vaccine for non-Hodgkin’s lymphoma
5. A vaccine against the HIV virus
6. Anthrax vaccine [(from tobacco plants) one acre of plants can produce 360 million doses in a year]
Application # 2.
RDT in Making our Agriculture more Productive:
Another major area of genetic engineering activity is manipulation of the genes of key crop plants. In plants the primary experimental difficulty has been identifying a suitable vector for introducing recombinant DNA.
Plant cells do not possess the many plasmids that bacteria do, so the choice of potential vectors is limited. The most successful results thus far have been obtained with the Ti (tumor inducing) plasmid of the plant bacterium Agrobacterium tumefaciens, which infects broadleaf plants such as tomato, tobacco, and soybean.
Part of the Ti plasmid integrates into the plant DNA, and researchers have succeeded in attaching other genes to this portion of the plasmid (Fig. 2.6).
The characteristics of a number of plants have been altered using this technique, which should be valuable in improving crops and forests. Among the features scientists would like to affect are resistance to disease, frost, and other forms of stress; nutritional balance and protein content; and herbicide resistance.
Unfortunately, Agrobacterium generally does not infect cereals such as corn, rice and wheat, but alternative methods can be used to introduce new genes into them.
A recent advance in genetically manipulated fruit is Calgene’s “Flavr Savr” tomato, which has been approved for sale by the USDA. The tomato has been engineered to inhibit genes that cause cells to produce ethylene. In tomatoes and other plants, ethylene acts as a hormone to speed fruit ripening.
In Flavr Savr tomatoes, inhibition of ethylene production delays ripening. The result is a tomato that can stay on the vine longer and that resists over ripening and rotting during transport to market.
(a) Herbicide Resistance:
Recently, broad leaf plants have been genetically engineered to be resistant to glyphosate, the active ingredient in Roundup, a powerful, biodegradable herbicide that kills most actively growing plants. Glyphosate works by inhibiting an enzyme called EPSP synthetase, which plants require to produce aromatic amino acids.
Humans do not make aromatic amino acids; they get them from their diet, so they are unaffected by glyphosate. To make glyphosate- resistant plants, agricultural scientists used a Ti plasmid to insert extra copies of the EPSP synthetase genes into plants.
These engineered plants produce 20 times the normal level of EPSP synthetase, enabling them to synthesize proteins and grow despite glyphosate’s suppression of the enzyme. In later experiments, a bacterial form of the EPSP synthetase gene that differs from the plant form by a single nucleotide was introduced into plants via Ti plasmids; the bacterial enzyme in these plants is not inhibited by glyphosate.
These advances are of great interest to farmers because a crop resistant to Roundup would never have to be weeded if the field were simply treated with the herbicide. And Roundup is a broad-spectrum herbicide, farmers would no longer need to employ a variety of different herbicides, most of which kill only a few kinds of weeds.
Furthermore, glyphosate breaks down readily in the environment, unlike many other herbicides commonly used in agriculture. A plasmid is actively being sought for the introduction of the EPSP synthetase gene into cereal plants, making them also glyphosate-resistant.
(b) Nitrogen Fixation:
A long-range goal of agricultural genetic engineering is to introduce the genes that allow soybeans and other legume plants to “fix” nitrogen into key crop plants. These so-called nif genes are found in certain symbiotic root-colonizing bacteria. Living in the root nodules of legumes, these bacteria break the powerful triple bond of atmospheric nitrogen gas, converting N2 into NH3 (ammonia).
The plants then use the ammonia to make amino acids and other nitrogen containing molecules. Other plants lack these bacteria and cannot fix nitrogen, so they must obtain their nitrogen from the soil. Farmland where these crops are grown soon becomes depleted of nitrogen, unless nitrogenous fertilizers are applied.
Worldwide, farmers applied over 60 million metric tons of such fertilizers in 1987, an expensive undertaking. Farming costs would be much lower if major crops like wheat and corn could be engineered to carry out biological nitrogen fixation.
However, introducing the nitrogen-fixing genes from bacteria into plants has proved difficult because these genes do not seem to function properly in eukaryotic cells. Researchers are actively experimenting with other species of nitrogen-fixing bacteria whose genes might function better in plant cells.
(c) Insect Resistance:
Humans are not the only species that consumes crop plants. Plants are subject to infections by viruses, bacteria, and fungi, but probably the most important crop pests are herbivorous insects. From the locusts of biblical (and modern) times to the cotton boll weevil, insects have continually eaten the crops people grow.
The development of insecticides has improved the situation somewhat, but insecticides have their own problems. Most, like organophosphates, are relatively nonspecific, killing not only pests in the field but beneficial insects in the ecosystem as well.
Some even have toxic effects on other organisms, including people. What’s more, insecticides are applied to the surface of crop plants and tend to be blown away to adjacent areas, where they may have unforeseen effects.
