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Higher Biology Metabolism & Survival Notes METABOLIC PATHWAYS AND THEIR CONTROL Metabolism is the sum of all chemical reactions taking place in a cell. Metabolic pathways are regulated by enzymes and can be affected by venoms, toxins and poisons. Metabolic reactions can be divided into two categories: ANABOLIC (synthesis); CATABOLIC (breakdown). Catabolic pathways bring about the breakdown (degradation)of complex molecules to simpler ones, releasing energy in the form of ATP and often producing building blocks. Examples: aerobic respiration; digestion Anabolic pathways bring about the biosynthesis of complex molecules from simpler building blocks and require energy from ATP. Examples: protein synthesis; photosynthesis Reversible and irreversible steps Metabolic pathways can have reversible and irreversible steps. They can also have alternative routes. A pathway often contains both reversible and irreversible steps e.g. glycolysis, the breakdown of glucose to pyruvate has reversible and irreversible steps as well as alternative routes. GLYCOLYSIS Glycolysis is the first stage in the process of respiration, where glucose is converted to pyruvate via a series of intermediate molecules, with different enzymes controlling each step. Some steps are reversible, some are not. Glucose Step 1 enzyme A intermediate 1 Step 1 Glucose diffuses from a high concentration outside the cell to a low concentration inside the cell and is then converted to intermediate 1. This benefits the cell by maintaining a low concentration of glucose outside the cell therefore allowing glucose to diffuse constantly into the cell. Step 2 The conversion of Intermediate 1 to Intermediate 2 is reversible. If more intermediate 2 is formed than the cell needs for the next step then some can changed back into intermediate 1 and used in an alternative pathway, e.g. to build glycogen in animal cells or starch in plant cells. Step 3 The conversion of Intermediate 2 to Intermediate 3 is irreversible. Intermediate 3 will always be converted to pyruvate (through many further steps). Step 2 enzyme B intermediate 2 Step 3 enzyme C intermediate 3 many enzyme-controlled steps pyruvate Alternative routes Pathways can be modified and contain alternative routes, so steps can be bypassed e.g. in glycolysis (used when cell has plenty of sugar). The alternative route bypasses the steps above and the glucose is converted to sorbitol that is eventually converted to pyruvate. Page 1 Higher Biology Metabolism & Survival Notes EFFECTS OF POISONS, TOXINS AND VENOMS ON METABOLIC PATHWAYS Poisons Definition Chemicals that can impair and damage the body; can be fatal. Action Disrupt metabolic pathways. Examples Paraquat (respiratory distress) Cyanide (inhibits respiratory enzymes; used in gas chambers) Potassium chloride (interferes with muscle contraction; USA executions) Arsenic (interferes with a respiratory enzyme) Toxins Poisons produced by living organisms Interfere with metabolic pathways. Tetanus toxin (acts on nervous system and muscle – causes spasms) Curare (acts on motor nerves – paralysis) Salmonella toxin (inflammation of gut lining) Botulinum toxin (neurotoxin – paralysis) Venoms Poisons produced by e.g. snakes and scorpions that are transmitted by a bite or sting. Affect transmission of nerve impulses causing spasms or paralysis. All scorpions and around 25% of snake species are venomous, as are some spiders. Page 2 Higher Biology Metabolism & Survival Notes COMPARTMENTALISATION Cells have different areas or compartments (organelles) for different functions, increasing efficiency. The cell itself, and all its organelles, are bounded by membranes. The cell membrane separates the internal contents of the cell from its surroundings and regulates entry and exit of materials. The membranes surrounding organelles such as mitochondria, chloroplasts and lysosomes keep chemical metabolites together or apart as required. Surface area to volume ratio Small organelles (and folds in the membrane) increase the surface area to volume ratio, leading to a greater surface area for reactions and a high concentration of reactants, increasing the chance of reaction. Mitochondrion The inner membrane is folded into cristae, increasing the surface area. The matrix contains enzymes that control the citric acid cycle (a metabolic pathway in respiration). Chloroplast Lysosomes The enzymes needed for ATP generation are bound together on flattened sacs containing chlorophyll. The Calvin cycle (a photosynthetic metabolic pathway) occurs in the fluid outside the sacs, where the required enzymes are also present. Lysosomes are membrane-bound organelles that contain powerful digestive enzymes. Their function is to break down large molecules e.g. proteins, carbohydrates and nucleic acids, for recycling of their building blocks. The enzymes must be kept apart from other parts of the cell so that they do not destroy them. Also, the enzymes work at around pH5, rather than at the cytoplasmic pH of 7.2. Page 3 Higher Biology Metabolism & Survival Notes Structure of the plasma membrane The cell membrane is made of protein and phospholipid molecules arranged in a FLUID MOSAIC structure. The phospholipids are arranged in a BILAYER, with their hydrophilic heads facing outwards, and their hydrophobic tails facing each other in the middle of the bilayer. The constantly moving phospholipid bilayer contains a mosaic of different protein molecules in, on or through it. TRANSPORT ACROSS MEMBRANES Transport across the membrane can be PASSIVE or ACTIVE. Passive transport is movement down a concentration gradient (from high to low) and does not require energy. Active transport requires energy as molecules move against a concentration gradient (from low to high). PASSIVE TRANSPORT • DIFFUSION Particles of a liquid or gas move down a concentration gradient from a region of high concentration to a region of low concentration until the concentration is equal. Small molecules such as water, oxygen and carbon dioxide can pass directly through the lipids in the cell membrane. Larger molecules such as glucose enter and leave via the pores of specific transport proteins (channel-forming proteins). • OSMOSIS Cells can gain or lose water by OSMOSIS. Osmosis is the diffusion of water from a region of high concentration to a region of low concentration across a selectively permeable membrane (a membrane that allows only small molecules through). ACTIVE TRANSPORT Active transport occurs when molecules are moved across the cell membrane against a concentration gradient. It is carried out by specific transport proteins. The transport proteins require an energy supply that is provided by the breakdown of ATP (Adenosine triphosphate) inside the cell. An example of active transport is the action of a transport protein called the sodium-potassium pump. The same protein pumps sodium ions out of the cells and potassium ions into the cells against their concentration gradients. This causes an electrical gradient that is needed for muscle contraction and nerve impulse transmission. ENZYMES IN THE MEMBRANE Some of the proteins in the plasma membrane are ENZYMES that catalyse some metabolic processes. For example, ATP synthase, present in the membrane of mitochondria, chloroplasts and prokaryotes, catalyses the synthesis of ATP. Multi-enzyme complexes ensure that steps in a metabolic pathway occur in the correct order. Page 4 Higher Biology Metabolism & Survival Notes Many membrane-bound enzymes are found in the cells of the small intestine, which are involved in the final stages of digestion and absorption. These enzymes are located on the ‘outside’ of the intestinal cells and break down small polysaccharides into single sugars, or polypeptides into amino acids. These small molecules can then be imported into the cells. By embedding these enzymes in the plasma membrane, the final products of digestion are produced close to the transport proteins that take them into the cell. ENZYME ACTION Revision Enzymes: • are proteins • are biological catalysts (speed up chemical reactions but remain unchanged) • have an active site on their surface • some build up (synthesise) small molecules to make larger molecules (anabolism) • some break down larger molecules (degrade) to make smaller molecules (catabolism) • bind with the substrate at the active site • are specific (act on one type of substance) • give the highest rate of reaction under optimum conditions. Enzyme active site and substrate molecules are complementary and fit to form an enzyme-substrate complex. Reaction takes place and end products are released. Enzymes are found in the cytoplasm; on membranes; in membrane-bound organelles such as the nucleus, lysosomes, mitochondria and chloroplasts. Activation energy and enzyme action A chemical reaction may involve the joining together of simple molecules into more complex ones or the splitting of complex molecules into simpler ones. Either way, energy (activation energy) is required to initially break the bonds in the reactants to form an unstable compound with molecules in a transition state. In non-living systems a high temperature is usually needed in order for reactions to proceed. Enzymes lower the activation energy needed for the formation of a transition state. Thus biochemical reactions are able to proceed rapidly at relatively low temperatures. Induced fit model Substrate molecules have an affinity for the active site and are complementary to it. However, the match between enzyme and substrate may not be an exact fit. The active site is flexible: when the substrate enters, the enzyme molecule and the active site change slightly making the active site fit very closely round the substrate molecule. The induced fit ensures that the active site comes into very close contact with the molecules of substrate and increases the chance of a reaction taking place. Page 5 Higher Biology Metabolism & Survival Notes Orientation of reactants When the reaction involves two (or more) substrates, the shape of the active site helps orientate the reactants in the right position so that a reaction can take place. 1. Reactants orientated to fit active site and held together in an induced fit. 2. Chemical bonds in reactants are weakened, activation energy reduced and reaction occurs. 3. Products now have a low affinity for the active site and are Released, leaving enzyme free to repeat the process. FACTORS AFFECTING ENZYME ACTION Enzyme action is affected by temperature and pH. It is also affected by: supply of the substrate(s); concentration of end-product; presence/absence of an inhibitor *. Effect of substrate concentration Low concentration: too few substrate molecules present to make use of all the active sites on the enzyme molecules. Increasing substrate concentration: increase in reaction rate as more active sites are involved. Further increase in substrate concentration does not increase rate of reaction further (graph levels off) since all the active sites are occupied. The enzyme concentration has become a limiting factor. Effect of end-product concentration End product inhibition is negative feedback used to regulate the production of a given molecule. In the example shown, the end product combines with enzyme 1 to stop the reaction so there will not be an excess production of the end product. Direction of enzyme action Enzyme 1 Enzyme 2 Enzyme 3 Metabolite W Metabolite X Metabolite Y Metabolite Z Page 6 Higher Biology Metabolism & Survival Notes Once metabolite W becomes available enzyme 1 is activated and converts W to X. When metabolite X becomes available enzyme 2 is activated and converts X to Y and so on. Most enzyme reactions are reversible: the actual direction of the reaction depends on the relative concentrations of reactants and products. In this way a balance of metabolites is always maintained. [*Note on inhibitors under ‘Control of metabolic pathways by regulation of enzyme action’.] CONTROL OF METABOLIC PATHWAYS The role of genes in the control of cell metabolism Consider this metabolic pathway: Gene(s) Gene(s) Enzyme 1 Metabolite A Enzyme 2 Metabolite B Each step is driven by a specific enzyme. Each enzyme is coded for by a gene or genes. If the correct enzyme is present then the pathway proceeds. If one enzyme is absent the pathway will stop. Metabolite C Enzyme action can be regulated by the level of gene expression. Some metabolic reactions e.g. respiration are always required; others may need to operate only in certain circumstances. To avoid wasting resources, the genes coding for enzymes are ‘switched on’ or ‘off’ as required. GENETIC CONTROL OF METABOLIC PATHWAYS Genetic control involves the switching of genes on and off. One example of a genetic switch was discovered by French biologist François Jacob and Jaques Monod, who studied the breakdown of lactose in the bacterium Escherichia coli (E. coli). There are hundreds of different strains of E. coli. Most are harmless and live in the intestines of mammals. Some can cause gastrointestinal infections. Lactose metabolism in E.coli Lactose is a sugar found in milk. E.coli’s normal environment (the gut) usually has glucose but not lactose. However, E.coli has a gene that enables it to digest lactose should any be present. This gene is only switched on when lactose is present. Background facts Glucose is used by E. coli for energy release. Lactose sugar – found in milk – is composed of glucose and galactose. Lactose is broken down by the enzyme ßgalactosidase. E. coli has a gene coding for ß-galactosidase. E. coli produces the enzyme only when lactose is present. The action of ß-galactosidase on lactose ß-galactosidase Lactose glucose + galactose Page 7 Higher Biology Metabolism & Survival Notes The gene for the enzyme is only switched on when lactose is present - it is otherwise switched off. This process of switching on a gene only when the enzyme it codes for is required is called ENZYME INDUCTION. In the case above, lactose is the INDUCER: its presence allows the expression of the genes coding for the enzyme that will break it down. These genes form part of a section of DNA called an OPERON. Operons An operon consists of one or more structural genes with an adjacent operator gene that controls it/them. The operator gene is regulated by a regulator gene that codes for a repressor molecule. The repressor prevents the operator gene from being transcribed. The Lac operon of E. coli An example of an operon is the lac (lactose) operon of E. coli. Structural gene is transcribed and translated into the enzyme ß- galactosidase which breaks down the sugar lactose. Operator gene switches on the structural gene Regulator gene controls the functioning of the operator through the production of a ‘repressor protein’ Lactose absent Operator mRNA for repressor protein transcribed and translated Repressor protein Operator blocked Repressor protein binds to operator gene structural genes switched off No galactosidase synthesised Lactose present Page 8 Higher Biology Metabolism & Survival Notes Operator mRNA for repressor protein transcribed and translated Repressor protein Operator is freed Structural genes allowing it to switch on switched on structural gene Lactose binds the repressor molecule Lactose enters cell mRNA for enzyme transcribed, and then translated into the protein -galactosidase synthesised Enzyme digests lactose until its supply runs out. Repressor then free to bind with operator and gene switched off. Lactose is called the inducer because its presence induces synthesis of the enzyme. Use of ONPG in investigations of the lac operon ß- galactosidase will also break down a colourless chemical called ONPG: ß- galactosidase ONPG galactose + yellow compound (ortho-nitrophenol) This is useful in experiments to indicate the activity of ß- galactosidase as the yellow compound is easily seen and its presence is an indicator of enzyme activity. Page 9 Higher Biology Metabolism & Survival Notes THE ARA OPERON E. coli can also use the sugar ARABINOSE in the absence of glucose. As in the case of lactose, arabinose is the inducer for the enzymes that break it