242 Biology Term 3 STPM Chapter 19 Biotechnology 19 100 COOH 191 Primary structure of HGH 150 NH2 1 50 3-D model pf HGH Figure 19.8 The primary structure and three dimensional model of HGH Application of Biotechnology in Medicine and Forensic Human insulin production 1. In 1978, Genentech Company has developed synthetic human insulin by joining its gene with a plasmid vector inserted into the bacterium E.coli. The procedure is discussed in Chapter 18. 2. Insulin, widely used for the treatment of diabetes, was previously extracted from the pancreas of abattoir animals (cattle and/or pigs). The resulting genetically engineered bacterium enabled the production of vast quantities of synthetic human insulin at relatively low cost. 3. According to the International Diabetes Federation, synthetic human insulin is considerably more expensive in most countries where both synthetic human and animal insulin are commercially available: e.g. within European countries the average price of synthetic human insulin was twice as high as the price of pork insulin. 4. There are advantages of treating diabetics with human insulin produced by gene technology because: (a) Chemically, they are identical to human insulin, little chance of an immune response. (b) Rapid response, exact fit to human insulin receptors of liver cells to lower blood sugar. (c) Duration of the response is much shorter compared to animal insulin. (d) No problems related to the development of tolerance to insulin from animals. (e) No objections from religion and ethical point of view. Human growth hormone (HGH) production 1. Human growth hormone (HGH) was synthesised by genetic engineering in 1970 and was commercially produced in the 1980s. (a) HGH has 191 amino acid residues, four alpha helixes, and two sulphide bonds. The primary structure and three dimensional model of HGH are shown in Figure 19.8. (b) In 1979, scientists at Genentech produced HGH by inserting DNA coding for HGH into a plasmid that was implanted in E.coli bacteria. The gene that was inserted into the plasmid was created by reverse transcription of the mRNA found in pituitary glands to complementary DNA. HaeIII, a type of restriction enzyme which acts at restriction sites in the 3’ non-coding region and at the 23rd codon in complementary DNA for HGH, was used to produce a DNA fragment of Language Check Language Check Abattoir = slaughterhouse
243 Biology Term 3 STPM Chapter 19 Biotechnology 19 551 base pairs which includes coding sequences for amino acids 24–191 of HGH. Then a chemically synthesised DNA ‘adaptor’ fragment containing an ATG initiation codon was produced with the codons for the first through 23rd amino acids in HGH. The two DNA fragments were combined to form a synthetic-natural ‘hybrid’ gene. (c) From the active cells that synthesise HGH many forms of mRNA of HGH can be extracted. These mRNA forms can be reversed transcribed into isoforms and given the name as shown in Table 19.4 HGH22k (or HGHN) is the main product of the HGH gene (hGH-N) in the anterior pituitary gland. It is the best known hormone and the only one of the HGH family that has been commercialised. Table 19.4 HGH-V isoforms generated by alternative splicing and processing Isoform Length Characteristic HGH-V22k 191aa Main isoform HGH-V25k 191aa Glycosylated version of HGH-V22 HGH-V2 230aa Retains the fourth intron HGH-V20K 176aa Deletion of aa residues 32 to 46 HGH-V3 219aa Alternate processing at level of exon 4 2. HGH is used to treat children who are born with a type of dwarfism caused by a failure of the brain to produce sufficient quantities of HGH. 3. Until the 1980s these children could only be treated with HGH extracted from the brains of people who had died. There are a number of problems with this treatment – only tiny amounts of HGH could be extracted from the brains of people who had died and hence only a few children could be treated. In addition, the extracted HGH could contain impurities that could increase the risk of children contracting a fatal brain disease. 4. Genetic engineering has enabled HGH to be produced in large quantities using bacteria. All children affected can now be treated with the artificial version of HGH that does not carry the risk of transmitting infectious diseases. 5. Approved indications for HGH therapy include treatment of growth hormone deficiency, Turner syndrome, Prader–Willi syndrome, chronic renal insufficiency, idiopathic short stature in children, AIDS-related wasting and fat accumulation associated with lipodystrophy in adults. 6. HGH is required for normal bodily growth and development – growth of bone, muscle and cartilage cells. It functions by promoting RNA and protein synthesis so that the cells can multiply and differentiate. Other functions are shown in Table 19.3.
