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МИНИСТЕРСТВО ОБРАЗОВАНИЯ И НАУКИ РЕСПУБЛИКИ КАЗАХСТАН ГОСУДАРСТВЕННЫЙ УНИВЕРСИТЕТ имени ШАКАРИМА г. Семей Кафедра «Стандартизация и биотехнология» МЕТОДИЧЕСКИЕ УКАЗАНИЯ к практическим занятиям по дисциплине «Профессионально-ориентированный иностранный язык» для специальности «5В070100» – «Биотехнология» Семей 2013 1 Разработано преподавателем кафедры «Стандартизация и биотехнология» Бепеевой А.Е. Утверждено на заседании кафедры «Стандартизация и биотехнология». Протокол № __ от _________________ Заведующая кафедрой_________________ Ж.Х. Какимова ПРАКТИЧЕСКИЕ ЗАНЯТИЯ 2 Микромодуль 1 – Biotechnology Практическое занятие №1 (2 часа) Тема: History and application of biotechnology Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. Biotechnology is not limited to medical/health applications (unlike Biomedical Engineering, which includes much biotechnology). Although not normally thought of as biotechnology, agriculture clearly fits the broad definition of "using a biotechnological system to make products" such that the cultivation of plants may be viewed as the earliest biotechnological enterprise. Agriculture has been theorized to have become the dominant way of producing food since the Neolithic Revolution. The processes and methods of agriculture have been refined by other mechanical and biological sciences since its inception. Through early biotechnology, farmers were able to select the best suited crops, having the highest yields, to produce enough food to support a growing population. Other uses of biotechnology were required as the crops and fields became increasingly large and difficult to maintain. Specific organisms and organism by-products were used to fertilize, restore nitrogen, and control pests. Throughout the use of agriculture, farmers have inadvertently altered the genetics of their crops through introducing them to new environments and breeding them with other plants—one of the first forms of biotechnology. Cultures such as those in Mesopotamia, Egypt, and India developed the process of brewing beer. It is still done by the same basic method of using malted grains (containing enzymes) to convert starch from grains into sugar and then adding specific yeasts to produce beer. In this process the carbohydrates in the grains were broken down into alcohols such as ethanol. Later other cultures produced the process of lactic acid fermentation which allowed the fermentation and preservation of other forms of food. Fermentation was also used in this time period to produce leavened bread. Although the process of fermentation was not fully understood until Pasteur's work in 1857, it is still the first use of biotechnology to convert a food source into another form. For thousands of years, humans have used selective breeding to improve production of crops and livestock to use them for food. In selective breeding, organisms with desirable characteristics are mated to produce offspring with the same characteristics. For example, this technique was used with corn to produce the largest and sweetest crops. In the early twentieth century scientists gained a greater understanding of microbiology and explored ways of manufacturing specific products. In 1917,. Chaim Weizmann first used a pure microbiological culture in an industrial process, that of manufacturing corn starch using Clostridium acetobutylicum, to produce acetone, which theUnited Kingdom desperately needed to manufacture explosives during World War I. Biotechnology has also led to the development of antibiotics. In 1928, Alexander Fleming discovered the mold Penicillium. His work led to the purification of the antibiotic by Howard Florey, Ernst Boris Chain and Norman Heatley penicillin. In 1940, penicillin became available for medicinal use to treat bacterial infections in humans.[7] The field of modern biotechnology is thought to have largely begun on June 16, 1980, when the United States Supreme Court ruled that a genetically modified microorganism could be patented in the case of Diamond v. Chakrabarty. Indian-born Ananda Chakrabarty, working for General Electric, had developed a bacterium (derived from the Pseudomonas genus) capable of breaking down crude oil, which he proposed to use in treating oil spills. Revenue in the industry is expected to grow by 12.9% in 2008. Another factor influencing the biotechnology sector's success is improved intellectual property rights legislation—and enforcement— 3 worldwide, as well as strengthened demand for medical and pharmaceutical products to cope with an ageing, and ailing, U.S. population. Rising demand for biofuels is expected to be good news for the biotechnology sector, with the Department of Energy estimating ethanol usage could reduce U.S. petroleum-derived fuel consumption by up to 30% by 2030. The biotechnology sector has allowed the U.S. farming industry to rapidly increase its supply of corn and soybeans—the main inputs into biofuels—by developing genetically modified seeds which are resistant to pests and drought. By boosting farm productivity, biotechnology plays a crucial role in ensuring that biofuel production targets are met. 1. Applications Biotechnology has applications in four major industrial areas, including health care (medical), crop production and agriculture, non food (industrial) uses of crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and environmental uses. For example, one application of biotechnology is the directed use of organisms for the manufacture of organic products (examples include beer and milk products). Another example is using naturally present bacteria by the mining industry in bioleaching. Biotechnology is also used to recycle, 4 treat waste, cleanup sites contaminated by industrial activities (bioremediation), and also to produce biological weapons. A series of derived terms have been coined to identify several branches of biotechnology; for example: Bioinformatics is an interdisciplinary field which addresses biological problems using computational techniques, and makes the rapid organization and analysis of biological data possible. The field may also be referred to as computational biology, and can be defined as, "conceptualizing biology in terms of molecules and then applying informatics techniques to understand and organize the information associated with these molecules, on a large scale."[12] Bioinformatics plays a key role in various areas, such as functional genomics, structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector. Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare. Green biotechnology is biotechnology applied to agricultural processes. An example would be the selection and domestication of plants via micropropagation. Another example is the designing of transgenic plants to grow under specific environments in the presence (or absence) of chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby ending the need of external application of pesticides. An example of this would be Bt corn. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate. Red biotechnology is applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures through genetic manipulation. White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods.[citation needed] The investment and economic output of all of these types of applied biotechnologies is termed as bioeconomy. Практическое занятие №2 (3 часа) Тема: Biotechnology in Medicine, Pharmacogenomics, Pharmaceutical products Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. 1. Medicine In medicine, modern biotechnology finds promising applications in such areas as drug production pharmacogenomics gene therapy genetic testing (or genetic screening): techniques in molecular biology detect genetic diseases. To test the developing fetus for Down syndrome, Amniocentesis and chorionic villus sampling can be used. 5 2. Pharmacogenomics DNA microarray chip – some can do as many as a million blood tests at once Pharmacogenomics is the study of how the genetic inheritance of an individual affects his/her body's response to drugs. It is a portmanteau derived from the words "pharmacology" and "genomics". It is hence the study of the relationship between pharmaceuticals and genetics. The vision of pharmacogenomics is to be able to design and produce drugs that are adapted to each person's genetic makeup. Pharmacogenomics results in the following benefits: 1. Development of tailor-made medicines. Using pharmacogenomics, pharmaceutical companies can create drugs based on the proteins, enzymes and RNA molecules that are associated with specific genes and diseases. These tailor-made drugs promise not only to maximize therapeutic effects but also to decrease damage to nearby healthy cells. 