Some bacteria have solved their own pest problem by producing proteins that kill insect larvae that eat them. For example, there are dozens of strains of Bacillus thuringiensis, each of which produces a protein toxic to the insect larvae that prey on it.
The toxicity of this protein is 80,000 times that of the usual commercial insecticides. When a hapless larva eats the bacteria, the toxin becomes activated, binding specifically to the insect’s gut to produce holes. The insect starves to death.
Dried preparations of B. thuringiensis have been sold for decades as a safe, biodegradable insecticide. But biodegradation is their limitation, because it means that the dried bacteria must be applied repeatedly during the growing season. A more permanent approach would be to have the crop plants made the toxin themselves.
The toxin genes from different strains of B. thuringiensis have been isolated and cloned, and they have been extensively modified by the addition of plant promoters and terminators, plant poly A addition sequences, plant codon usage, and plant regulatory elements.
These modified genes have been introduced into plant cells in the laboratory using the Ti plasmid vector, and transgenic plants have been grown and tested for insect resistance in the field. So far, transgenic tomato, corn, potato, and cotton crops have been shown to have considerable resistance to their insect predators.
(d) Grains with Improved Nutritional Characteristics:
To remain healthy, humans must eat foods (or supplements) containing an adequate amount of a-carotene, which the body converts into vitamin A. About 400 million people worldwide suffer from vitamin A deficiency, which makes them susceptible to infections and blindness.
One reason is that rice grains, which do not contain a-carotene, but only a precursor molecule for it, make up a large part of their diet. Other organisms, such as the bacterium Erwinia and daffodil plants, have enzymes that can convert the precursor into a-carotene.
The genes for this biochemical pathway are present in the bacterial and daffodil genomes, but not in the rice genome. Scientists isolated two of the genes for the a-carotene pathway from the bacterium and the other two from daffodil plants. They added promoter signals for expression in the developing rice grain, and then added each gene to rice plants by using the Ti plasmid vector from (Agrobacterium tumefaciens).
The resulting rice plants produce grains that look yellow because of their high a-carotene content (Fig. 2.7). About 300 grams of this cooked rice a day can supply all the α- carotene a person needs. This new transgenic strain is now being crossed with more locally adapted strains, and it is hoped that the diets of millions of people will be improved as a result.
(e) Crops that Adapt to the Environment:
Throughout human history, agriculture has involved ecological management—tailoring the environment to the needs of crop plants. A farm field is an unnatural, human-designed system, and when conditions in that field become intolerable, the crops die.
The Fertile Crescent, the region between the Tigris and the Euphrates rivers in the Middle East where agriculture probably originated 10,000 years ago, is no longer fertile. It is now a desert, largely because the soil has a high salt concentration.
Few plants can grow on salty soils, primarily because the environment is hypertonic to the plant roots, and water leaves them, resulting in wilting. Recently, a gene was discovered in the shale cress (Arabidopsis thaliana) that allows this tiny weed to thrive in salty soils.
The gene codes for a protein that transports Na+ into the vacuole. When this gene was added to tomato plants, they too grew in soils four times as salty as the normal lethal level (Fig. 2.8). This finding raises the prospect of growing useful crops on what were previously unproductive soils.
More importantly, this example illustrates what could become a fundamental shift in the relationship between crop plants and the environment.
Instead of manipulating the environment to suit the plant, biotechnology may allow us to adapt the plant to the environment. As a result, some of the negative effects of agriculture, such as water pollution, could be lessened.
Application # 3.
RDT in the Generation of Transgenic Animals:
Transgenic animals are genetically modified organisms and thus carry a transgene in their genome. There are many reasons for the development of transgenic animals.
In some cases transgenic animals may be designed simply to be visually interesting, to study, to yield more meat, or to perform a specific task better while in other cases they can be used as bioreactors for the production of certain products (like milk and meat) containing pharmaceutical molecules.
Transgenic animals have had their DNA altered specifically by having the DNA of another animal inserted into their own code.
(a) Medicine in the Milk:
A transgene can be inserted into an animal, and if the appropriate promoter is present, the gene can be expressed in a readily available tissue. People with one type of emphysema have lung damage because they lack adequate amounts of a protein called α-l-antitrypsin (α-1AT).
This protein inhibits elastase, an enzyme that breaks down connective tissue. Thus, using an inhibitor of elastase could alleviate these symptoms in these patients.
The problem is that only minuscule amounts of α-1AT can be purified from human serum. To overcome this problem, the gene for human α-1AT was introduced into the eggs of sheep, next to the promoter for lacto globulin, a protein found in large amounts in milk.
The resulting transgenic sheep made large amounts of α-1AT in their milk. Since female sheep produce large amounts of milk all over the year, this natural “bioreactor” produced a large supply of α-1AT, which was easily separated from the other components of the milk.
Goats, sheep, and cows are all being used for what has come to be called pharming: the production of medically useful products in milk. These products include blood clotting factors for treating hemophilia and antibodies for treating colon cancer.