244 Biology Term 3 STPM Chapter 19 Biotechnology 19 Table 19.3 New functions attributed to HGH22k Factors Functions Immunisation and healing • Resistance to common diseases • Healing of old lesions • Ability to heal • Healing of other lesions Skin and hair • Skin elasticity • Growth of new hair • Skin thickness • Disappearance of wrinkles • Skin texture • Skin hydration Sexual factors • Duration of an erection • Regulation and control of the menstrual cycle • Increase in libido • Positive effects in the reproductive system • Potential/frequency of sexual activity • Increase in breast-milk volume Bone • As treatment for bone fractures • Increases flexibility of the back and joints • Osteoporosis treatment Mental function • Emotional stability • General aspect and attitude • Memory • Mental energy and clarity Muscle strength and tone • Increase in energy in general • Promotion of muscle mass gain • Increase muscle strength Circulatory system • Improvement in circulation • Improvement in cardiac function • Stabilization of blood pressure Fats • Increases “good” cholesterol (HDL) levels • Reduces fat Gene therapy 1. Gene therapy may be used for treating, or even curing, genetic and acquired diseases like cancer and AIDS by using normal genes to supplement or replace defective genes or to bolster a normal function such as immunity. 2. Gene therapy can be used to target somatic cells (i.e., those of the body) or gamete (i.e., egg and sperm) cells. In somatic gene therapy, the genome of the recipient is changed, but this change is not passed along to the next generation. In contrast, in germ line gene therapy, the egg and sperm cells of the parents are changed for the purpose of passing on the changes to their offspring. 3. There are basically two ways of implementing a gene therapy treatment: (a) Ex vivo (“outside the body”) Cells from the patient’s blood or bone marrow are removed and grown in the laboratory. They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the cell DNA. The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein. The procedure is summarised in Figure 19.9. 2013 2016
245 Biology Term 3 STPM Chapter 19 Biotechnology 19 DNA formed isolated Harmful genes removed ADA gene inserted Retrovirus formed extracted DNA into new host cell Inoculated into stem cell Stem cells tested for ADA Cultured rat cells Inoculated with retrovirus Figure 19.9 Steps of ex vivo gene therapy Steps of ex vivo gene therapy: i. Cultured rat cells are first inoculated with retrovirus. ii. The DNA formed by reverse transcription by the virus is isolated and harmful genes are removed. iii. The adenosine deaminase (ADA) gene is inserted into the DNA and the recombinant DNA is injected back into the host cell. iv. The recombinant DNA is stimulated to form retrovirus particles. v. The retrovirus is then used to attack bone marrow stem cells extracted from the baby. vi. After the RNA of the virus is changed to DNA, the recombinant DNA is incorporated into the stem cells. vii. The stem cells are tested for the presence of the ADA enzyme. Then, they are injected back into the bone marrow. viii. The stem cells form white blood cells that can produce the enzyme. So, the baby becomes normal but more often the procedure has to be repeated, as it is only effective for a short period. (b) In vivo (“inside the body”) No cells are removed from the patient’s body. Instead, vectors are used to deliver the desired gene to cells in the patient’s body. There are two delivery systems for in vivo gene therapy of cystic fibrosis, a genetic disorder producing sticky mucus especially in the lungs. Viral delivery systems: i. Adenoviruses infect lung can be used as the vector. ii. Their virulence (ability to cause disease) is removed. Info Bio Children born with severe combined immunodeficiency disorder (“SCID”) were reported to have been cured after being given genetically engineered cells. They were born with homozygous recessive defective adenosine deaminase alleles causing no white blood cells can be formed.
246 Biology Term 3 STPM Chapter 19 Biotechnology 19 iii. Engineered adenoviruses are to carry the functional human CFTR (cystic fibrosis trans-membrane regulator) gene. iv. Aerosol is sprayed directly into the lungs. v. The adenoviruses release the genetic material into the cells where it is expressed Non-viral delivery systems i. Lipid spheres or liposomes containing the DNA are made. ii. An aerosol of liposomes carrying the DNA is sprayed into the lungs for the gene to express. iii. DNA also can be compressed into a very small volume which may directly enter cells. 4. Gene therapy faces many obstacles before it can become a practical approach for treating disease. At least four of these obstacles are as follows: (a) Gene delivery tools. Genes are inserted into the body using gene carriers called vectors. The most common vectors now are viruses, which have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. They can introduce problems like toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, in order for gene therapy to provide permanent therapeutic effects, the introduced gene needs to be integrated within the host cell’s genome. Some viral vectors affect this in a random fashion, which can introduce other problems such as disruption of an endogenous host gene. (b) High costs. Since gene therapy is relatively new and at an experimental stage, it is an expensive treatment to undertake. This explains why current studies are focused on illnesses commonly found in developed countries, where more people can afford to pay for treatment. (c) Limited knowledge of the gene functions. Scientists currently know the functions of only a few genes. Hence, gene therapy can address only some genes that cause a particular disease. (d) Multi-gene disorders and effect of environment. Most genetic disorders involve more than one gene. Moreover, most diseases involve the interaction of several genes and the environment. For example, many people with cancer not only inherit the disease gene for the disorder, but may have also failed to inherit specific tumour suppressor genes. Diet, exercise, smoking and other environmental factors may have also contributed to their disease.
