4.1 Chromosomes, genes, alleles and mutations 42 Meiosis 43 Theoretical genetics 44 — Genetic engineering and biotechnology are 4.1 CHROMOSOMES, GENES, ALLELES AND MUTATIONS rokaryotic chromosomes are circular. Prokaryotes ‘also have some DNA in the form of small loops called plasmids. Prokaryotic DNA is found in the nucleoid area of the cell, not in a nucleus. Prokaryotic chromosomes contain DNA but no protein. Refer to Figure 215. Eukaryotic chromosomes are found inside @ nucleus. “There are usually more than one chromosome and they are linear. Eukaryotic chromosomes contain DNA and proteins. Refer to Figure 222. A gene is a heritable factor that controls a specific characteristic (such as eye colour), consists of a length of DNA and occupies a position on a chromosome known asa locus ~¢ term allele refers to one specific form of a gene, «fering from other alleles by one or a few bases only and, occupying the same gene locus as other alleles ofthe gene. ‘Refer to Figure 401. A genome isthe total genetic material ‘of an organelle, cell or organism, The gene pool refers to ‘the total of the genes carried by the individual members Ale for purple Mowers o—_ Locustor~ — HomSiogous Floweeoleur — pairot Alle or white flowers Figure 401 Alleles for flower colour of a population. A gene mutation may be defined as a permanent change in the sequence of base pairs in the DNA that makes up a gene. A mutation may involve just ‘one nucleotide or it may affect a large section of the gene. ‘An example of a mutation occurs in the Manx cat shown in Figure 402. Due to a mutation, some Manx cats were born without tails. This was selected for by breeders and a tailless breed of cats was created. Figure 402 The Manx cat, an example ofa mutation 53It is often thought that a mutation which involves a smaller number of nitrogenous bases in the DNA will be less significant than one where a larger number of bases is changed. This is not necessarily true and a good example is found in the gene that controls sickle-cell anemia where a change of only one base leads to a protein with one amino acid changed and the resulting disease of sickle-cell Hemoglobin is made of four polypeptide chains: two alpha chains and two beta chains. When an A to T base substitution occurs in the region of the gene coding for the 6th amino acid in the beta chain, the codon GAG {glutamic acid) becomes GTG (valine). A urepaced by Sees DA bs 8 @ yp Deorygenaion RBCS Sickled se c Normal m, ae : Necrotic, saat Figure 403 Sickle-cell anemia: cause and consequences “The resulting polypeptide is different and the hemoglobin formed is commonly known as HbS: the ‘normal’ hemoglobin is known as HbA. PARENTS The result of this is a slightly different structure, by one amino acid of the hemoglobin molecule which makes it crystallise at low oxygen levels (eg, in the capillaries). The erythrocyte in which the hemoglobin can be found will then change from a biconcave shape into a sickle-cell ‘cHLoREN 34 Figure 404 Sickle-cells and normal red blood cells shape (see Figure 404) and can block the small capillaries, and is less efficient at transporting oxygen. Even when the oxygen concentration increases again, the cells keep their sickle shape. ‘The symptoms of sickle-cell anemia are acute anemia, which causes physical weakness. The lack of oxygen ‘ay be severe enough to cause damage to the heart and kidneys or even death (in homozygous individuals) ‘The gene for sickle-cell anemia is codominant with the “normal” allele although the latter is expressed more strongly in the heterozygous individual. Heterozygous individuals (carriers) have some HbS but more normal hemoglobin. They may suffer from mild anemia, The selective advantage of being a cartier is found in malar, infested areas. Plasmodium (the protist causing malaria) cannot reproduce in erythrocytes with HbS, This means that individuals heterozygous forthe sickle-cell trait have a reduced chance of contracting malaria Natural selection has ensured that the sickle-cell trait is more common among people living in malaria-infested areas such as West Africa, As the African-American population largely originates from this area, the trait is found in frequencies higher than usual in this group. Carriers may not be aware ofthe fact that they possess the sickle-cell allele and are capable of passing it on to their children. Iftwo carriers have a child, here is 1 in 4 (25%) chance of the child having the disease. See Figure 405. HOA nes Hoa Hes eater carrer HbA Ho bans | [HOA HBS Hos HS nowmal cater carter person wth sickle | celanaemia KEY. Hos thenormal alle Hiss the sickle gene Figure 405 The inheritance of sickle-cell anemiaTherefore, itis important that people who may be cartier, are tested to confirm the presence or absence ofthe sickle- cell allele. Ifa female carrier is pregnant and the father is + a cartier, itis possible to test using amniocentesis or worionic villi sampling (refer to Topic 4.2.6) to see if the child will have sickle-cell anemia, Should this be the case, then the parents may decide to discontinue the pregnancy. ‘There are no easy answers to this problem. A great deal of research is being done about treating sickle-cell anemia including bone marrow transplant and/or blood stem cell treatment. Gene therapy is also possibility but research is in an early stage. Refer also to Topic 4.3.2 and D.2.11, TOK Mat i and sickle-cell anemia he