F215 - Cellular Control & Biotechnology and Gene Technologies

Use genetic diagrams to solve problems involving sex linkage and codominance

(Sex Linkage) Haemophilia A - a protein, factor VIII, is among the factors that are needed in order for the blood to clot following a wound. It is coded for on the X chromosome. The recessive allele expresses an altered protein that does not function. This leads to an increase in the time it takes for blood to clot. Males only have one X chromosome and if it has the allele Haemophilia A they will suffer from haemophilia. Such males are hemizygous - they only have one allele for a particular characteristic. Haemophilia A shows a recessive inheritance pattern. If a carrier mother (XhXH) and unaffected father (XHY) reproduce, the possible offspring phenotypes are 25% carrier female, 25% unaffected female, 25% affected (haemophiliac) male ad 25% unaffected male. Duchenne muscular dystrophy - the DMD gene for a muscle protein (dystrophin) is on the X chromosome in humans. It is a large protein, involved in structures needed for muscle contraction. Mutations of the gene usually result in a severely truncated dystrophin protein or no dystrophin. (Codominance) Sickle-Cell Anaemia - all individuals with the disease have the same mutation. The B-strands of haemoglobin differ by one amino acid at position 6. In normal haemoglobin, glutamic acid is at position 6, but in sickle-cell haemoglobin, valine is present instead. When this abnormal haemoglobin is deoxygenated it is not soluble and becomes crystalline and aggregates into linear and less globular structures. This deforms the red blood cells, making them inflexible and unable to squeeze through the capillaries. After many cycles of oxygenation and deoxygenation, some cells become irreversibly sickled and some are destroyed. If enough sickle-cells become lodged in capillaries blood flow is impeded. Organs, particularly bones, do not receive enough oxygen, leading to a painful crisis. Eventually organs, especially heart, lungs and kidneys, become damaged. The genotype of people with normal haemoglobin is HAHA. The genotype of people with sickle-cell anaemia is HSHS. The genotype of symptomless heterozygotes is HAHS. In heterozygotes, RBCs are made in the bone marrow with half their haemoglobin normal and half sickled. The presence of normal haemoglobin prevents sickling in the RBC when they are in circulation and deoxygenated. Thus heterozygotes are symptomless carriers and at whole-organism level SCA could be considered to be recessive, but at a molecular level, because both alleles contribute to the phenotype as observed in RBCs, it is codominant. If two parents are carriers, (both HAHS), 25% of offspring would have all normal haemoglobin, 50% to be heterozygous carriers and 25% to have SCA. Roan Cattle - one of the genes for coat colour in shorthorn cattle have two alleles: CR codes for red hairs and CW codes for white hairs. Homozygous individuals with the genotype CRCR have red (chestnut) coats. Homozygous individuals with the genotype CWCW have white coats. Heterozygotes, genotype CRCW, have red and white hairs and are roan. If two roan shorthorn cattle are interbred, the probable progeny phenotypes are 25% red, 50% roan and 25% white.

State that genes code for polypeptides, including enzymes

A gene is a length of DNA that codes for one (or more) polypeptides. The genome of an organism is the entire DNA sequence of that organism.

Define the term recombinant DNA;

A section of DNA, often in the form of a plasmid, which is formed by joining DNA from two different sources

Describe the interactions between loci (epistasis).

Epistasis is the interaction of different gene loci so that one gene locus masks or suppresses the expression of another gene locus. The genes involved may control the expression of one phenotypic characterisitc either by: a) working against each other (antagonistically) resulting in masking or b) they may work together in a complimentary fashion. The homozygous precense of a recessive allele may prevent the expression of another allele at a second locus. The alleles at the first locus are epistatic to the alleles at the second locus, which are described as hypostatic. Epistasis is not inherited, it is an interaction of gene loci. It reduces phenotypic variation. A 9:3:3:1 ratio suggests recessive epistasis, a 12:3:1 or a 13:3 ratio suggests dominant epistasis and a 9:7 ratio suggests epistasis by complimentary action.

Outline the steps involved in sequencing the genome of an organism;

Genomics refers to the study of the whole set of genetic information in the form of DNA base sequences that occur in the cells of organisms of a particular species. The sequenced genomes of organisms are placed on public access databases. The sequencing reaction can only operate on a length of DNA of about 750 base pairs. This means that the genome must be broken up and sequenced in sections. In order to ensure that the assembled code is accurate, sequencing is carried out a number of times on overlapping fragments, with the overlapping regions analysed and put back together to from the completed code. The stages are: 1) the genomes are first mapped to identify which part of the genome (i.e. which chromosome or which section of a chromosome) they have come from. Information that is already known is used - e.g. using the location of micro satellites (short runs of repetitive sequences of 3-4 base pairs found in several thousand locations of the genome. 2) samples of the genome are sheared (mechanically broken) into smaller sections of around 10 000 base pairs. This is sometimes referred to as the 'shotgun' approach. These sections are placed into bacterial artificial chromosomes (BACs) and transferred to E.Coli (bacterial) cells. As the cells grow in culture, many copies (clones) of the sections are produced. These cells are referred to as clone libraries. In order to sequence a BAC section: 1) cells containing specific BACs are taken and cultured. The DNA is extracted from the cells and restriction enzymes used to cut it into smaller fragments. The use of different restriction enzymes on a number of samples gives different fragment types. 2) the fragments are separated using electrophoresis. 3) each fragment is sequenced using an automated process. 4) computer programmes then compare overlapping regions from the cuts made by different restriction enzymes in order to reassemble the whole BAC segment sequence. Automated DNA sequencing is based on interrupted PCR and electrophoresis. Sequencing fragments of DNA was initially carried out slowly using radioactively labelled nucleotides. The development of automated sequencing has led to a rapid increase in the number of organism genomes sequenced. The reaction mixture (as with PCR) contains the enzyme DNA polymerase, many copies of the single-stranded template DNA (the bit of DNA to be copied), free DNA nucleotides and primers. Within the sequencing mixture, some of these free nucleotides carry a fluorescent marker. These nucleotides are modified and, if they are added to the growing chain, the DNA polymerase is 'thrown off' and the strand cannot have any further nucleotides added. Each nucleotide type has a different coloured fluorescent marker. The reaction proceeds: 1) the primer anneals at the 3' end of the template strand, allowing DNA polymerase to attach. 2) DNA polymerase adds free nucleotides according to base pairing rules so the strand grows - essentially the same as natural DNA replication and PCR. 3) if a modified nucleotide is added, the polymerase enzyme is thrown off and the reaction stops on that template strand. 4) as the reaction proceeds, many molecules of DNA are made. The fragments generated vary in size. In some of them the template strand has only one additional nucleotide added before the polymerase is thrown off, in others the template strand is completed. In each case, the final nucleotide added is tagged with a specific colour. 5) as these strands run through the machine (in the same way that DNA strands move in electrophoresis) a laser reads the colour sequence, from the strand with only a single nucleotide added, to the one with two nucleotides added, then three, then four etc. The sequence of colours, and so the sequence of bases, can then be displayed.

State that biotechnology is the industrial use of living organisms (or parts of living organisms) to produce food, drugs or other products

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State that cyclic AMP activates proteins by altering their three-dimensional structure;

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Explain why microorganisms are often used in biotechnological processes

1. microorganisms grow rapidly in favourable conditions, with a generation time (time taken for numbers to double) as little as 30 minutes. 2. often produce proteins or chemicals that are given out into the surrounding medium and can be harvested. 3. can be genetically engineered to produce specific products. 4. grow well at relatively low temperatures, much lower than those required in chemical engineering of similar processes. 5. can be grown anywhere in the world and are not dependent of climate change. 6. tend to generate products that are more pure in form than those generated via chemical processes. 7. can often be grown using nutrient materials that would otherwise be useless or toxic to humans.

Describe how DNA probes can be used to identify fragments containing specific sequences;

A DNA probe is a short single-stranded section of DNA that is complimentary to the section of DNA being investigated. The probe is labelled in one of two ways: a. using a radioactive marker so that the location can be revealed by exposure to photographic film. b. using a flourescent marker that emits colour when exposed to UV light. Copies of the probe are added to a sample of DNA fragments and will anneal to any fragment where a complimentary base strand is present.

