A-Level Biology: Gene Expression and DNA Technology Notes PDF

2 A-Level Biology Gene Expression and DNA Technology  Gene Mutations Gene mutations are changes in the sequence of nucleotide bases in DNA. Gene mutations occur spontaneously. Gene mutations may occur during DNA replication. The mu...

2 A-Level Biology Gene Expression and DNA Technology  Gene Mutations Gene mutations are changes in the sequence of nucleotide bases in DNA. Gene mutations occur spontaneously. Gene mutations may occur during DNA replication. The mutation rate is increased by mutagenic agents e.g. X-rays, benzene Mutations can result in a different amino acid sequence in the polypeptide that the genes code for. This is due to the altered DNA base sequence coding for a different sequence of amino acids. Types of Gene mutations Some gene mutations change only one triplet code. Due to the degenerate nature of the genetic code, not all mutations result in a change to the amino acid coded for. Some gene mutations change the nature of all base triplets downstream from the mutation, i.e. result in a frame shift (see below). There are several types of mutation: Substitution Substitution is the replacement of one or more bases by one or more different bases. The substitution of a single base may result in: a new triplet coding for a different amino acid in the polypeptide chain which may result in a non- functional protein being formed. the same amino acid may be coded for due to the degeneracy of the DNA code - e.g. CGA and CGG both code for the same amino acid, alanine - so that the polypeptide remains unchanged. Deletion Deletion is the removal of one or more bases. This results in: a frame shift, which is the alteration in all the base triplets from the point of deletion. the sequence of amino acids is altered from the point of deletion, and the protein formed will be non-functional. 2 Addition Addition is the adding of one or more bases e.g. ATCCGT becomes ATCCTCGT. This also results in a frame shift. Duplication Duplication is where one or more bases is repeated, e.g., CCGTAA becomes CCGCGTAA. This also results in a frame shift. Inversion Inversion is where a sequence of bases is reversed e.g., TAGCAGCT becomes TCGACGAT. Translocation Translocation is where a sequence of bases is moved from one location in the DNA molecule to another part of the genome. The effects of this depend upon where the sequence came from and where it is moved to. Mutations and the Development of Tumours The rate of cell division is controlled by two main sets of genes: Proto-oncogenes – these genes code for proteins that stimulate cell division Tumour suppressor genes – these genes code for proteins that slow cell division Mutations of these genes can lead to rapid, uncontrolled cell division (by mitosis) resulting in the development of a tumour. This can occur in two ways. A mutated proto-oncogene, called an oncogene stimulates cells to divide too quickly. This results in rapid uncontrollable cell division. A mutation in a tumour suppressor gene. The tumour suppressor protein is not made, or is non- functional. This results in rapid uncontrollable cell division. 3 Gene Expression and Cancer Cancer is a group of diseases caused by alterations in the genes that regulate mitosis and the cell cycle. Tumours are masses of dividing cells. There are two types of tumours, benign and malignant. Malignant tumours are cancerous. Benign tumours Malignant tumours Grow slower than malignant tumours Grow faster than benign tumours Non-cancerous - cells do not spread Cancerous - cells can break off and spread (metastasize) to other tissues as the tumour to other parts of the body, as the tumour is is enclosed by fibrous tissue not enclosed. Cells often remain differentiated Cells often become undifferentiated (non- (specialised) specialised) Cell nucleus has a relatively normal Cell nucleus is larger and darker appearance 4  Stem Cells and Cell Specialisation Stem cells are undifferentiated cells that can divide by mitosis and differentiate into different types of cells. There are several types of stem cells. Totipotent stem cells Totipotent cells occur for a limited time in early mammalian embryos. Totipotent cells can differentiate into any type of cell Pluripotent stem cells Pluripotent cells are found in embryos (e.g. embryo stem cells and fetal cells) and develop from totipotent cells. Pluripotent cells can differentiate into almost any type of cell. Multipotent stem cells Multipotent cells are found in mature mammals. Multipotent cells can differentiate into a few, limited types of specialised cells e.g. multipotent stem cells in the bone marrow can produce any type of blood cell. Unipotent stem cells Unipotent cells are found in mature mammals. Unipotent cells can only differentiate into one type of cell e.g. cardiomyocyte stem cells can only differentiate into heart (cardio) muscle cells (myocytes). 5 Induced pluripotent stem cells (iPS cells) These are a type of pluripotent stem cell produced from unipotent stem cells. Appropriate transcription factors are used to make a unipotent stem cell pluripotent. Then transcription factors bring about expression of some genes, and inhibit other genes, so that these cells differentiate into a particular type of cell. These iPS cells can develop into a wide range of different types of tissue which could potentially be used to treat people with certain diseases. 