247 Biology Term 3 STPM Chapter 19 Biotechnology 19 DNA fingerprinting 1. Electrophoresis is used to form a pattern using an individual's DNA profile in genetic fingerprinting. The pattern is from a special marker called variable number of tandem repeats (VNTR) sequence e.g. GTGTGT which is found in the introns or between the genes of DNA. 2. It usually is a polymer chain reaction (PCR) related method. The starting material for such a PCR reaction can be a single hair or a tiny sample of blood. PCR is used to increase the concentration of the VNTR so as the pattern formed can be clearly observed. 3. Usually the VNTR is cut by specific restriction enzymes into different lengths depending on the number of repeats. More repeats will be cut into longer lengths and vice versa. 4. The different fragment lengths are separated by gel electrophoresis (Figure 19.10). Primers for PCR Separate PCR products on gel amplification Repeated squences of a VNTR locus Homologous chromosomes Paternal Maternal Electrophoresis VNTR 1 individual A Individual B Individual C Forensic sample F 3 pairs of homologous chromosomes VNTR 2 VNTR 3 0 5 10 15 20 25 30 35 A B C AF Number of repeats Electrophoresis PCR PCR PCR PCR Figure 19.10 Steps in making and understanding genetic fingerprint Info Bio Polymerase chain reaction (PCR) is to clone or produce millions copies of DNA with identical sequence (STPM 2014 essay question). Summary of PCR 1. A strand in buffer solution heated to 98°C. 2. DNA is seperated into two strands as hydrogen bonds are broken. 3. Cooled to 60°C and primer added to 3' ends. 4. Hotspring bacteria DNA polymerase added togather with A, C, G and T nucleotide triphosphates. 5. Two identical DNA is formed. 6. Heated to 98°C again followed by cooling and process repeated. 2014
248 Biology Term 3 STPM Chapter 19 Biotechnology 19 5. The gel appears solid but is actually a colloid. Electrodes are placed, DNA molecules move under the influence of an electric current. Rate depends on the mass of the fragment molecule. Smaller fragments move further and faster in a given time while larger DNA fragments move slower forming a DNA fingerprint. 6. DNA must be stained to make them visible. This is done by using ethidium bromide, methylene blue or Nile blue A. 7. Another way is by creating a gene probe that is complementary to the VNTR. The gene probe is a single stranded labelled piece of DNA and is detected by its ability to expose the photographic film that is used to make X-ray photographs. 8. The pattern is compared with other samples of DNA as in forensic investigations. Samples can be derived from related individuals and yet have similarities in number of tandem repeats. A similar approach is now routinely used for paternity testing. Application of Biotechnology in Public Health Genetic screening 1. Genetic screening is a technique used to test a group of people for genes and defective karyotypes. 2. Such testing is to identify the presence or absence of a particular allele especially deleterious ones. The testing is also to determine risk of having or passing on particular genetic conditions. 3. It is carried out in three situations namely predictive, prenatal and carrier diagnosis. 4. Predictive diagnosis is to determine if the individual already has the allele or alleles, which have not been expressed. One example is dominant Hungtington’s chorea where the on set of the disease typically occurs in around thirty-five years old. 5. Prenatal screening can detect the following genetic abnormalities: (a) Chromosomal abnormalities, such as Down,s Syndrome (trisomy 21), trisomy 13 also called Patau syndrome, and trisomy 18 also known as Edwards syndrome with many affected foetuses do not survive to term. All these chromosomal conditions are associated with severe intellectual disability and physical abnormalities in many parts of the body. (b) Single gene disorders e.g. haemophilia (lack of blood clotting factor), sickle cell anaemia (red blood cells become sickle-shaped) and cystic fibrosis (producing sticky mucus especially in the lungs).
249 Biology Term 3 STPM Chapter 19 Biotechnology 19 (c) Neural tube defects, such as spina bifida and anencephaly. Spina bifida is a birth defect in which the backbone and spinal canal do not close before birth. A newborn may have a sac sticking out of the mid to lower back. Anencephaly is the absence of a large part of the brain and the skull. 6. There are three ways to do this namely amniocentesis and chorionic villus sampling (CVS). (a) Amniocentesis i. It is a procedure in genetic screening using foetal cells obtained from the amniotic fluid. This is done after the amniotic cavity is formed from 12th to the 18th week of pregnancy as shown in Figure 19.11. Foetus (8 – 10 weeks) Amniotic fluid withdrawn Uterus wall Amniotic fluid Foetus (14 – 16 weeks) Supernatant Biochemical tests Several weeks later Cell culture Foetal cells Chorionic villus sampling Amniocentesis Chorionic villi Catheter Chorion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Y Figure 19.11 Amniocentesis and chorionic villus sampling ii. A long hollow needle is pierced through the abdominal wall and a syringe is used to suck the fluid. iii. About 20 ml of the fluid is obtained and it is centrifuged to obtain the cells shed from the skin or alimentary canal. iv. Dead cells may be separated to use for sex determination and abnormal genes. v. Living cells are cultured to increase their numbers and to analyse their cell division. vi. About 40 types of genetic diseases of metabolism can be analysed and chromosome defects can be obtained through karyotype analysis. Exam Tips Student should be able to differentiate between amniocentesis and its use. (STPM 2015 essay question) 2014
250 Biology Term 3 STPM Chapter 19 Biotechnology 19 (b) Chorionic villus sampling i. It is a procedure in genetic screening using cells obtained from the outermost layer of the embryo. This is done as early as 8th week of pregnancy. ii. A long hollow needle is pierced through the abdominal wall and a syringe is used to suck the cells. Another way is a catheter through the vagina. iii. The cells obtained are living and dividing. They can be used directly to test for genetic diseases or karyotype analysis. iv. This method has an advantage to obtain the result in earlier pregnancy and shorter time is required. It runs a higher risk of miscarriage as it has a higher risk of injuring the embryo. (c) Intra-uterine blood test Foetal blood is sampled at 16 – 18 weeks to test for an array of both infectious and genetic diseases. 7. Carrier analysis also known as gene probe or DNA analysis is to determine or identify recessive defective allele in the heterozygous state. (a) This is done when one member of the family is affected by a genetic disease such as cystic fibrosis. (b) Depending on the type of genetic disease being analysed, the sequence of amino acid of the abnormal protein formed in the family member has to be known. (c) Based on the abnormal sequence of the amino acids, the base sequence of the gene is worked out using the genetic code table. (d) A radioactive probe usually of RNA or single stranded DNA is artificially made in the laboratory. The RNA sequence is complementary to the defective gene. The numbers of probe molecules are amplified by a method called polymerase chain reaction (PCR). (e) DNA is isolated from cells obtained from the suspected carrier. It is reacted with a restriction endonuclease to cut it into fragments. The fragments are separated using gel electrophoresis. (f) The bands are transferred to nylon membrane using Southern Blotting so that the DNA become single stranded. The probe is added and washed to see if there is any hybridisation. (g) If hybridisation of the probe with the DNA fragment occurs, then the suspect must be a carrier. This can be shown by autoradiography when photographic film is exposed to the bands. 2014