down. ara operon Arabinose absent group of structural genes coding for enzyme section of chromosome Regulator gene Transcription and translation to form inactive regulator molecule promoter Regulator binds to DNA but remains inactive Structural genes remain switched off No arabinose-digesting enzyme synthesised Regulator molecule Arabinose present group of structural genes coding for enzyme section of chromosome Regulator gene Transcription and translation to form inactive regulator molecule promoter arabinose combines with regulator, changing its shape and making it act on promoter RNA polymerase begins transcription Regulator molecule Transcription and translation of arabinosedigesting enzyme arabinose enters cell Transformation of the ara operon The DNA of E. coli can be artificially transformed e.g. by having particular genes inserted into a plasmid. The structural genes of the ara operon can be replaced with a gene coding for green fluorescent protein (GFP) and another that confers resistance to ampicillin. [This is done using pGLO plasmids.] gene for ampicillin resistance section of chromosome Regulator gene promoter GFP gene in place of gene for enzyme Bacteria transformed in this way will produce GFP instead of arabinose-digesting enzymes (in the presence of arabinose) and will be ampicillin-resistant. These features allow the operation of the operon to be studied more easily. Page 10 Higher Biology Metabolism & Survival Notes CONTROL OF METABOLIC PATHWAYS BY REGULATION OF ENZYME ACTION Some metabolic pathways must operate continuously – their genes are always ‘on’ and the enzymes they code for are always present in the cell. Such pathways are controlled by regulation of the rates of reaction of their enzymes by signal molecules and inhibitors Signal molecules A signal molecule causes a gene to be switched on by combining with the product of the regulator gene so that the structural gene produces the required enzyme. Signal molecules can be INTRACELLULAR or EXTRACELLULAR. • Intracellular signal molecules come from within the cell itself. • Extracellular signal molecules come from outside the cell e.g. adrenaline is made by the adrenal glands and acts on liver cells to activate the enzyme that converts glycogen to glucose. Inhibitors An inhibitor is a substance that reduces the rate of an enzyme-controlled reaction. Inhibitors can occur naturally or be man-made e.g. drugs, pesticides. There are three kinds of inhibition: competitive, non-competitive, and feedback inhibition. Competitive inhibition Effect of increasing substrate concentration on competitive inhibition Competitive inhibitors are molecules that have a similar structure to the substrate and can therefore fit the enzyme’s active site. This reduces the rate of reaction. Non-competitive inhibition A non-competitive inhibitor does not combine with the enzyme’s active site but to a non-active site, thus changing the shape of the molecule. This indirectly alters the shape of the active site so that the substrate molecule cannot bind with it. Non-competitive inhibitors reduce the amount of active enzyme and their effect is permanent. Increasing substrate concentration does not increase reaction rate in presence of noncompetitive inhibitors. Examples: cyanide, heavy metal ions, some insecticides. Blockage of some active sites by the inhibitor reduces reaction rate when the substrate concentration is not at a high level. However, an increase in substrate concentration increases the chance that it will bind to the enzyme and reaction rate returns to normal. Increasing substrate concentration in the presence of different inhibitors Competitive: concentration of inhibitor and substrate control the degree of inhibition. Non-competitive: concentration of inhibitor alone controls the degree of inhibition. Page 11 Higher Biology Metabolism & Survival Notes Active and inactive forms of enzyme molecules Some enzymes are made up of several polypeptide units, each with its own active and non-active (allosteric) sites. The enzyme may exist as an active or inactive form, each form having a different shape. The enzyme changes shape if a regulatory molecule (activator or inhibitor) binds to a nonactive site. Non-active site (one of four) Active form Activator: causes the enzyme to take its active form, stimulating enzyme activity. Non-competitive inhibitor: causes a change to an inactive form of enzyme, inhibiting its activity. Inactive form End product inhibition As the concentration of the end product builds up, some of it binds to molecules of enzyme A. This slows down the conversion of substrate to the intermediate 1 metabolite. Substrate As the concentration of the end product drops, fewer molecules of enzyme A are affected and more of intermediate 1 is converted to intermediate 2 and so on. Intermediate 1 Intermediate 2 This is called negative feedback control and avoids the wasteful conversion and accumulation of intermediates. Intermediate 3 Example: Effect of phosphate on the enzyme phosphatase Phosphatase releases phosphate from its substrate for use in cell metabolism. Phosphatase acts on the chemical phenolphthalein phosphate, releasing its phosphate. phosphatase phenolphthalein phosphate phenolphthalein + phosphate Phenolphthalein is pink in alkaline conditions. This colour change clearly shows the activity of the enzyme (the more activity, the more pink the result). Increasing phosphate concentration leads to a decrease in enzyme activity – the phosphate acts as an endproduct inhibitor of phosphatase. Page 12 Higher Biology Metabolism & Survival Notes CELL RESPIRATION Respiration is the process occurring in every living cell by which chemical energy is released from food (usually carbohydrates, although fats and proteins can also be used as respiratory substrates); the energy is used to regenerate the high-energy molecule adenosine triphosphate (ATP). Word equation: glucose + oxygen carbon dioxide + water ATP formed Respiration is a series of catabolic reactions, catalysed by enzymes, in which 6-carbon glucose is oxidised* to form carbon dioxide. The energy released due to the oxidation of glucose is used to synthesize ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi): ADP + Pi ATP [*oxidation - loss of hydrogen] ATP ATP is a high-energy molecule made during cellular respiration by the addition of inorganic phosphate to ADP. The energy for this reaction comes from glucose. high energy bond ATP structure ATP is made up of one adenosine and three phosphate molecules. The terminal phosphate is held by a high energy bond: the energy is released when the bond is broken. When ATP is broken down into ADP + Pi it releases its energy. This energy is released as heat but can also be used in e.g. chemical reactions, muscular contractions, active transport, nerve impulses, DNA replication, protein synthesis. ADENOSINE P P P 3 PHOSPHATE GROUPS breakdown releasing energy ATP (high energy state) ADP + Pi (low energy state) build-up requiring energy ATP as energy carrier Phosphorylation Phosphorylation is an enzyme-controlled process by which a phosphate group is added to a molecule e.g. when low energy ADP and Pi combine to form high energy ATP. Similarly, if ATP donates a phosphate to a reactant in a metabolic pathway, the reactant becomes phosphorylated and gains energy, becoming more reactive. ATP (high energy state) Glucose (low energy state) ADP + Pi (low energy state) Glucose-6-phosphate (high energy state) Page 13 Higher Biology Metabolism & Survival Notes ATP in cells An active cell needs about 2 million molecules of ATP per second to satisfy its energy requirements. This is made possible by the rapid turnover of ATP: as fast as ATP is broken down to release its energy it is being regenerated from ADP and Pi (using the energy from respiration). Only about 50g of ATP is stored in the body at any one time, but the body may be using it up and regenerating it at about 400g/hr. ATP synthase ATP synthesis The respiratory pathway produces a flow of high-energy electrons that the cell uses to pump hydrogen ions (H+) ATP synthase