incidence of sickle-cell anemia is correlated with the incidence of malaria in parts of Aftica and South Asia. It Is @ case of heterozygote advantage, where those ho are heterozygous for the sickle-cell anemia allele re batter suited to the environment than homozygotes. ickle-cell heterozygotes can be infected with malaria, ut it does not effect them as severely es those who do jt have the allele. Heterozygotes do not express sickle- cell disease, Those who are homozygous recessive for the allele do express the disease which Is often fatal. ‘balance between the risk of death from malaria and jeath from the homozygous form of sickle-cell anaemia, is played out in the population of individuals in the parts cf the world where both these disease are present. This is a caso of a causal link forthe continuation of sickle-cell snemia alleles in the population In other cases causel links may be difcult to find oF simply just not there. Making the decision that there is & causal requires a large body of evidence to support the ink asin the incidence of sicKe-col disease and malaria here was al one stage a suggested causal Ink betwen the Measles, Mumps and Rubella (MMR) Vaccine and tem. Several large-scale studies have since confmed hat tere is no causal lnk between the MMR vaccine nd autism, 4.2 MEIOSIS Meiosis iscalleda “reduction division’ becqusethedaughter cells have only half of the number of chromosomes as the original parent cel. ‘The purpose of meiosis is to produce gametes. A (haploid) gamete (sex cell, sperm or egg cell) has half the number of chromosomes compared to a (diploid) somatic cell (body cell). A male and a female gamete may then fuse (fertilisation) to form a zygote, which will have the same ‘number of chromosomes asa somatic cel. ‘The number of chromosomes in a haploid cell is often given as ‘n! The number of chromosomes in a diploid cell is usually given as 2r’. For example: + inahuman somatic cell -2n = 46 + ina gamete of a camel n=35 + ima somatic cell ofan apple 2n= 34 Homologous chromosomes (or a homologous pair) are two chromosomes, one from each parent, that look the same. They are the same size and they will show the same banding pattern in a karyotype (see Figure 409). They carry the same genes but not necessariy the same alleles, for example, both homologous chromosomes will carty the gene for eye colour but one may have the allele ‘brown! while the other may have the allele ‘blue. Homologous chromosomes will pair up and split up during meiosis. “The process of meiosis can be divided into two main stages: + Meiosis 1 + Meiosis IL CORECORE cee. Both Meiosis [and Il are subdivided into: + Prophase + Metaphase + Anaphase + Telophase ‘The key to the process of meiosis is the pairing of ‘homologous chromosomes in Prophase I and the splitting of the homologues in Anaphase I. See Figure 406. ‘THEN FOREACH CELA FURTHER DIVISION OCCURS cela Nee Promise! SMeEPS peomasel soil _ MeWpnaseT weneursen TG fi a z aunase) anaowase ro coer % recent Tera Figure 405 The process of meiosis ‘One event that can take place during meiosis is crossing over,as seen in Figure 407. Crossing over isthe exchange of genetic material between two homologous chromosomes which have paired up during Prophase 1. The result of crossing over is genetic recombination: an exchange of genes. The points where the homologous chromosomes cross over are called chiasmata, 56 S50 1 oer: mewphet a Figure 407 Crossing over Recombinant ‘chromosomes ‘The pairing up of homologous chromosomes during Prophase I is called synapsis. Each resulting pair of homologous chromosomes (both chromosomes consisting of two identical ‘sister’ chromatids) is called tetrad or bivalent. The separation of homologous chromosomes during Anaphase 1 is called disjunction. Failure of chromosomes to separate can lead to aneuploidy, which ‘means having one extra chromosome or missing one chromosome. In extreme cases, all homologous pairs fail to separate, This is called total non-disjunction and will lead to polyploidy, where the organisms has one complete extra set of chromosomes and becomes, for example, 3: One of the best known examples of aneuploidy is Down Syndrome. Ifnon-disjunction occurs in either parent, oneof the two gametes will carry two copies of chromosome 21, When the zygote is formed, it will contain three copies of chromosome 21 and will be aneuploid Itis also referred as trisomy 21. The resulting symptoms are called Down syndrome and are accompanied by (varying degrees of) disability. The karyotype ofa person with Down syndrome is illustrated in Figure 408. (1 i PKI Te) tat) 1 (s t \e (es y wm» a Figure 408. The cause of Down syndrome Unlike the production of sperm cells in the male (which begins at puberty), the female gametes develop before birth. At birth, the future female gametes are present as primary oocytes in Prophase I of meiosis. They remain in this stage until ovulation. This means that for a twenty year old woman, her egg cells have collected twenty years of damage (chemicals, radiation, etc). But for a forty year ‘old woman, the cells have collected forty years of damage and the chance of non-disjunction, resulting in a baby ‘with Down Syndrome is increased. Since the sperm cells ofthe male are produced constantly, the age ofthe father seems to have less effect than the age of the mother. Genetic factors can play a role in either parent: generally if a person has a relative