251 Biology Term 3 STPM Chapter 19 Biotechnology 19 8. Newborn screening is now a standard practice in all hospitals where newborns are screened for many genetic diseases including phenylketonuria (pku) (the baby is born not to be able to tolerate too high protein concentration). If detected, remedial action not to feed the baby with too high protein for one year can cure the fatal condition of too high phenyketones in the brain in the past. Diagnostic kits 1. Diagnostic kits are usually in the form of dipsticks used in a diagnosis serving to identify a particular disease or a characteristic symptom. 2. One of the uses of dipsticks is in the quantitative measurement of glucose in the urine or blood. (a) The enzyme glucose oxidase is immobilised with a solid medium on the surface of the strip of paper. (b) This enzyme will oxidise the glucose into hydrogen peroxide and gluconic acid, if there is glucose in the urine. (c) Hydrogen peroxide in the strip will oxidise an indicator chemical which changes colour to varying intensity. (d) The exact colour change is related to the concentration of the glucose. (e) A specially prepared calibrated colour chart gives a reading for the glucose concentration. 3. Another is a pregnancy testing kit comprised of the immunoassay strip and the housing. (a) The kit is to test hormone called human chorionic gonadotrophin (HCG). (b) HCG is produced by the outer layer of a developing embryo after conception. (c) The ‘sampler’ or dipstick with an absorbent pad whose surface is coated with monoclonal antibodies specific to HCG. (d) Coloured latex particles are attached to indicate reaction. (e) When dipped into urine if HCG is present will bind to the antibodies. (f) HCG molecules will bind with these antibodies to give colour. (g) Another spot is lined with immobilised HCG and antibodies as control. (h) No coloured line indicates faulty pad as shown in Figure 19.12. Oil-decomposing bacteria 1. Oil is made up of hydrocarbons, organic compounds comprised of hydrogen and carbon. While some hydrocarbons evaporate from or form “mousse” and tar balls on the ocean surface, hydrocarbons in the deep sea, whether released by human-caused spills or natural seeps, are eliminated almost entirely by bacteria. 1. POSITIVE: Three distinct pinkcolored bands appear, one in the control region (C) one in the reference region (R), and a third distinct pink-colored band in the patient test region (T). A Test line with a color density less than that of the reference band indicates an hCG level likely below 25 mlU/ml. 2. NEGATIVE: Only two pinkcoloured bands appear, one in the control region (C) and one in the reference region (R). No distinct pink-colored band appears in the patient test region (T). 3. INVALID: A total absence of pinkcolored bands in all three regions is an indication of procedural error or that test reagent deterioration has occured. Transfer Pipet Test Device C T CRT CRT CRT Sample Well Test Region Control Region Positive Negative Invalid Interpretation of results Figure 19.12 Pregnancy test kit and result VIDEO Bacteria that Help and Harm
252 Biology Term 3 STPM Chapter 19 Biotechnology 19 Info Bio Alcanivorax borkumensis were used against the ongoing Deepwater Horizon oil spill in the Gulf of Mexico. These tiny billions of hydrocarbon breaking-up bacteria were used together with more than 830,000 gallons of chemical dispersants on the oil slick both above and below the surface of the sea. 2. Vibrio and Pseudomonas bacterial colonies have been helpful in facilitating the cleanup and degradation of oil spills that occur in the ocean. A genetically engineered bacterium, Pseudomonas is known as one of the “oil-eating bacteria.” Used to assist cleaning up the oil spill from the Exxon Valdez in 1989, it has been used several times since. 3. Another bacterium that contributes to the decomposition of oil is Alcanivorax borkumensis (Figure 19.13), a marine bacterium that depends on oil to live. It is almost undetectable in uncontaminated water, but when the water becomes oily the bacterium aggressively begins to attack and break down the oil immediately. Sea water Alcanivorax secretes natural emulsifiers which help to break up oil droplets Oil droplet Biofilm of Alcanivorax at oil water interface Figure 19.13 Alvcanivorax naturally ‘feed’ on oil 4. Bacteria adapt to gradual exposure to oil. The more oil a bacterial community has been exposed to in the past, the greater its capacity and availability to biodegrade oil in the future. In one study, bacteria together with other microbes from sediments previously contaminated with oil were able to metabolise oil 10 to 400 times faster than those from sediments that had never been contaminated. Once a species of bacteria is exposed to oil and metabolises it, the next generations inherit that ability, a concept known as genetic adaptation. This has been studied in a particular species of Vibrio in the northwestern Gulf of Mexico. 5. In essence, bacteria break down the ring structures of the hydrocarbons in seaborne oil using enzymes and oxygen in the seawater. The bacteria can grow exponentially in days on the excess oil. Indigenous population of microbes are adapted to oil in the Gulf of Mexico. Measurements of oxygen depletion in the Gulf were as much as 30 percent suggesting that the microbes are hard at work eating oil. Over time, specialised bacteria in deep water have adapted to the presence of hydrocarbons, including polycyclic aromatic hydrocarbons like benzene, toluene and naphthalene. Some marine bacteria species, including some in the genus Vibrio, depend on oil. Hydrocarbons from oil are their only source of carbon. VIDEO Exxon Valdex
