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SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Cloning in animals and plants Learning objectives: Outline the differences between reproductive and non-reproductive cloning; Describe the production of natural clones in plants using the example of vegetative propagation in elm trees; Describe the production of artificial clones of plants from tissue culture; Discuss the advantages and disadvantages of plant cloning in agriculture; Describe how artificial clones of animals can be produced; Discuss the advantages and disadvantages of cloning animals; Key definitions: Compile a glossary by writing your own definitions for the following key terms related to the learning objectives above. Key term vegetative propagation tissue culture explant callus totipotent stem cells cloned animal Definition SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Vegetative propagation of plants Many flowering plants are able to reproduce asexually and spread quickly through vegetative means e.g. through runners and suckers. Humans exploit this ability in the vegetative propagation of plants by methods such as cutting and grafting. Such methods result in genetically identical plants (clones) year after year. Clones are produced for several reasons, e.g. to obtain a uniform plant performance (as in fruit trees), to multiply sterile or seedless species, or to propagate species with flowers in which the stamens have changed to petals and there is no pollen produced. In general, artificial propagation is a more efficient way to multiply certain kinds of plants because it produces a larger plant faster than one raised from seed and it avoids seed dormancy. New varieties can be developed by grafting, which combines the favourable characteristics of two existing varieties. Cutting: cutting is a method of propagation where a vegetative structure is removed from a parent plant and grown as a new individual. Cuttings are successfully used to propagate herbaceous plants, but can be used on woody plants with the use of hormones that promote root growth. Grafting: grafting is a procedure by which the structures of two or more plants are joined. Typically a twig section (scion) from one plant is joined to the shoot of another (the rootstock). Grafting is used for many fruit and landscape trees because it avoids juvenility, and the special properties of the rootstock and the scion are able to be incorporated into the same plant. Plant tissue culture Plant tissue culture, or micropropagation, is a method used for cloning plants. It is widely used for the rapid multiplication of commercially important plant species with superior genotypes, as well as in the recovery programmes for endangered plant species. Plant productivity and quality may be rapidly improved, and resistance to disease, pollutants and insects increased. Continued culture of a limited number of cloned varieties leads to a change in the genetic composition of the population (genetic variation is reduced). New genetic stock may be introduced into cloned lines periodically to prevent this reduction in genetic diversity. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Micropropagation is possible because differentiated plant cells have the potential to give rise to all the cells of an adult plant. It has considerable advantages over traditional methods of plant propagation, but it is very labour intensive. In addition, the optimal conditions for growth and regeneration must be determined and plants propagated in this way may be genetically unstable or infertile, with chromosomes structurally altered or in unusual numbers. The success of tissue culture is affected by factors such as selection of explant material, the composition of the culturing media, plant hormone levels, lighting and temperature. 1. Stock plants are kept as free from pests and pathogens as possible. 2. Small pieces are cut (excised) from the plant. These pieces, called explants, may be stem tissue with nodes, flower buds, leaves or tiny sections of shoot tip meristems. 3. The surfaces of explants are sterilised using solutions such as sodium hypochlorite. 4. The explants are transferred to a culture vessel under sterile conditions. 5. Incubation of culture vessels: Duration: 3-9 weeks Temperature: 15-30°C Light regime: 10-14 hours per day NOTE: Different kinds of hormones in culture media produce different growth responses. By changing the relative levels of several plant hormones, the formation of callus, roots and shoots can be initiated. 6. An undifferentiated mass of cells known as a callus develops. Growth medium: contains nutrients and growth regulators (plant hormones such as auxins, gibberellins and cytokinins) set in an agar gel. 7. New shoots that develop are removed from the explant and placed on new culture medium. The process is repeated every few weeks so that a few plants can give rise to millions of plants. 8. Tissue culture plants must be acclimatised in special glasshouses before they can be planted outside. 9. Plant cell culture: if the callus is suspended in a liquid nutrient medium and broken up mechanically into individual cells it forms a plant cell culture that can be maintained indefinitely. Advantages of tissue culture Possible to create large numbers of clones from a single seed or explant. Selection of desirable traits is possible directly from the culturing setup (in vitro), decreasing the amount of space required for field trials. Reproduction of plants is possible without having to wait for the onset of seed production. Rapid propagation is possible for species that have long generation times, low levels of seed production, or seeds that do not readily germinate. Enables the preservation of pollen and cell collections from which from which plants may be propagated (like a seed bank). Allows the international exchange of sterilised plant materials (eliminating the need for quarantine). Helps to eliminate plant diseases through careful stock selection and sterile techniques during propagation. Overcomes seasonal restrictions for germination. Enables cold storage of large numbers of viable plants in a small space. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Cloning animals Cloning by embryo splitting Livestock breeds frequently produce only one individual per pregnancy and all individuals in a herd will have different traits. Cloning (by embryo splitting or other means) makes it possible to produce high value herds with identical traits more quickly. This technique also has applications in the medical field e.g. in the cloning of embryonic stem cells. Such applications demonstrate the advances made recently in cloning technology. Some of the most ambitious medical projects now being considered involve the production of universal human donor cells. Scientists know how to isolate undifferentiated stem cells from early embryos in mice. They are also learning how to force stem cells to differentiate into different tissues. Such techniques may make it possible to manufacture cells or replace tissues damaged by illness e.g. muscular dystrophy or diabetes. Individually matched stem cells could be made by transferring the nucleus from one of the patient’s cells into a human egg to create an embryo. The embryo would be allowed to develop only to a stage where stem cells could be separated and cultured from it. Although the embryo would consist of only a few hundred cells, there would be many ethical issues raised by this technique. Egg cells are removed from an animal and fertilised in a Petri dish. At the first stage of development, one of these fertilised eggs divides into two. The cloning process (forming the twin) begins here. The zona pellucida is removed with an enzyme and the two cells are separated. An artificial zona is added, allowing development to proceed. The cells continue to divide, forming genetically identical embryos. These are implanted into surrogates. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Cloning by nuclear transfer Clones are genetically identical individuals produced from one parent. Cloning is not new; it has been used in plant breeding for years. In recent years clones have been produced from both embryonic and non-embryonic cells using standard nuclear transfer techniques. In 2004, Australian genetic researchers successfully cloned a cow (called Brandy) using serial nuclear transfer (SNT) which involves an extra round of nuclear transfer to improve the reprogramming of the fused donor cells. In animal reproductive technology, cloning has facilitated the rapid production of genetically superior stock. These animals may then be dispersed among commercial herds. The primary focus of the new cloning technologies is to provide an economically viable way to rapidly produce transgenic animals with very precise genetic modification. Creating Dolly using standard nuclear transfer Dolly, the Finn Dorset lamb born at the Roslin Institute (near Edinburgh) in July 1996, was the first mammal to be cloned from non-embryonic cells. Nuclear transfer has been used successfully to clone cells from embryonic tissue, but Dolly was created from a fully differentiated udder cell from a six year old ewe. This cell was made quiescent and then ‘tricked’ into re-entering an embryonic state. Dolly’s birth was a breakthrough, because it showed that the processes leading to cell specialisation are not irreversible; even specialised cells can be ‘reprogrammed’ into an embryonic state. While cloning seems relatively easy to achieve using this method, Dolly’s early death has raised concerns that the techniques could have caused premature ageing. Although there is, as yet, no evidence for this, the long term viability of animals cloned from non-embryonic cells has still to be established. Dolly the sheep was euthanased on February 14th, 2003 after examinations showed she had developed progressive lung disease. Dolly was six years old; half the normal life expectancy of sheep. A post-mortem examination showed that she succumbed to a viral infection, not uncommon in older sheep, especially those housed inside. Despite the concerns of some scientists, there is no evidence that cloning was a factor in Dolly contracting the disease. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Donor cells taken from udder: cells from the udder of a Finn Dorset ewe were cultured in low nutrient medium for a week. The nutrient deprived cells stopped dividing, switched off their active genes, and became dormant. Unfertilised egg has nucleus removed: in preparation for the nuclear transfer, an unfertilised egg was taken from a Scottish Blackface ewe. Using micromanipulation techniques, the nucleus containing the DNA, was removed. This left a recipient egg cell with no nucleus, but an intact cytoplasm and the cellular machinery for producing an embryo. Cells are fused: the two cells (the dormant donor cell and the recipient egg cell) were placed next to each other and a gentle electric pulse causes them to fuse together (like soap bubbles). Cell division is triggered: a second electric pulse triggers cellular activity and cell division, effectively jump-starting the cell into production of an embryo. This reaction can also be triggered by chemical means. After six days, the resulting embryo was surgically implanted into the uterus of the surrogate mother; another Scottish Blackface ewe. Of the hundreds of reconstructed eggs, only 29 successfully formed embryos, and only one Dolly survived to birth. Birth: after a gestation of 148 days, the pregnant blackface ewe gave birth to Dolly, the Finn Dorset lamb that is genetically identical to the original donor. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 1. Human populations have herded cattle for milk for around 9000 years. Artificial selection over this time has resulted in the modern dairy cow. (a) State three phenotypic traits (characteristics) that have been selected for in dairy cows. 1 ____________________________________________________________ 2 ____________________________________________________________ 3 _________________________________________________________ [3] (b) Fig. 1.1 shows the pattern of variation of a phenotypic trait in a herd of dairy cows. The shaded part of the graph indicates those cows that are chosen to breed. Draw, on Fig. 1.1, a second curve to show the pattern of variation in the next generation. (c) In recent years, artificial selection of dairy cows has been helped by modern reproductive technology. Name two modern techniques or procedures that can be used in the selective breeding of dairy cows. 1 ____________________________________________________________ 2 _________________________________________________________ [2] (d) Lactase is an enzyme that is necessary to digest lactose sugar in milk. In some parts of the world, animals are not farmed for milk and no dairy products are eaten. Adult humans that are native to these parts of the world do not produce lactase. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY In areas where animals are farmed for milk, native adult humans do not produce lactase. In these populations, a new allele has arisen by gene mutation. State what is meant by gene mutation. _____________________________________________________________ ___________________________________________________________ [1] (e) Over time, the frequency of this new allele increased in the gene pool of the human populations, whose diet included milk. Name the process by which this increase occurred. ___________________________________________________________ [1] (f) All human babies produce the enzyme lactase. The genetic change that allows adults to produce this enzyme is thought to involve a mutation in a regulatory gene. This mutation causes the structural gene to be expressed in adults. Distinguish between the terms ‘regulatory gene’ and ‘structural gene’. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (g) Adult humans who cannot produce the enzyme lactase are described as lactose intolerant and cannot drink milk without experiencing health problems. However, lactose intolerant people can safely eat yoghurt. Yoghurt is produced from milk that is fermented by bacteria. These bacteria perform anaerobic respiration, using carbohydrate as their respiratory substrate. Suggest why yoghurt is a suitable food for lactose-intolerant people. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (h) The control of the expression of the lac operon genes, which allow uptake and digestion of lactose in the bacterium Escherichia coli, is well known. Fig. 1.2 shows the arrangement of the elements of the lac operon. Describe how genes Z and Y are switched on in bacteria that are moved to a nutrient medium that contains lactose. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [3] 2. Molecular evidence has shown that all specimens of the English Elm tree, Ulmus procera, form a genetically isolated clone. English elms developed from a variety of elm brought to Britain from Rome in the first century A.D. Although English Elm trees make pollen, they rarely produce seeds. Instead they spread by developing structures known as suckers from their roots. Each sucker can grow into a new tree. This tendency of elms to create suckers has been exploited by humans, who have separated the suckers, with roots attached, and used them to plant hedges and establish new woodlands. (a) Suggest a technique that could be used to provide molecular evidence that all English Elm trees form a clone. ___________________________________________________________ [1] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (b) State why the English Elm clone is genetically isolated from other varieties of elm. _____________________________________________________________ ___________________________________________________________ [1] (c) State the name given to the process in which plants reproduce asexually by means such as suckers. ___________________________________________________________ [1] (d) In 1967, a new, virulent strain of elm disease fungus arrived in Great Britain on imported timber. Beetles that lived under the bark of elm trees spread the fungus. The saws used to cut down dead branches were not sterilised after use. When the saws were used to prune healthy trees, these trees became infected. Approximately 25 million elm trees, most of the English population, died within a few years of the arrival of this fungus. Explain why there was such a rapid loss of elm trees in Britain as a result of this elm disease. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [4] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (e) Elm trees respond to fungal infection by plugging their xylem vessels. The leaves on the upper branches of the tree then turn yellow and die. When most of the branches have lost their leaves and died, the roots are weakened and my also die. Explain why the plugging of xylem vessels will result in the leaves of the upper branches turning yellow. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (f) Explain why the loss of leaves from the tree may result in the death of the tree’s roots. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (g) Many ornamental plants for gardens can be cloned by tissue culture. Describe the process of cloning plants by tissue culture. In your answer, you should use appropriate technical terms, spelled correctly ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [7] 3. Rhubarb, Rheum x hybridum, is a plant that is grown for its edible stems. In spring, the stems and leaves grow from fleshy roots which have survived the winter underground. Growers have developed many new varieties of rhubarb by growing plants from seed, choosing the best young plants and then asexually reproducing them. Seeds are produced by sexual reproduction and the rhubarb plants that grow from seed show variation in characteristics such as stem colour, dormancy period and the concentration of oxalic acid in their leaves. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (a) Outline the events that lead to genetic variation in gametes and in the plants grown from seed. ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [5] (b) Traditionally rhubarb plants have been produced by vegetative propagation. The best young rhubarb plants are allowed to grow for three seasons until their underground root systems are large enough. Then they are dug up in winter, the roots are cut into pieces and the pieces are replanted. Each piece is then able to grow into a new rhubarb plant that is identical to the parent. State the biotechnological term for this type of vegetative propagation. ___________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (c) A gardener wished to multiply his rhubarb plants using the traditional method, but he discovered that his plants were infected by a virus. Name the modern technique which allows commercial growers to produce large numbers of genetically identical plants that are also virus-free. ___________________________________________________________ [1] (d) Rhubarb plants must spend seven to nine weeks at a temperature below 3°C in order to break their winter dormancy and allow them to start growing stems and leaves again. The length of the cold period that is required depends on the variety of rhubarb. In the variety ‘Timperley Early’, the length of the cold period is shorter, so the plants grow and produce a crop earlier in the year than the variety ‘Victoria’. Suggest two ways in which the varieties may differ from one another biochemically to account for the difference in the length of the cold period required by each. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (e) Rhubarb leaves contain oxalic acid, a relatively strong acid which is soluble in water and in alcohol. High concentrations of oxalic acid makes rhubarb leaves poisonous to humans and other animals. The amount of oxalic acid in the leaves varies according to the variety of rhubarb, the age of the plant and environmental factors. Suggest and plan an experiment to compare how the variety of rhubarb affects the amount of oxalic acid in rhubarb leaves. Include in your plan: the variables that you could control an outline of the experimental procedure you would use any measurements that you would make In your answer, you should use appropriate technical terms, spelled correctly ________________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [6] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (f) As rhubarb leaves are poisonous, they are cut off when the stems are harvested and may be left to decompose on the compost heap. Outline the role of decomposers in the decomposition of leaves. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [3] (g) An early harvest of rhubarb stems can be obtained by placing an upturned bin over the root when it comes out of dormancy, so the emerging shoots are kept in the dark. The shoots then grow more quickly to a height suitable for picking. Use your knowledge of plant growth regulators (plant hormones) to suggest why shoots kept in the dark grow taller than those left in the light. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Biotechnology Learning objectives: State that biotechnology is the industrial use of living organisms (or parts of living organisms) to produce food, drugs or other products; Explain why microorganisms are often used in biotechnological processes; Describe, with the aid of diagrams, and explain the standard growth curve of a microorganism in a closed culture; Describe how enzymes can be immobilised; Explain why immobilised enzymes are used in large-scale production; Compare and contrast the processes of continuous culture and batch culture; Describe the differences between primary and secondary metabolites; Explain the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield of product required; Explain the importance of asepsis in the manipulation of microorganisms; Key definitions: Compile a glossary by writing your own definitions for the following key terms related to the learning objectives above. Key term biotechnology culture primary metabolites secondary metabolites batch continuous asepsis aseptic technique Definition SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY The growth curve A small number of organisms placed in a fresh ‘closed culture’ environment will undergo population growth in a very predictable, standard way (see diagram above). A closed culture refers to the growth of microorganisms in an environment where all conditions are fixed and contained. No new materials are added and no waste products or organisms removed. Lag phase: organisms are adjusting to the surrounding conditions. This may mean taking in water, cell expansion, activating specific genes and synthesising specific enzymes. The cells are active but not reproducing so population remains fairly constant. The length of this period depends on the growing conditions. Log (exponential) phase: the population size doubles each generation as every individual has enough space and nutrients to reproduce. In some bacteria, for example, the population can double every 20-30 minutes in these conditions. The length of this phase depends on how quickly the organisms reproduce and take up the available nutrients and space. Stationary phase: nutrient levels decrease and waste products like carbon dioxide and other metabolites build up. Individual organisms die at the same rate at which new individuals are being produced. Note: in an open system, this would be the carrying capacity of the environment. Decline or death phase: nutrient exhaustion and increased levels of toxic waste products and metabolites lead to the death rate increasing above the reproduction rate. Eventually, all organisms will die in a closed system. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Industrial scale fermenters An industrial-scale fermenter is essentially a huge tank which may have a capacity of tens of thousands of litres. The growing conditions in it can be manipulated and controlled in order to ensure the best possible yield of the product. The precise growing conditions depend on the microorganisms being cultured, and on whether the process is designed to produce a primary or secondary metabolite. They are: temperature – too hot and enzymes will be denatured; too cool and growth will be slowed; type and time of addition of nutrient – growth of microorganisms requires a nutrient supply, including sources of carbon, nitrogen and any essential vitamins and minerals. The timing of nutrient addition can be manipulated, depending on whether the process is designed to produce a primary or secondary metabolite. oxygen concentration – most commercial applications use the growth of organisms under aerobic conditions, so sufficient oxygen must be made available. A lack of oxygen will lead to the unwanted products of anaerobic respiration and a reduction in growth rate. pH – changes in pH within the fermentation tank can reduce the activity of enzymes and so reduce growth rates; Such large cultures need large ‘starter’ populations of the microorganism. These are obtained by taking a pure culture and growing it in sterile nutrient broth. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY A batch culture, where the microorganism starter population is mixed with a specific quantity of nutrient solution, then allowed to grow for a fixed period with no further nutrient added. At the end of this period, the products are removed and the fermentation tank is emptied. Penicillin is produced using batch culture of Penicillium fungus. A continuous culture, where nutrients are added to the fermentation tank and products removed from the fermentation tank at regular intervals – or even, as the name suggests, continuously. Human hormones such as insulin are produced from continuous culture of genetically modified Escherichia coli bacteria. The nutrient medium in which the microorganisms grow could also support the growth of many unwanted microorganisms. Any unwanted microorganism is called a contaminant. Unwanted microorganisms: compete with the culture microorganisms for nutrients and space; reduce the yield of useful products from the culture microorganisms; may cause spoilage of the product; may produce toxic chemicals; may destroy the culture microorganisms and their products; in processes where foods or medicinal chemicals are being produced, contamination means that all products must be considered unsafe and so must be discarded. The term aseptic technique refers to the measures taken to ensure asepsis, that is, that contamination of the culture does not occur at any point from isolation of the initial culture, through scaling up, fermentation and product harvesting. Immobilising enzymes SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Depending on the way in which the desired end-product is produced, enzymes may be used as crude whole cell preparations or as cell-free enzyme extracts. Whole cell preparations are cost effective, and appropriate when the processes involved in production of the end product are complex, as in waste treatment and the production of semi-synthetic antibiotics. Cell free enzyme extracts are more expensive to produce, but can be a more efficient option overall. To reduce costs and improve the efficiency of product production, enzymes are sometimes immobilised within a matrix of some kind and the reactants are passed over them. Advantages of immobilised enzymes The enzymes can be used repeatedly and recovered easily (this reduces costs). The enzyme-free end-product is easily harvested. The enzymes are more stable due to the protection of a matrix. The life of some enzymes e.g. proteases, is extended by immobilisation. Disadvantages of immobilised enzymes The entrapment process may reduce the enzyme activity (more enzyme will be needed). Some methods offering high stability(e.g. covalent bonding) are harder to achieve. Immobilisation can be costly. Methods of enzyme immobilisation Micro-encapsulation: the enzyme is held within a membrane, or within alginate or polyacrylamide capsules. Lattice entrapment: enzyme is trapped in a gel lattice e.g. silica gel. The substrate and reaction products diffuse in and out of the matrix. Covalent attachment: enzyme is covalently bonded to a solid surface e.g. collagen or a synthetic polymer. Direct cross-linking: glutaraldehyde is used to cross-link the enzymes. They then precipitate out and are immobilised without support. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 1. The antibiotic penicillin is produced by batch culture of the fungus Penicillium chrysogenum. (a) Fig. 4.1 shows the concentration of penicillin, lactose and ammonia as well as the fungal biomass over time when penicillin is being produced by batch culture. With reference to Fig. 4.1, describe and explain the changes in concentration of lactose and ammonia. description ___________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY explanation ___________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [4] (b) A student incorrectly suggested that penicillin might be produced by continuous culture fermentation instead of by batch culture. Suggest how the curves for lactose, ammonia and biomass on Fig. 4.1 might differ in continuous culture. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (c) A second student said that continuous culture would not be suitable, as penicillin is a secondary metabolite. What evidence is there in Fig. 4.1 that penicillin is a secondary metabolite? _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (d) Explain the importance of maintaining aseptic conditions in manufacturing penicillin by fermentation. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [3] (e) State three physical or chemical factors within the fermenter, other than nutrient levels, that need to be monitored and controlled. For each factor, explain why it must be controlled. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [3] 2. Microorganisms are often used in biotechnological processes. Fig. 6.1 shows the standard growth curve for a culture of bacteria. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (a) Identify the phases labelled P, Q and R in Fig. 6.1. P _________________________ Q _________________________ R _________________________ [3] (b) Metabolic processes taking place in bacteria grown in a batch culture produce primary and secondary metabolites. Explain what is meant by a primary metabolite. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (c) With reference to the information in Fig. 6.1, state the phase or phases, P, Q, R or S, when: primary metabolite production is at its highest rate: ________________________________________________________ [1] most secondary metabolites are produced: ________________________________________________________ [1] the concentration of secondary metabolites reach a maximum: ________________________________________________________ [1] (d) Some aerobic recombinant bacteria were grown in a fermenter. They synthesised the protein human growth hormone (HGH). Suggest two ways in which named factors inside the fermenter could be adjusted in order to maximise the yield of HGH. 1 ____________________________________________________________ _____________________________________________________________ _____________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 2 ____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [4] (e) HGH made in this way is given by injection to some children who have a genetic mutation. The mutation means that they do not produce enough HGH to enable them to grow at the normal rate. Explain why injecting recombinant HGH in this way is not an example of gene therapy. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [3] 3. Microorganisms include fungi and bacteria. Fungi are eukaryotes. Bacteria are prokaryotes. (a) Describe one distinctive feature of the cell structure of each of these microorganisms. fungal cell _____________________________________________________ ______________________________________________________________ bacterial cell ___________________________________________________ ___________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (b) The use of microorganisms in biotechnology involves aseptic technique. Aseptic technique prevents pathogens contaminating products. What is meant by the term pathogen? ______________________________________________________________ ___________________________________________________________ [1] (c) State what is meant by biotechnology using suitable examples from different areas of biotechnology and explain why microorganisms are used in biotechnological processes. In your answer, you should give examples of products and the microorganisms used to make them, as well as the advantages of using microorganisms. ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [8] 4. Fig. 5.1 is a crossword that should contain five words relating to the use of microorganisms by humans. Use the clues below to write the five appropriate words in the correct spaces on Fig. 5.1. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 5. Enzyme immobilisation is an important technique in biotechnology. Figs 1.1 and 1.2 show two stages in making a bioreactor to remove lactose sugar from milk. In Fig. 1.1 the enzyme lactase is immobilised in alginate beads. In Fig. 1.2 milk flows over the beads and the lactose sugar is hydrolysed to two other sugars. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (a) Suggest and explain how you might use the method shown in Fig. 1.2 to obtain milk that was lactose-free. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (b) Fig. 1.1 and Fig. 1.2 show that alginate beads can be used to immobilise an enzyme. Outline two other methods of immobilising enzymes. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (c) Enzyme immobilisation is used in the biotechnology industry for the largescale production of materials. Discuss the benefits of using immobilised enzymes for large-scale production. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [4] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Genomes and gene technologies Learning objectives: Outline the steps involved in sequencing the genome of an organism; Outline how gene sequencing allows for genome-wide comparisons between individuals and between species; Define the term recombinant DNA; Explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product; Describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes; Outline how DNA fragments can be separated by size using electrophoresis; Describe how DNA probes can be used to identify fragments containing specific sequences; Outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments; Explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase; State other vectors into which fragments of DNA may be incorporated; Explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product; Describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment; Outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid; Outline the process involved in the genetic engineering of bacteria to produce human insulin; Outline the process involved in the genetic engineering of ‘Golden Rice TM’; Outline how animals can be genetically engineered for xenotransplantation; Explain the term gene therapy; Explain the difference between somatic cell gene therapy and germ line cell gene therapy; Discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms; SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Key definitions: Compile a glossary by writing your own definitions for the following key terms related to the learning objectives above. Key term downstream processing immobilisation genomics DNA profiling (genetic fingerprinting) genomic sequencing genetic engineering gene therapy coding DNA non-coding DNA clone libraries electrophoresis annealing primers amplification recombinant DNA technology Definition SACKVILLE SCIENCE DEPARTMENT Key term vector restriction enzymes restriction site sticky end DNA ligase recombinant DNA transgenic recombinant plasmid replica plating gene therapy genetically modified organism (GMO) liposomes xenotransplantation Definition A2 BIOLOGY SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Gel electrophoresis Gel electrophoresis is a method that separates large molecules (including nucleic acids or proteins) on the basis of size, electric charge, and other physical properties. Such molecules possess a slight electric charge. To prepare DNA for gel electrophoresis the DNA is often cut up into smaller pieces. This is done by mixing DNA with restriction enzymes in controlled conditions for about an hour. Called restriction digestion, it produces a range of DNA fragments of different lengths. During electrophoresis, molecules are forced to move through the pores of a gel (a jelly-like material), when the electrical current is applied. Active electrodes at each end of the gel provide the driving force. The electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules. The frictional force of the gel resists the flow of the molecules, separating them by size. Their rate of migration through the gel depends on the strength of the electric field, size and shape of the molecules, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated molecules in each lane can be seen as a series of bands spread from one end of the gel to the other. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY The polymerase chain reaction – PCR Many procedures in DNA technology (such as DNA sequencing and DNA profiling) requires substantial amounts of DNA to work with. Some samples, such as those from a crime scene or fragments of DNA from a long extinct organism, may be difficult to get in any quantity. The diagram below shows the laboratory process called polymerase chain reaction (PCR). Using this technique, vast quantities of DNA identical to trace samples can be created. This process is often termed DNA amplification. PCR can be used to make billions of copies in only a few hours. It is repeated for about 25 cycles. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 1. A DNA sample (called target DNA) is obtained. It is denatured (DNA strands are separated) by heating at 98°C for 5 minutes. 2. The sample is cooled to 60°C. Primers are annealed (bonded) to each DNA strand. In PCR, the primers are short strands of DNA; they provide the starting sequence for DNA extension. 3. Free nucleotides and the enzyme DNA polymerase are added. DNA polymerase binds to the primers and, using the free nucleotides, synthesises complementary strands of DNA. 4. After one cycle, there are now two copies of the original DNA. 5. This is repeated for about 25 cycles – repeat cycle of heating and cooling until enough copies of the target DNA have been produced. Restriction enzymes One of the essential tools of genetic engineering is a group of special restriction enzymes (also known as restriction endonucleases). These have the ability to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites. These enzymes are the ‘molecular scalpels’ that allow genetic engineers to cut up DNA in a controlled way. Although first isolated in 1970, these enzymes were discovered earlier in many bacteria. The purified forms of these bacterial restriction enzymes are used today as tools to cut DNA. Enzymes are named according to the bacterial species from which they were first isolated. By using a ‘tool kit’ of over 400 restriction enzymes recognising about 100 recognition sites, genetic engineers can isolate, sequence, and manipulate individual genes derived from any type of organism. The sites at which the fragments of DNA are cut may result in overhanging ‘sticky ends’ or non-overhanging ‘blunt ends’. Pieces may later be joined together using an enzyme called DNA ligase in a process called ligation. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ‘Sticky end’ restriction enzymes A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site. The cuts produce a DNA fragment with two sticky ends (ends with exposed nucleotide bases at each end). The piece it is removed from is also left with sticky ends. Restriction enzymes may cut DNA leaving an overhang or sticky end, without its complementary sequence opposite. DNA cut in such a way is able to be joined to other exposed end fragments of DNA with matching sticky ends. Such joins are specific to their recognition sites. ‘Blunt end’ restriction enzymes A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site. The cuts produce a DNA fragment with two blunt ends (ends with no exposed nucleotide bases at each end). The piece it is removed from is also left with blunt ends. It is possible to use restriction enzymes that cut leaving no overhang. DNA cut in such a way is able to be joined to any other blunt end fragment, but tends to be non-specific because there are no sticky ends as recognition sites. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Ligation DNA fragments produced using restriction enzymes may be reassembled by a process called ligation. Pieces are joined together using an enzyme called DNA ligase. DNA of different origins produced in this way is called recombinant DNA (because it is DNA that has been recombined from different sources). The combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering (also known as recombinant DNA technology). Creating a recombinant DNA plasmid If two pieces of DNA are cut by the same restriction enzyme, they will produce fragments with matching sticky ends (ends with exposed nucleotide bases at each end). When two such matching sticky ends come together, they can join by base-pairing. This process is called annealing. This can allow DNA fragments from a different source, perhaps a plasmid, to be joined to the DNA fragment. The joined fragments will usually form either a linear molecule or a circular one, as shown in the diagram above for a plasmid. However, other combinations of fragments can occur. The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Gene cloning using plasmids Gene cloning is a process of making large quantities of a desired piece of DNA once it has been isolated. The purpose of this process is often to yield large quantities of either an individual gene or its protein product when the gene is expressed. Methods have been developed to insert a DNA fragment of interest e.g. a human gene for a desired protein into the DNA of a vector, resulting in a recombinant DNA molecule or molecular clone. A vector is a self-replicating DNA molecule e.g. plasmid or viral DNA used to transmit a gene from one organism into another. To be useful, all vectors must be able to replicate inside their host organism, they must have one or more sites at which a restriction enzyme can cut, and they must have some kind of genetic marker that allows them to be easily identified. Organisms such as bacteria, viruses and yeasts have DNA that behaves in this way. Large quantities of the desired gene can be obtained if the recombinant molecule is allowed to replicate in an appropriate host. The host e.g. bacterium may then go on to express the gene and produce the desired protein. Two types of vector are plasmids and bacteriophages (viruses that infect bacteria). Cloning a human gene 1. A gene of interest (DNA fragment) is isolated from cells that have been grown in laboratory culture. 2. An appropriate plasmid vector is isolated from a bacterial cell. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 3. Both the human DNA and the plasmid are treated with the same restriction enzyme to produce identical sticky ends. 4. The restriction enzyme cuts the plasmid DNA at its single recognition sequence, disrupting the tetracycline resistance gene. Antibiotic resistant marker genes may be used to identify the bacteria that have taken up the foreign e.g. human DNA. The plasmid used often carries two genes that provide the bacteria with resistance to the antibiotics ampicillin and tetracycline. Without this plasmid, the bacteria have no antibiotic resistance genes. A single restriction enzyme recognition sequence lies within the tetracycline resistance gene. A foreign gene, spliced into this position, will disrupt the tetracycline resistance gene, leaving the bacteria vulnerable to this antibiotic. It is possible to identify the bacteria that successfully take up the recombinant plasmid by growing the bacteria on media containing ampicillin, and transferring colonies to media with both antibiotics. 5. The DNA fragments are mixed together and the complementary sticky ends are attracted to each other by base-pairing. The enzyme DNA ligase is added to bond the sticky ends. 6. The recombinant plasmid, or molecular clone, is introduced into a bacterial cell by adding the DNA to a bacterial culture. Under the right conditions, some bacteria will take up the plasmid from solution by the process of transformation. Preparation of the clone up to this point Cloning the gene starts here 7. The actual gene cloning process (making multiple copies of the human gene) occurs when the bacterium with the recombinant plasmid is allowed to reproduce. 