with Down Syndrome, this person may have an increased chance of wing a baby with Down Syndrome. pc narey Karyotyping is the process of finding the chromosomal characteristics of a cell, Chromosomes can be stained to show banding, This is used in the process of karyotyping Chromosome structure and banding can be used to arrange the chromosomes in their pairs, Figure 409 shows an idealised picture of the human chromosomes. To make a karyotype, the chromosomes in the picture would be cut ‘out and arranged in pairs, starting with the largest a as a’ : I py Hi i ti it i i Hf : Wo wm we boi 7 of wp mn ae oi 8 oR hae ep Figure 409{a) and (b) The process of karyotyping ‘One application of karyotyping can be found in an amniocentesis. The risk of having a child with Downs syndromeinereases withthe mother'sage.Non-disjunction, which can cause Down syndrome, also seems to have a genetic component so that it can be said that people with relatives with Down syndrome have an increased chance of| having a baby with this genetic disorder, as have mothers over the age of 35, 37 CORECORE In either situation, itis possible to do pre-natal testing ‘There are two common tests: + chorionic villus sampling + amniocentesis, "To explain both of these techniques, itis helpful to brielly outline human embryonic development. Human fertilisation takes place in the oviduct. The zygote will travel to the uterus and undergo many divisions. ‘When it arrives in the uterus, its a ball which consists of an inner cell mass and an outer cell mass. The outer cells ‘vill come into contact with the mucus lining of the wall of the uterus and develop into chorionic villi. They will eventually become the placenta. Chorionic villus sampling is a pre-natal test that can be done at 11-12 weeks of pregnancy. It involves taking a sample of the chorionic villi in order to obtain cells from tissue that originally came from the aygote. The cells will therefore have the same genetic composition as the cells of the unborn baby so a karyotype can be made. This should take fewer than two weeks, ‘The risks associated with chorionic villus sampling (around 19) are slightly larger than of amniocentesis (around 0.5%). Figure 410 shows chorionic villus sampling (CVS) and where the tissue will be removed. Utasound transducer Chote wit Pracenta Fetus (toweeks) “catheter Uterine cavity Figure 410 Chorionic villus sampling Amniocentesis can be done around the 16th week of the pregnancy. A sample of the amniotic fluid (containing fetal cells) is taken and a culture is made. When sufficient cells have been obtained, a karyotype can be done to detect chromosome abnormalities, The dividing cells ate photographed and, using these pictures, the chromosomes are arranged in homologous pairs. Some genetic disorders can be detected this way (eg. Down syndrome). ‘This process takes approximately 3 weeks. A diagram of the process of amniocentesis is shown in Figure 411 58 Uttasonic probe. Placenta Fetus cervix Amniocentesis Figure 411 TOK Karyotyping {All medical procedures involve some risk. Those who Undergo procedures nead to balance the risk against the benefit, Karyotyping increases the risk of miscarriage but can reveal the presence of major chromosomal abnormaities. Knowledge is power. The knowiedge gained from keryotyping can be used to perform an abortion of allow life to continue. There are significant responsbbilties and consequences in such decisions, 80 ite no surprise that there is disagreement as to who should have the power to authorise such testing and take action on the outcome of the test. Who should make such decisions: the individual (mother or father) the couple > together, their wider family, health care professionals, government? There are cultural and religious opinions to consider. This will always be an area of significant cebate, ‘When analysing a human karyotype to determine gender and the occurrence of non-disjunction, the karyotype must be carefully examined and pairs of chromosomes need to be identified and placed together, This can be done using pictures on paper or on a computer screen. If non- disjunction has occurred there will either be one, oF three copies, ofa particular chromosome. See also Topic 4.3.5.ries 4.3, THEORETICAL GENETICS Genotype refers to the alleles of an organism. This is usually written using upper and lower case leters, eg, Tt Phenotype includes all the characteristics of an organism. This is written as a word, eg, tall. A dominant allele cone which has the same effect on the phenotype whether it is present in the homozygous or heterozygous state A recessive allele is one which only has an effect on the phenotype when present in the homozygous state, Codominant alleles are a pair of alleles that both affect the phenotype when present in a heterozygote. The terms ‘incomplete’ and ‘partial dominance are no longer used, ‘A locus is the particular position on homologous chromosomes of a gene. Homozygous means having the two identical alleles of gene. Heterozygous meanshaving two different alleles ofa gene. A carrier is a heterozygous ‘individual that has one copy ofa recessive allele that causes \__ genetic disease in individuals that are homozygous “Tor this allele, A Test cross refers to testing a suspected heterozygote by crossing with a known homozygous recessive. (The term ‘backcross is no longer used.) A Punnett grid isa way of finding the expected ratio