253 Biology Term 3 STPM Chapter 19 Biotechnology 19 6. The bacteria work best near the surface, whether at land or sea, where warm-water bacteria such as Thalassolituus oleivorans can thrive but colder, deeper waters inhibit microbial growth. In sediment and the deep ocean seafloor that suffers from a dearth of oxygen, anaerobic bacteria can survive but the rate of oil degradation is slow. These bacteria use sulphate rather than oxygen to break down the oil spill. 7. Microbes do not eat enough nor quickly enough; it takes a lot of them a few days to go through an eyedropper full of oil. Research has to go on to investigate the effects of environment so as to produce a fast digesting bacterial variety. This bacterial variety can work in cold temperature and anaerobic conditions. 8. Too fast-eating bacteria are good in getting rid of waste oils. Ideal release of engineered bacteria that die fast after eating the waste oils must be well controlled. Oil companies might not want organisms literally eating the natural oil or crude store. 9. There is also concern over what the environmental impact of releasing engineered bacterial strains might be. The United States Environmental Protection Agency does not permit seeding with Genetically Engineered Microbes (GEMs) due to potential unintended effects of introducing new microbes into an ecosystem. For example, one unintended effect could be a harmful algal bloom. In addition, currents and winds are unpredictable and may carry the new microbes beyond the target area into other ecosystems. Info Bio In 1981, Dr. Chakrabarty received a patent on a genetically modified Pseudomonas bacterium that would eat up oil spills, the first patent of its kind; he was the first person to win a patent on a living organism. Dr. Chakrabarty and his team inserted plasmids from all four species of the oil eaters and put them into a single microbe. While these plasmids would usually not operate together in the same cell, exposing the cell to ultraviolet light caused the plasmids to join into one that could express components of all four pathways of the original plasmids so that several oil components could be broken down. As more research is done, super oil decomposing bacterial species might be developed one day. STPM PRACTICE 19 Objective Questions 1. What is the main factor that makes biotechnology can be applied to almost all aspects of human life? A Genetic engineers are able to understand how living organisms function at cellular and molecular levels. B All organisms synthesise various organic molecules that can be used for a specific application. C All organisms share the same basic genetic material with an almost universal genetic code. D Organisms could be manipulated to express foreign genes. 2. Pharmacogenomics deal with genes that A cause cancer B cause differences in drug response C code for different drug productions D effect economics of drug productions
254 Biology Term 3 STPM Chapter 19 Biotechnology 19 3. Which bacteria can be used to make cheese? A Rhizopus C Saccharomyces B Asperigillus D Acetobacter 4. Which of the following is a correct match between the type of biotechnology and the field involved? Type Field (a) Green biotechnology (i) Aquatic (b) White biotechnology (ii) Agriculture (c) Red biotechnology (iii) Industry (d) Blue biotechnology (iv) Medical (a) (b) (c) (d) A (i) (ii) (iv) (iii) B (ii) (iii) (iv) (i) C (iii) (iv) (ii) (i) D (iv) (iii) (i) (ii) 5. Which enzyme is not involved in the conversion of biomass? A Celullulase B -glucosidase C -galactosidase D Renin 6. Fermentation enriches food because the microorganisms A release carbon dioxide B break down chlorophyll C secrete vitamins D add aroma 7. Why is vitamin K not emphasised in vitamin-enriched eggs? A Vitamin K cannot be increased B Vitamin K is not essential to human body C Vitamin K is too low in eggs D Vitamin K is already high in regular eggs 8. The seeds of hybrid rice cannot be used to plant for the next generation because A most alleles present are homozygous B must alleles present are heterozygous C the hybrid is pure D the hybrid has foreign genes 9. The development of a variety of rice before hybrid is produced takes a long time because A many generations of selfing are required B the variety must be heterozygous is every gene C the variety must all have homozygous dominant alleles D the variety must all have homozygous recessive alleles 10. Which are true of disadvantages of transgenic plants? I Resistant to insecticides II Indiscriminate use of herbicide III Delayed ripening of fruits IV Creation of super weeds A I and III C II and III B I and IV D II and IV 11. Why is a promoter gene of a growth hormone gene required to be transfered into the zygote of a transgenic fish? A The promoter gene produces a promoter protein to induce growth B The promoter gene is needed for growth hormone to bind C The promoter gene will produce the hormone D The promoter gene is required to start the transcription of the growth hormone gene 12. A herbicide resistant transgenic plant can be produced by A crossing a herbicide sensitive plant with a herbicide resistant plant B injecting deactivated herbicide into the plant
255 Biology Term 3 STPM Chapter 19 Biotechnology 19 C continuously exposing the palnt to the herbicide of low concentration for a long period of time D inserting a gene which is able to produce an enzyme for herbicide degradation in its genome 13. The transgenic plant is different from the wild-type plant because A it contains antibiotic resistance plasmid B it is a triploid C it contains foreign gene in their genomes D its DNA is transcribed from 3’ to 5’ direction 14. An undentified human skeleton is discovered. Which of the following steps could be done to identify the victim? I Isolating RNA sample from the victim II Collecting tissue sample from the victim III Collecting fingerprints from probable family members IV Collecting blood samples from probable family members A I and III B I and IV C II and III D II and IV 15. The diagram below shows a plasmid molecule having restriction sites for EcoR1 and SmaI. EcoRI SmaI EcoRI Plasmid What will happen if the plasmid is cut with EcoR1? A Three DNA fragments will be produced B Two DNA fragments will be produced C A linear plasmid molecule will be produced D The plasmid will remain intact because SmaI disrupts the activity of EcoR1 16. Bacteria carrying a plasmid with an antibiotic resistance gene are important in cloning because A they can kill other bacteria B they help in screening C they can produce antibiotics for commercial purpose D they protect themselves after being released into the environment 17. The following are transgenic organisms except A the cloned sheep (Dolly) B the herbicide-resistant maize C the bacteria producing human insulin D the sheep producing human protein in milk 18. Which combination is the roles of biotechnology in our life? I To produce human growth hormone in medical field II To trace diseases in foetus by using amniocentesis III To detect abnormalities in the body by using X-ray IV To treat sewage to reduce pollution A I and III C II and III B I and IV D II and IV 19. Which transgenic microorganism is successfully used in bioremediation of oil spills? A Bacillus sp. B Agrobacterium sp. C Pseudomonas sp. D Trichoderma sp.