across the inner mitochondrial membrane against a concentration gradient. The H+ ions flow back down a concentration gradient across a membrane protein ATP synthase, causing part of it to rotate. ATP synthase then catalyses the synthesis of ATP from ADP and Pi. ATP synthase molecules are found in the membranes of mitochondria and chloroplasts. Page 14 Higher Biology Metabolism & Survival Notes THE BIOCHEMISTRY OF RESPIRATION Respiration can be aerobic (using oxygen) or anaerobic (without oxygen). Aerobic respiration occurs in three main stages: Glycolysis, the Citric Acid Cycle and the Electron Transport Chain. GLYCOLYSIS Glycolysis (‘glucose-splitting’) consists of a series of chemical reactions, each catalysed by a specific enzyme. It occurs in the cytoplasm and does not require oxygen. During glycolysis, glucose is split into 2 pyruvate molecules. Energy investment phase The phosphorylation of ATP phosphorylation at step 1 intermediates at the beginning of ADP the glycolysis pathway uses 2 other metabolic molecules of ATP. INTERMEDIATE 1 pathways GLUCOSE energy investment phase INTERMEDIATE 2 ATP ADP phosphorylation at step 3 INTERMEDIATE 3 _________________________________ 2NAD 2NADH energy pay-off phase ADP ATP ADP ATP Energy pay-off phase Later reactions in glycolysis result in regeneration of 4 ATP molecules, giving a net gain of 2 ATP. During this phase, H+ ions are released from glucose by a dehydrogenase enzyme. The H ions are passed to a hydrogen carrier, the coenzyme NAD, forming NADH. NADH carries the hydrogen on to a later stage of respiration, the electron transport system. PYRUVATE After glycolysis If oxygen is available, pyruvate passes to the next stage in aerobic respiration, the Citric Acid Cycle (also known as the Krebs Cycle, after its major discoverer). This cycle of reactions takes place in the matrix of mitochondria. site of electron transport chain site of citric acid cycle Page 15 Higher Biology Metabolism & Survival Notes CITRIC ACID CYCLE The reactions of the citric acid cycle take place in the matrix of the mitochondrion. 1. Pyruvate is broken down to carbon dioxide and an acetyl group. 2. The acetyl group combines with coenzyme A to form acetyl coenzyme A. As this happens H ions are released and become joined to NAD forming NADH. 3. The acetyl group of acetyl coenzyme A combines with oxaloacetate to form citrate and enters the citric acid cycle. 4. After several enzyme-controlled steps oxaloacetate is regenerated. 5. At 3 steps in the cycle dehydrogenase enzymes remove H ions along with associated high-energy electrons. These H ions and high-energy electrons are passed to the coenzyme NAD to form NADH. 6. A similar reaction at one other step uses the coenzyme FAD which becomes FADH. 7. In addition, ATP is produced at one step and carbon dioxide is released at another two. Pyruvate NAD NADH CO2 Acetyl group Coenzyme A Acetyl coenzyme A Citrate 2CO2 Oxaloacetate Citric acid cycle 3NAD 3NADH FADH2 FAD ATP ADP + Pi Page 16 Higher Biology Metabolism & Survival Notes THE ELECTRON TRANPORT CHAIN The electron transport chain consists of a group of protein molecules attached to the inner membrane of the cristae of mitochondria. There are many of these chains in a cell. 4 5 6 1. NADH and FADH, from glycolysis and the citric acid cycle, release high-energy electrons and pass them to the electron transport chains. 2. The electrons begin in a high-energy state. As they flow along a chain of electron acceptors, they release energy. 3. This is used to pump hydrogen ions across the membrane from the matrix side to the inter-membrane space to maintain a higher concentration of hydrogen ions. 4. When the hydrogen ions flow back down the concentration gradient to the matrix they pass through molecules of ATP synthase. 5. This drives this enzyme to synthesise ATP from ADP and Pi. 6. When the electrons come to the end of the electron transport chain they combine with oxygen – the final hydrogen acceptor. At the same time, the oxygen joins to a pair of hydrogen ions to form water. Most of the ATP generated by cellular respiration is produced in mitochondria in this way. In the absence of oxygen the electron transport chains do not proceed and there is no production of ATP except for that produced during glycolysis. Page 17 Higher Biology Metabolism & Survival Notes SUBSTRATES FOR RESPIRATION CARBOHYDRATES starch glycogen sucrose glucose maltose Starch and glycogen are broken down to glucose as required. Sugars such as maltose and sucrose can be converted to glucose or to intermediates in the glycolysis pathway. fructose pyruvate FATS glucose Fats can be broken down into glycerol and fatty acids. Glycerol is converted into one of the intermediates in glycolysis Fatty acids are eventually converted into acetyl coenzyme A for use in the citric acid cycle. intermediate Glycerol Fat pyruvate Fatty acids acetyl co-A Citric acid cycle PROTEINS glucose pyruvate amino acid urea protein acetyl co-A amino acid Proteins are broken down into amino acids. Excess amino acids are deaminated, forming urea (waste product) and respiratory pathway intermediates. urea amino acid intermediate urea Citric acid cycle Page 18 Higher Biology Metabolism & Survival Notes METABOLIC RATE Metabolic rate is the quantity of energy used by the body over a given time. It is measured in kilojoules (or kilocalories). Metabolic rate can be measured as: oxygen consumption per unit time; carbon dioxide production per unit time; energy production (as heat) per unit time. [Glucose + oxygen carbon dioxide + water; energy released ATP] Measuring metabolic rate Metabolic rate can be measured in different ways e.g. using respirometers and calorimeters. Coloured water A respirometer is a chamber with a continuous airflow that allows the measurement of differences in oxygen, carbon dioxide and temperature in air entering and leaving. A calorimeter measures the heat generated by an organism by comparing the temperature of water entering and leaving a well-insulated container. Basal metabolic rate Basal metabolic rate (BMR) is the minimum rate of energy release required to maintain essential body processes when an animal is at complete rest. BMR is expressed as kilojoules of heat released per square metre of body surface per hour (kJmˉ²hˉˡ). BMR is low compared to the metabolic rate when the body is exercising. Comparative metabolic rates Generally, the greater the mass of an organism the higher its metabolic rate. Animal Sea anemone Octopus Eel Frog Human Mouse Hummingbird Volume of oxygen consumed (mm³ g⁻ˡ body mass h¯ˡ) 13 80 128 150 200 1500 3500 Page 19 Higher Biology Metabolism & Survival Notes OXYGEN DELIVERY Oxygen is required for aerobic respiration. High metabolic rates require efficient delivery of oxygen to cells. Organisms with high metabolic rates need efficient transport systems for the delivery of oxygen. In vertebrates, oxygen is delivered in blood, pumped by a heart. Prior knowledge Main blood vessels involved in the circulation of blood around the body: Arteries – carry blood away from the heart (under high pressure). Capillaries – smallest blood vessels; exchange nutrients, gases, and waste products between blood and body tissue. Veins – carry blood back to the heart (under low pressure). The heart The heart has two types of chamber: atria, where blood enters the heart; ventricles , where blood leaves the heart. Circulatory systems in vertebrates All vertebrates have closed circulatory systems where the blood is contained in a continuous circuit of blood vessels and is kept moving by a muscular pump (the heart). In closed systems a drop in pressure occurs when blood passes through the capillaries because the narrow tubes offer resistance to the flow of blood. Single circulatory system (in fish) Gas exchange occurs in the gills. As water flows over the gill filaments oxygen diffuses down a concentration gradient to the blood. Fish have a single circulatory system: blood passes through the 2-chambered heart only once for each circuit of the body. The blood flows to the gills at high pressure