6 Cell Differentiation by Regulation of Transcription An organism develops by mitosis from a single fertilised egg. Therefore, all the body cells of an organism are genetically identical. During development of an organism, some genes are transcribed and expressed, and other genes are not expressed. This is controlled by transcription factors found in the cytoplasm of cells. Transcription factors are proteins. Transcription factors move into the nucleus and attach to a promoter region close to the target gene or genes that it affects. Part of each transcription factor is complementary in shape to a particular sequence of nucleotides in a promoter sequence. This gives specificity. Transcription factors work by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase. The expression of different genes results in different proteins being coded for resulting in different specialised cells being produced - i.e. cell differentiation. The diagram above shows a transcription factor promoting the binding of RNA polymerase and thereby stimulating gene expression (via transcription). Other transcription factors can inhibit the binding of RNA polymerase and prevent gene expression. 7 Hormonal Regulation of Gene Expression Some hormones are able to enter their target cell. Once inside the cell, they function by stimulating the expression of a particular gene in the target cell. Oestrogen The diagram opposite shows the role of oestrogen in gene expression. The following events correspond with the numbers on the diagram: 1. Oestrogen is lipid soluble as it is a steroid hormone (derived from cholesterol). It can therefore easily diffuse across the cell membrane. 2. Oestrogen binds specifically to a receptor protein that is part of a transcription factor. 3. The transcription factor enters the nucleus. 4. The binding of oestrogen changes the shape of the transcription factor and allows it to bind specifically to the promoter sequence of a particular gene. 5. This allows RNA polymerase to attach to the gene and catalyse the transcription of the gene. 6. mRNA is transcribed from the gene. 7. This mRNA is translated into protein. Thus oestrogen increases the expression of particular genes. 8 Oestrogen and Breast cancer In some tissues, oestrogen increases the expression of genes associated with cell division. This means that high blood concentrations of oestrogen over a period of time can increase the risk of uncontrollable cell division therefore cancer, especially breast cancer. Tamoxifen is a drug that has been effective in treating some forms of breast cancer linked to high oestrogen concentrations. In the body, tamoxifen is converted to endoxifen, a molecule similar in structure to oestrogen. Endoxifen competes with oestrogen for binding to the oestrogen receptor, inhibiting the effect of oestrogen. 9 Regulation of Translation In eukaryotes and some prokaryotes, translation of the mRNA produced from target genes can be inhibited by RNA interference (RNAi). This often involves small interfering RNA or siRNA siRNA molecules are short, double-stranded sections of RNA, usually only 20-25 base pairs long. siRNA regulates gene expression by causing mRNA to be broken down after transcription, thereby preventing translation. The diagram shows how siRNA prevents translation. 1. Longer, double-stranded molecules of RNA are hydrolysed (by an enzyme) into shorter molecules. 2. RNA becomes single-stranded siRNA 3. siRNA binds to an enzyme that hydrolyses mRNA 4. The siRNA binds to a specific molecule of mRNA by complementary base-pairing. Thus siRNA ‘guides’ the hydrolytic enzyme to a target molecule of mRNA. 5. The enzyme hydrolyses the mRNA molecule. This prevents the translation of mRNA into protein. 10 11  Epigenetics and Gene Expression in Eukaryotes Epigenetics refers to changes in gene function without changes in the base sequence of DNA. These changes in gene function may be caused by aspects of environment - e.g. stress, diet, exposure to toxins etc. Epigenetic can either increase or decrease gene expression. Epigenetic changes include: increased methylation of DNA or decreased acetylation of associated histones. Increased methylation of DNA This occurs when a methyl group (CH3) attaches to the DNA sequence of a gene. The methyl group always attaches to a cytosine (C) base when it is next to guanine (G), (known as a CpG site, the p representing the phosphate between the 2 bases) Increased methylation inhibits transcription by preventing the binding of transcription factors to the promoter sequence so that the gene is not expressed. Decreased acetylation of Histones In eukaryotes, the DNA is wrapped around proteins called histones to form chromatin. These histones can be epigenetically modified by the addition or removal of acetyl (COCH3) groups. When histones are more acetylated (acetyl groups added), the chromatin is less condensed (more loosely packed). Transcription of genes is more likely as the genes (DNA) are now more accessible to transcription factors. When histones are less acetylated (removal of acetyl groups) the chromatin is more condensed. Transcription is inhibited as the genes are not accessible to transcription factors. 