2. More accurate methods of determining appropriate drug dosages. Knowing a patient's genetics will enable doctors to determine how well his/ her body can process and metabolize a medicine. This will maximize the value of the medicine and decrease the likelihood of overdose. 3. Improvements in the drug discovery and approval process. The discovery of potential therapies will be made easier using genome targets. Genes have been associated with numerous diseases and disorders. With modern biotechnology, these genes can be used as targets for the development of effective new therapies, which could significantly shorten the drug discovery process. 4. Better vaccines. Safer vaccines can be designed and produced by organisms transformed by means of genetic engineering. These vaccines will elicit the immune response without the attendant risks of infection. They will be inexpensive, stable, easy to store, and capable of being engineered to carry several strains of pathogen at once. 3. Pharmaceutical products Most traditional pharmaceutical drugs are relatively small molecules that bind to particular molecular targets and either activate or deactivate biological processes. Small molecules are typically manufactured through traditional organic synthesis, and many can be taken orally. In contrast, Biopharmaceuticals are large biological molecules such as proteins that are developed to address targets that cannot easily be addressed by small molecules. Some examples of biopharmaceutical drugs include Infliximab, a monoclonal antibody used in the treatment of autoimmune diseases, Etanercept, a fusion protein used in the treatment of autoimmune diseases, and Rituximab, a chimeric monoclonal antibody used in the treatment of cancer. Due to their larger size, and corresponding difficulty with surviving the stomach, colon and liver, biopharmaceuticals are typically injected. Modern biotechnology is often associated with the use of genetically altered microorganisms such as E. coli or yeast for the production of substances like synthetic insulin or antibiotics. It can also refer to transgenic animals or transgenic plants, such as Bt corn. Genetically altered mammalian cells, such as Chinese Hamster Ovary cells (CHO), are also used to manufacture certain pharmaceuticals. Another promising new biotechnology application is the development of plant-made pharmaceuticals. Biotechnology is also commonly associated with landmark breakthroughs in new medical therapies to treat hepatitis B, hepatitis C, cancers, arthritis, haemophilia, bone fractures, multiple 6 sclerosis, and cardiovascular disorders. The biotechnology industry has also been instrumental in developing molecular diagnostic devices that can be used to define the target patient population for a given biopharmaceutical. Herceptin, for example, was the first drug approved for use with a matching diagnostic test and is used to treat breast cancer in women whose cancer cells express the protein HER2. Modern biotechnology can be used to manufacture existing medicines relatively easily and cheaply. The first genetically engineered products were medicines designed to treat human diseases. To cite one example, in 1978 Genentech developed synthetic humanized insulin by joining its gene with a plasmid vector inserted into the bacterium Escherichia coli. Insulin, widely used for the treatment of diabetes, was previously extracted from the pancreas of abattoir animals (cattle and/or pigs). The resulting genetically engineered bacterium enabled the production of vast quantities of synthetic human insulin at relatively low cost. According to a 2003 study undertaken by the International Diabetes Federation (IDF) on the access to and availability of insulin in its member countries, synthetic 'human' insulin is considerably more expensive in most countries where both synthetic 'human' and animal insulin are commercially available: e.g. within European countries the average price of synthetic 'human' insulin was twice as high as the price of pork insulin. Yet in its position statement, the IDF writes that "there is no overwhelming evidence to prefer one species of insulin over another" and "[modern, highly purified] animal insulins remain a perfectly acceptable alternative. Modern biotechnology has evolved, making it possible to produce more easily and relatively cheaply human growth hormone, clotting factors for hemophiliacs, fertility drugs, erythropoietin and other drugs. Most drugs today are based on about 500 molecular targets. Genomic knowledge of the genes involved in diseases, disease pathways, and drug-response sites are expected to lead to the discovery of thousands more new targets. Микромодуль 2 – Application of biotechnology Практическое занятие №3 (2 часа) Тема: Biotechnology in Genetic testing, Controversial questions, Gene therapy Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. 1. Genetic testing Genetic testing involves the direct examination of the DNA molecule itself. A scientist scans a patient's DNA sample for mutated sequences. There are two major types of gene tests. In the first type, a researcher may design short pieces of DNA ("probes") whose sequences are complementary to the mutated sequences. These probes will seek their complement among the base pairs of an individual's genome. If the mutated sequence is present in the patient's genome, the probe will bind to it and flag the mutation. In the second type, a researcher may conduct the gene test by comparing the sequence of DNA bases in a patient's gene to disease in healthy individuals or their progeny. Genetic testing is now used for: Carrier screening, or the identification of unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest; Confirmational diagnosis of symptomatic individuals; Determining sex; Forensic/identity testing; Newborn screening; 7 Prenatal diagnostic screening; Presymptomatic testing for estimating the risk of developing adult-onset cancers; Presymptomatic testing for predicting adult-onset disorders. Some genetic tests are already available, although most of them are used in developed countries. The tests currently available can detect mutations associated with rare genetic disorders like cystic fibrosis, sickle cell anemia, and Huntington's disease. Recently, tests have been developed to detect mutation for a handful of more complex conditions such as breast, ovarian, and colon cancers. However, gene tests may not detect every mutation associated with a particular condition because many are as yet undiscovered. 2. Controversial questions The absence of privacy and anti-discrimination legal protections in most countries can lead to discrimination in employment or insurance or other use of personal genetic information. This raises questions such as whether genetic privacy is different from medical privacy.[18] 1. Reproductive issues. These include the use of genetic information in reproductive decision-making and the possibility of genetically altering reproductive cells that may be passed on to future generations. For example, germline therapy changes the genetic make-up of an individual's descendants. Thus, any error in technology or judgment may have far-reaching consequences (though the same can also happen through natural reproduction). Ethical issues like designed babies and human cloning have also given rise to controversies between and among scientists and bioethicists, especially in the light of past abuses with eugenics (see reductio ad hitlerum). 2. Clinical issues. These center on the capabilities and limitations of doctors and other health-service providers, people identified with genetic conditions, and the general public in dealing with genetic information. 3. Effects on social institutions. Genetic tests reveal information about individuals and their families. Thus, test results can affect the dynamics within social institutions, particularly the family. 4. Conceptual and philosophical implications regarding human responsibility, free will visà-vis genetic determinism, and the concepts of health and disease. 