but is delivered to the systemic capillaries at low pressure. Fish have a two-chambered heart with an atrium, a ventricle and a valve in between. Double circulatory system [Systemic means ‘of the body’] Double circulatory system Other vertebrate groups have a double circulatory system: blood passes through the heart twice for each circuit of the body. Blood is pumped to both the lungs and the body both at high pressure ensuring vigorous flow, making a double circulation more efficient than single systems. Double circulatory system: incomplete Reptile and amphibian circulatory systems are described as incomplete because there is only one ventricle and some mixing of oxygenated blood from the lungs and deoxygenated blood from the body occurs. In amphibians, some gas exchange occurs through the moist skin, so blood returning from the body is partially oxygenated. In most reptiles, the ventricle is partly divided by a septum. Page 20 Higher Biology Metabolism & Survival Notes Double circulatory system: complete Birds and mammals (and crocodiles!) have complete circulatory systems: the heart has two atria and two ventricles completely separated by a septum. A complete double circulation is the most efficient: it ensures that oxygenated and deoxygenated blood is not mixed. This allows endothermic (‘warm-blooded’) birds and mammals to access enough oxygen for respiration, releasing enough heat to keep their bodies warm. Lung complexity Essential features of a gas exchange system: large surface area; moist surface to allow gases to dissolve; thin structures to allow rapid diffusion into the tissues of the organism; good supply of blood vessels. Amphibians Amphibians usually exchange gases though skin and mouth lining, only using their lungs when very active. Lungs: small, thin-walled sacs with few alveoli, if any. Reptiles and mammals Reptiles and mammals depend entirely on lungs for gas exchange. Their lungs have a system of repeatedly branching tubes ending in many alveoli. Alveoli have a thin, moist lining and give a large surface area (100m² in humans) to provide enough oxygen for aerobic respiration. Birds Flight requires a high metabolic rate and consequently a high supply of oxygen. Birds have relatively small lungs and several large air sacs that act as bellows, moving air in one direction through the lungs. Birds’ lungs have no alveoli but have parabronchi (tiny channels) through which oxygenated air passes in one direction. . Page 21 Higher Biology Metabolism & Survival Notes Physiological adaptations for low-oxygen niches There are two main low oxygen niches: high altitudes and deep-diving marine habitats. Humans at high altitude We function best at oxygen concentration around 20%. At lower concentrations (e.g. at altitude) we need more red blood cells (rbcs) in order to function normally. This is brought about by: an increase in the level of erythropoietin, the hormone that stimulates red blood cell production, leading to an increase in rbc count, thus improving oxygen transport and allowing normal activity. Deep-diving mammals Physiological adaptations of mammals such as whales and seals include: decrease in heart rate when submerged (less oxygen used by cardiac muscle); partial collapse of lungs, forcing air into upper respiratory system and compressing it, making animal less buoyant; high volume of blood per kg body mass; high concentration of haemoglobin; myoglobin in muscles. Concentration of atmospheric oxygen over geological time The Earth’s atmosphere has changed over geological time, with oxygen increasing from none to 1% (possibly produced by the first photosynthetic cyanobacteria), then to the present 21% (reason not known). A high level of atmospheric oxygen is necessary to maintain the metabolism of large air-breathing animals. Oxygen uptake and fitness Maximum oxygen uptake: the maximum volume of oxygen that a body can take up during gradually-increasing intense exercise. VO2 max: has been reached when oxygen consumption remains steady; is calculated using breathing rate and concentrations of respiratory gases in inhaled and exhaled air is measured in cm3kg-1min-1. VO2 max. score is an indicator of fitness and improves with training. Page 22 Higher Biology Metabolism & Survival Notes METABOLISM IN CONFORMERS AND REGULATORS The ability of an organism to maintain its metabolic rate is affected by abiotic factors such as temperature and pH. Organisms can be categorised by the way in which they are able to control their internal environment: CONFORMERS are organisms that are directly dependent on their external environment. REGULATORS can control their internal environment and maintain a steady state regardless of their surroundings. CONFORMERS Internal environment is directly dependent upon the external environment; not a problem if environment is stable. Advantage: low metabolic costs Disadvantages: • narrow range of ecological niches; • less adaptable to change. • many conformers use behavioural responses to help to maintain an optimum metabolic rate e.g. lizards bask in sunshine to warm up. REGULATORS Regulators control their internal environment by physiological means. Regulation of the internal environment within tolerable means is called HOMEOSTASIS. Advantage: increases the range of possible ecological niches. Disadvantage: energy is required to achieve homeostasis. PHYSIOLOGICAL HOMEOSTASIS The cells in the body have to be kept in a stable environment even when there are changes in the body’s rate of activity. This is necessary so that cells can function efficiently. Maintaining conditions in the body within tolerable limits is called physiological homeostasis. Homeostasis is necessary for the control of e.g. water concentration, blood sugar level and body temperature. In homeostasis, the control of the body’s internal environment is brought about by NEGATIVE FEEDBACK CONTROL. Negative feedback involves a corrective mechanism that opposes any deviation from the normal optimal level (norm or set point) e.g. rise in body temperature, fall in blood sugar concentration. Mechanism of negative feedback control Changes from the norm (or set point) are detected by receptors. Messages are sent via nerves or hormones to effectors which return conditions to normal by negative feedback control. Receptors Corrective response by effector Increase in factor Decrease in factor Factor increased Factor decreased Normal Condition THERMOREGULATION Animals can be classified as ECTOTHERMS or ENDOTHERMS according to their ability to regulate their body temperatures. Ectotherms are unable to regulate their body temperature and derive their body heat from the environment e.g. invertebrates, fish, amphibians and reptiles. Some regulate their body temperature by behavioural means e.g. lizard basking in sunshine. Endotherms such as mammals and birds derive most of their body heat from their own metabolism. The temperature of an endotherm’s internal environment must be regulated to ensure that enzyme-controlled metabolic processes in the body are kept within tolerable limits i.e. 35-40°C. Endotherm body temperature is independent of external conditions and is under homeostatic control. Page 23 Higher Biology Metabolism & Survival Notes Homeostatic control of temperature Body temperature is monitored by the HYPOTHALAMUS, a gland in the brain. It receives information about surface temperature from the thermoreceptors in skin and its own thermoreceptors monitor blood temperature. The hypothalamus coordinates the response to these signals with priority being given to blood temperatures (indicate temp. of body core). The hypothalamus sends nerve impulses directly to effectors, triggering corrective feedback mechanisms. Effectors Effector Increase in temp. Decrease in temp. Sweat glands Skin arterioles Hair erector muscles sweat glands stimulated; heat energy from the body is used during evaporation of water from surface of skin and lowers body temperature. causes vasodilation, leading to increased heat loss by radiation body hairs lowered Skeletal muscles no increase in activity to cause