(b) High Yielding Farm Animals:
The gene encoding the growth hormone somatotropin was one of the first to be cloned successfully. In 1994, Monsanto received federal approval to make its recombinant bovine somatotropin (BST) commercially available, and dairy farmers worldwide began to add the hormone as a supplement to their cows’ diets, increasing the animals’ milk production (Fig. 2.9).
Genetically engineered somatotropin is also being tested to see if it increases the muscle weight of cattle and pigs, and as a treatment for human disorders in which the pituitary gland fails to make adequate levels of somatotropin, producing dwarfism.
BST ingested in milk or meat has no effect on humans, because it is a protein and is digested in the stomach. Nevertheless, BST has met with some public resistance, primarily due to generalized fears of gene technology.
Some people mistrust milk produced through genetic engineering, even though that milk is identical to other milk. Problems concerning public perception are not uncommon as gene technology makes an even greater impact on our lives.
Transgenic animals engineered to have specific desirable genes are becoming increasingly available to breeders. Now, instead of selectively breeding for several generations to produce a racehorse or a stud bull with desirable qualities, the process can be shortened by simply engineering such an animal right at the start.
Application # 4.
RDT in Forensics:
Genetic engineering has had an enormous impact on the field of forensic science. In 1987, the first court case was heard in Britain in which RFLP analysis was used. The DNA from a suspect was compared with that of the DNA extracted from the semen left behind at the scene of a rape.
The suspect was convicted of the crime and sent to prison as a result of the DNA match, along with overwhelming corroborating evidence. In a short time, DNA fingerprinting has become one of the most valuable tools investigators can use to determine whether or not the evidence originated from the suspect.
DNA evidence alone cannot convict a suspect, for it cannot support on its own the claim that the suspect committed the crime. The only conclusion that can be made with very close certainty is that the DNA source came from the suspect in question.
Either RFLP analysis or PCR can be used in criminal investigations, depending on the state of the DNA sample. RFLP analysis requires large samples that are un-degraded, whereas PCR can be performed with minute quantities that are degraded.
Both techniques produce a “fingerprint” or pattern of bands on a gel that can be compared with the evidence found at the scene. Both techniques take advantage of the non-coding regions of the human genome.
It is in these non-coding regions that most of the variability lies. The non-coding regions differ in the quantity of variable number tandem repeats and, therefore, can be used as a basis for discriminating between individuals.
In fact, the probability is very low of matching in six to seven areas of non-coding regions with another person. In some cases, depending on the population and the frequency of the given site, it could be as low as one in 1 billion.
It is important to note that DNA fingerprinting is most often used to illustrate innocence as opposed to guilt. Many cases do not even go to trial because of the lack of DNA evidence, and Ontario has saved large sums in legal costs as a result.
Numerous people have been exonerated using this technology. Guy Paul Morin spent approximately 10 years in custody before his conviction of murder was overturned with DNA evidence. The murder victim had been raped before being killed. Luckily, the evidence had been kept at the Centre for Forensic Sciences in Toronto. Using PCR, the DNA sample was tested in seven different target regions.
Morin did not match up in three of those regions. If he had not matched in only one of the seven regions, that would have been enough evidence to release him, because identical DNA samples should produce identical banding patterns when subjected to the same analysis.
What is unfortunate about Morin’s case and others like it is that these wrongfully convicted individuals had to wait for the technology to be developed before they could be released.
Currently, DNA databases are being shared among criminal institutions across the United States and Canada. In fact, it is now illegal in Canada to refuse to provide a DNA sample to police on arrest if requested. In the past, traditional fingerprints were collected, and the future holds the possibility of DNA fingerprints being collected.
Many argue that this is an invasion of privacy. The same arguments were made a century ago when police started to collect traditional fingerprints.
Application # 5.
RDT in Keeping Our Environment Pollution Free:
A vast majority of applications of environmental biotechnology use naturally occurring micro-organisms (bacteria, fungi, etc.) to identify and filter manufacturing waste before it is introduced into the environment. Bioremediation program involving the use of micro-organisms are currently in progress to clean up contaminated air, tracks of land, lakes and waterways.
Recombinant technology helps in improving the efficacy of these processes so that their basic biological processes are more efficient and can degrade more complex chemicals and higher volumes of waste materials.
Recombinant DNA technology also is being used in development of bio-indicators where bacteria have been genetically modified as ‘bioluminescors’ that give off light in response to several chemical pollutants.
These are being used to measure the presence of some hazardous chemicals in the environment. Other genetic sensors that can be used to detect various chemical contaminants are also undergoing trials, and include sensors that can be used to track how pollutants are naturally degrading in ground water.
For example, when gene, such as the mercury resistance gene (mer) or the toluene degradation (tol) gene, is linked to genes that code for bioluminescence within living bacterial cells, the biosensor cells can signal extremely low levels of inorganic mercury or toluene that are present in contaminated waters and soils by emitting visible light, which can be measured with fibre-optic flurometers.