8. Colonies of bacteria that carry the recombinant plasmid can be identified by the fact that they are resistant to ampicillin but sensitive to tetracycline. Most often today, another gene plays the role of the tetracycline resistance gene, but the principle remains the same; the inserted DNA disrupts the activity of the gene whose activity is easily determined. Preparing a gene for cloning Double stranded DNA of a gene from a eukaryotic organism e.g. human containing introns. As a normal part of the cell process of gene expression, transcription creates a primary RNA molecule. The introns are removed by splicing enzymes to form a mature mRNA (now excluding the introns) that codes for the making of a single protein. The introns are removed as it makes the DNA (the human gene) shorter, and therefore easier to insert into plasmids. It also allows the bacterial enzymes to properly translate the human gene from the ‘reassembled’ DNA (bacterial enzymes cannot cope with the introns). Reverse transcriptase is added which synthesises a single stranded DNA molecule complementary to the mRNA. The second DNA strand is made by using the first as a template, and adding the enzyme DNA polymerase. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Gene therapy Gene therapy refers to the application of gene technology to correct or replace defective genes. It was first envisioned as a treatment, or even a cure, for genetic disorders, but it could also be used to treat a wide range of diseases, including those that resist conventional treatments. Gene therapy may operate by providing a correctly working version of the faulty gene or by adding a novel gene to perform a corrective role. In other cases, gene expression may be blocked in order to control cellular (or viral) activity. About two thirds of currently approved gene therapy procedures are targeting cancer, about one quarter aim to treat genetic disorders, such as cystic fibrosis, and the remainder are attempting to provide relief for infectious diseases. Gene therapy requires a gene delivery system; a way to transfer the gene to the patient’s cells. This may be achieved using an infectious agent such as a virus; a technique called transfection. A promising development has been the recent approval for gene therapy to be used in treating tumours in cancer patients. Severe combined immune deficiency syndrome (SCIDS) has also shown improvement after gene therapy. Infants treated for this inherited, normally lethal condition have become healthy young adults. Gene therapy involving somatic cells may be therapeutic, but the genetic changes are not inherited. The transfection of stem cells, rather than mature somatic cells, achieves a longer persistence of therapy in patients. In the future, the introduction of corrective genes into germline cells will enable genetic corrections to be inherited. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Gene delivery using extracted cells 1. Body cells from patient are isolated. These cells are homozygous for the defective allele. 2. A copy of the normal human allele is inserted into the DNA of a viral vector using restriction enzymes and DNA ligase. 3. Isolated body (somatic) cells are infected with virus containing the recombinant DNA. 4. Viral DNA carrying the normal allele inserts itself into the patient’s somatic cell chromosome. 5. Somatic cells containing the introduced normal allele are cultured in a nutrient medium. In this way, the desired gene is cloned. 6. Cultured cells are injected into the patient. 7. Symptoms are relieved in the patient by the expression of the normal allele. By using the techniques of recombinant DNA technology, medical researchers insert a functional gene into a patient’s body (somatic) cells. This should make the patient capable of producing the protein encoded in that allele. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 1. Fig. 1.1 is a flow diagram showing the main stages involved in making cheese. The starting material is milk, which contains the protein, casein. (a) Explain why making cheese can be described as a biotechnological process. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (b) Suggest two benefits of the pasteurisation stage. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [2] (c) Rennin is a protein that can be obtained from the stomach lining of calves. It is used in the cheese-making process in the ratio one part rennin to 10 000 parts milk. Suggest what type of protein rennin is and explain how a very small quantity of rennin is able to convert a large quantity of milk. _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ ___________________________________________________________ [3] (d) Rennin could, in theory, be immobilised for use in cheese-making. List two potential advantages of this. 1 ____________________________________________________________ 2 _________________________________________________________ [2] (e) Rennin can now be made by genetically modified microorganisms. Outline the process by which bacteria can be genetically modified to produce rennin. In your answer, you should make clear how the steps in the process are sequenced. ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ___________________________________________________________ [8] 2. This question is about genetic engineering and the techniques used for making multiple copies of genes (gene cloning). (a) Genetic engineering uses the following: A an enzyme that synthesises new DNA B an enzyme that cuts DNA at specific sequences C an enzyme that reseals cut ends of DNA D small circular pieces of DNA found in bacteria; these pieces of DNA have antibiotic resistance genes E an enzyme found in some viruses with an RNA genome; this enzyme converts RNA into DNA SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY Name A to E. A ____________________________________________________________ B ____________________________________________________________ C ____________________________________________________________ D ____________________________________________________________ E _________________________________________________________ [5] (b) Genes are cloned for a number of reasons. For example: one group of research scientists at a hospital wanted to sequence a disease-causing mutation to learn more about a human disease; these scientists started their research using white blood cells; another group of scientists at a biotechnology company wanted to clone the insulin gene in order to manufacture its protein product to treat diabetes; these scientists started their research using cells from the pancreas; Suggest and explain the biological reasons why the two groups each started with a different cell. ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ___________________________________________________________ [4] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (c) A gene can be cloned in vitro (in a test tube) by the polymerase chain reaction (PCR). Alternatively, a gene can be cloned in vivo (in living cells) by introducing the gene into bacterial host cells. Table 5.1 identifies some of the key steps in each process. Compare the two processes of gene cloning by explaining the advantages of each. In your answer, you should ensure that clear comparisons between the two processes are made and explained. ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [8] 3. Describe the differences between: somatic cell gene therapy germ line cell gene therapy ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT 4. A2 BIOLOGY Genetic modification of organisms uses a ‘toolkit’ that includes: enzymes that cut DNA enzymes that join sections of DNA together vectors that introduce DNA into new host cells (a) Some of the enzymes and vectors that are important in genetic modification are given an identifying letter in Table 4.1. Select one correct letter from Table 4.1 to fit each of the following statements: An enzyme that cuts DNA ________ An enzyme that joins sections of DNA together ________ A vector to introduce foreign DNA into bacteria ________ A vector to introduce foreign DNA into plant cells ________ A vector to introduce foreign DNA into animal cells ________ [5] (b) Discuss the potential benefits to mankind and the ethical concerns raised by the following examples of genetically modified organisms: rice modified for increased vitamin A content (‘Golden RiceTM’) humans having somatic gene therapy treatment for a genetic disease SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY In your answer, you should give a balanced account of the benefits and concerns for each example of genetic modification. ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [9] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 5. Transgenic goats, containing a gene from a spider that codes for spider web silk protein, have been produced by genetic modification. The silk protein can be harvested from the milk of the female transgenic goats. Spider silk protein is lightweight but has very high tensile strength. It is used to make items such as bullet-proof vests. (a) A vector containing recombinant DNA is needed to produce transgenic goats. Define the term recombinant DNA. ______________________________________________________________ ______________________________________________________________ ___________________________________________________________ [1] (b) Complete Table 3.1 by suggesting one example of a suitable vector for each of the following applications of genetic modification. [4] (c) In order to make spider silk protein on a commercial basis, many transgenic goats will be needed. Outline the process by which an animal, such as the first transgenic goat, may be cloned to produce a population. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ___________________________________________________________ [5] (d) An alternative method for producing a population of more transgenic goats is to breed the transgenic goat with normal goats. Discuss the advantages and disadvantages of cloning the transgenic goat compared with breeding the transgenic goat with normal goats. advantages ____________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ disadvantages __________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ___________________________________________________________ [5] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 6. A number of new techniques for manipulating cells and genomes are now available, and it is hoped this manipulation will allow cures for diseases to be developed. (a) Five goals that scientists would like to achieve are described below and are listed A to E: A producing large numbers of genetically identical ‘model’ transgenic mice that show symptoms of diabetes B growing a replacement kidney identically tissue-matched to an individual patient C obtaining replacement hearts from transgenic pigs, partially tissue-matched to humans D genetically manipulating cells of one adult to cure a genetic disease in that individual E altering a prokaryotic pathogen for use as a vaccine The names of the procedures corresponding to four of the five goals A to E are written below. Match the correct letters to the names. No letter should be used more than once. xenotransplantation __________ somatic gene therapy __________ non-reproductive cloning __________ animal reproductive cloning__________ (b) Table 7.1 shows four different combinations of techniques used to achieve goals A to E. Write the letters A, B, C, D or E in the first column of the table to match each goal to the appropriate combination of techniques needed to achieve it. Use each letter only once. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 7. The Oxford Botanic Garden was founded in 1621 to grow plants for the teaching of medicine. Since that time it has seen many changes. When the ideas of Linnaeus were adopted in the 18th Century, the plants were dug up and re-planted in family groups according to his new system of taxonomy. (a) Recently the plants have once again had to be reorganised: DNA sequencing techniques, together with cladistic analysis, have provided a radical new view of plant evolutionary relationships. The same techniques have also improved the ability of researchers to pinpoint new cures for diseases, by examining the closest relatives of plants already known to have medicinal properties. Comment on what the different arrangements of plants in the Oxford Botanic Garden over time tell us about the nature of scientific knowledge. ______________________________________________________________ ___________________________________________________________ [1] (b) Suggest two purposes of a plant collection in a modern botanic garden. ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ___________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY (c) DNA sequencing techniques have provided new information about plant relationships. Outline the roles of each of the following procedures in sequencing a genome: the polymerase chain reaction (PCR) ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ __________________________________________________________ [2] electrophoresis ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ __________________________________________________________ [2] digestion of DNA by restriction enzymes ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ __________________________________________________________ [2] (d) Suggest why a genome has to be fragmented before sequencing ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ __________________________________________________________ [2] SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY 8. Table 5.1 lists some plants considered for genome sequencing by the ‘Floral Genome Project’. The chromosome numbers and genome sizes in mega base pairs (Mbp) are shown. One Mbp is equal to 1 000 000 base pairs of DNA. (a) The sequencing method that will be used is only able to sequence fragments of DNA with a maximum length of 750 base pairs. Calculate the minimum number of DNA fragments that would need to be sequenced to read the genome of Amborella. Show your working. Answer = ____________________ [2] (b) Monkey flower and blueberry belong to the same taxonomic group within the plant kingdom. Only one pair was chosen for further sequencing work. Using the data in Table 5.1, suggest reasons why monkey flower was chosen instead of blueberry. SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ __________________________________________________________ [2] (c) Use your knowledge of the effects of polyploidy in bread wheat to suggest one way in which the fruit of a hexaploid (6n) blueberry might differ in appearance from that of a diploid (2n) blueberry. __________________________________________________________ [1] (d) DNA sequence information is most useful when used with the phylogenetic (cladistic) approach to classification. How does the phylogenetic approach to classifying species differ from the biological species concept? ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ __________________________________________________________ [2] 9. Steroid hormones are one example of molecules that can switch genes on and off in mammalian cells. Other molecules involved in genetic control have been studied in eukaryotes and prokaryotes. Describe one other example of genes being switched on or being switched off by a molecule that binds directly to DNA. ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ SACKVILLE SCIENCE DEPARTMENT A2 BIOLOGY ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ ________________________________________________________________ _____________________________________________________________ [4]