256 Biology Term 3 STPM Chapter 19 Biotechnology 19 Structured Questions 1. DNA sample Extraction of DNA Cleavage of DNA by restriction enzyme Gel electrophoresis to separate DNA fragments Southern blotting technique Autoradiography to obtain DNA bands Develop and visualise DNA fingerprint (a) How is DNA extracted? [1] (b) The cleaved DNA fragments are usually treated with a primer and started a polymerase chain reaction (PCR) for only the variable tandem repeat (VNTR) markers. (i) What is a primer? [1] 20. Which are true about gene therapy? I To complement a defective gene with a normal gene II To use a normal gene to destroy a defective gene III To replace a defective gene with a normal gene IV To detect a defective gene A I and III C II and III B I and IV D II and IV 21. Which of the following techniques can identify a criminal that has left a cell at the scene of a crime? A Southern blotting B DNA fingerprinting C DNA sequencing D DNA cloning 22. Which are true of the advantages of transgenic plants? I Create superweeds II Delay the ripening of fruits III Reduce weed species on farmlands IV Resistant to insecticides A I and III B I and IV C II and III D II and IV 23. A bloodstained knife was found by the police at a murder scene. Analysis of DNA profiles from the victim, the bloodstain and the suspects were shown below. DNA DNA from DNA DNA DNA DNA victim knife suspect w suspect x suspect y suspect z The most probable killer is A w B x C y D z
257 Biology Term 3 STPM Chapter 19 Biotechnology 19 (ii) What is PCR? [2] (iii) What is a VNTR? Where is it located? [2] (iv) How is VNTR used as DNA fingerprint? [2] (c) What is Southern blotting? [2] 2. The diagram below shows two types of pre-natal testing. Foetus (8 – 10 weeks) Amniotic fluid withdrawn Uterus wall Amniotic fluid Foetus (14 – 16 weeks) Supernatant Biochemical tests Several weeks later Cell culture Foetal cells Chorionic villus sampling Amniocentesis Chorionic villi Catheter Chorion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Y (a) State two advantages of chorionic villus testing over amniocentesis. [2] (b) State one disadvantage of chorionic villus testing. [1] (c) State two recessive traits that can be tested using biochemical tests. [2] (d) How does one get the chromosome arrangement as shown in the diagram? [2] (e) What is trisomy 21? Why is it always tested? [3] Essay Questions 1. (a) What is amniocentesis? Give one of its uses. [8] (b) State three characteristics of cloning vector and four characteristic of bacteria host cell. [7] 19
258 Biology Term 3 STPM Chapter 19 Biotechnology 19 STPM Practice 19 Objective Questions 1. D 2. B 3. C 4. B 5. D 6. C 7. D 8. B 9. A 10. D 11. D 12. D 13. C 14. B 15. B 16. D 17. A 18. B 19. C 20. A 21. B 22. C 23. D Structured Questions 1. (a) Selective digestion of protein and RNA leaving DNA to be strung out. (b) (i) A short RNA chain complementary to DNA to start the PCR replication. (ii) A chain reaction to start DNA replication to make more DNA molecules of the same type so the required DNA molecules are increased. (iii) A repeated sequence of two or more bases e.g. GTGTGTGT. Normally located in the introns and between genes. (iv) They are formed by DNA mutations of ansectors so they are very specific. The number of repeats is unique to each individual. (c) Southern bloting is a process to separate the double strands of DNA into single strands. Then, the single strands are transferred from gel into a special nitro-cellulose paper. 2. (a) Chorionic villus testing can be used earlier (8 –11 weeks) when cells obtained are alive so can straight away be tested or observed for mitosis. (b) Risks of injuring of foetus is higher. (c) • Haemophilia • Cystic fibrosis • Phenylketouria (d) By taking photograph of a mitotic metaphase. The photograph is enlarged, the chromosomes are cut from the photograph and homologous chromosomes and arranged. (e) Down's syndrome. It has a higher chance of occurence in children of older mothers. Down's syndrome child is both physically and mentally retarded so it needs special care and mental preparation. Essay Questions 1. (a) • Amniocentesis is a procedure in genetic screening using a long needle to withdraw amniotic fluid for prenatal diagnosis. • This is done through the belly after the amniotic sac is formed after 14 to 16 weeks of pregnancy. • A sample of 20 ml of fluid is taken and then centrifuged to obtain cells shed by the foetus. • Cells are cultured to observe mitosis and micrographs are taken during metaphase. • The chromosomes are counted for any changes in number like Downs syndrome. • Each of the chromosome is studied for chromosomal structural abnormalities like cri-du-cat syndrome. • It can be used to determine the sex of foetus. (b) Characteristics of cloning vector: • It must be able to replicate if stimulated • It must carry a regular gene so that transcription of the structural genes is possible when stimulated • It must not carry any harmful gene that may kill the host cell • It must carry a marker with a restriction site in the middle so as a gene can be spliced into the middle of the gene • It must be able to enter host cell with or without stimulation (Any three answers) Characteristics of bacteria host: • It must be able to grow and divide fast in normal minimum medium. • It must be able to allow to cloning vector to replicate independent of its own main DNA replication. • It must be able to grow and divide fast in normal minimum medium. • It must be able to allow the genes brought in by the vector to express themselves. • It must be able to store proteins produced by vector such as insulin or allow them to be released. ANSWERS