of the offspring, given certain parental phenotypes. We can study one of the characteristics Gregor Mendel (1822-1884) used in his experiments. He studied the size of pea plants and found that ‘all’ is dominant over ‘short, If we start with 2 pure breeding (homozygous) plants of contrasting traits (tall and short), we will obtain an FL (Firs filial generation) which has the dominant phenotype (tall) but is heterozygous. When self-fertiising the Fl, we will obtain an F2 (Second filial generation) which will appear 3/4 dominant (tll) and 1/4 recessive (short) sing a certain format will help to solve a question. Start by writing down what phenotypes exist for the relevant, gene and what the corresponding genotypes are, as in Figure 412. Possible phenetypes. | Gorrespe Tal Trertt Shor t Figure 412 Table of phenotypes and genotypes ‘Then write out the cross. Make sure to include both genotype and phenotype of the parents (P), as well as the genotype and phenotype of the offspring (F,). P: tall ox short (phenotype) TT tt (genotype) T vec (gametes) F; Tt tall ‘The next generation is called F, whch could be created by self-fertlising the F, tall tall (phenotype) n Tt (genotype) Tort Tort (gametes) TT + 27 + tt (genotype) 3 tall + Ishort (phenotype) A Punnett grid (also known as a Punnett square) can be used to find the genotypes of the offspring, (Figure 413) [Punnett square | emreype geroyne | comers] ot) | samen 7 7 Tt genotype offspring | ‘ar_|_sat_| phenoxpe ofspring 0 | | & | crop oferng tat | ston | phenosype ofspring Figure413- APunnett square (ao) 1‘CORE The example in Topic 44.2 used the gene for size which had two alleles, tall and short. It is possible to have more than two alleles for one gene. A good example of this is found in blood groups. The gene is blood type and the alleles are IP, and i, ‘The ABO blood group system is based on 4 different phenotypes (group A, B, AB and ©) caused by different combinations of 3 different alleles (I, and i). The alleles and IP are codominant so both will affect the phenotype. ‘The allele i is recessive and will only affect the phenotype when homozygous. Refer to Figure 414 Po 8 PP oF Fi AB He ° ii Figure414 Blood groups Using a Punnett square, it can be worked out how a {female with blood group A and a male with blood group B can have four children, each with a different blood group. Refer to Figure 415, ee are : “genotype parent Seren |aaretes | |G ‘gamatos e ae Fi | genotype offspring a p08 | ype | phenotype ofspring pe | |_| concise otscxing type | 90.0 | chenatype oping Figure 415. First blood group cross Both parents would have to be heterozygous in order to produce a type O child, It also explains why a female with blood group O and a male with blood group AB cannot have children with either of the parent’ blood group. Refer to Figure 416. conn loamees | a) | re RAT cone. otra ei co Ee PB | | oenpeotsina ‘p28 | s926 | pnenoype ong Figure 416 Second blood group cross Humans have 46 chromosomesin somatic cells These cells, are diploid, so there are pais of homologous chromosomes ‘which carry thesamegenesand|ookthesameinakaryotype. “This is indeed the case for chromosome pairs 1-22, The remaining two chromosomes are the sex chromosome Males have one X and one ¥ (XY) chromosome, while females have two X chromosomes (XX) as shown in Figures 417 and 418, respectively. A diagram of these chromosomes can be seen in Figure 419. Figure 417 ‘Male sex chromosomes oy on) Figure 418 Female sex chromosomes [As shown in Figures 417 to 419, the X chromosome is much larger than the Y chromosome. It contains some genes that are not found on the ¥ chromosome. ‘A Punnett square can be used to predict the chances the gender of a child. So the gender-determining factot-~ (the X and ¥ chromosomes) can be treated like any other trait and predictions can be made accordingly. Refer to Figure 420.Figure 419. Adiagram of the human sex chromosomes (X,Y) genotype parent| » | @ gameles % | 2% | 2% | senotvee otsring tomate | female | phenotype offspring xy ye | 20 | ee | genoryneotspring mele _|_male_| phenotype ofring aenotyee |, ot parent |ametes {gure 420 Using o Punnett square to determine gender ‘The X chromosome is relatively large; the Y chromosome is much smaller. Several genes are located on the X chromosome such as the ability to see colours and ‘hemophilia (also spelled haemophilia) but are absent from the Y chromosome. These genes are said to be ‘X-linked. ‘This means that a male with one allele for colour blindness ‘on the X chromosome, will be colour blind since there is no locus on the ¥ chromosome. The same applies for hemophilia. Both of these conditions are therefore found much more commonly in males than in females. Only a few genes are located exclusively on the ¥ chromosome (eg hairy ears) Conditions like colour blindness and hemophilia are much more common in men than in women and are said to be sex-linked. rates Sex linkage occurs when the genes carried on the sex chromosomes, most often on the X chromosome. ‘An example of inheritance of a sex-linked trait is shown, in Figure 421. oni ff Unafeced Unc Carer auger daughter on Figure421 Inheritance ofan X-linked, recessive characteristic CoLour BLINDNESS Colour blindness is a condition that can be caused by genetic factors, Human eyes contain cells with different pigments that absorb different wavelengths (colours) of light. If this pigment absorbs light, a