3.Gene therapy Gene therapy may be used for treating, or even curing, genetic and acquired diseases like cancer and AIDS by using normal genes to supplement or replace defective genes or to bolster a normal function such as immunity. It can be used to target somatic cells (i.e., those of the body) or gamete (i.e., egg and sperm) cells. In somatic gene therapy, the genome of the recipient is changed, but this change is not passed along to the next generation. In contrast, in germline gene therapy, the egg and sperm cells of the parents are changed for the purpose of passing on the changes to their offspring. There are basically two ways of implementing a gene therapy treatment: 1. Ex vivo, which means "outside the body" – Cells from the patient's blood or bone marrow are removed and grown in the laboratory. They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the DNA of the cells. The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein. 2. In vivo, which means "inside the body" – No cells are removed from the patient's body. Instead, vectors are used to deliver the desired gene to cells in the patient's body. As of June 2001, more than 500 clinical gene-therapy trials involving about 3,500 patients have been identified worldwide. Around 78% of these are in the United States, with Europe having 18%. These trials focus on various types of cancer, although other multigenic diseases are being studied as 8 well. Recently, two children born with severe combined immunodeficiency disorder ("SCID") were reported to have been cured after being given genetically engineered cells. Gene therapy faces many obstacles before it can become a practical approach for treating disease.[18] At least four of these obstacles are as follows: 1. Gene delivery tools. Genes are inserted into the body using gene carriers called vectors. The most common vectors now are viruses, which have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists manipulate the genome of the virus by removing the disease-causing genes and inserting the therapeutic genes. However, while viruses are effective, they can introduce problems like toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, in order for gene therapy to provide permanent therapeutic effects, the introduced gene needs to be integrated within the host cell's genome. Some viral vectors effect this in a random fashion, which can introduce other problems such as disruption of an endogenous host gene. 2. High costs. Since gene therapy is relatively new and at an experimental stage, it is an expensive treatment to undertake. This explains why current studies are focused on illnesses commonly found in developed countries, where more people can afford to pay for treatment. It may take decades before developing countries can take advantage of this technology. 3. Limited knowledge of the functions of genes. Scientists currently know the functions of only a few genes. Hence, gene therapy can address only some genes that cause a particular disease. Worse, it is not known exactly whether genes have more than one function, which creates uncertainty as to whether replacing such genes is indeed desirable. 4. Multigene disorders and effect of environment. Most genetic disorders involve more than one gene. Moreover, most diseases involve the interaction of several genes and the environment. For example, many people with cancer not only inherit the disease gene for the disorder, but may have also failed to inherit specific tumor suppressor genes. Diet, exercise, smoking and other environmental factors may have also contributed to their disease. Практическое занятие №4 (3 часа) Тема: Biotechnology in Human genome project, Cloning Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. 1. Human genome project The Human Genome Project is an initiative of the U.S. Department of Energy ("DOE") and the National Institutes of Health ("NIH") that aims to generate a high-quality reference sequence for the entire human genome and identify all the human genes. The DOE and its predecessor agencies were assigned by the U.S. Congress to develop new energy resources and technologies and to pursue a deeper understanding of potential health and environmental risks posed by their production and use. In 1986, the DOE announced its Human Genome Initiative. Shortly thereafter, the DOE and National Institutes of Health developed a plan for a joint Human Genome Project ("HGP"), which officially began in 1990. The HGP was originally planned to last 15 years. However, rapid technological advances and worldwide participation accelerated the completion date to 2003 (making it a 13 year project). Already it has enabled gene hunters to pinpoint genes associated with more than 30 disorders. 2. Cloning Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed. There are two types of cloning: 9 1. Reproductive cloning. After a few divisions, the egg cell is placed into a uterus where it is allowed to develop into a fetus that is genetically identical to the donor of the original nucleus. 2. Therapeutic cloning. The egg is placed into a Petri dish where it develops into embryonic stem cells, which have shown potentials for treating several ailments. In February 1997, cloning became the focus of media attention when Ian Wilmut and his colleagues at the Roslin Institute announced the successful cloning of a sheep, named Dolly, from the mammary glands of an adult female. The cloning of Dolly made it apparent to many that the techniques used to produce her could someday be used to clone human beings. This stirred a lot of controversy because of its ethical implications. Микромодуль 3 – Agriculture and biotechnology Практическое занятие №5 (2 часа) Тема: Crop yield, Reduced vulnerability of crops to environmental stresses, Improved taste, texture or appearance of food, Reduced dependence on fertilizers, pesticides and other agrochemicals Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. 1. Agriculture a) Crop yield Using the techniques of modern biotechnology, one or two genes (Smartstax from Monsanto in collaboration with Dow AgroSciences will use 8, starting in 2010) may be transferred to a highly developed crop variety to impart a new character that would increase its yield. However, while increases in crop yield are the most obvious applications of modern biotechnology in agriculture, it is also the most difficult one. Current genetic engineering techniques work best for effects that are controlled by a single gene. Many of the genetic characteristics associated with yield (e.g., enhanced growth) are controlled by a large number of genes, each of which has a minimal effect on the overall yield. There is, therefore, much scientific work to be done in this area. b) Reduced vulnerability of crops to environmental stresses Crops containing genes that will enable them to withstand biotic and abiotic stresses may be developed. For example, drought and excessively salty soil are two important limiting factors in crop productivity. Biotechnologists are studying plants that can cope with these extreme conditions in the hope of finding the genes that enable them to do so and eventually transferring these genes to the more desirable crops. One of the latest developments is the identification of a plant gene, At-DBF2, from Arabidopsis thaliana, a tiny weed that is often used for plant research because it is very easy to grow and its genetic code is well mapped out. When this gene was inserted intotomato and tobacco cells (see RNA interference), the cells were able to withstand environmental stresses like salt, drought, cold and heat, far more than ordinary cells. If these preliminary results prove successful in larger trials, then At-DBF2 genes can help in engineering crops that can better withstand harsh environments. Researchers have also created transgenic rice plants that are resistant to rice yellow mottle virus (RYMV). In Africa, this virus destroys majority of the rice crops and makes the surviving plants more susceptible to fungal infections. 10 c) Increased nutritional qualities Proteins in foods may be modified to increase their nutritional qualities. Proteins in legumes and cereals may be transformed to provide the amino acids needed by human beings for a balanced diet. A good example is the work of Professors Ingo Potrykus and Peter Beyer in creating Golden rice (discussed below). d) Improved taste, texture or appearance of food Modern biotechnology can be used to slow down the process of spoilage so that fruit can ripen longer on the plant and then be transported to the consumer with a still reasonable shelf life. This alters the taste, texture and appearance of the fruit. More importantly, it could expand the market for farmers in developing countries due to the reduction in spoilage. However, there is sometimes a lack of understanding by researchers in developed countries about the actual needs of prospective beneficiaries in developing countries. For example, engineering soybeans to resist spoilage makes them less suitable for producing tempeh which is a significant source of protein that depends on fermentation. The use of modified soybeans results in a lumpy texture that is less palatable and less convenient when cooking. The first genetically modified food product was a tomato which was transformed to delay its ripening. Researchers in Indonesia, Malaysia, Thailand, Philippines and Vietnam are currently working on delayed-ripening papaya in collaboration with the University of Nottingham and Zeneca. Biotechnology in cheese production: enzymes produced by micro-organisms provide an alternative to animal rennet – a cheese coagulant – and an alternative supply for cheese makers. This also eliminates possible public concerns with animal-derived material, although there are currently no plans to develop synthetic milk, thus making this argument less compelling. Enzymes offer an animalfriendly alternative to animal rennet. While providing comparable quality, they are theoretically also less expensive. About 85 million tons of wheat flour is used every year to bake bread. By adding an enzyme called maltogenic amylase to the flour, bread stays fresher longer. Assuming that 10–15% of bread is thrown away as stale, if it could be made to stay fresh another 5–7 days then perhaps 2 million tons of flour per year would be saved. Other enzymes can cause bread to expand to make a lighter loaf, or alter the loaf in a range of ways. Практическое занятие №6 (3 часа) Тема: Production of novel substances in crop plants, Animal biotechnology, Bioremediation and biodegradation Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. 