Describe the production of natural clones in plants using the example of vegetative propagation in elm trees;

A clone describes genes, cells or whole organisms that carry identical genetic material because they are derived from the same original DNA. When plants reproduce asexually by producing runners, the new plants are clones. In this process identical copies of the 'original' DNA generate new organisms with the same cloned DNA. The production of cloned DNA, cells and organisms is a natural process for growth and reproduction that can also be achieved by artificial means. Prokaryotes divide by binary fission. Their DNA replicates and the cell divides into two. Provided there are no mutations, the two resulting cells are genetically identical to each other and to the parent cell. In multicellular organisms, such as plants, some of the cells produced by mitosis can grow into new, separate organisms with DNA that is identical to the parent plant, so they are clones of the parent plant. The advantages of asexual reproduction are that: it is quick, allowing organisms to reproduce rapidly and so take advantages of resources in the environment, it can also be completed if sexual reproduction fails or is not possible, all offspring have the same genetic information to enable them to survive in their environment. The disadvantage of asexual reproduction is that: it does not produce any genetic variety, so any genetic parental weakness will be in all offspring. If the environment changes, e.g. with the introduction of a new disease-causing organism, then all genetically identical organisms will be equally susceptible. e.g in the 20th century, Dutch Elm disease spread through Europe's elms; the leaves withered, followed by death of the branches and trunks, as a result of a fungal disease carried by a beetle. The English Elm responded with the production of growing root suckers. However, once the new trees got to about 10cm in diameter, they became infected and died. Because the new trunks were clones of the old one, they did not have any resistance to the fungal attack so they remained just as vulnerable as the original tree. There is genetic variation within a cloned population, so natural selection cannot occur. Asexual reproduction in plants takes place naturally in a variety of different ways. E.g. a number of plant species like the English Elm are adapted to reproduce asexually following damage to the parent plant. This allows the species to survive catastrophes such as disease or burning. New growth in the form of root suckers, or basal sprouts, appear within 2 months of the destruction of the main trunk. These suckers grow from meristem tissue in the trunk close to the ground, where least damage is likely to have occurred. This is vegetative propagation, the production of structures in an organism that can grow into individual new organisms. These offspring contain the same genetic information as the parent and so are clones of the parent. Root suckers help the elm spread, because they grow all around the original trunk. When the tree is stressed or the trunk dies, e.g. when felled as part of the coppice cycle, the suckers grow into a circle of new elms called a clonal patch. This in turn puts out new suckers so that the patch keeps expanding as far as resources permit.

Describe how artificial clones of animals can be produced

A cloned animal is one that has been produced using the same genetic information as another animal. Such an animal has the same genotype as the donor organism. In animals, only embryonic cells are capable of undergoing stages of development in order to generate a new individual. These are totipotent stem cells, i.e capable of differentiating into any type of adult cell found in an organism. These cells are able to 'switch on' any of the genes present on the genome. There are 2 methods of artificially cloning animals: 1. Splitting embryos, 'artificial identical twins' - cells from a developing embryo can be separated out, with each one then going on to produce a separate genetically identical organism. e.g. a high value female, such as one with a high milk yield, has eggs collected from her, to be used to undergo in vitro fertilisation with the collected sperm of a high value male, i.e one that is known to produce daughters that have a high milk yield. The in vitro is grown to a 16 cell embryo, which is then split into several separate segments, which are individually implanted into surrogate mothers. Each calf is an genetically identical clone. 2. Nuclear transfer, 'using enucleated eggs' - a differentiated cell from an adult can be taken, and its nucleus placed in an egg cell which has had its own nucleus removed (enucleated). The egg then goes through the stages of development using genetic information from the inserted nucleus. The first animal cloned by this method was Dolly the Sheep in 1996. The cell was taken from a mammary gland of a 6 year old ewe, its nucleus transplanted into a cell from a second sheep and then inserted into the uterus of a third sheep, and then a fourth, to develop. This was the only success from 277 attempts.

State that mutations cause changes to the sequence of nucleotides in DNA molecules

A mutation is a change in the amount of, or arrangement of, the genetic material in a cell. Random change to the structure of a chromosome is called a chromosome mutation. 1) inversion - a section of a chromosome turns through 180. 2) deletion - a part of a chromosome is lost. 3) translocation - a piece of one chromosome becomes attached to another. 4) non-disjunction - homologous chromosomes fail to separate properly at meiosis I or chromatids fail to separate at meiosis II; if this happens to a whole set of chromosomes polyploidy results). The shuffling of alleles in phrophase I is NOT an example of mutation. DNA mutation may also occur during interphase when DNA replicates (this can also occur in mitosis or binary fission). Mutation increases genetic variation, even if the effect is negative. If mutation occurs in the sperm/ egg that are used in fertilisation, then the mutated gene will be present in every cell of the offspring.

Outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid;

A plasmid is used which carries genes which makes any bacteria recieving them resistant to two different antibiotics (usually ampicillin and tertacycline). The resistance genes are known as genetic markers. The plasmids are cut by an enzyme which has its restriction site in the middle of one of the resitance genes (tertacycline G1) so that if the required gene is taken up, the resistance gene for one of the antibiotics (teracylcine) does not work, but the other (G2 ampicillin) does. Then a process called replica plating is used - the process of growing bacteria on an agar plate, then transferring a replica of that growth to other plates, usually containing different growth promoters or inhibitors. Analysis of growth patterns on the replica plates gives information about the genetic properties of the growing bacteria. The DNA is placed in the plasmids, and the plasmids in bacterial cells. The bacteria are grown on an agar plate to produce a colony. Some cells from the colonies are transferred onto agar that has been made from the second antibiotic. Only the bacteria that has been made from the antibiotic that remains intact, meaning that all bacteria that have taken up a plasmid will grow. Some cells are transferred onto agar that has been made from the second antibiotic will grow. By keeping track of which colonies are which, we now know that any bacteria which grow on agar containing the first antibiotic, but not on the agar containing the second antibiotic, must have taken up the recombinant plasmid. The required colonies can now be identified and grown on a large scale.

Describe, with the aid of diagrams, and explain the standard growth curve of a microorganism in a closed culture

A small number of organisms placed in a fresh 'closed culture' (the growth of microorganisms in an environment where all conditions are fixed and contained. No new materials are added and no waste products are removed) grow in a very predictable, standard way. 1. Lag phase - microorganisms are adjusting to their surrounding conditions. This may mean taking in water, cell expansion, activating specific genes, and synthesising specific enzymes. The cells are active but not reproducing so population remains fairly constant. The length of this period depends on the growing conditions. 2. Log (exponential) phase - the population size doubles every generation as every individual has enough space and nutrients to reproduce. In some bacteria, the population can double every 20-30 minutes in these conditions. The length of this phase depends on how quickly the microorganisms reproduce and take up the available nutrients and space. 3. Stationary phase - nutrient levels decrease and waste products like carbon dioxide and other metabolites build up. Individual organisms die at the same rate at which new individuals are being produced. In an open system, this would be the carrying capacity of the environment. 4. Decline or death phase - nutrient exhaustion and increased levels of toxic waste products and metabolites lead to the death rate increasing above the reproduction rate. Eventually, all organisms will die in a closed system.

Discuss the advantages and disadvantages of cloning animals

Advantages: high value animals, e.g. cows giving high milk yields, can be cloned in large numbers. Rare animals can be cloned to preserve the species. Genetically modified animals - e.g. sheep that produce pharmaceutical chemicals in the milk - can be quickly reproduced. Disadvantages - high value animals are not necessarily produced with animal welfare in mind. Some strains of meat-producing chickens have been developed that are unable to walk. As with plants, excessive genetic uniformity in a species makes it unlikely to be able to cope with, or adapt to, changes in the environment. It is still unclear whether animals cloned using the nuclear material of adult cells will remain healthy in the long term. e.g. there was controversy over Dolly the Sheep's post mortem results as it was wrongly reported that her death was due to premature aging caused by cloning.

Discuss the advantages and disadvantages of plant cloning in agriculture

Agriculture had sought to provide high-quality crops in terms of yield and resistance to environmental conditions such as drought, pests or weeds. Selective breeding over generations has resulted in crop plants having reduced genetic variation. This is because farmers have identified and grown only these crops with useful features. Some crops such as fruit trees cannot be grown from seed because the new tree will have a combination of genes that will not give the correct fruit. Bananas have to be grown by cloning because all cultivated bananas are sterile. Propagation using callus culture means: farmers know what the crop plant produced will be like because it is cloned from plants with known features such as high yield, taste, colour and disease resistance. Farmers costs are reduced because all the crop is ready for harvest at the same time. This is essentially a 'refinement' of selective breeding. Micropropagation is much faster than selective breeding, because huge numbers of genetically identical plants can be generated from a small number of plants or a single valuable plant. The disadvantages of using cloned plants in agriculture are the same as those for asexually reproducing organisms. Genetic uniformity means that all plants are equally susceptible to any new pest, disease or environmental change. e.g. the Irish potato famines are examples of problems associated with genetic monoculture, as much of the potato was lost due to infection by a fungus like protoctist. Although the potato crop at the time was not produced using plant culture, the plants were genetically uniform so they were all susceptible to the same disease. Farming methods are now more regulated and although genetically uniform crops are grown, the areas given to a specific crop and the distance between areas of the same crop are controlled in order to limit the effects of the arrival of new pathogens.

Explain the terms allele, locus, phenotype, genotype, dominant, codominant and recessive

Allele - an alternative version of a gene locus with a specific position on a chromosome, occupied by a specific gene. Phenotype -observable characteristics of an organism. Genotype - alleles present within cells of an individual, for a particular trait/characteristic. Dominant - a characteristic in which the allele responsible is expressed in the phenotype, even in those with heterozygous genotypes. Codominant- a characteristic where both alleles contribute to the phenotype. Recessive - a characteristic in which the allele responsible is only expressed in the phenotype is there is no dominant allele present.

Explain that both genotype and environment contribute to phenotypic variation.