message is sent to the brain and we see a colour. The ability to produce the different pigments is mainly found as genes on the X chromosome. The ability to make the pigment is a dominant allele; the recessive allele will not allow the pigment to be made. A female has two X chromosomes. In order for a female to be colour blind, she would have to have two copies of the recessive allele. A male has only one X chromosome so will only have one copy of the gene. If this is the recessive allele, then the man is colour blind. Existing alleles X# for normal vision, X* for colour blindness. Afemale can be XEXB orXPX! or XX genorpes coordina (phenotypes) 6 CORE(ao) 1 Amale canbe XY or xY (genotypes) sera ison colourtina (phenotypes) HEMOPHILIA Hemophilia is a blood disorder. Normally, there is a very fine balance for blood clotting. Blood should not clot when it is inside blood vessels or it will block the vessels {possibly causing a stroke) but it should clot when there is injury so that not too much blood is los. ‘The process of blood clotting involves 2 number of different proteins, each having their own gene. If even ‘one of these genes has an allele that does not code for the proper protein, the entire process of blood clotting can be disturbed and even very small wounds will not clot. In humans the locus for the gene thet controls the production of a blood clotting factor is on the X chromosome (ie. and not on the ¥ chromosome). This ‘means that ifa male has one defective allele he will have the hemophilia condition. However a female will need to have two copies of the defective allele in order to have the condition. Statistically this is much less likely and with the advent of menstruation and child birth, a woman will need blood transfusions containing the clotting factors to survive, Prior (o this technique hemophilia was thought to be homozygous lethal in all cases and some examples in some books are still based on this information. Existing alleles X* for normal, 3 for hemophilia. Afemale can be XEXM or XX! or = XEX! (genotype) opis verre (Phenotype) Amale canbe xy or XY Genotype) oc emilicre (phenotype) Sex-linked genes are found on the X chromosome. Since females have two X chromosomes, they can have two dominant alleles (homozygous dominant), two recessive alleles (homozygous recessive) or one dominant and one recessive allele (heterozygous). Males only have one X chromosome. This means that the terms homozygous or heterozygous do not apply. a In humans the locus for the gene that controls the production of a blood clotting factor is on the X chromosome (ie. and not on the ¥ chromosome). This means that if male has one defective allele he will have the hemophilia condition. However a female will need to hhave 2 copies of the defective allele in order to have the condition. Statistically this is much less likely and with the advent of menstruation and child birth a woman will need blood transfusions containing the clotting factors to survive. Women can be carriers for trait. In that case, they ae heterozygous and will not show the trait but are capable of passing it on Since men only have one X chromosome, the allele on this chromosome wall always be expressed, Men can have the allele for colour blindness and be colour blind or have the allele for normal vision and have normal vision, The same applies for hemophilia. Men cannot have the allele ‘without expressing it so they can never be carriers for X- linked traits. ‘The accepted notation for the alleles for this characteriste is shown in Figure 422. [Sekeeal | Hot normat [Ho®= sicke-cot ies [>= lunes [vol vion oo ao ‘X¥= normal bloodeloting Figure 422. Accepted notation Codominance, the main letter should relate to the gene, while the superscript should relate to the allele. Both should be capital letters. eg. CH =red flowers CW white flower Drosophila melanogaster (vinegar fy) a+=dominantallele a eg vgt=normalwing recessive alle -vestigial wing” ‘Mendels two laws are as followsMendel's first law Law of segregation ‘Parental factors (genes) are in pairs and split so that one retor is present in each gamete? Mendel’s second law Principle of independent assortment ‘Any of one pair of characteristics may combine with either cone of another pair! (dihybrid inheritance) TOK Aparadigm shift ‘Blending inheritance’ was the most widely accepted exolanation of inheritance in the nineteenth. contury ‘The offspring of parents usually seemed to cisplay characteristics of both parents. Mendel's theories of inhertance involved a paradigm shift in the understanding ‘of the basis of inheritance, Mendel progosed that inheritance was governed by discrete structures that he called elemente. Mendel's published results ‘explaining pattems of inheritance but could not provide ‘an explanation of the mechanism by which his elements ‘were passed from one generation to the next, Without a mechanism his model was forgotten. ‘Today we know Mendel’ elemente as alloles. The really of the existence of alleles and genes was not contirmed nti the early twentieth century, more than twenty years after Mendel’ death, It was only then that 2 mechanism was discovered that allowed an. explanation of how Mendes model of hertance could take place. Mendels place in genetics today stands upon the fact that his model was based on simple, easily eproducible experiments, for which a large amount of data was collected, processed and