259 STPM Model Paper (964/3) 1. An unknown marine organism has the following characteristics. • Diploblastic • Radial symmetry • Outerlayer hasstinging cells Which is the most probable phylum for the organism? A Platyhelminthes C Cnidaria B Porifera D Mollusca 2. One of the special characteristics of sexlinked gene in mammals is A only fathers can become carriers B only females show special characteristics C fathers commonly have the dominant traits D mothers commonly have the recessive traits 3. The table below shows three levels of biological diversity and their explanation. Level of biological diversity Explanation • Ecosystem diversity I Wild orchids can grow on the ground as terrestrial orchids, on trees as epiphytes or on rocks as lithophytes. • Genetic diversity II Different types of wild orchids can be found in peat swamp forests and on rocky limestone hills or from highland to lowland forests. Section A [15 marks] Answer all questions in this section. • Species diversity III The shape, size and flowers of wild and hybrid orchids vary within their respective species. Which combination is correct? Genetic diversity Species diversity Ecosystem diversity A I II III B II III I C II I III D III I II 4. The gross primary productivity of producers in an ecosystem is 5000 kJ m–2y–1. The net primary productivity of producers is 2200 kJ m–2y–1. If the efficiency of energy transfer from producers to herbivores is 10% and to the next level is 20%, what is the amount of energy obtained by secondary consumers? A 44 kJ m–2y–1 B 88 kJ m–2y–1 C 122 kJ m–2y–1 D 440 kJ m–2y–1 5. A dam separates individuals of species X upstream from the individuals downstream. After prolonged separation, the individuals in the upstream and downstream become two new species. What type of speciation is this? A Intraspecific and sympatric B Intraspecific and allopatric C Interspecific and sympatric D Interspecific and allopatric
260 Biology Term 3 STPM STPM Model Paper (964/3) 6. What is the term given to the end product of a metabolic pathway that activates the repressor of the operon to produce enzymes for the pathway? A Corepressor C Promoter B Supressor D Inducer 7. A population of birds live in the same geographical area. One of the group is sexually active in the evening, while the other in the morning. Which type of isolation mechanism is involved? A Gametic B Behavioral C Temporal D Mechanical 8. A cross between two homozygous flowers of different colours is carried out. The genotypic and phenotypic ratios for the F2 generation are both 1 : 2 : 1. What do the ratios indicate? A The presence of lethal gene B Incomplete dominance C Dominant and recessive interactions D The presence of linked genes 9. Which about genetic code is not correct? A It is non-overlapping B It is a triplet codon C Most organisms share the same genetic code D One amino acid is coded by one genetic code only 10. Which of the following characteristics indicates Coniferophyta is more advanced than Filicinophyta? I Ovary II Unisexual strobili III Megaspores and microspores IV Double fertilisation V Alternation of generations A I and II B II and III C II, III and IV D II, III, IV and V 11. Whose experiment proved that DNA replication is semiconservative? A Hershey and Chase B Beadle and Tatum C Meselson and Stahl D Avery, MacLeod and McCarty 12. Brown eye (B) in cats is dominant to blue eye, while black coat (B) is dominant to white coat. Cats that are heterozygous for both characteristics were test crossed and produced 100 offspring. How many offspring would have brown eyes and black coat? A 100 C 45 B 80 D 25 13. Which technique is used when the amount of DNA is very small for DNA fingerprinting? A Restricton fragment length polymorphisms B Polymerase chain reaction C Gel electrophoresis D DNA cloning 14. What is the approximate percentage of net primary productivity of a terrestrial ecosystem that is passed on to the herbivore? A 1% C 40% B 10% D 90% 15. Which organism has the highest mortality rate in the young? 0 Age D C B A 100 Percent survived 50
261 Biology Term 3 STPM STPM Model Paper (964/3) Section B [15 marks] Answer all questions in this section. 16. The diagram below shows DNA fingerprints of a cell obtained under the fingernails of a murder victim together with three suspects, P, Q and R. The cells under the victim’s fingernail Suspect P Suspect Q Suspect R (a) With reference to the diagram above, (i) state the technique used to obtain the DNA fingerprinting of the suspect. [1 mark] (ii) state the factors that influence the separation of the DNA bands. [3 marks] (iii) which suspect, P, Q and R was the murderer? Why? [2 marks] (b) State one other situation that can use the same technique named in (a)(i). [1 mark]
262 Biology Term 3 STPM STPM Model Paper (964/3) 17. The following diagram shows the pairing of a mutated and normal chromosome during metaphase I of meiosis. C C B B D D Crossing over E A E A (a) What type of chromosomal mutation is shown? [1 mark] (b) Why do the chromosomes pair in such a way? [1 mark] (c) Draw what will happen after a crossing-over occurrs within the loop? Label your drawing. [3 marks] (d) Explain how this mutation is formed. [3 marks] Section C [30 marks] Answer any two questions in this section. 18. (a) Give an account of the characteristics of phylum chordata. [5 marks] (b) How in situ and ex situ conservation are carried out for conservation of species diversity in Malaysia? [10 marks] 19. (a) Describe how energy flows through an ecosystem. [6 marks] (b) Explain how an error during meiotic cell division could lead to Down,s syndrome. [9 marks] 20. Explain the meaning and components of ecosystems, biomes and biosphere. [15 marks]