Although a plant with the genotype AABBCC has the genetic potential to produce ears of grain of 12cm, some plants may not produce such long ears. This could be because they are short of water, minerals such as nitrates and phosphates, or light. Any of these environmental factors may limit the expression of the genes. In humans, intelliegence is partly determined by genes and partly determined by environment. Children inherit many genes, with alleles from each parent, giving a genetic potential. However that potential is only realised with the help of a stimulating learning environment both at home and at school. It is also aided by good nutrition for growth and development of organs, including the brain and nervous system. The expression of polygenic traits (characteristics that are coded for by many genes) is influenced more by the environment than the expression of monogenic traits (characteristics that are coded for by one gene).

Explain how mutations can have beneficial, neutral or harmful effects on the way a protein functions

An allele is an alternative version of a gene with the same locus on the chromosome and codes for the same polypeptide but the alteration to the DNA base sequence may alter the protein's structure. Neutral effects: an allele may produce no change to the organism if: the mutation is in a non coding region of DNA or if it is a silent mutation - although the base triplet has changed, it still codes for the same amino acid so the protein is unchanged. Beneficial/Harmful effects: Early humans is Africa almost certainly had dark skin. Melanin protected them from the harmful effects of UV light yet could still synthesise vitamin D from the action o fthe inense sunlight on their skin - this is an importnant source of Vitamin D as we eat food that contains very little Vitamin D. Humans with mutations of the genes determining skin colour, producing paler skin, would have burned and suffered from skin cancer. As human migrated to more temperate climes, the sunlight was not intense enough to cause Vitamin D to be made by those with darker skins. Humans with mutations producing paler skin or lack of pigmenwould have an advantage over those with darker skin as they could synthesise more vitamin D. Lack of vitamin D = rickets and a narrow pelvis, producing difficulties in childbirth Vitamin D helps protect against cancer and heart disease. The inuit people have not lost all their skin pigments, although they do not live in an environment that has intense sunlight because they eat a lot of fish and seal meat, both rich sources of dietary Vitamin D.

Predict phenotypic ratios in problems involving epistasis

An example of recessive epistasis is the inheritance of flower colour in Salvia. Two gene loci, A/a and B/b, on different chromosomes are involved. A pure breeding pink-flowered variety of Salvia, genotype AAbb, was crossed with a pure-breeding white-flowered variety, genotype aaBB. All the F1 generation, genotype AaBb, had purple flowers. Interbreeding the F1 to give the F2 generation resulted in purple, pink and white flowers in the ratio 9:3:4. The homozygous aa is epistatic to both alleles of the gene B/b. Niether the allele B for purple or b for pink can be expressed in there is no dominant allele, A, present. Dominant epistasis occurs when a dominant allele at one gene locus masks the expression of the alleles at a second gene locus. e.g. fruit colour in summer squash. Two gene loci, D/d and E/e are involved. The precense of one D allele results in white fruits, regardless of the alleles present at the second locus (E/e). In homozygous dd individuals, the precense of one E allele produces yellow frutis and the presence pf two e alleles produces green fruits. If two white-coloured, double heterozygotes (DdEe) are crossed, the offspring show the phenotype ratio 12 white (D-E or D-ee) : 3 yellow (ddE-) : 1 green (ddee). The feather colour of chickens is also dominant epistasis. There is an interaction between two gene loci, I/i and C/c. Individuals carrying the dominant allele, I, have white feathers, even if they also have the dominant allele, C, for coloured feathers. Birds that are homozygous for c (genotype IIcc, Iicc or iicc) are also white. Pure-breeding White Leghorn chickens have the genotype IICC. Pure-breeding white chickens have the genotype iicc. If two such birds are mated, all the progeny are white, with the genotype IiCc. If the progeny interbreed, they produce white-feathered chickens and coloured-feathered chickens, in a ratio of 13:3. Working antagonistically: if two strains of white flowered sweet peas are crossed (ccRR x CCrr), all the F1 generation have purple flowers. Allowing the F1 generation to interbreed produces an F2 generation with purple and white flowers in a ration 9:7, suggesting that at least one dominant allele for both gene loci (C-R-) has to be present for flowers to be purple. All other genotype combinations, such as ccR- or C-rr, produce white flowers. This is because the homozygous recessive condition at either locus masks the expression of the dominant allele at the other locus. The way the twp gene loci may produce these results is if they complement each other - if one gene codes for an intermediate colourless pigment and the second locus codes for an enzyme that converts the intermediate compound to the final purple pigment. Coat colour in mice may be agouti (alternating bands of pigment on each hair so it looks grey), black or albino. The gene for agouti has to alleles, A/a. Allele a is a mutation; when found in homozygous individuals it produces a black coat. A gene B/b at a separate locus controls the formation of pigment. Individuals of genotype B- can produce pigment, but those with the genotype bb cannot, and are albino. When several pairs of agouti individuals with the genotype AaBb are crossed, the total offspring are 28 agouti, 10 black and 12 albino, giving a ratio very close to 9 : 3 :4. There are 4 types of comb shape in domestic chickens (single, rose, walnut, pea). Two gene loci, P/p and R/r, interact to affect comb shape. The effect of P/p alleles depends on which of the R/r alleles are present in the bird's genotype. When true-breeding pea-combed chickens, genotype PPrr, are bred with true-breeding rose-combed chickens, genotype ppRR, the progeny have walnut combs, PpRr. When walnut-combed chickens are interbred, the progeny show four phenotypes in the 9 : 3 : 3 : 1 ratio for the dihybrid for F2 progeny.

Explain genetic control of protein production in a prokaryote using the lac operon

An operon is a length of DNA, made up of structural genes and control sites. The structural genes code for proteins. The control sites are the operator and promoter region, which are both genes. E. coli can synthesise about 3000 different polypeptides. It normally respires glucose but can also use lactose as a respiratory substrate. E. coli grown in a culture medium with no lactose (disaccharide sugar) can be placed in a medium with lactose. At first, E.coli can't metabolise the lactose as it only has small amounts of the enzymes beta-galactosidase (which catalyses the hydrolysis of lactose to glucose and galactose) and lactose permease (which transports lactose into the cell). A few minutes after the lactose is added, E. coli increases the rate of synthesis of these enzymes by about 1000 times, so lactose triggers the production of the enzymes, and is called an inducer. The lac operon - the regulator gene (I): This is not part of the operon and is some distance away from it. It is important as it produces the repressor protein. The control sites (P and O): P is the promoter region. It's a length of DNA to which the enzyme RNA polymerase binds to in order to begin the transcription of the structural genes. The operator region can switch the structural genes on and off. The structural genes (Z and Y): Z codes for beta galactosidase and Y codes for the enzyme lactose permease. Each consists of a sequence of base pairs that can be transcribed into a length of mRNA. When lactose is absent: the regulator gene is expressed and the repressor protein is synthesised. It has two binding sites. One binds to lactose and one that binds to the operator region. In binding to the operator region, it covers part of the promoter region where RNA polymerase normally attaches. RNA polymerase cannot bind to the promoter region so the structural genes cannot be transcribed into mRNA. Without mRNA the genes cannot be translated and the enzymes cannot be synthesised. When lactose is added: lactose binds to the other site on the repressor protein, causing the molecule to change shape. This prevents the other binding site from binding to the operator region. The repressor dissociates from the operator region. This leaves the promoter region unblocked. RNA polymerase can now bind to it and initiate the transcription of mRNA.The operator- repressor- inducer system acts as a molecular switch. It allows synthesis of the structural genes. As a result, the bacteria can now use the lactose permease enzyme to take up lactose from the medium into their cells. They can then convert it to glucose and galactose using the β-galactosidase enzyme. These sugars can then be used for respiration, thus gaining energy from lactose.

Explain the term gene therapy;

Any therapeutic technique where the functioning allele of a particular gene is placed in the cells of an individual lacking the functioning alleles of thay particular gene. Can be used to treat some recessive conditions, but not dominant conditions.

Outline how apoptosis (programmed cell death) can act as a mechanism to change body plans.

Apoptosis is an integral part of plant and animal tissue development. It is a series of biochemical events that leads to an orderly and tidy cell death, in contrast to cell necrosis, an untidy and damaging cell death that occurs after trauma which leads to the release of harmful hydrolytic enzymes. There is extensive division and proliferation of a particular cell type followed by pruning through programmed cell death. The excess cells shrink, fragment and are phagocytosed so that the components are reused and no harmful hydrolytic enzymes are releasing into the surrounding tissue. It is tightly regulated during development, and different tissues use different signals for inducing it. It weeds out ineffective or harmful T lymphocytes during the development of the immune system. Apoptosis ensures that the rate of cells produced by mitosis is the same as the rate of cells dying, so the number of cells remains constant. Not enough apoptosis leads to cancer, whereas too much leads to cell loss and degeneration. Apoptosis causes the digits (toes and fingers) to separate from each other during development. Cell signalling plays a vital role in maintaining the right balance. The process is controlled by a diverse range of cell signals, some of which come from the outside and some the inside. The signals include cytokines made by cells of the immune system, hormones, growth factors, and nitric oxide. Nitirc oxide can induce apoptosis by making the inner mitochondrial membrane more permeable to hydrogen ions and dissipating the proton gradient. Proteins are released into the cytosol. These proteins bind to apoptosis inhibitor proteins and allow the process to take place. First enzymes break down the cell cytoskeleton. The cytoplasm becomes dense with organelles tightly packed. The cell surface membrane changes and blebs form.The chromatin condenses and the nuclear envelope breaks. DNA breaks into fragments. The cell breaks down into vesicles that are taken up by phagocytosis. The cellular debris is disposed of so that it does not damage other cells or tissue.

Describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment;

Bacteria are capable of a process known as conjugation, where genetic material may be exchanged. In this process, copies of plasmid DNA are passed between bacteria, sometimes even of different species. Since plasmids often carry genes associated with resistance to antibiotics, this swapping of plasmids is a concern because it speeds the spread of antibiotic resistance between bacterial populations. The advantage to bacteria of conjugation is that it may contribute to genetic variation, and in the case of antibiotic resistance genes, survival in the precense of those chemicals.

State other vectors into which fragments of DNA may be incorporated;

Plasmids, liposomes, viral DNA e.g. bacteriophages, or hybrid vectors with the properties of both plasmids and bacteriophages

Explain the basis of continuous and discontinuous variation by reference to the number of genes which influence the variation

Both types of variation may be the result of more than one gene. However, in discontinuous variation, if there is more than one gene involved, the genes interact in an epistatic way, where one gene masks or influences the expression of another gene. In many examples of discontinuous variation there may only be one gene involved, e.g. CF occurs when a person has two faulty alleles of the CFTR gene. In discontinuous variation: different alleles at a single gene locus have large effects on the phenotype, different gene loci have quite different effects on the phenotype, e.g. codominance, dominance, and recessive patterns of inheritance. In continuous variation: traits exhibiting continuous variation are controlled by two or more genes, each gene provides an additive component to the phenotype, different alleles at each gene locus have a small effect on the phenotype, and a large number of different genes may have a combined effect on the phenotype. These are known as polygenes and they are unlinked - they are on different chromosomes. The characteristic they control is described as polygenic.

Explain the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield of product required

Commercial applications of biotechnology often require the growth of a particular microorganism on a huge scale. An industrial-scale fermenter can have its conditions manipulated and controlled in order to ensure the best possible yield of the product. The precise growing conditions depend on the microorganisms being cultured and on whether the process is designed to produce a primary or secondary metabolite. These are: 1. temperature - too hot and enzymes will be denatured; too cold and growth will be slowed. 2. type and time of addition of nutrient - growth of microorganisms requires a nutrient supply, including sources of carbon, nitrogen and any essential vitamins and minerals. The timing of nutrient addition can be manipulated, depending on which metabolite the process is designed to produce. 3. oxygen concentration - most commercial applications use the growth of organisms under aerobic conditions, so sufficient oxygen must be made available. A lack of oxygen will lead to the unwanted products resulting from anaerobic respiration and a reduction in growth rate. 4. pH - changes in pH within the fermentation tank can reduce the activity of enzymes and so reduce growth rates. Such large cultures need 'starter; populations of the microorganism. These are obtained by taking a pure culture and growing it in a sterile nutrient broth. General features of a large scale fermenter: pressure vents to prevent any gas, e.g. carbon dioxide build up, air inlet - sterile air provides oxygen in aerobic fermenters, mixing blades (impellers), water jacket inlet - allows circulation of water around the fermenter to regulate temperature, an outlet tap for draining fermenter, a motor that rotates the blades to mix the culture evenly, an inlet for the addition of nutrients, a water jacket outlet, electronic probes for measuring oxygen, pH and temperature levels, air outlets, often in a ring - air bubbles come out from outlets, mixing with the culture (known as sparging), all inlets and outlets are fitted with filters to prevent contamination.

Outline how DNA fragments can be separated by size using electrophoresis

DNA samples are treated with restriction enzymes to cut them into fragments. The DNA samples are placed into cells cut into the negative electrode end of the gel. The gel is immersed in a tank of buffer solution and an electric current is passed through the solution for a fixed period of time, usually around two hours. DNA is negatively charged because of its phosphoryl groups. It is attracted to the positive electrode. Shorter lengths of DNA move faster than longer lengths, so move further in the fixed time that current is passed through the gel. The position of the fragments can be shown using a dye that stains DNA molecules.

Describe the differences between continuous and discontinuous variation

Discontinuous variation describes qualitiative differences between phenotypes. Qualitative differences fall into clearly distinguishable categories. There are no intermediate cateogries - you are either male or female, you have blood group O, A, B or AB; pea plants may be tall or dwarf etc. Continuous variation describes quantitative differences between phenotypes. These are phenotypic differences where there is a wide range of variation within the population, with no distinct categories. e.g. height and mass in humans, cob length in maize varieties, grain colour in wheat, seed length in broad beans, milk yield in cattle and egg size in poultry etc.

Explain why immobilised enzymes are used in large-scale production;

Enzymes are specific - they catalyse reactions between specific chemicals, even in mixtures of many different chemicals. This means that fewer by-products are formed and less purification of products is necessary. Most enzyme function well at relatively low temperatures, much lower than those needed for many industrial chemical processes. This saves money on fuel costs. However, enzymes from thermophilic bacteria (bacteria that thrive at high temperatures) have been extracted and used in reactions that need a high temperature. In some biotechnological processes, whole organisms are cultured on a large scale to produce particular products. This means that the product of a single chemical reaction is required, so it is often more efficient to use isolated enzymes to carry out the reaction rather than growing the whole organism or using an inorganic catalyst. Isolated enzymes can be produced in large quantities in commercial biotechnological processes. The extraction of enzyme from the fermentation process is known as downstream processing, used to describe the processes involved in the separation and purification of any product of large-scale fermentations. In order for the product of an enzyme-controlled reaction to be generated, enzyme and substrate must be able to collide and form enzyme-substrate complexes. This is most easily achieved by mixing quantities of substrate and isolated enzyme together, under suitable conditions for the enzyme to work. The product generated then needs to be extracted from the mixture. This can be a costly process. It is possible to immobilise enzymes so that they can continue to catalyse the enzyme-controlled reaction but do not mix freely with the substrate as they would normally in a cell or isolated system. Advantages of using immobilised enzymes: 1. enzymes are not present with products so purifications/ downstream processing costs are low. 2. enzymes are immediately available for reuse. This is particularly useful in allowing for continuous processes. 3. immobilised enzymes are more stable because the immobilising matrix protects the enzyme molecules. Disadvantages: 1. immobilisation requires additional time, equipment and materials and so it is more expensive to set up. 2. immobilised enzymes can be less active because they do not mix freely with the substrate. 3. any contamination is costly to deal with because the whole system would need to be stopped.

Describe the production of artificial clones of plants from tissue culture

For many years, farmers and growers have been able to artificially The two main methods are: 1. taking cuttings - a section of stem is cut between leaf joints (nodes). The cut end of the stem is then often treated with plant hormones to encourage root growth, and planted. The cutting forms a new plant which is a clone of the original parent. Large numbers of plants, such as geraniums, can be produced quickly in this way. 2. grafting - a shoot section of a woody plant (often a fruit tree or a rosebush) is joined to an already growing root and stem (known as a rootstock). The graft grows and is genetically identical to the parent plant, but the rootstock is genetically different. Although useful, cutting and graftings cannot produce huge numbers of cloned plants very easily. Also some plants do not reproduce well either from cutting or grafting. More modern methods of artificial propagation use plant tissue culture in order to generate huge numbers of genetically identical plants from a very small amount of plant material. Tissue culture can be used to generate large stocks of a particularly valuable plant very quickly, with the added advantage that these stocks are known to be disease free. Micropropagation by callus tissue culture - the most common method used in large-scale cloning of plants. Many houseplants are produced in this way, notably orchids. 1. A small piece of tissue is taken from the plant to be cloned, usually from the shoot tip. This is called an explant. 2. The explant is placed on a nutrient growth medium. 3. Cells in the tissue divide, but they do not differentiate. Instead they form a mass of undifferentiated cells called a callus. 4. After a few weeks, single callus cells can be removed from the mass and placed on a growing medium containing plant hormones that encourage shoot growth. 5. After a further few weeks, the growing shoots are transferred onto a different growing medium containing different hormone concentrations that encourage root growth. 6. The growing plants are then transferred to a greenhouse to be acclimatised and grown further before they are planted outside.

Outline the process involved in the genetic engineering of 'Golden Rice'

Functions of vitamin A; forms part of the visual pigment rhodopsin, involved in the synthesis of many glycoproteins, maintenance and differentation of epithelial cells, helping to reduce infection, and essential for growth of bones. Rice plants contain the genes that code for the production of beta-carotene. This molecule is a photosynthetic pigment molecule so is required in the green parts of the plant. However if that part of the plant is eaten, the endosperm, the genes for beta-carotene production are switched off. A gene from the Daffodil (Phytoene synthtase) and one from the bacterium Erwina urefovora (Crt 1 enzyme) The genes were inserted into TI plasmids and taken up by the bacterium Argobacterium tumfaciens. This introduces the genes into rice embryos. The genes were inserted into the rice genome near to a specific promotor sequence that switches on the genes associated with endosperm development. This meant they were expressed as the endosperm grew. The resulting rice plants produce seeds with beta-carotene in the endosperm, which is yellow. Vitamin A is produced in our bodies from beta-carotene. Beta-carotene is a precursor to Vitamin A. Golden Rice is said to be biofortified becuase it contains higher than normal concentrations of a particular nutrient, in this case beta-carotene.