analysed ‘appropriately. His research was [serrate 80 thelr theories are remembered as pre-scientfc, fat all oo "ABO Phenoiypes | ABO Genotypes _ A Port 8 FF or 2B ne © i Rhesus Phenoiypes. a Rhesus postive RNR" or RARE Pa Rhesusnegatve | RARE o u Figure 423 Human blood Groups Pedigree charts are offen used to record blood lines in royal families. Figure 424 is a pedigee showing Queen Victoria and her descendants. tis very likely that Queen ‘Victoria was a carrier with respect to hemophilia because ‘one of her sons was affected and two of her daughters were carriers. “The genotype of Queen Victoria must have been X# X*, sehile her husband was XY. Leopold must have received Y from his father and X" from his mother which caused him to have hemophilia. Alice received X from her father and X* from her mother, making her a carrier. The same happened to Alice daughter, Alexandra. She subsequently passed on her X® to herson Alexis, who obviously received his Y chromosome from his father Czar Nicolas II of Russia, Alexis had hemophilia Orman @ caserrente eo published allowing tto be re-dlscovered Vers and’ tous | Ace Aad Hine Live Atbur teopls setce and stand up to sorutny as is required according to the scientific method. 1 ol Mende!’ contemporaries cid not carry eae ¥ ee eee Mears | eos out experiments that could withstand the scrutiny ofthe scientific community, ous see OR nl Figure 424 Part of the European royal family pedigree 8CORE 4.4 GENETIC ENGINEERING AND BIOTECHNOLOGY PCR stands for polymerase chain reaction. When researchers want to study a particular sequence of DNA, they need many (identical) copies of it. The traditional ‘method of cloning (using plasmids) takes alot of time and work, Instead, 2 ‘photocopier for DNA’ is used, which is essentially what the PCR does. It uses enzymes to replicate DNA so no living organisms (e.g, bacteria or yeast) are required. The process for PCR was developed in 1983 by Kary Mullis who received the Nobel prize for Chemistry in 1993 for his work, PCR is carried out in a thermal cycler which is shown in Figure 425. sxiguaraetaeg" Seon loo a LT, orn tconlemenes . aero rome { ae om Figure 426 The PCR process sonnet a wT Figure 425. APCR machine THE PRINCIPLE OF PCR The desired DNA is heated which breaks the hydrogen bonds between the strands of the double helix so that they separate. Primers are added to start the process of DNA replication (see Topic 7.2). AS the mixture is cooled, the primers bond to the original, but now single stranded, DNA molecules (through hydrogen bonding between complementary base pairs). Nucleotides and > thermostable DNA polymerase are added. ‘The nucleotides will bond with the ‘exposed? organic bases of the single stranded DNA (again through hydrogen bonding between complementary base pairs) and the DNA polymerase will then join them into a DNA strand. This way, each of the original strands has formed a new complementary strand. These strands can be heated and separated and will function as a template for ‘more DNA strands to be formed, A large amount of identical copies ofthe original DNA can therefore be made quickly. The DNA is exponentially amplified. This is shown in Figure 426, ’Gel electrophoresis is a process commonly used in biochemistry. Electrophoresis is a technique used to separate large molecules (nucleic acids or proteins) based on their different rates of movement in an electrie Field caused by a combination of their charge and their size. Agelis preparedintoa thin ayerand placedin thecontainer as shown in Figure 427. Then some holes are made on one side where the molecular biologist will place the samples. Often at least one of them will bean unknown sample. The others can contain (mixtures of) known molecules. Then the equipment is connected to a source of electricity: The gel will conduct electricity. Depending on the charge ofthe ‘molecules, they will be attracted more or less to the other side, DNA fragments are slightly negative and will move towards the positive electrode (anode). So they will start to move through the gel. The speed with which they move will depend on how strong the attraction force is, but also on the size of the molecule. Larger molecules will move more slowly through the gel than smaller molecules. samespot os layer oF ; da bulferslston of kaon pe Figure 427, Gelelectrophoresis When the molecules have moved through the gel, the equipment is turned off and the gel removed. Sometimes the gel then needs to be stained to show the spots to where the molecules have travelled. If a spot of the unknown sample has travelled exactly the same distance as one of the known molecules, they are likely to be the same. PCR can be used to increase the size of a sample and then gel clectrophoresis can be used to compare the sections of DNA with the DNA of various suspects in an investigation. tosrey DNA profiling is also known as DNA fingerprinting. It is a technique that compares DNA from different sources out mapping the entire genome since that would take along time. i DNA profiling is used to compare the QNA of a suspect ‘with, for example, blood found at a crime scene or to compare the DNA of a child with that of an adult who could be the parent. ‘When a small amount of DNA is found, for example, in a spot of blood on a crime scene, the polymerase chain reaction (PCR) can be used to increase the amount of DNA. The two strands of the DNA double helix