shivering Metabolic rate decrease in metabolic rate sweating reduced to a minimum causes vasoconstriction, leading to decrease in heat loss causes erector muscles at the base of hairs to contract; hairs raised from the surface trap a layer of insulating air and reduce heat loss. increase in activity and production of additional heat by shivering increase in metabolic rate leading to increased heat production Page 24 Higher Biology Metabolism & Survival Notes METABOLISM AND ADVERSE CONDITIONS An organism’s environment can change very quickly e.g. rain, wind, sunlight, or more slowly e.g. seasons. Some changes can cause stress to the organism, when the conditions are beyond the limits of its metabolic rate. Organisms must maintain a constant internal environment and are therefore either adapted to survive change or are able to avoid it. Organisms can have adaptations to help them survive. These can be: Structural e.g. body size, appendages, insulation, colour Physiological e.g. dormant states such as hibernation (‘winter sleep’); aestivation (‘summer sleep’); brumation (less deep hibernation in ectotherms); diapause (suspension of growth in insects) Behavioural: collective den, snow roost Dormancy Some organisms survive extreme conditions by entering dormancy, when their metabolic rate is reduced to the minimum level necessary to sustain life. Dormancy can be predictive or consequential. Predictive dormancy happens in advance of adverse conditions so the organism is prepared. Examples • Hibernation in some animals. • Deciduous trees drop their leaves in Autumn, triggered by decreasing photoperiod, and winter buds remain closed until Spring. Consequential dormancy happens after the conditions have arrived e.g. in areas of unpredictable climate. Allows longer period of activity but may cause death. Example: many seeds remain dormant during drought and germinate only when sufficient water is available. Dormancy in seeds Dormancy can be result of: • a physical barrier e.g. thick seed coat – has to be broken down by micro-organisms; • chemical inhibitors – need heavy rain to remove them or long period of cold to halt production. Both of these can be shown experimentally by scarification of seeds or subjection to cold conditions. Dormancy ensures that seeds germinate only when conditions are right for plant growth e.g. in the Spring. Seed banks Seed banks have been established as a means of preserving seeds from many plant species, especially those that • are used as food crops (and wild relatives); • have medicinal properties; • are rare/threatened. Seeds must be kept under the best conditions to retain their viability: orthodox seeds are desiccation tolerant and are kept at low temperature and low humidity; recalcitrant seeds are desiccation intolerant so the whole plant has to kept alive and growing. Dormancy in animals Main types: HIBERNATION and AESTIVATION; also DAILY TORPOR HIBERNATION is a period of inactivity in mammals to enable them to survive winter conditions. Before hibernation, extra food consumption allows the build-up of a fat store. Occurs in e.g. hedgehogs, dormice. Physiological changes: • drop in metabolic rate; • decrease in body temperature; • slower heart rate; • slower breathing rate. Page 25 Higher Biology Metabolism & Survival Notes AESTIVATION allows animals to survive periods of heat and drought by remaining in a state of torpor (deep and prolonged sleep) with reduced metabolic rate. Examples Mediterranean land snails: climb bushes and seal shells with mucus to conserve moisture Nile crocodiles: stay in burrows, drawing on fat reserves, until the rains arrive. DAILY TORPOR Some animals, such as small birds and mammals, enter dormancy on a 24 hour cycle. This is called daily torpor, when heart, breathing rate and body temperature all decrease. Small endotherms need a high metabolic rate to maintain body temperature: torpor allows a decrease in overall energy consumption. Examples Hummingbirds feed by day and are in torpor by night whereas nocturnal bats and shrews feed by night and enter torpor by day. Migration Migration is the regular movement of species members from one place to another. Migration avoids metabolic adversity by expending energy to relocate to a more suitable environment. The expenditure of energy to relocate is a disadvantage to the organism in the short term but is beneficial in the long term. Organisms that migrate include many birds including swallows and corncrakes; mammals such as whales and fish such as salmon. Long-distance migrants usually make an annual round trip between the regions e.g. corncrakes breed in Scotland in Spring but winters in Africa. Some invertebrates also migrate long distances e.g. Monarch butterflies breed in North America but overwinter in Mexico (no butterfly makes a return journey as they are short-lived). Migration study techniques It is of interest to find out details of migration of species e.g. time of outward and return migration; overwintering habitat; place returned to; lifespan of animal etc. Animals are caught and labelled in various ways in order to identify and track them. • Ringing, usually of birds’ legs, with unique number and contact details; recapture necessary when birds return, in order to read rings. • Tagging for small creatures e.g. invertebrates. • Colour marking of birds – allows them to be seen easily. • Transmitters glued to body allows satellite tracking; no need to recapture. Migration triggers Photoperiod (day length) This is the primary trigger for migration in many birds, causing hormonal changes that lead to behavioural and physiological changes such as restlessness and fat storage. The Sun The position of the Sun is used as a compass to locate the correct direction for migration. Stars Experiments at night (and in planetaria) show that birds respond to particular patterns of stars to direct their migration. Magnetic field Some birds can sense changes in the Earth’s magnetic field and use these as a compass, perhaps even ‘seeing’ the magnetic field. [Birds with magnets attached become disorientated and lose their way easily.] Some long-distance migrants may use a combination of Sun and magnetic field compasses. Internal clock Many animals have an internal ‘clock’ that allows them to go in the right direction regardless of other stimuli. Page 26 Higher Biology Metabolism & Survival Notes Migration: influences Migratory behaviour is thought to be influenced by both innate and learned behaviour. Innate behaviour is inherited from parents to offspring and is likely to be the biggest influence on successful migration e.g. direction of travel. It is common to all members of a species. Learned behaviour is gained by experience e.g. specific stop points on a long journey. It may come from parents or other members of a social group. It is more flexible and has a lesser role in migration. Experiments Displacement studies with European starlings have shown that both innate and learned behaviours are used by birds: young birds use purely innate behaviour and fly according to genetic instructions but experienced birds also use knowledge of geographical features. Studies with blackcap warblers show that young birds will fly in the direction typical of their population, showing innate behaviour. Cross-fostering of herring gulls (non-migratory) and black-headed gulls (migratory) show that black-headed gulls migrate even if brought up by herring gulls (innate behaviour) and herring gulls migrated with their foster parents (learned behaviour). Migration: genetic control Genetic studies of the blackcap warbler have shown a link between a gene and migratory behaviour. The gene codes for a peptide that influences preparations for migration such as metabolic rate and fat usage. It is thought that this gene causes 3% of migratory behaviour: many other genes must also be involved. EXTREMOPHILES An extremophile is an organism that lives in conditions that are ‘extreme’. They are generally they are found in the domain Archaea. They have enzymes that are able to function under these unusual