Explain that the genes that control development of body plans are similar in plants, animals and fungi, with reference to homeobox sequences

Genetic control of Drosophilia development - genetically mediated by Homeobox genes. Some (maternal-effects) genes, determine the embryo's polarity. Other (segmentation) genes specify the polarity of each segment. Homeotic selector genes specify the identity of each segment and direct the development of individual body segments. These are the master genes in the control networks of regulatory genes. There are 2 gene families: the complex that regulates development of abdomen and thorax segments and the complex that regulates the development of head and thorax segments. Mutations of these genes can change one body part to another. This can be seen in the condition antennapedia - where the antennae of Drosophilia look more like legs. Homeotic genes are similar in plants, animals and fungi. These genes control the development of body plans, including the polarity (head and tail ends) and positioning of the organs, and are expressed in specific patterns and in particular stages of development depending on when they are activated. They specify the identities and fates of embryonic cells. The homeobox is a sequence of DNA that codes for a region of 60 amino acids, and the resulting protein is found in most, if not all, eukaryotes. The region binds to DNA so that they can regulate transcription. The homeobox genes are arranged in clusters known as Hox clusters. Nematodes have one Hox cluster, Drosophilia has two Hox clusters, and vertebraes have four clusters, of 9-11 genes, located on separate chromosomes. The increase in the number of Hox clusters probably arose by duplication of a single complex present in segmented worms (annelids) and has allowed for more complex arthropods to evolve from the simpler annelids.

Explain how genetic drift can cause large changes in small populations

Genetic drift is a change in allele frequency that occurs by chance because only some of the organisms in each generation reproduce. It is particularly noticeable when a small number of individuals are separated from the rest of the large population. They form a small sample of the original population and so are unlikely to be representative of the large population's gene pool. Genetic drift will alter the allele frequency still further.

Explain the role of isolating mechanisms in the evolution of new species, with reference to ecological (geographic), seasonal (temporal) and reproductive mechanisms;

If two sub-populations are separated from each other, they will evolve differently as they have different selection pressures, so different alleles will be eliminated or increased within each sub population. Eventually the sub populations will not be able to interbreed and so will be different species. A large population of organisms may be split into sub-groups by various isolating mechanisms: geographic (ecological) barriers, such as a river or mountain range. Seasonal (temporal) barriers such as climate change throughout a year, or reproductive mechanisms - members may no longer be able to physically mate - their genitals may be incompatiable or their breeding seasons or courtship behaviours may vary. This leaves two sub-populations, isolated from each other. In each case different alleles will be eliminated or increased within each sub-population. Eventually the sub-populations will not be able to interbreed to produce fertile offspring and will be different species (speciation).

Describe the differences between primary and secondary metabolites;

Primary metabolites - substances produced by an organism as part of its normal development. e.g. amino acids, proteins, enzymes, nucleic acids, ethanol and lactate. The production of primary metabolites mirrors the growth in population of the organism. Secondary metabolites - substances produced by an organism that are not part of its normal growth e.g. antibiotic chemicals. Production usually begins after the main growth period of the organisms and so does not match in population of the organism.

Describe how enzymes can be immobilised;

Immobilisation of enzymes refers to any technique where enzyme molecules are held, separated from the reaction mixture. Substrate molecules can bind to the enzyme molecules and the products formed go back into the reaction mixture leaving the enzyme molecules in place. Methods of immobilsing enzymes: 1. Adsorption - enzyme molecules are mixed with the immobilsing support and bind to it due to a combination of hydrophobic interactions and ionic links. Adsorbing agents include: porous carbon, glass beads, clays and resins. Because the bonding forces are not particularly strong, enzymes can become detached (known as leakage). However provided the enzyme molecules are held so that their active site is not changed and is displayed, adsorption can give very high reaction rates. 2. Covalent bonding - enzyme molecules are covalently bonded to a support, often by covalently linking to enzymes together to an insoluble material (e.g. clay particles) using a cross-linking agent like gluteraldehyde or sepharose. This method does not immobilise a large quantity of enzyme but binding is very strong so there is very little leakage of enzyme from the support. 3. Entrapment - enzymes may be trapped, e.g. in a gel bead or network of cellulose fibres. The enzymes are trapped in their natural state (i.e. not bound to another molecule so their active site will not be affected). However, reaction rates can be reduced because substrate molecules need to get through the trapping barrier. This means the active site is less easily available than with adsorbed or covalently bonded enzymes. 4. Membrane separation - enzymes may be physically separated from the substrate mixture by a partially permeable membrane. Most simply, the enzyme solution is held at one side of a membrane whilst substrate solution is passed along the other side. Substrate molecules are small enough to pass through the membrane so that the reaction can take place. Product molecules are small enough to pass back through the membrane.

Compare and contrast the processes of continuous culture and batch culture

Industrial-scale fermenters can be operated in either a batch or a continuous culture. In a batch culture, where the microorganism starter population is mixed with a specific quantity of nutrient solution, then allowed to grow for a fixed period. At the end of the fixed period, the products are removed and the fermentation tank is emptied. Penicillin is produced using batch culture of Penicullium fungus. In a batch culture, the growth rate is slower because nutrient levels decline with time, it is easy to set up and maintain, if contamination occurs only one batch is lost, less efficient as the fermenter is not in operation all of the time, very useful for processes involving the production of secondary metabolites. In a continuous culture, where nutrients are added to the fermentation tank and products are removed from the fermentation tank at regular intervals or even continuously. Human hormones such as insulin are produced from continuous culture of genetically modified E. Coli bacteria. In continuous culture, the growth rate is higher as nutrients are continuously added, the set up is more difficult and the maintenance of required growing conditions can be more difficult to achieve, if contamination occurs huge volumes of product may be lost, it is more efficient as the fermenter operates continuously, and is very useful for processes involving the production of primary metabolites.

Use the chi-squared (χ2) test to test the significance of the difference between observed and expected results.

It is a statistical test to find out if the difference between observed cateogrical data and expected data is small enough to be due to chance. The X2 test can be used for categorical data, where there is a strong biological theory that we can use to predict expected values. Other criteria must also be met: the sample size must be relatively large, only raw counts, not percentages or ratios, can be used and there are no zero scores. The chi squared test tests the null hypothesis (there is no statisically significant difference between the observed and expected numbers, and any difference is due to chance). If there is no significant difference between observed and expected data, we can accept the null hypothesis. If there is a significant difference between the observed and expected data, we can accept the experimental hypothesis and reject the null hypothesis, that the difference is significant and not due to chance. The formula for caluclating chi squared is: the sum of (observed numbers O) - expected numbers (E)2) / expected numbers (E). The differences may be positive or negative so they are squared to stop any negative values cancelling out the positive values, and dividing by E takes into account the size of the numbers and the sum sign takes into account the number of comparisions being made. The bigger the value of chi squared the more certain we are that there is a significant difference between observed and expected values or the smaller the value the more certain we are that the difference is due to chance and not due to a significant difference. We look up the calculated value of chi squared in a distribution table showing values of chi squared. e.g. the critical value of X2 with three degrees of freedom (degrees of freedom = number of cateogries - 1, so in a test with 4 cateogries, there are 3 degrees of freedom) and a p for probability of 0.05 is 7.82. If our calculated value of X2 is smaller than that critical value then we say that the difference is due to chance and is not statistically significant. If it is greater than the critical value, then the difference is probably not due to chance and we have to think again about our genetic explantation for the results.

Use the Hardy-Weinberg principle to calculate allele frequencies in populations for traits with dominant and recessive alleles

It predicts how gene frequencies will be inherited from generation to generation given a specific set of assumptions: the population is very large (this eliminates sampling error), the mating within the population is random, there is no selective advantage for any genotype, there is no mutation, migration or genetic drift. The equation is: p2+2pq+q2 = 1. Where 'p' and 'q' represent the frequencies of alleles. p added to q always equals one (100%). Consider the MN blood group. Humans inherit either the M or the N antigen which is determined by two different alleles at the same gene locus. If we let the frequency of allele M=p and the frequency of the other allele N=q, then the next generation's genotypes will occur as follows: •Frequency of MM genotype = p2 •Frequency of MN genotype = 2pq •Frequency of NN genotype = q2 We can take a sample of the population and count the number of people with each genotype. For example, a sample of 5000 has; 1460 individuals of type MM, that is 1460/5000 or 29.2%, 2550 of type MN, that is 2550/5000 or 51% and 990 of type NN, that is 990/5000 or 19.8%. If we apply the Hardy-Weinberg equation (p2 + 2pq + q2 = 1) we can calculate the allele frequencies as: Frequency of M = p2 + 0.5 (2pq) = 0.292 + (0.5 x 0.51) = 0.547 Frequency of N = q = 1 - p = 1 - 0.547 = 0.453 We can now calculate our expected genotype frequencies: •MM = p2 = 0.5472 = 0.299, or 1496 individuals in the sample •MN = 2pq = 2 x 0.547 x 0.453 = 0.496, or 2478 individuals •NN = q2 = 0.4532 = 0.205, or 1026 individuals

Explain the terms linkage and crossing-over

Linkage - genes for different characteristics that are present at different loci on the same chromosome are linked. Crossing-Over - where non-sister chromatids exchange alleles during prophase I of meiosis

Describe the behaviour of chromosomes during meiosis, and the associated behaviour of the nuclear envelope, cell membrane and centrioles.