are separated, Restriction enzymes (or endonucleases, see Topic 4.4.8), which will cut between specific sequences of organic bases, are used to cut the DNA into sections. The sections will differ in size and charge and are separated using gel electrophoresis, The same treatment is used on. every sample. The gel electrophoresis will separate the sections of DNA according to size and charge. This creates a pattern of stripes and bands, detemined by the sequence of the organic bases. As every persons DNA is unique (except ‘monozygotic or idential twins), it would be highly unlikely that two different people would produce the exact same pattern of DNA sections on the gel. PATERNITY PROFILING DNA profiling to determine paternity uses the concept that all of the child’s DNA comes from its parents. Each band shown on the DNA profile of child must correspond with a band in the profile of the father or the mother. See also Topic 4.4.5. FORENSIC INVESTIGTIONS Compare DNA from the suspect with DNA from the crime scene sample This technique breaks down DNA into sections which are separated by gel electrophoresis. If the DNA bands of a suspect are @ match with those found in a sample (eg, blood, semen, hairs) at the crime scene, then the 65CORE suspect probebly was at the scene although itis not always, this simple. See_http://en.wikipedia.org/wiki/Genetic_ fingerprinting for some ideas of problems when using, DNA profiling as evidence. Use a relative’s DNA to determine the identity of a victim This technique has also been used to try to determine the identity of the remains of dead people, People claimed that the Tsar of Russia and his family were shot during the Russian Revolution and bodies were shown to prove this. However, not everyone believed that they were really the remains of the Romanovs. The identity of these bodies could not be proven until DNA fingerprinting was brought in, By taking blood samples of (distant) relatives of the Romanovs, DNA patterns could be established Samples from the bodies showed similar DNA patterns and the conclusion was that the bodies were likely to be the Romanov family TOK DNA Profiles ood typing was used in paternity and forensic testing bofore the advent of DNA profling. The outcome of tests sing blood typing is so broad that itis only useful in ‘excluding an individual (thats, tcansay thatthe individual {d098 not have the same blood type as the one searched $ for. To say that they do have the same blood type would not be useful unless the pool of possible suspects was very small and all wth ferent blood types) DNA profing, on the other hand, compares parts of non= coding DNA that are unique to each individual (except Identical twins). Profiles constructed using appropriate procedures have a statistically extremely high reliability rato and as such, are widely used in patomity and forensic cases with great success. FORENSIC INVESTIGATION A crime has been committed and two suspects are under investigation. DNA profiling has been carried out and the results are shown in Figure 428, It can be seen that the two bands visible in the “sperm DNA” match the DNA bands of Suspect 1 but not Suspect 2 66 Sexual Assault Case K562 Control evo Spormona’ Even 00 000.980 688 LADDER ‘Suspoet 1 ‘Suspect 2 Female Celts Vieum 08 -006-008 888 LADDER mK Figures 428 Evidence from a sexual assault case PATERNITY INVESTIGATION ‘When the paternity of a child is investigated, the ban? found in the childs DNA are compared to those of t. mother and the alleged father. Since all ofthe child’s DNA comes from its parents all bands should be found in either parent. Of course, a parent only gives half of its DNA to the child, so not al the parent’ bands can be found in the child, Pater Paternity Eecusion Inclusion LADDER, Mother CAF mix. LADDER Alleged Fé e- = = = = 2 - * - _ * * oi =_ —— = Figure 429(a) Exclusion __-429(b)_InclusionIn Figure 429 (a), it can be seen that there are some DNA. bands in the child’s profile that cannot be attributed to vither the father of the mother. Hence the alleged father sot the father of this child, In Figure 429 (b), you can See that both of the child’s bands can be attributed to its parents; one to the mother and one to the alleged father. ‘This person is likely to be the father of the child. TOK Human Genome Project ‘At one level the human genome project maps the DNA sequence of @ human and could be seen as a recipe for what itis to be human, This reductionist view does not account for the complexity of life that stems from the myriad of Interactions between our genes, the environment end our ability to transcend our genetic basis, Studies of identical and conjoined twins ilustrate the limitations of this reductionist approach. Such twins have many similares that stem from an identical ‘genetic code, but are without doubt unique individuals, Conjoined twins have had an identical environment since ‘conception. Clearly tobe human is more than simply the ‘expression of genetic code and its interaction with our ‘environment. How can this be? What makes us human? ‘The Human Genome Project was a commitment undertaken by the scientific community across the world to determine the location and structure of al genes in the human chromosomes. Groups of scientists sequenced the genes of a particular (section of) a chromosome and all the information was pooled. It is part of the international Human Genome Organisation