conditions, allowing the organisms to thrive in environments that would be lethal to almost all other species. Some of these enzymes are used for scientific purposes e.g. heat-tolerant DNA polymerase used in PCR. Types of extremophiles Extremophile Environment Example Cryophile cold: temps. as low as -15°C sea ice; Arctic and Antarctic ice packs. Thermophile very hot: 80°C -100°C deep sea vents and volcanic lakes Alkaliphile pH levels 9 or more soda lakes Acidophile pH levels 3 or less sulphur springs Halophile salt conc. at least 0.2M salt lakes and salt mines Xerophile very dry desert Page 27 Higher Biology Metabolism & Survival Notes ENVIRONMENTAL CONTROL OF METABOLISM Micro-organisms - introduction Microbiology is a specialised area of biology that studies organisms that are too small to be seen without magnification: these are known as microorganisms or microbes. Microbiology makes up one of the largest and most complex biological sciences because it deals with microbehuman and microbe-environmental interactions. These interactions are relevant to disease (in both plants and animals), drug therapy, immunology, genetic engineering, industry, agriculture and ecology. Microorganisms include archaea, bacteria and some species of eukaryote e.g. yeasts and filamentous fungi. Some of these species use a wide variety of often cheap substrates for metabolism and produce a range of products from their metabolic pathways. Microorganisms are also used in research and industry because of their adaptability, ease of cultivation, speed of growth and ease of manipulation of metabolism. Archaea are single-celled and have no nucleus or organelles. While outwardly appearing similar to bacteria, several of their metabolic pathways are similar to eukaryotes. Many have been classified as extremophiles and the properties that allowed them to exploit these niches make them of potential use to industry. Bacteria are divided into three main groups. Each group is further divided until the species level is reached. Different types within a species are called strains: that is, groups of different cells derived from a single cell. Many species of bacteria are of economic importance to humans. Bacteria are involved in the production of yoghurt, cheese, biofuels and many other products and are used in genetic engineering. Fungi are eukaryotic cells and can be sub-divided into single-celled yeasts and multicellular moulds. Some fungi are beneficial and some are harmful. Beneficial • decomposers • essential to many industrial processes that involve fermentation e.g. bread, beer, wine production • manufacture of antibiotics Harmful • major cause of plant disease. • cause of many animal diseases Protozoa and Algae Protozoa are unicellular eukaryotic cells that contain organelles and, like animal cells, lack cell walls. Algae are plants, often unicellular, growing in a wide variety of moist habitats. Growth of micro-organisms (cell culture) Micro-organisms can be cultured relatively easily in a laboratory. They must be given an appropriate growth medium and the environmental conditions must be carefully controlled to ensure successful growth. Growth media A growth medium is a solid or liquid substance used to grow, transport or store micro-organisms. A liquid medium is called broth and solid media are made of agar jelly with added nutrients. Growth media can be composed of specific substances or can contain complex ingredients such as beef extract. Two types of media are commonly used: • complex media contain one or more crude sources of nutrients and their exact chemical composition and components are unknown; • defined media, or synthetic media, are media in which the components of the medium are chemically known and are present in relatively pure form. Page 28 Higher Biology Metabolism & Survival Notes Requirements for growth of micro-organisms Microorganisms require an energy source (which may be chemical or light) and simple chemical compounds for biosynthesis. Many microorganisms can produce all of the complex molecules required for life, including amino acids for protein synthesis. Other microorganisms require more complex compounds to be added to the growth media, including vitamins and fatty acids. Environmental conditions In order to grow cells in culture, they must be supplied with a growth medium and the correct environmental conditions, including: sterility to eliminate contamination e.g. using aseptic techniques; heat sterilisation by autoclaving temperature - controlled using an incubator pH - controlled by the use of buffers or addition of acid/alkali gaseous environment - some microorganisms are anaerobic and will not grow in the presence of oxygen; others will require a good oxygen supply light (if it is a photosynthetic microorganism) Industrial production methods Production of micro-organisms on an industrial scale requires the use of large fermenters that are computercontrolled. Culture conditions are monitored by sensors and conditions are maintained at optimum levels. Patterns of growth Growth is the irreversible increase in dry biomass. Growth occurs when rate of synthesis of organic materials exceeds rate of breakdown. Measuring dry biomass is not always practicable: investigation of bacterial growth involves measuring the increase in cell number over time. The time taken for a cell to divide into two cells (to double) is called generation time. Under ideal conditions, some species of bacteria are capable of doubling in number every 20 minutes. The four main stages in growth are: 1. Lag phase - micro-organisms adjust to the conditions of the culture by producing enzymes that metabolise the available substrates. 2. Exponential or log (logarithmic) phase - rate of growth is at its highest 3. Stationary phase - culture medium becomes depleted and secondary metabolites are produced. 4. Death phase - lack of substrate and toxic accumulation of metabolites causes death of cells If bacterial growth during the exponential (log) phase is plotted on normal graph paper, there is not enough space on the y axis, or the scale is so reduced that it makes plotting or reading with accuracy almost impossible. The solution is to use semi-logarithmic graph paper. This has been printed in a specific way to allow data which has a very wide range to be plotted. Page 29 Higher Biology Metabolism & Survival Notes Primary and Secondary metabolism Filamentous fungi (and some bacteria) exhibit two types of metabolism: Primary and Secondary. Primary metabolism Metabolism during lag and exponential (log) phases of growth: breakdown of substrate and production of primary metabolites e.g. amino acids and nucleotides for synthesis of microbial cells. Primary metabolites also include end products of fermentation e.g. ethanol, a waste product but one of the first microbial metabolites used by humans. Secondary metabolism Metabolism at end of log phase and during entire stationary phase: production of secondary metabolites that are not used for growth (some may be toxic) but may confer an advantage e.g. antibiotics inhibit bacteria. Humans grow micro-organisms on an industrial scale in order to harvest many secondary metabolites e.g.: Antibiotics Cyclosporin (immunosuppressant) Gibberellin (plant hormone) Glutamic acid (amino acid used to make monosodium glutamate) Citric acid Manipulation of metabolism Micro-organisms naturally control their metabolic pathways by several processes e.g. induction or inhibition of enzymes; end-product inhibition. In industrial processes, micro-organisms are used to produce large amounts of a desired compound. This involves manipulation of microbial metabolism as the desired compound is often an intermediate of a metabolic pathway which, under normal circumstances, would not accumulate in large quantities. The production of these useful substances is often stimulated by the addition of precursors, inducers or inhibitors. Precursor: an earlier metabolite in the pathway. Inducer: a metabolite that induces the formation of an enzyme. Inhibitor: reduces the activity of an enzyme by acting