Meiosis I -- Prophase I (1. the chromatin condenses and supercoils, 2. the chromosomes come together in their homologous pairs to form a bivalent. Each member of the pair has the same genes at the same loci. Each pair consists of one maternal and one paternal chromosome 3. the non sister chromatids wrap around each other and attach at points called chiasmata, 4. they may cross over and swap sections of chromatids with each other, 5. the nucleolus disappears and the nuclear envelope breaks down, 6. a spindle forms, made of protein microtubules). Prophase I may last for days, months or even years, depending on the species and on the type of gamete (male or female). Metaphase I (1. bivalents line up across the equator of the spindle, attached to spindle fibres at the centromeres, 2. the bivalents are arranged randomly (random assortment) with each member of the homologous pair facing opposite poles) Anaphase I (1. the homologous chromosomes in each bivalent are pulled by the spindle fibres to opposite poles, 2. the centromeres do not divide, 3. the chiasmata separate and the lengths of chromatid that have been crossed over remain with the chromatid to which they have become newly attached) Telophase I (1. in most animal cells two new nuclear envelopes form- one around each set of chromosomes at each pole and the cell divides by cytokenesis. There is a brief interphase and the chromosomes uncoil, 2. in most plant cells the cell goes straight from Anaphase I to Meiosis II. Meiosis II -- this occurs at right angles to Meiosis I Prophase II (1. If a nuclear envelope has reformed, it breaks down again, 2. the nucleolus disappears, chromosomes condense and spindles form) Metaphase II (1. the chromosomes arrange themselves on the equator of the spindle. They are attached to spindle fibres at the centromeres, 2. the chromatids of each chromosome are randomly assorted) Anaphase II (1. the centromeres divide and the chromatids are pulled to opposite poles by the spindle fibres. The chromatids randomly segregate) Telophase II (1. nuclear envelopes reform around the haploid daughter nuclei, 2. in animals, the two cells now divide to give four daughter cells, 3. in plants, a tetrad of four haploid cells if formed)

Outline how the polymerase chain reaction (PCR) can be used to make multiple copies of DNA fragments;

PCR (polymerase chain reaction) is basically artificial DNA replication, carried out on tiny amounts of DNA to replicate lots of copies of the DNA sample. This is used by forensic science to create enough of a DNA sample for genetic profiling. The sequencing reaction relies of the fact that DNA is made up of antiparallel backbone strands and is made up of strands that have a 5' end and a 3' end, grows only from the 3' end and that base pairs pair up according to the complementary base pairing rules. PCR is not identical to natural DNA replication. Can replicate short sequences of DNA, not entire chromosomes.The addition of primer molecules is required in order for the process to start. Heating and cooling is used to separate and bind strands.PCR is a cyclic reaction.The DNA sample is mixed with a supply of DNA nucleotides and the enzyme DNA polymerase.The mixture is heated to 95C. This breaks the hydrogen bonds holding the complementary strands together making them single stranded. Short lengths of single-stranded DNA are added. These are called primers (around 10-20 bases long. They are needed in sequencing reactions and polymerase chain reactions to bind to a section of DNA because the DNA polymerase enzymes cannot bind directly to single-stranded DNA fragments. The temperature is then reduced to around 55C allowing the primers to bind and form small sections of double-stranded DNA at either end of the sample. The DNA polymerase can bind to these double-stranded sections. Temperature is raised to 72C as to extend the double stranded section by adding free nucleotides to the unwound DNA. The whole process can be repeated as many times so the amount of DNA increases exponentially. DNA polymerase in the PCR is described as thermophillic because it is not denatured by extreme temperatures.The enzyme is derived from a thermophillic bacterium which grows in hot springs at a temperature of 90C.

Explain how meiosis and fertilisation can lead to variation through the independent assortment of alleles

Meiosis: crossing over during prophase I 'shuffles' alleles - the homologous chromosomes par and come together to form bivalents. On average between 2-3 cross-over events occur on each pair of human chromosomes. Non-sister chromatids wrap around each other very tightly and attach at chiasmata. The chromosomes may break at these points. The broken ends of the chromatids rejoin to the ends of non-sister chromatids in the same bivalent. This leads to similar sections of non-sister chromatids being swapped over. These sections contain the same genes but often different alleles. This is crossing over. It produces new combinations of alleles on the chromatids (which will eventually become chromosomes in the daughter cells). The chiasmata will remain in place during metaphase and they hold the maternal (the set of chromosomes in an individual's cells which have come from the egg) and the paternal (the set of chromosomes in an individual's cells which have come from the sperm) homologues together on the spindle, facing the way they will migrate. Holding the homologous pairs on the spindle equator ensures that when segregation occurs at anaphase I, one member of each pair goes to each pole. Reassortment of chromosomes - is the consequence of the random distribution of maternal and paternal chromosomes on the spindle equator at metaphae I, and the subsequent segregation into two daughter nuclei at anaphase I. Each gamete acquires a different mixture of maternal and paternal chromosomes. From this process one individual could produce 2n genetically different gametes where n = the haploid number of chromosomes. However the actual number is much larger due to crossing over and subsequent genetic recombination during prophase I. Reassortment of chromatids - is the result of the random distribution on the spindle equator of the sister chromatids at metaphase II. Because of crossing over, the sister chromatids are no longer genetically identical. How they align at metaphase II determines how they segregate at anaphase II. Fertilisation - in humans only one ovum (actually it is a seconday oocyte and has not completed the second meiotic division) is usually released from an ovary at a time. There are about 300 million spermatozoa, all genetically different, and any one of them can fertilise the secondary oocyte. Whichever one fertilises the ovum, genetic material from two unrelated individuals is combined to make the zygote.

Describe how artificial selection has been used to produce the modern dairy cow and to produce bread wheat (Triticum aestivum)

Modern dairy cow: the original wild cattle which were first domesticated were thought to have looked like the modern Chillingham White cattle. By repeatedly selecting cows with high milk yields and allowing them to breed over many generations, humans have artificially selected improved breeds with higher milk production (though we also select animals for docility, meat production and to survive in the environment). Today, breeders still practice artificial selection: 1. each cow's milk yield is measured and recorded, 2. the progeny of bulls is tested to find which bulls have produced daughters with high milk yields. 3. only a few good quality bulls need to be kept as the semen from one bull can be collected and used to artificially inseminate many cows. 4. some elite cows are given hormones so they produce many eggs. 5. the eggs are fertilised in vitro and the embryos and implanted into surrogate mothers. 6. these embryos could also be cloned and divided into many more identical embryos. In this way a few elite cows can produce more offspring than they would naturally. How artificial selection has produced bread wheat: the genus Triticum includes wild and domestic species of wheat. The genus Aegilops, or wild goat grass, has contributed its genome to the modern bread wheat. Most wild species of wheat are diploid with 14 chromosomes; n = 7. Grasses, like many other domesticated plants, are able to undergo polyploidy - their nuclei can contain more chromosomes in the nucleus of each cell. Because the nuclei need to be larger to contain the extra chromosomes their cells are also bigger. Genetic analysis of modern species of domesticated wheat has shown that it is a hybrid containing three distinct genomes, AuAuBBDD. The genome AuAu has come from a wild wheat species such as T.uratu. The genome BB has come from wild emmer wheat, T. turgidum (a tetraploid, 4n, a species thought to be a hybrid of T. uratu and a wild goat grass like Ae. speltoides). The genome DD has come from a wild goat grass such as Ae. tauschii or Ae. squarrosa. Using the Linnaean system of classification, all wheats that can interbreed are classified as being in the same species. However a more recent genetic classification has been used. Both classifications are valid. Wild einkorn AuAu (2n = 14) --> Einkorn AuAu (2n =14). This resulted from domestication and artificial selection, which altered the phenotype but not the chromosome number. Einkorn AuAu (2n =14) x wild grass BB (2n = 14) --> sterile hybrid P, AuB. A mutation then doubled the chromosome number, producing Emmer wheat AuAuBB (4n = 28). This then bred with Goat grass DD (2n =14) ---> producing a sterile hybrid Q AuBD. Another mutation then occurred that doubled the chromosome number again --> common wheat AuAuBBDD (6n = 42). Wheat is a very important crop. Breeders continue to carry out selection programmes to produce improved varieties. Characteristics they focus on include: resistance to fungal infections, high protein content, straw stiffness, resistance to lodging (stems bending over in wind and rain) and increased yield. Each year the wheat varieties are classified according to their suitability for making bread or biscuits or for the use as animal feed.

Compare and contrast natural selection and artificial selection

Natural selection is a mechanism for evolution. Those organisms that are best adapted to their environments are more likely to survive to reproductive age and pass on thier favourable characteristics (via alleles) to their offspring. The environment (nature) is doing the selecting. Artifical selection (selective breeding) is the process by which humans breed other animals and plants for particular traits. In artifical selection: humans select the organisms with useful characteristics, humans allow those with useful characteristics to breed and prevent those organisms without the useful characteristics from breeding. Thus humans have a significant effect upon the evolution of these populations or species.

Explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product;

Once a gene has been identified to be placed in another organism, it can be cut from DNA using a restriction enzyme and then placed in a vector, e.g. bacterial plasmid. A plasmid is a small circular piece of DNA, and are found in many types of bacteria that are seperate from the main bacterial chromosome. Plasmids often carry genes that code for resistance to antibiotic chemicals. If plasmids are cut with the same restriction enzyme as that used to isolate the gene, then complimentary sticky ends will be formed. Mixing quantities of plasmid and gene in the precense of ligase enzyme mean that some plasmids will combine with the gene, which then becomes sealed into the plasmid to form a recombinant plasmid. Many cut plamids will, in the precense of DNA ligase, simply reseal to form the orignial plasmid. Large quantities of plasmid are mixed with bacterial cells, some of which will take up the recombinant plasmid. The addition of calcium salts and 'heat shock', where the temperature of the culture is lowered to around freezing, then quickly raised to 40C, increase the rate at which plasmids are taken up by bacterial cells. Even so the process is very inefficent. Less than a quarter of 1% bacterial cells take up a plasmid. Those that do are known as transformed bacteria. This transformation results in bacteria containing new DNA. By definition the bacteria are thus transgenic.

Describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes;

Recombinant DNA techniques often involve the cutting and sticking together of DNA strands. e.g. a useful gene may need to be cut out of the chromosome on which it has been found, and then sealed into a plasmid vector. Enzymes known as restriction enzymes (restriction endonucleases) are used to cut through DNA at specific points. A particular restriction enzyme will cut DNA wherever a specific base sequence occurs and only where that sequence occurs. This sequence is called the restriction site, and is usually less than 10 base pairs long. In most of the restriction enzymes in use, the enzyme catalyses a hydrolysis reaction which breaks the phosphate-sugar backbones of the DNA double helix in different places. This gives a 'staggered cut' which leaves some exposed bases known as a sticky end.

Outline the differences between reproductive and non reproductive cloning.

Reproductive cloning is done to generate new organisms. One of the most significant potential developments in cloning is the possibility of using cloned cells to generate new cells, tissues and organs to replace those damaged by diseases or accidents. The are numerous advantages of using cloned cells: 1. being genetically identical to the individual's own cells means they will not be 'rejected' because the immune system will not recognise them as foreign. 2. cloning and cell culture techniques could mean an end to the current problems of waiting for donor organs to become available for transplant. 3. cloned cells can be used to generate any cell type because they are totipotent. 4. damage caused by some diseases and accidents cannot currently be repaired by transplantations or other treatments. 5. using cloned cells is likely to be less dangerous than a major operation such as a heart transplant. There are many possibilities for therapeutic cloning: the regeneration of heart muscle cells following a heart attack, the repair of nervous tissue destroyed by disease such as multiple sclerosis or repairing the spinal cord of those paralysed by an accident that resulted in a broken back or neck. However, there are ethical objections to the use of human embryonic material and some scientific concerns about a lack of understanding of how cloned cells will behave over time.

Explain the differences between somatic cell gene therapy and germ line cell gene therapy;

Somatic gene cell therapy - the functioning allele of the gene is introduced into target cells - therefore techniques to get the gene to the target location are needed or specific cells must be removed, treated and then replaced (ex vivo therapy). Introduction into somatic cells means that any treatment is short-lived and has to be repeated regularly. The specalised cells containing the gene will not divide to pass on the allele. There are difficulties in getting the allele into the genome in a functioning state. Genetically modified viruses have been tried but the host becomes immune to them so cells will not accept the virus vector on second and subsequent treatments. Liposomes (small spheres of lipid bilayer containing a functioning allele) are used but these may be inefficient. Genetic manipulations are restricted to the actual patient. Germline cell gene therapy - the functioning allele of the gene is introduced into germline cells - delivery techniques are more straightforward. Introduction into germline cells means that all cells derived from these germline cells will contain a copy of the functioning allele, the offspring may also contain the allele. Although more straightforward, it is considered unethical to engineer human embryos. It is not possible to know whether the allele has been successfully introduced without any unintentional changes to it, which may cause damage to the embryo. Genetic manipulations could be passed on to the patient's children and is currently banned.

Explain the significance of the various concepts of the species, with reference to the biological species concept and the phylogenetic (cladistic/evolutionary) species concept

The biological species concept is a group of similar organisms that can interbreed and produce fertile offspring and is reproductively isolated from other such groups. The concept is problematic when biologists want to classify living organisms that do not reproduce sexually. Also some members of the same species may look very different from each other. In some species males and females look very different. Some isolated populations may appear to be very different from each other.The phylogenetic species concept is a group of organisms that have similar morphology (shape), physiology (biochemistry), embryology (stages of development) and behaviour, and occupy the same ecological niche. Closely related organisms have similar molecular sturctures for DNA, RNA and proteins. With improved methods of DNA sequencing, biologists have used systematic molecular analysis to compare particular base sequences (haplotypes) on chromosomes of specfic organisms. Analysis of the base pair sequences is carried out and the differences, cuased by base substitutions, are expressed as % divergence. Any group of organisms with haplotypes that are more similar to each other than to those in an other group is called a clade - the use of molecular analysis is a cladistic approach to classification. It assumes that the classification of living organisms corresponds to their phylogenetic descent and that all valid taxa (groups) must be monophyletic (a group that includes an ancestral organism and all of its descendent species). Cladisitics is the hierachical classification of species based on their evolutionary ancestry. It is different from the taxonomic classification system as: 1) it focuses of phylogenetic relationships (evolution) rather than on similarities between species. 2) it places great importance on molecular analysis 3) it uses DNA and RNA sequencing 4) makes no distinction between extinct and extant species and both may be included in cladograms. 5) uses computer programming and information of nucleic acids to generate dendograms. 6) does not use the groups kingdom, phyllum or class as it regards the evolutionary tree as very complex. However the Linnean system aslo reflects the phylogenies (evolutionary relationships) between the different species of organisms. Unlike cladistics, it shows both monophyletic and paraphletic groups (includes the most recent ancestor but not all of its descendents. It is a monophyletic group with one or more clades excluded) as taxa. The cladisitc approach has often confirmed the Linnean system of classification but has sometimes led to organisms being reclassified. It has helped biologists to understand the evolutionary relationships between species.

Explain, how environmental factors can act as stabilising or evolutionary forces of natural selection

The environmental factors that limit the growth of a population may include space (for plants to grow, or for animals to defend their feeding territory and to rear young), availability of food, light, minerals and water, predation or infection by pathogens. These factors offer environmental resistance. Some are abiotic (caused by non-living components of the environment) and some are biotic (caused by other living organisms). Over a period of time, the population size will fluctuate around a mean level. If the environmental resistance is great enough, the population size will decrease. This reduces competition and the population will grow, etc. Variation in a population determines which individuals in the population will survive. e.g. some members will be better adapted and able to out-compete other members. They will then reach reproductive age and produce young with the alleles for the favourable characterisitcs. With prey animals, the predator is the selection pressure. e.g. a rabbit that has an agouti coat can hide from a predator and reach reproductive age, increasing the chances of the alleles for agouti coat being passed on to the young. It reduces the chances of alleles for a white or black coat being passed on to the next generation. Here, natural selection keeps things the way they are. Thisis stabilising selection. If a new phenotype does arise it is unlikely to confer an advantage and will not be selected. However, if the environment changes, the selection pressure changes - e.g. should the climate change and the ground be covered with snow, then those rabbits with white fur would now have a selective advantage. Those rabbits would then be more likely to survive and breed to pass on the alleles for white fur. The frequency of these alleles in the gene pool would change, as would the frequency of individuals with white coats in the population. This is directional selection and leads to evolutionary change. It is an evolutionary force of natural selection.

Outline how gene sequencing allows for genome-wide comparisons between individuals and between species

The identification of genes for proteins found in all or many living organisms gives clues to the relative importance of such genes to life. Comparing the DNA of different species shows evolutionary relationships. Modelling the effects of changing DNA can be carried out. Comparing genomes from pathogenic and similar but and non pathogenic organisms can be used to identify the genes or base-pair sequences that are more important in causing disease, so more effective drugs can be developed. The DNA of individuals can be analysed to reveal the presence of alleles associated with particular diseases, such as heart diseases or cancer. Helps us to understand the role of genetic information in a range of areas including health, behaviour and evolutionary relationships between organisms.