1. Production of novel substances in crop plants Most of the current commercial applications of modern biotechnology in agriculture are on reducing the dependence of farmers on agrochemicals. For example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein with insecticidal qualities. Traditionally, a fermentation process has been used to produce an insecticidal spray from these bacteria. In this form, the Bt toxin occurs as an inactive protoxin, which requires digestion by an insect to be effective. There are several Bt toxins and each one is specific to certain target insects. Crop plants have now been engineered to contain and express the genes for Bt toxin, which they produce in its active form. When a susceptible insect ingests the transgenic crop cultivar expressing the Bt protein, it stops feeding and soon thereafter dies as a 11 result of the Bt toxin binding to its gut wall. Bt corn is now commercially available in a number of countries to control corn borer (a lepidopteran insect), which is otherwise controlled by spraying (a more difficult process). Crops have also been genetically engineered to acquire tolerance to broad-spectrum herbicide. The lack of herbicides with broad-spectrum activity and no crop injury was a consistent limitation in crop weed management. Multiple applications of numerous herbicides were routinely used to control a wide range of weed species detrimental to agronomic crops. Weed management tended to rely on preemergence—that is, herbicide applications were sprayed in response to expected weed infestations rather than in response to actual weeds present. Mechanical cultivation and hand weeding were often necessary to control weeds not controlled by herbicide applications. The introduction of herbicidetolerant crops has the potential of reducing the number of herbicide active ingredients used for weed management, reducing the number of herbicide applications made during a season, and increasing yield due to improved weed management and less crop injury. Transgenic crops that express tolerance toglyphosate, glufosinate and bromoxynil have been developed. These herbicides can now be sprayed on transgenic crops without inflicting damage on the crops while killing nearby weeds. From 1996 to 2001, herbicide tolerance was the most dominant trait introduced to commercially available transgenic crops, followed by insect resistance. In 2001, herbicide tolerance deployed in soybean, corn and cotton accounted for 77% of the 626,000 square kilometres planted to transgenic crops; Bt crops accounted for 15%; and "stacked genes" for herbicide tolerance and insect resistance used in both cotton and corn accounted for 8%. Production of novel substances in crop plants Biotechnology is being applied for novel uses other than food. For example, oilseed can be modified to produce fatty acids for detergents, substitute fuels and petrochemicals. Potatoes, tomatoes, rice tobacco, lettuce, safflowers, and other plants have been genetically engineered to produce insulin and certain vaccines. If future clinical trials prove successful, the advantages of edible vaccines would be enormous, especially for developing countries. The transgenic plants may be grown locally and cheaply. Homegrown vaccines would also avoid logistical and economic problems posed by having to transport traditional preparations over long distances and keeping them cold while in transit. And since they are edible, they will not need syringes, which are not only an additional expense in the traditional vaccine preparations but also a source of infections if contaminated.[33] In the case of insulin grown in transgenic plants, it is wellestablished that the gastrointestinal system breaks the protein down therefore this could not currently be administered as an edible protein. However, it might be produced at significantly lower cost than insulin produced in costly bioreactors. For example, Calgary, Canada-based SemBioSys Genetics, Inc. reports that its safflower-produced insulin will reduce unit costs by over 25% or more and approximates a reduction in the capital costs associated with building a commercial-scale insulin manufacturing facility of over $100 million, compared to traditional biomanufacturing facilities. 2. Animal biotechnology In animals, biotechnology techniques are being used to improve genetics and for pharmaceutical or industrial applications. Molecular biology techniques can help drive breeding programs by directing selection of superior animals. Animal cloning, through somatic cell nuclear transfer (SCNT), allows for genetic replication of selected animals. Genetic engineering, using recombinant DNA, alters the genetic makeup of the animal for selected purposes, including producing therapeutic proteins in cows and goats. There is a genetically altered salmon with an increased growth rate being considered for FDA approval. 3.Bioremediation and biodegradation Biotechnology is being used to engineer and adapt organisms especially microorganisms in an effort to find sustainable ways to clean up contaminated environments. The elimination of a wide range of pollutants and wastes from the environment is an absolute requirement to promote a 12 sustainable development of our society with low environmental impact. Biological processes play a major role in the removal of contaminants and biotechnology is taking advantage of the astonishing catabolic versatility of microorganisms to degrade/convert such compounds. New methodological breakthroughs in sequencing, genomics, proteomics, bioinformatics and imaging are producing vast amounts of information. In the field of Environmental Microbiology, genome-based global studies open a new era providing unprecedented in silico views of metabolic and regulatory networks, as well as clues to the evolution of degradation pathways and to the molecular adaptation strategies to changing environmental conditions. Functional genomic and metagenomic approaches are increasing our understanding of the relative importance of different pathways and regulatory networks to carbon flux in particular environments and for particular compounds and they will certainly accelerate the development of bioremediation technologies andbiotransformation processes. Marine environments are especially vulnerable since oil spills of coastal regions and the open sea are poorly containable and mitigation is difficult. In addition to pollution through human activities, millions of tons of petroleum enter the marine environment every year from natural seepages. Despite its toxicity, a considerable fraction of petroleum oil entering marine systems is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCCB). МИКРОМОДУЛЬ 4 – Food biotechnology Практическое занятие №7 (2 часа) Тема: Biotechnology- Significance in food production. Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. The significance of the application of modern biotechnology in the area of food production and its resultant impact in terms of human health and development can not be undermined. As the world is faced with ever increasing population and more and more food shortage and regional imbalances, new technologies and techniques are being developed to enhance production and increase the shelf life of perishable items. It is in this direction that new research initiatives in the field of green biotechnology are being made to enhance productivity and nutrition value of food items. Foods produced through modern biotechnology can be categorized as follows: 1. Foods consisting of or containing living/viable organisms, e.g. maize. 