competitively or non-competitively or by binding to the operator region of the coding gene. Microbial metabolic pathway: In this metabolic pathway, metabolite C is the desired product. C can be mass-produced by adding: a precursor - in this case metabolite A, providing a continuous supply of metabolite B; an inducer – such as metabolite B – that would induce the formation enzyme 2; an inhibitor of enzyme 3, allowing metabolite C to accumulate. • • • • • • Example of industrial process: production of penicillin Spores of Penicillium chrysogenum germinated. Mycelium used to inoculate liquid medium in fermenter. Optimum conditions promote rapid growth – vegetative phase. Balance of nutrients altered: slows down growth and increases antibiotic production – antibiotic production phase. Fungal mycelium filtered out. Penicillin recovered by series of chemical processes giving 99.5% pure crystals of penicillin. Page 30 Higher Biology Metabolism & Survival Notes GENETIC CONTROL OF METABOLISM IN MICRO-ORGANISMS Use of micro-organisms Wild strains of micro-organisms have been used in industry for many years. These wild strains can be cultivated so that: • they produce required substances more efficiently; • useful mutant strains can be isolated and cultivated. Wild mutants may need to be improved in order to confer e.g. • genetic stability • ability to grow on low-cost nutrients • ability to overproduce required product • ease of harvesting Strain improvement Strain improvement - to alter a wild strain’s genome - can be brought about by: • mutagenesis • selective breeding • recombinant DNA technology Mutagenesis Mutations occur naturally but can also be induced artificially by mutagenesis. Mutagenesis is caused by the use of mutagenic agents such as radiation and mutagenic chemicals. Most mutations are deleterious but some may produce an improved strain which is beneficial to the organism or makes it more useful to humans. Many micro-organisms used in industry have been improved by repeated mutagenesis and selection for the desired trait. The improved strain usually lacks an inhibitory control mechanism, allowing it to produce more of the required end-product. Mutant strains are often genetically unstable: they regress to the wild type after generations in culture by undergoing a reverse mutation. Improved strains must be monitored to ensure that this has not occurred. Selective breeding Eukaryotic micro-organisms can reproduce sexually and asexually. Sexual reproduction in fungi leads to variation and can be used in breeding programmes to produce new strains. Bacterial reproduction is asexual but horizontal transfer of genetic material can lead to variation and new strains. Bacteria can pass plasmids or pieces of chromosomal DNA to each other in this way, leading to variation when different strains are cultured together. Horizontal transfer can occur in several ways: • transformation • transduction • conjugation Transformation Bacterial cell takes up a piece of DNA from the remains of another bacterium and incorporates it into its own DNA. Page 31 Higher Biology Metabolism & Survival Transduction Foreign DNA (from another bacterial cell) is introduced by a bacteriophage and incorporated into host bacterium’s DNA. Notes Conjugation Conjugation tube formed between two different bacterial cells allows transfer of replicated plasmid. Transduction Recombinant DNA technology Recombinant DNA technology is a technique used in genetic engineering where a required plant or animal gene sequence is transferred into a micro-organism. The micro-organism has been artificially transformed so that it will: • produce more of a target product; • secrete the product into the surrounding medium; • not survive in an external environment (safety mechanism). Gene sequences are transferred from donor cells to the host bacterium using a vector such as a plasmid. The transformed host will express the new gene, producing e.g. insulin. The host cell has recombinant DNA - a combination of its own and foreign DNA. Recombinant DNA technology techniques Recombinant DNA technology requires: • donor cells with required gene; • restriction endonucleases to cut up DNA; • vector e.g. plasmid to carry DNA from donor to host; • DNA ligase to seal DNA fragment into plasmid • host cells to receive the altered vector. Restriction endonucleases Restriction endonucleases are a group of enzymes that can recognise and cut specific sequences of DNA into fragments with ’sticky ends’ (pieces of DNA that have unpaired nucleotides at each end). They allow specific genes to be cut out of a source chromosome as well as the cutting of bacterial plasmids. Each endonuclease recognises a different restriction site (DNA sequence 4-8 nucleotides long). Page 32 Higher Biology Metabolism & Survival Notes Vectors To be effective, a vector must have: • restriction site that can be cut open by the same restriction endonuclease used to the cut the donor DNA; • marker gene to indicate whether host cell has taken up the vector e.g. gene for penicillin resistance (cells that have no marker are killed by penicillin); • origin of replication controlling self-replication of plasmid and regulatory sequences controlling gene expression. DNA ligase DNA ligase is an enzyme that can join two different fragments of DNA together. It seals DNA fragments into a plasmid to form a recombinant plasmid. The sticky ends of the DNA fragments and the plasmids are complementary to each other, allowing ligase to bind them together. Steps in the process • • Identification of gene of interest in donor • Isolation of gene of interest • Insertion of gene into vector • Introduction of vector to host (transformation). • Selection of transformed host cells • Expression of introduced gene in host Artificial chromosomes Artificial chromosomes, constructed by scientists, can also act as vectors. They are usually made by adding nonbacterial DNA to bacterial chromosomes. They are useful in that they carry larger fragments of DNA than is possible using plasmids. Use of bacteria – limitations Eukaryote DNA Prokaryote DNA • introns and exons • exons but no introns • primary transcripts modified by splicing • no modification by splicing • proteins undergo post-translational modification. • no post-translational modification These differences may lead to the production of an inactive polypeptide due to incorrect folding or lack of posttranslational modification. Some proteins are therefore more successfully produced by using genetically transformed eukaryotic cells e.g. yeast. Hazards and control of risks Requirements for licence Product must be: • safe for manufacturing staff • safe for consumers • pure • uncontaminated by micro-organisms • fit for purpose Manufacturing process must: • maintain standards of product purity • use safe, well-designed facilities Risk assessment Many micro-organisms used in biotechnology can act as allergens, irritants or pathogens and their use requires stringent risk assessment. A risk assessment must: 1. assess the risk 2. identify potential hazards 3. construct and apply control measures 4. review effectiveness and adopt improvements 5. regularly repeat the process Page 33 Higher Biology Metabolism & Survival Notes Ethical considerations Ethics can be defined as ‘moral principles that govern a person's behaviour or the conducting of an activity ‘(Oxford dictionary). Regarding recombinant DNA technology, it deals with issues of what is right and wrong in research and development in this field. There are many arguments on both sides. Arguments in favour Recombinant DNA technology leads to improvement of: • nutrition and food security (more and better food) • the environment, by reducing use of pesticides and fertilisers • health, by efficient drug production Arguments against Recombinant DNA technology could lead to: • uncontrolled dispersal of altered cells into the natural environment; • sideways transfer of genetic material to different species; • unforeseen and difficult to control metabolic modifications; • creation of new pathogenic micro-organisms. Page 34