263 Biology Term 3 STPM STPM Model Paper (964/3) Objective Questions 1. C 2. B 3. A 4. A 5. A 6. A 7. C 8. B 9. D 10. B 11. C 12. D 13. B 14. B 15. D Structured Questions 16. (a) (i) Gel electrophoresis (ii) pH used, quality of the gel, relative similarity in the length of the fragments (iii) Suspect Q. This is due to the band patterns of the suspect’s DNA is identical to the DNA found under the victim fingernail. (b) It can be used to identify the remains of a dead body if damaged beyond recognition by comparing the DNA fingerprint with that of close relative. 17. (a) Inversion (b) The homologous parts of the homologous chromosomes come into contact side by side. (c) C C B B D D Crossing over E A A B C B A A B D C C D B E D C Acentric chromosome Dicentric chromosome B E E E A E A (d) During pairing in prophase I, a thin thread of chromatid chromosome forms a loop. There is a breakage at the crossing of the loop and when rejoined wrongly, it can be inverted. 18. (a) • They have notochord, a vacuolated cell chord above the spinal cord in certain stage of their life • They have post anal tail • They have pharyngeal clefts at certain stage of their life • They have segmented muscle blocks on both sides of their bodies • Their blood flows forward ventrally and backwards dorsally (b) • For in situ, there are national parks designated to allow plant and animal live naturally with least disturbances. • Besides, specific sanctuaries are set up in certain areas where animals or plants under threats are kept naturally. • There are marine parks where certain area around islands are declared specifically for marine animals. • Similarly, costal mangrove swamp is declared as conservation park for marine breeding ground. • Wet land areas are also designated as parks to conserve migratory birds. • Ex situ conservation is the setting up of zoos to breed endangered animals. • Similarly, marine and freshwater aquaria are set up to conserve and release aquatic animals. • Botanical gardens gather endangered plants and breed for conservation. • Besides, seed banks are set up to keep the seeds of endangered plants. • Similarly, egg and sperm banks are set up to conserve endangered animals. 19. (a) • Energy first comes from sunlight in most terrestrial or aquatic ecosystem and receives by producers. • The producers are mostly photosynthetic organisms including green bacteria, cyanobacteria, algae and terrestrial plants. • These plants absorb sunlight of certain wavelengths where light energy is converted into chemical energy. • The primary consumers than obtain their energy in chemical form from the producers. • The primary producers like herbivores feed on the leaves, flowers or fruits. • From the primary consumers the energy is passed into the secondary consumers like carnivores. (b) • An error in meiosis known as non-disjunction may occur in the first or second meiotic division. • Non-disjunction in the production of egg cells seems more common in older mothers due to the failure of the production of spindle fibre. • This usually involves the smallest autosome chromosome 21 resulting the paired chromosome failure to separate. • If this occurs in the first meiotic division two products of meiosis will have an extra chromosome 21 and the other two products will be lacking. • If this occurs in the second meiotic division only one product will have an extra chromosome 21. ANSWERS
264 Biology Term 3 STPM STPM Model Paper (964/3) 20. • Ecosystems are natural units consisting of different communities in certain areas, including the abiotic factors or physical environment. • Examples are pond, forest or the whole world or biosphere. • Within the ecosystem, the biotic components are all the different communities living in it. • The abiotic components are the physical environment, the earth and the climatic factors. • Energy flows through the ecosystem in one direction, i.e. enters as light and lost as heat. • Elements recycle within the ecosystem. Examples of biogeochemical cycles are water, carbon, nitrogen, sulphur and phosphorus cycles. • Biomes are gatherings of major communities. Each community covers a wide-spread area with characteristic features determined mostly by climatic factor. • Each biome occupies a geographical region with a unique set of conditions and supporting communities of plants and animals. • Each biome is determined by characteristic dominant plants. This can be seen in tropical rain forest that has dense, lush vegetation with immense variety of species. • Each biome also has major communities of animals adapted within. Some organisms are endemic and found in no other region, such as orang-utans in Borneo. • Each biome has a characteristic ecosystem. However, it has sub-sets of other ecosystems, especially near the shore, river, lake or on mountain. • There are eight major terrestrial biomes in the world i.e. tropical rain forest, temperate deciduous forest, temperate evergreen forest, taiga, savannah, temperate grassland, desert and tundra. • Terrestrial biomes compose of zones such as underground zone, ground zone and canopy zone. • Biosphere or ecosphere is the whole world, including the atmosphere where all organisms can live. • It extends up to an altitude of several kilometres, also several kilometres down in the ocean and 300 m to water bearing rock on land. • It composes of the land and ocean with the different organisms that live in the whole earth. • It consists of all the different biomes, each with its populations organised into communities and ecosystems. • The distribution of species in the biosphere is a result of past changes of earth structure, climate and interaction of among the species. • Human activities affect the biosphere through pollution with chemicals, increase of carbon dioxide concentration and depletion of the atmospheric ozone layer.