and an excellent example of how collaboration of scientists across the world can benefit all of us. ‘Mapping the human genome was first suggested in 1985 and the expected financial and time investment was staggering. Finally, in 1990, the project of sequencing all (approximately 3 x 10°) base pairs in the human DNA ‘was started. It was expected to take 15 years, Some new developments in DNA sequencing and software made the process faster and cheaper than originally expected and “rough draft” was finished in 2000. In 2003, 50 years after Watson and Crick described the DNA double helix structure, the sequencing of the human DNA was 99.9% complete. Having a map of the sequence of the nucleotides of the ‘human DNA then lead to mapping the genes, i. listing and finding the locus of each human gene. They are not the same thing: knowing the sequence of A, T, C and G. does not tell you if you are looking at a gene for eye colour or the ability to roll your tongue. Outcomes of having sequenced the entire human genome ‘+ an improved understanding of mang genetic diseases: now many more genes causing disease are known than before the Human Genome Projects «+ theproduction of medicines (based on DNA sequences) to cure diseases and/or genetic engineering to remove the genes which cause the diseases + todeterminefully which genetic diseasesany individual is prone to (genetic screening leading to preventive ‘medicines + research into a particular disease can now focus on only the gene(s) that are relevant to the disease; + it can provide more information about evolutionary paths by comparing similarities and differences in genes between species. This information could be valuable, but it could also be abused (eg. by insurance companies or prospective employers) and society faces the challenge of coming to terms with these ethical issues. Genetic engineering refers to the deliberate manipulation of genetic material. It is possible to move genetic material between species because the genetic code is universal. ‘This means tha, for every organism, the same RNA codon codes for the same amino acid in an mRNA strand, UU, and UUC both code for the amino acid phenylalanine while CUU, CUC, CUA and CUG all code for leucine, regardless of the species Since the genetic code is universal, itis possible to transfer genetic material from one species to another. Because the code is universal, it is possible to introduce a human gene for making insulin into 2 bacterium. The bacterium will then produce the human protein hormone insulin, which is responsible for making cells take up more glucose and convert it to glycogen. or fete) 13‘Gene transfer involves the following elements: + avector + ahost cell + restriction enzymes + DNA ligase. “The vector is what is needed to carry the gene into the host cell. Plasmids are often used as vectors, Bacteria carry all the required genetic information on one large circular DNA. However, most bacteria also possess extra DNA in the form of plasmids. Plasmids are circular bits of genetic material carrying 2-30 genes. Plasmids may replicate at the time the chromosome replicates, or at other times Itis therefore possible fora cell to possess several identical plasmids. Plasmids are used to clone a desired gene. Thisisillustrated in Figure 430. First, you splice or introduce the desired, gene into a plasmid and transfer it into a bacterial cel. ‘Then, culture these bacteria; many of them will have a plasmid with the desired gene. Usea section of nucleotides, complementary to the desired gene, but also attached toa (eg: radioactive) label to find out which plasmids have the gene and which do not. Use restriction enzymes to cut the desired gene out of the plasmids and purify the gene using gel electrophoresis (see Topic 4.4.2). Refer to Figure 430. ‘The host cell is the cell which is to receive the genetic ‘material, A bacterium may be the host cel and produce a protein desired by humans. Restriction enzymes (endonucleases) are used to cit a desired section of the DNA. Bacteria produce these enzymes naturally as a defense against invading viruses. One commonly used restriction enzyme recognises the sequence GAATTC and cuts both strands of the DNA between G and A (see Figure 434). When preparing to transfer DNA, the same restriction enzyme is used for the host and the donor so the cuts are made in the same way. This way, the same ‘sticky ends are created so that the donor DNA can fit in between the host DNA. Refer to GAATTC C ‘The result isan uneven cut in the DNA and the ends of the DNA are referred to as “sticky ends’ Ifthe same sequence is found on another section of DNA and Is cut in the same way, the two strands can be combined as shown in Figure 432. ‘To attach the two cut sections of DNA, the enzyme DNA ligase is used to create the required covalent bonds. aoa umes as Lt ‘ring freign gene © thepiasmig ibrehserted Restriction) into the bacterium. @ the bacteriumis ceavage Used to insert the sie “FONAcaring the 1 foreign gene nto the a Sronoseresta.” © thepans cet plant ce aregrown imelitre Recombinant © The plasmidis removed aad from the bacterium and, the -DNAscutoy 3 Festiction enzyme © rorsign DNAs cut by the same enzyme, iS © The foreign ONAis ofthe plasmic Inserted inte the T-DNA @ A plantis generated from acell lone All fs cells cary the foreign gene and may express itase new tat Figure 430. The general technique of gene transfer, 68