Explain the importance of asepsis in the manipulation of microorganisms

The nutrient medium in which the microorganisms grow could also support the growth of many unwanted microorganisms. Any unwanted microorganism is called a contaminant. Unwanted microorganisms: compete with the culture microorganisms for nutrients and space, reduce the yield of useful products from the culture microorganisms, may cause spoilage of the product, may produce toxic chemicals, may destroy the culture microorganisms and their products. In processes where foods or medicinal chemicals are being produced, contamination means that all products must be considered unsafe and so must be discarded. Aseptic technique refers to any measure at any point in a biological process, to ensure asepsis (that unwanted microorganisms do not contaminate the culture that is being grown or the products that are extracted). Aseptic techniques and measures at laboratory and starter culture level: all apparatus for carrying/moving microorganisms is sterilised before and after use, e.g. heating in a flame until glowing or by UV light. Some equipment is steam sterilised at 121C for 15 mins in an autoclave. Work can be carried out in a fume cupboard or laminar flow cabinet where air circulation carries any airborne contaminants away from the bench space. Cultures of microorganisms are kept closed where possible and away from the bench surface when open and in use. Aseptic techniques and measures at large-scale culture level: washing, disinfecting and steam-cleaning the fermenter and associated pipes when not in use removes excess nutrient medium and kills microorganisms. Fermenter surfaces made of polished stainless steel prevent microbes and medium sticking to the surfaces. Sterilsing all nutrient media before adding to the fermenter prevents the introduction of contaminants. Fine filters on inlet and outlet pipes avoid microorganisms entering or leaving the fermentation vessel.

Explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product

The required gene is obtained. Use a restriction enzyme (endonuclease) to cut the gene coding for the protein required or to fragment (digest) the DNA and use a gene probe, or obtain mRNA and then use reverse transcriptase to make cDNA, or sequence the protein and work out the base code, then make this DNA sequence. This produces sticky ends. A copy of the gene is placed in a vector, e.g. plasmid. Cut open the plasmid using the same restriction enzyme that was used to cut out the DNA. Allow the base pairing of sticky ends (annealing). Then join the sugar-phosphate backbones using DNA ligase. The result is a recombinant. Mix the plasmid with bacteria and use Ca2+ ions or lower the temperature to freezing before quickly raising to 40C ('heat shock'). The plasmid is now transformed. The vector carries the gene to the recipent cell. The recipent expresses the gene through protein synthesis.

Explain the meaning of the term genetic code

The sequence of nucleotide bases on a gene provides a code. The genetic code has a number of characteristics: it is a triplet code (three nucleotide bases codes for an amino acid), it is a degenerate code (all amino acids except methionine have more than one code), some codes don't correspond to an amino acid but indicate 'stop' - end of the polypeptide chain, and the genetic code is widespread but not universal.

Discuss the ethical concerns raised by the genetic manipulation of animals (including humans), plants and microorganisms

The virus vector may cause disease if the genes are introduced into a human, the procedure may be invasive, dangerous, painful or stressful to the human or other animal, it is temporary and needs to be repeated. It has limited success with somatic gene therapy. The immune system could cause rejection and problems with genetically engineered animal organ transplants. There is also a 'yuk' factor with animal organ trnasplants, an instinctive revulsion. Some claim that it is 'speciest' - it implies that humans are superior to other species, when morally we should grant all species equal rights. There are animal testing concerns. The consumption of genetically modified foods may have unforseen effects on humans, the technology increases the risk of biological warfare, reduces the genetic diversity of plants, e.g. golden rice. Clones may all suffer from one disease or one envrionmental change. There is the possibility of hybridisation with wild plants and the spread of genes to wild populations, with the potential to disturb the balance of the ecosystem and is a threat to biodiversity. Possible danger when interferring with an organism's genome, e.g. may have unexpected effects like releasing toxic byproducts. The seeds are expensive and need to be bought each year. The plants, e.g. rice may not grow in all areas where it is needed.

Describe the way in which a nucleotide sequence codes for the amino acid sequence in a polypeptide

Transcription is the first stage of protein synthesis. It is the creation of a single-stranded mRNA copy of the DNA coding strand. A messenger RNA molecule is made. For this, one strand (the template strand) of the length of DNA is used as a template. There are free DNA nucleotides in the nucleoplasm and free RNA molecules in the nucleolus. The nucleotides are activated - they have two extra phosphoryl groups attached. There are four different activated RNA nucleotides: ATP, GTP, CTP and UTP. A gene to be transcribed unwinds and unzips. To do this the length of DNA that makes up the gene dips into the nucleolus. Hydrogen bonds between the complimentary bases break. Activated RNA nucleotides bind, with hydrogen bonds, to their exposed complimentary bases. A binds with U, G with C and A with T on the template strand. This is catalysed by the enzyme RNA polymerase. The two extra phosphoryl groups are released. This releases energy for bonding adjacent nucleotides. The mRNA produced is complimentary to the nucleotide base sequence on the template strand of the DNA and is therefore a copy of the base sequence on the coding strand of the length of DNA. The mRNA is released from the DNA and passes out of the nucleus, through a pore in the nuclear envelope, to a ribosome.

Describe how the sequence of nucleotides within a gene is used to construct a polypeptide, including the roles of messenger RNA, transfer RNA and ribosomes

Translation is the assembly of polypeptides at ribosomes. It is the second stage of protein synthesis. They are assembled in the sequence dictated by the sequence of codons (triplets of nucleotide bases) on the mRNA. The genetic code, copied from DNA into mRNA, is now translated into a sequence of amino acids. This chain of amino acids is called a polypeptide. It happens at ribosomes, which may be free in the cytoplasm but many are bound to the rough endoplasmic reticulum. Ribosomes are assembled in the nucleolus of eukaryotic cells, from ribosomal RNA (rRNA) and protein. Each is made of two subunits and there is a groove into which the length of mRNA, with the code for the sequence of amino acids, can fit. The ribosome can then move along the mRNA, which can slide through the ribosomal groove, reading the code and assembling the amino acids in the correct order to make a functioning protein. The sequence of amino acids in a protein is critical because: it forms the primary structure of a protein, which determines the tertiary structure - how the protein folds up into its 3D shape and is held in that 3D shape by hydrogen or ionic bonds and hydrophobic interactions forming between the R groups of amino acids. The shape is what allows the protein to function, and is dependent on the primary structure which is dependent on the genetic code. If the tertiary structure is altered, the protein can no longer function so effectively, e.g. active site of an enzyme is changed, so substrate ions will no longer fit or if a chloride ion channel protein in cell surface has a different shape it won't allow ions to pass through it. Another form of RNA, tRNA, is made in the nucleus and passes into the cytoplasm. These are lengths of RNA that fold into hairpin shapes and have 3 exposed bases at one end where a particular amino acid can bind. At the other end of the molecule are 3 unpaired nucleotide bases, known as an anticodon. Each anticodon can bind temporarily with its complimentary codon. A molecule of mRNA binds to a ribosome. Two codons are attached to the small subunit of the ribosome and exposed to the large subunit. The first exposed mRNA codon is always AUG. Using ATP energy and an enzyme, a tRNA molecule with the amino acid methionine and the anticodon UAC forms hydrogen bonds with this codon. A seconds tRNA molecule, bearing a different amino acid, binds to the second exposed condon with its complementary anticodon. A peptide bonds forms between the two adjacent amino acids. This is catalysed by an enzymein the small ribosomal sub unit. The ribosome now moves along the mRNA reading the next codon. A third tRNA brings another amino acid and a peptide bonds forms between it and the dipeptide. The first tRNA leaves and is able to collect and bring another of its amino acids.The polypeptide chain grows until a stop codon is reached, for which there are no corresponding tRNAs and the polypeptide chain is complete

Explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase;

When sepearate fragments of DNA need to be stuck together, an enzyme known as DNA ligase is used to catalyse a condensation reaction which joins the phosphate-sugar backbones of the DNA double helix together. This enzyme is the same as that used in natural DNA replication to seal DNA nucleotides together to form new DNA strands. In order to join DNA fragments from different sources both need to have originally been cut with the same restriction enzyme. This means that the sticky ends are complimentary and allows the bases to pair up and hydrogen bond together. DNA ligase can then seal the backbone.

Explain why variation is essential in selection

When the environment changes, those individuals that are well adapted will survive and reproduce, passing on their advnatageous alleles to their offspring, thus allowing the species to continue. This is the basis for evolution by natural selection.

Outline how animals can be genetically engineered for xenotransplantation

Xenotransplantation is the transplantation of cell tissues or organs between animals of different species whereas allotransplantation refers to transplantation between animals of the same species. Pigs have been engineered to lack the enzyme a-1-3 transferase, which is a key trigger for graft rejection of organs in humans. The human nucleotidase enzyme has been grafted into pig cells in culture. It reduces the number of immune cell activities involved in xenotransplant rejection.

Outline the process involved in the genetic engineering of bacteria to produce human insulin;

mRNA from human insulin is extracted from pancreas cells. Reverse transcriptase uses mRNA as a template to make single stranded cDNA, and this is made double stranded by DNA polymerase. A single strand of nucleotides (GGG) is added to each end of the DNA to make sticky ends. Plasmids are cut open with a restriction enzyme. Cut plamsids have a single sequence of nucleotides (CCC) to each make sticky ends. Plasmids and the insulin gene are mixed so that the sticky ends form base pairs. DNA ligase links sugar-phosphate backbones of plasmid and insulin gene. Plasmids are mixed with bacteria in the presence of calcium ions. Bacteria take up the plasmids and multiply to form a clone. The genetically engineered bacteria transcribe and translate the human gene to make human insulin. The bacteria then produce insulin on a large scale which can be harvested for use.