2. Foods derived from or containing ingredients derived from Genetically Modified Organisms (GMOs), e.g. flour, food protein products, or oil from GM soybeans, wheat etc. 3. Foods containing single ingredients or additives produced by GM microorganisms (GMMs), e.g. colours, vitamins and essential amino acids. 4. Foods containing ingredients processed by enzymes produced through GMMs, e.g. highfructose corn syrup produced from starch, using the enzyme glucose isomerase (product of a GMM). The first genetically modified food item -GM food (delayed-ripening tomato) was introduced on the US market in the mid-1990s. Since then, GM strains of maize, soybean, rape and cotton have been adopted by a number of countries and marketed internationally. In addition, GM varieties of papaya, potato, rice, squash and sugar beet have been trialed or released. It is estimated that GM crops cover almost 4% of total global arable land. The development of GM organisms has revolutionized the scenario of world food production. It has also offered the potential for increased agricultural productivity or improved nutritional value that can contribute directly to enhancing human health and development. From a health perspective, there 13 may also be indirect benefits, such as reduced agricultural chemical usage and enhanced farm income, and improved crop sustainability and food security, particularly in developing countries. While the introduction of GM crops has undoubtedly changed the agricultural scenario and led to significant impact on human development, it has also raised various social, cultural and ethical issues and reluctance on the part of various countries and governments to accept the GM food even in times of grave needs such as famine and drought. While some countries have established premarket regulatory standards for risk assessment of each and every food item before being launched in the market for application, there may be a case of a consistent and uniform international regulatory structure so as to ensure that such food items conform to a set of standards which are fair and equitable. This will also help quell a number of misgivings and doubts in the minds of countries yet to benefit from these food items. Практическое занятие №8 (2 часа) Тема: How Biotech Has Changed What We Eat. Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. Developments in food biotechnology have had numerous impacts on how food is produced, packaged, tested and preserved. Many of the changes have meant undisputable improvements to our safety and health, while others are more controversial. 1. Potable Water Testing With so many people worldwide suffering from diseases caused by contaminated water, any improvements to potable water testing make the top of the list. Recombinant gene technology methods are being developed to test water for safe drinking. Cryptosporidium parvum (Crypto), is a waterborne pathogen that produces spores, making it difficult to remove by boiling or chemical treatments. However, Crypto can be detected using bioassays that incorporate monoclonal antibodies. 2. Increased Nutritional Value Certain food crops are being altered, using methods to control gene expression, so they produce higher concentrations of known nutrients and disease-fighting compounds. An example of this is tomatoes bred to produce higher amounts of lycopene, a compound that has been linked to lower blood cholesterol levels, and lower risk of breast and prostate cancers. 3. Higher Quality Crops Agricultural biotechnology research has resulted in the development of many Pathogen-resistant crops, able to fight disease and produce increase yields and/or improved quality. While some quality enhancements are purely cosmetic, others that increase yields could result in more food for impoverished nations. Since the introduction of the controversial transgenic BT-corn, a multitude of new genetically altered crops have been developed for resisting disease caused by fungi, molds and insects. Some of the means of engineering resistance include cloning of genes for recombinant or pathogen-related proteins into plants, or for antisense and siRNAs that block pathogenesis. 4. Packaging To Reduce Spoilage 14 Plastic wraps that prevent food from spoiling inhibit the growth of bacteria, and some are even edible! Natural antibiotic substances derived from sources such as cloves, oregano, thyme and paprika have been combined with controlled-release biodegradable polymers (smart polymers) to create plastics that can prevent biofilm formation. 5. Reduced Health Risk Some plants that are used to produce vegetable oils are being genetically modified so the fatty acids we extract from them are better for our health. Plants have been altered to produce more linoleic acid, the beneficial fatty acid found in fish. In others, genetic modifications have been done to reduce the saturated fatty acids they produce. One example of a plant with altered gene expression to improve the quality of the product, is the soybean that has been developed to produce more stearic acid, thus giving soybean oil better heat stability, to match the properties of trans-hydrogenated fatty acids. With this alteration, less hydrogenated oils can be utilized for the same traditional purposes as hydrogenated oils. Практическое занятие №9 (3 часа) Тема: BIOENGINEERED PLANTS Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. Genetic engineering methods have been extensively used to increase the quantity of different nutrients (vitamins, essential amino acids, minerals, and phytochemicals) and enhance their availability in plants. There are two main methods to transfer genes into plants for production of transgenic plants: Agrobacterium-mediated transformation and microprojectile bombardment. In the Agrobacteriummediated transformation method, a genetically engineered strain of Agrobacterium tumefaciens is used to transfer the transgene into the plants. Some strains of A. tumefaciens have the natural ability to transfer a segment of their own DNA into plants for inducing crown-gall tumors. These crown gallinducing wild-type strains of A. tumefaciens have a Ti (tumor inducing) plasmid that carries the genes for tumor induction. During the process of infection, Agrobacterium transfers a segment of Ti plasmid, known as T-DNA, to plant cells (Willmitzer et al. 1983). The Ti plasmid can be engineered into a twoplasmid (binary) system containing a “disarmed” Ti plasmid in which the T-DNA has been deleted, and a small plasmid, which is referred to as a binary cloning vector, containing an “engineered” TDNA segment. The disarmed Ti plasmid, which is maintained in an A. tumefaciens strain, serves as a helper, providing the transfer function for the engineered T-DNA, which contains a target gene and a plant selectable marker gene inserted between the T-DNA left and right borders. When the A. tumefaciens containing the disarmed Ti plasmid and the binary cloning vector is grown in the presence of acetosyringone, the Agrobacterium vir (virulence) gene proteins are produced, which help to transfer the engineered T-DNA region of the binary cloning vector to the plant cells (Zambryski 1988). The Agrobacterium-mediated transformation is the most commonly used method for genetic engineering of plants. The microprojectile bombardment method, also known as the gene gun or biolistic transformation method, involves the delivery and expression of foreign DNA in individual plant cells directly (Klein et al. 1987). It has been proven to be a powerful method for transforming a large number of plant species, including monocots, which are often difficult to transform using A. tumefaciens (Vain et al. 1995). In this method, tungsten or gold spherical particles, approximately 4 μm in size, are coated with DNA and accelerated to high speed into plant cells using a biolistic particle delivery system or a gene gun. Once the DNA gets inside a cell, it integrates into the plant DNA through some unknown process. It is not known whether integration of DNA into the chromosome 15 requires the delivery of the microprojectiles into the plant nucleus. The microprojectile bombardment method has been used to transfer genes into various plant sections used in tissue culture regeneration, calli, cell suspensions, immature embryos, and pollens in a wide range of plant species. This method can also be used to transfer genes into chloroplasts and mitochondria, which cannot be accomplished by the A. tumefaciens-mediated gene transfer (Southgate et al. 1995). Микромодуль 5 – ESSENTIAL VITAMINS Практическое занятие №10 (2 часа) Тема: Vitamin A, Vitamin C, Vitamin E. Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. Nearly two-thirds of the world population depends on rice as their major staple, and among them an estimated 300 million suffer from some degree of vitamin A deficiency (WHO 1997). This is a serious public health problem in a number of countries, including highly populated areas of Asia, Africa, and Latin America. The rice endosperm (the starchy interior part of the rice grain) does not contain any ß-carotene, which is the precursor for vitamin A. Vitamin A is a component of the visual pigments of rod and cone cells in the retina, and its deficiency causes symptoms ranging from night blindness to total blindness. In Southeast Asia, it is estimated that a quarter of a million children go blind each year because of this nutritional deficiency. Plant foods such as carrots and many other vegetables contain ß-carotene. Each ß-carotene molecule is oxidatively cleaved in the intestine to yield two molecules of retinal, which can be then reduced to form retinol or vitamin A (Fig. 3.1). Ingo Potrykus from the Swiss Federal Institute of Technology, Zurich, Switzerland and Peter Beyer from the University of Freiburg recently developed transgenic rice, expressing genes for ßcarotene biosynthesis in rice grains (Potrykus 2001). Rice endosperm naturally contains geranlylgeranyl pyrophosphate (GGPP), which is a precursor of the pathway for β-carotene biosynthesis. GGPP can be converted into β-carotene in four steps (Bartley et al. 1994) (Fig. 3.2). The bacterial enzyme phytoene desaturase (EC 1.14.99.30) encoded by the crtI gene can substitute the functions of both phytoene desaturase and ζ-carotene desaturase (EC 1.14.99.30) in plants (Armstrong 1994). To reduce the number of genes transformed into rice for the β-carotene pathway, the researchers used the crtI gene from the bacterium Erwinia uredovora (Ye et al. 2000). The psy gene encoding phytoene synthase (EC 2.5.1.32) and the lcy gene encoding lycopene β-cyclase used for transformation originated from the plant daffodil. The plant psy gene (cDNA) and the bacterial crtI gene were placed under the control of the endosperm-specific rice glutelin (Gt1) promoter and the 35S CaMV promoter, respectively, and introduced in the binary plasmid pZPsC. Another plasmid, pZLcyH, was constructed by inserting the lcy gene from daffodil under the control of rice Gt1 promoter and the aphIV gene, for hygromycin resistance, under the control of 35S CaMV promoter. Plasmids pZPsC and pZLCyH were co-transformed into immature rice embryos by Agrobacteriummediated transformation (Ye et al. 2000). All hygromycin-resistant transformants were screened for the presence of the psy, crtI, and lcy genes by Southern hybridization. A few of the transformed plants produced β-carotene in the endosperm, which caused the kernel to appear yellow. The selected line contained 1.6-μg β-carotene per gram of endosperm, and was established as “golden rice.” Vitamin C or ascorbic acid, found in many plants, is an important component in human nutrition. It has antioxidant properties, improves immune cell and cardiovascular functions, prevents diseases linked to the connective tissue (Davey et al. 2000), and is required for iron utilization (Hallberg et al. 16 1989). Most animals and plants are able to synthesize ascorbic acid, but humans do not have the enzyme, L-gulono-1,4-lactone oxidoreductase (EC 1.1.3.8), necessary for the final step in ascorbic acid biosynthesis. For this reason, ascorbic acid needs to be consumed from dietary sources, especially from plants (Davey et al. 2000). The recent identification of ascorbic acid pathway in plants opened the way to manipulating its biosynthesis and allowed the design of bioengineered plants that produce ascorbic acid at significantly higher levels. The biosynthetic pathway of ascorbic acid differs from animals to plants. In plants, vitamin C biosynthesis can be accomplished in two ways. First, Dgalacturonic acid, which is released upon the hydrolysis of pectin (a major cell wall component), is converted into L-galactonic acid with the help of the enzyme D-galacturonic acid reductase (EC 2.7.1.44). L-galactonic acid is then readily converted into L-galactono-1,4-lactone, which is the immediate precursor of ascorbic acid (Fig. 3.3; Wheeler et al. 1998; Smirnoff et al. 2001). Researchers in Spain (Agius et al. 2003) isolated and characterized galUR, a gene in strawberry that encodes the enzyme D-galacturonic acid reductase. The galUR gene was amplified by PCR as a 956-bp fragment and cloned into a binary vector behind a 35S CaMV promoter. The resulting plasmid was transformed into E. coli and delivered to Agrobacterium by triparental mating. Finally, the GalUR gene was introduced into Arabidopsis thaliana plants via Agrobacterium-mediated transformation. The expression of the strawberry GalUR gene in A. thaliana allowed the bioengineered plants to increase the biosynthesis of ascorbic acid by two to three times compared with the wild-type plants (Agius et al. 2003). The second way by which plants synthesize vitamin C is through the recycling of used ascorbic acid (Fig. 3.4). During the first step of this recycling, ascorbic acid is oxidized forming a radical called monodehydroascorbate (MDHA). Once MDHA is formed, it can be readily converted back into ascorbic acid by the enzyme monodehydroascorbate reductase (MDHAR) (EC 1.6.5.4), or further oxidized forming dehydroascorbate (DHA). DHA can then undergo irreversible hydrolysis or be recycled to ascorbic acid by the enzyme dehydroascorbate reductase (DHAR) (EC 1.8.5.1), which uses the reductant glutathione (GSH) (Washko et al. 1992; Wheeler et al. 1998; Smirnoff and others 2001). Researchers from the University of California, Riverside, hypothesized that by enhancing the expression of DHAR in plants, they could increase ascorbic acid synthesis, because a more efficient ascorbate recycling process would be achieved (Chen et al. 2003). To test their hypothesis, they isolated DHAR cDNA from wheat and expressed the gene in tobacco and maize plants. Tobacco plants were transformed by using Agrobacterium. A His tag was added to DHAR, which was then introduced in the binary vector pBI101, behind a 35S CaMV promoter. For maize, a DHAR without a His tag was placed under the control of the maize ubiquitin (Ub) promoter or the Shrunken 2 (Sh2) promoter in the pACH18 vector. Transgenic maize was generated by particle bombardment of the embryogenic callus. DHAR expression was amplified up to 32 times in tobacco, and up to 100 times in maize, resulting in an increased ascorbic acid levels of up to four-fold in the bioengineered plants (Chen et al. 2003). Vitamin E is a broad term used to describe a group of eight lipid-soluble antioxidants in the tocotrienol and tocopherol families that are synthesized by photosynthetic organisms, mainly plants (Hess 1993). Both tocotrienol and tocopherol families can be distinguished into four different forms each (α, β, γ, δ), based on the number and position of methyl groups in the aromatic ring (Kamal-Eldin and Appelqvist 1996). Tocotrienols and tocopherols protect plants against oxidative stresses and the antioxidant property of these molecules adds functional qualities to food products (Andlauer and Furst 1998). Vitamin E is an important component of mammalian diet, and excess intake has been shown to produce many beneficial therapeutic properties, including reduction of cholesterol levels, inhibition of breast cancer cell growth in vitro, decrease risk of cardiovascular diseases, and decrease incidence of many human degenerative disorders (Theriault et al. 1999). Tocotrienols have more powerful antioxidant properties than tocopherols but are not absorbed as readily. The predominant forms of vitamin E in leaves and seeds are α-tocopherol and γ-tocotrienol, respectively (Munne´-Bosch and 17 Alegre 2002). While the biosynthesis of tocopherols and tocotrienols has been known for many years, the particular genes that encode for the different enzymes in the pathway have only recently been discovered. Researchers are trying to develop plants with increased vitamin E levels and some positive results have already been achieved. The first step in the pathway for the biosynthesis of both tocopherols and tocotrienols is the formation of homogentisic acid (HGA) from p-hydroxyphenyl-pyruvate, catalyzed by the enzyme phydroxyphenyl-pyruvate dioxygenase (HPPD) (EC 1.13.11.27) (Fig. 3.5) (Grusack and DellaPenna 1999). Tocotrienol and tocopherol biosynthesis in plants originates from two different precursors. Tocotrienols are produced from the condensation of HGA and geranylgeranyl diphosphate (GGDP), catalyzed by HGA geranylgeranyl transferase (HGGT) (EC 2.5.1.32), and tocopherols are formed from the condensation of HGA and phytyl diphosphate (PDP), catalyzed by HGA phytyl transferase (HPT) (EC 2.5.1.62) (Fig. 3.5) (Soll et al. 1980; Schultz et al. 1985; Collakova and DellaPenna 2001). Researchers from the Institute of Botany in Germany described the effects of constitutive expression of HPPD cDNA from barley (Hordeum vulgare) in tobacco plants. The HPPD gene was cloned into the pBinAR binary vector, in a SmaI cloning site located between the 35S CaMV promoter and the octopine synthase (EC 1.5.1.11) polyadenylation signal. The construct was then introduced into Agrobacterium GV3101, which was used to transform tobacco explants. The results showed that transgenic lines had a greater capacity for overall biosynthesis of homogentisic acid and produced a two-fold increase in the amount of vitamin E in the seeds. Vitamin E content in leaves was not affected (Falk et al. 2003). In another approach towards vitamin E enhancement, Cahoon et al. (2003) reported the identification and isolation of a novel monocot gene that encodes HGGT, which is so far the only known enzyme specific for the synthesis of tocotrienols. These researchers found that the expression of the barley HGGT enhanced the tocotrienol synthesis by 10- to 15-fold in the leaves of A. thaliana and by six-fold in the seeds of corn. The barley HGGT cDNA was placed under the control of the 35S CaMV promoter and the nopaline synthase terminator. The construct was inserted into the binary vector pZS199 to generate plasmid pSH24. The plasmid was then introduced into Agrobacterium for transformation into tobacco and A. thaliana (Cahoon et al. 2003). A third way by which vitamin E content in plants can be manipulated involves the last enzyme in the final step of tocotrienols and tocopherols biosynthetic pathway, in which γ-tocotrienol and γtocopherol are converted to α-tocotrienol and α-tocopherol, respectively. This step is catalyzed by the enzyme γ-tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) (Fig. 3.5) (Shintani and DellaPenna 1998). α-tocopherol has the highest oxidative property among the members of the vitamin E family (Kamal-Eldin and Appelqvist 1996). Unfortunately, plant oils, which are the main dietary source of vitamin E, contain only a fraction amount of α-tocopherol but a high level of its precursor, γtocopherol. Shintani and DellaPenna overexpressed endogenous A. thaliana γ-TMT to enhance conversion of γ-tocopherol into α-tocopherol. They introduced the γ-TMT cDNA construct under the control of a 35S CaMV promoter in a binary vector into A. thaliana plants by Agrobacterium-mediated transformation. α-tocopherol content of bioengineered seeds was nine-fold greater than that of the wild-type seeds (Shintani and DellaPenna 1998). Практическое занятие №11 (2 часа) Тема: Essential minerals. Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. 18 To maintain a well functioning, healthy body, humans require 17 different essential minerals in their diet. Minerals are inorganic ions found in nature and cannot be made by living organisms. They can be divided into two classes: macronutrients and micronutrients. Macronutrients are the minerals that we need in large quantity, including calcium, phosphorus, sodium, magnesium, chlorine, sulfur, and silicon. Micronutrients, or trace minerals, are the minerals that are required in small amounts, of which iron is the most prevalent, followed by fluorine, zinc, copper, cobalt, iodine, selenium, manganese, molybdenum, and chromium. Although a balanced consumption of plant-based foods should naturally provide these nutrients, mineral deficiency, especially of iron, is widespread among the world population. Iron Even though iron is required in trace amounts, it is the most widespread nutrient deficiency worldwide. It is believed that about 30% of the world population suffers from serious nutritional problems caused by insufficient intake of iron (WHO 1992). Iron is an important constituent of hemoglobin, the oxygen-carrying component of the blood, and is also a part of myoglobin, which helps muscle cells to store oxygen. Low iron levels can cause the development of iron deficiency anemia. In an anemic person the blood contains a low level of oxygen, which result in many health problems including infant retardation (Walter et al. 1986), pregnancy complication (Murphy et al. 1986), low immune function (Murakawa et al. 1987), and tiredness (Basta et al. 1979). Iron is present in food in both inorganic (ferric and ferrous) and organic (heme and nonheme) forms. Heme iron, which is highly bioavailable, is derived primarily from the hemoglobin and myoglobin of flesh foods such as meats, fish, and poultry (Taylor et al. 1986). In humans, reduced iron (ferrous) is taken up more readily than oxidized (ferric) iron. Several approaches have been used in the fight against iron deficiency including nutraceutical supplementation, food fortification, and different methods of food preparation and processing (Maberly et al. 1994). So far, none of these approaches have been successful in eradicating iron deficiency, especially in developing countries. A new tool in the fight against nutrient deficiency is the use of biotechnology to improve essential mineral nutrition in staple crops. At this time, there are basically two ways in which genetic engineering can be used for this purpose: (1) by increasing the concentration of the iron-binding protein ferritin and (2) by reducing the amount of iron-absorption inhibitor phytic acid. Although iron intake is important for human health, it can be toxic, so the ability to store and release iron in a controlled manner is crucial. The 450 kDa ferritin protein, found in animals, plants, and bacteria, can accumulate up to 4500 atoms of iron (Andrews et al. 1992). This protein consists of 24 subunits assembled into a hollow spherical structure within which iron is stored as a hydrous ferric oxide mineral core (Fig. 3.6). The two main functions of ferritin in living organisms are to supply iron for the synthesis of proteins such as ferredoxin and cytochromes and to prevent free radicals damage to cells. Studies have shown that ferritin can be orally administrated and is effective for treatment of rat anemia (Beard et al. 1996), suggesting that increasing ferritin content of cereals may solve the problem of dietary iron deficiency in humans. Japanese researchers (Goto et al. 1999) introduced soybean ferritin cDNA into rice plants, under the control of a seed specific promoter, GluB-1, from the rice seed-storage protein gene encoding glutelin. The two advantages of this promoter are the accumulation of iron specifically in the rice grain endosperm, and its ability to induce ferritin at a high level. The ferritin cDNA was isolated from soybean cotyledons, inserted into the binary vector pGPTV-35S-bar, and transferred into rice using Agrobacterium. The iron content of the rice seed in the transgenic plants was three times greater than that of the untransformed wild-type plants. Phytic acid, or phytate, is the major inhibitor of many essential minerals, including iron, zinc, and magnesium, and is believed to be directly responsible for the problem of iron deficiency (Ravindran et al. 1995). In cereal grains, phytic acid is the primary phosphate storage and it is 19 deposited in the aleurone storage vacuoles (Lott 1984). During seed germination, phytic acid is catalyzed into inorganic phosphorous, by the action of the hydrolytic enzyme phytase (EC 3.1.3.8) (Fig. 3.7). There is little or no phytase activity in the dry seeds or in the digestive tract of monogastric animals (Gibson and Ullah 1990; Lantzsch et al. 1992). In a recent study, it has been shown that phytase activity can be reestablished in mature dry seeds under optimum pH and temperature conditions (Brinch-Pedersen et al. 2002). A reduction in the amount of phytic acid in staple foods is likely to result in a much greater bioavailability of iron and other essential minerals. Lucca et al. (2002) inserted a fungal (Aspergillus fumigatus) phytase cDNA into rice to increase the degradation of phytic acid. Rice suspension cells, derived from immature zygotic embryos, were used for biolistic transformation with the A. fumigatus phytase gene. Phytase from A. fumigatus was the enzyme of choice because it is heat stable and thus can refold into an active form after heat denaturation (Wyss et al. 1998). The main purpose of this research was to increase phytase activity during seed germination and to retain the enzyme activity in the seed after food processing and in the human digestive tract. Although the researchers achieved high expression levels of phytase in the rice endosperm, by placing it under the control of the strong tissue-specific globulin promoter, the thermotolerance of the transgenic rice was not as high as expected. It has been speculated that the reason for this unexpected low thermostability of the A. fumigatus phytase in transgenic rice is due to the interference of the cellular environment of the endosperm to maintain the enzyme in an active configuration (Holm et al. 2002). Further studies are needed to develop an endogenous phytase enzyme that is thermostable and maintains high activity in plant tissues. Практическое занятие №12 (2 часа) Тема: Essential amino acids. Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. Proteins are organic molecules formed by amino acids. The digestive system breaks down proteins into single amino acids so that they can enter into the bloodstream. Cells then use the amino acids as building blocks to form enzymes and structural proteins. There are two types of amino acids, essential and nonessential. Essential amino acids cannot be synthesized by animals, including humans, therefore, need to be acquired in the diet. The nine essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The body can synthesize nonessential amino acids as long as there is a proper intake of essential amino acids and calories. Proteins are present in foods in varying amounts, some foods have all nine essential amino acids in them, and they are referred to as complete proteins. Most animal products (meat, milk, eggs) provide a good source of complete proteins. Vegetables sources, on the other hand, are usually low on or missing certain essential amino acids. For instance, grains tend to lack lysine while pulses are short in methionine (Miflin et al. 1999). In order to provide better nutrition from plant sources, it is essential to increase the content of essential amino acids in seed and tuber proteins. This is particularly important for countries where certain crops, such as rice, potatoes, and corn, are the main dietary source. Lysine Rice is one of the most important staple crops and is consumed by 65% of the world population on a daily basis (Lee et al. 2003). It is a good source of essential nutrients such as vitamins B1 (thiamin), B2 (riboflavin), B3 (niacin), but it is low in the essential amino acids, lysine and isoleucine (Fickler 1995). Adequate intake of lysine is essential because it serves many important functions in the body including aiding calcium absorption, collagen formation, and the production of antibodies, hormones, and enzymes. A deficiency in lysine may result in tiredness, inability to concentrate, 20 irritability, bloodshot eyes, retarded growth, hair loss, anemia, and reproductive problems (Cooper 1996). Zheng et al. (1995) developed a transgenic rice with enhanced lysine content. They accomplished this by expressing the seed storage protein β-phaseolin from the common bean (Phaseolus vulgaris) in the grain of transgenic rice. The genomic and cDNA sequences of the βphaseolin gene from P. vulgaris was placed under the control of either a rice seed-specific glutelin Gt1 promoter or the native β-phaseolin promoter. The vectors containing the β-phaseolin gene were transferred into the rice chromosome by protoplast-mediated transformation. Four percent of total endosperm protein in the transgenic rice was phaseolin, which resulted in a significant increase in the lysine content in rice (Zheng et al. 1995). Methionine and Tyrosine In terms of global food production, potato (Solanum tuberosum) is only behind rice, wheat, and corn on the list of the crop species that are most important for human nutrition worldwide (Chakraborty et al. 2000). There are four main purposes for the production of potatoes: for the fresh food market, for animal feed, for the food processing industry, and for nonfood industrial uses such as manufacture of starch and alcohol (Chakraborty et al. 2000). Potato is a good source of potassium, iron, vitamin C and B, but it is not a rich protein source. Potato proteins are limited in nutritive value for the lack of the amino acids lysine, methionine, and tyrosine (Jaynes et al. 1986). A lack of methionine in a person’s diet may result in an imbalanced uptake of other amino acids, as well as retardation in growth and development. Methionine is also the main supplier of sulfur, which prevents disorders of the hair, skin, and nails, helps lower cholesterol levels by increasing the liver’s production of the phospholipid lecithin, and is a natural chelating agent for heavy metals (Cooper 1996). Scientists from the National Center for Plant Genome Research in India isolated and cloned a gene that encodes for a seed-specific protein from Amaranthus hypocondriacus called amaranth seed albumin (AmA1) (Chakraborty et al. 2000). The advantages of using the AmA1 seed-protein to improve crops nutritional value are that (1) it is well-balanced in the composition of all essential amino acids, (2) it is a non-allergenic protein, and (3) it is encoded by a single gene AmAl. This gene was cloned into a binary vector, under the control of a constitutive 35S CaMV promoter (plasmid pSB8) and a tuber specific granule-bound starch synthase (EC 2.4.1.21) promoter (plasmid pSB8G). The AmAl gene constructs from these two binary plasmids were introduced into potato through Agrobacterium-mediated transformation. The amino acid contents in the pSB8-transgenic potato showed a 2.5- to 4-fold increase in lysene, methionine, and tyrosine, while the tissue-specifc pSB8Gtransgenic potatoes showed a four to eight-fold increase in these amino acids (Fig.3.8). Практическое занятие №13 (2 часа) Тема: Modified milk in transgenic dairy cattle. Задание: 1. Прочитать текст. 2. Перевести текст. 3. Выписать новые слова. Bovine milk has been described as an almost perfect food, because it is a rich source of vitamins, calcium, and essential amino acids (Karatzas and Turner 1997). Some of the vitamins found in milk include vitamin A, B, C, and D. Milk has greater calcium content than any other food source, and daily consumption of two servings of milk or other dairy products supplies all the calcium requirements of 21 an adult person (Rinzler et al. 1999). Caseins represent about 80% of the total milk protein and have high nutritional value and functional property (Brophy et al. 2003). The caseins have a strong affinity for cations such as calcium, magnesium, iron, and zinc. There are four types of naturally occurring caseins in milk, αS1, αS2, β, and κ (Brophy et al. 2003). They are clumped in large micelles, which determine the physicochemical properties of milk. Even small variations in the ratio of the different caseins influence micelles structure, which in turn can change the milk’s functional properties. The amount of caseins in milk is an important factor for cheese manufacturing, since greater casein content results in greater cheese yield and improved nutritional quality (McMahon and Brown 1984). It has been estimated that by enhancing the casein content in milk by 20% would result in an increase in cheese production, generating an additional $190 million/year for the dairy industry (Wall et al. 1997). Dairy cattle have only one copy of the genes that encode α (s1/s2), β, and κ-casein proteins, and out of the four caseins, κ and β are the most important (Bawden et al. 1994). Increased milk κ-casein content reduces the size of the micelle resulting in improved heat stability. β-caseins are highly phosphorylated and bind to calcium phosphate, thus influencing milk calcium levels (Dalgleish et al. 1989; Jimenez Flores and Richardson 1988). Research on modification of milk composition to improve nutritional or functional properties has been mostly done in transgenic mice. Mice are good models for the study of protein expression in mammary glands, but they do not always reflect the same protein expression levels as ruminants (Colman 1996). Brophy et al. (2003), using nuclear transfer technology, produced transgenic cows carrying extra copies of the genes CSN2 and CSN3, which encode bovine β- and κcaseins, respectively. Genomic clones containing CSN2 and CSN3 were isolated from a bovine genomic library. Previous studies conducted with mice revealed that CSN3 had very low expression levels (Persuy et al. 1995). In order to enhance expression of CSN3, the researchers created a CSN2/3fusion construct, in which the CSN3 gene was fused with the CSN2 promoter. The CSN2 genomic clone and the CSN2/3-fusion construct were co-transfected into bovine fetal fibroblast (BFF) cells, where the two genes showed coordinated expression. The transgenic cells became the donor cells in the process of nuclear transfer, generating nine fully healthy and functional cows. Overexpression of CSN2 and CSN2/3 in the transgenic cows resulted in an 8-20% increase in β-casein and 100% increase in κ-casein levels (Brophy et al. 2003). 22