12  Epigenetics and Disease Epigenetic changes can lead to disease by causing abnormal activation or inhibition of genes. Epigenetic changes can be reversed. New drugs are being developed to counteract the epigenetic changes which may cause disease. These drugs are often designed to target specific cells, e.g. cancer cells, in which the epigenetic changes have taken place. Abnormal Methylation of Cancer-Related Genes Abnormal changes in the level of methylation can lead to development of cancers. Cancers can develop from: The hypermethylation (too much methylation) of tumour suppressor genes so that these genes are not transcribed. The proteins that slow down cell division are not produced leading to uncontrolled cell division and the development of a tumour. The hypomethylation (too little methylation) of proto-oncogenes so these genes are continually transcribed. This increases production of proteins involved in stimulating cell division. This leads to rapid, uncontrolled cell division and the development of a tumour 13  DNA Technology ‘Tools’ Gene technologies allow the study and alteration of gene function, allowing a better understanding of organism function and the design of new industrial and medical processes. DNA Technology typically uses one or more of the following ‘tools’: Restriction Enzymes Electrophoresis PCR DNA Primers & DNA Probes Restriction Enzymes, or Restriction Endonucleases These enzymes hydrolyse the phosphodiester bonds in DNA or RNA, producing smaller fragments. They hydrolyse the phosphodiester bonds in both strands of DNA. Restriction enzymes hydrolyse the DNA or RNA at specific base sequences, known as recognition sequences or recognition sites. Some restriction enzymes hydrolyse the DNA at different locations, producing ‘sticky ends’. Some hydrolyse at the same position in both strands, producing ‘blunt ends’: Sticky ends enable the DNA to be joined or spliced onto a different piece of DNA more easily because complementary base pairing can occur between the sticky ends. 14 Gel Electrophoresis ‘Tools’ DNA or RNA fragments can be separated by gel electrophoresis. This procedure is based on the principle that smaller DNA fragments will travel faster and therefore further through the gel when an electric charge is applied. Negatively charged DNA fragments move towards the positively charged terminal. The example below shows the separation of DNA fragments in three different samples. The three DNA samples are placed in separate wells at the top of the gel. The DNA fragments in each sample are separated according to size as smaller fragments moved further in the gel when an electric charge is applied. After electrophoresis, the DNA fragments are transferred to a nylon membrane, then radioactively labelled DNA probes are added. The nylon membrane is placed on X-ray or photographic film and the position of the radioactively labelled fragments is revealed as dark bands on the film. The appearance of radioactively labelled compounds on X-ray or photographic film is known as autoradiography. Alternatively, the DNA fragments can be identified using fluorescent labelled DNA probes. DNA ‘Ladders’ Often when separating DNA fragments a DNA ladder is also ‘run’ alongside the unknown DNA sample/s. A DNA ladder has DNA fragments of known sizes (base number) and can be used to calculate the size of the DNA fragments in the unknown sample/s. 15 The Polymerase Chain Reaction (PCR) ‘Tools’ This technique enables multiple copies of identical fragments of DNA or genes to be produced (or amplified) from a small sample. Stage 1 Reactants are mixed together and heated at 95 ºC for 5 minutes. This breaks hydrogen bonds in the DNA. Stage 2 The mixture is cooled to 50-60 ºC for 2 minutes. This allows the primers to join (anneal) to their specific complementary target sequence. ‘Free’ DNA nucleotides align to the DNA strands by complementary base-pairing. Stage 3 The temperature is increased to 72 ºC, which is the optimum for DNA polymerase. This enzyme joins the individual nucleotides of a strand together to form a new complementary strand. Many cycles of heating and cooling produce exponential numbers of DNA molecules. Each cycle of PCR doubles the number of DNA molecules. Therefore, the number of molecules produced is: 2n where n = number of cycles 16 DNA Primers ‘Tools’ DNA primers are short, single-stranded molecules of DNA. They provide a starting sequence for DNA polymerase. This is important because DNA polymerase cannot begin at a single-stranded starting point. They also help to prevent the original DNA strands from simply joining back together. DNA Probes DNA probes are short, single-stranded molecules of DNA that are radioactively or fluorescently labelled. They are used to identify or locate known sequences of DNA. Primers are used for starting; Probes are used for finding. 17 Recombinant DNA Technology ‘Processes’ Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another. Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism. A transgenic organism is an organism that has received transferred DNA. Obtaining the required fragment or gene. Fragments of DNA e.g. genes can be obtained by several methods, including: 1. Using Reverse Transcriptase Rather than obtaining the specific gene or fragment of DNA from an organism, mRNA which has been transcribed from the gene is removed from cells and used. The mRNA is used as a template to produce the required gene or required fragment of DNA. For example, to produce a gene for insulin production, mRNA complementary to the insulin gene is isolated from pancreatic cells. This mRNA is mixed with free DNA nucleotides and the enzyme reverse transcriptase. The free DNA nucleotides align next to their complementary bases on the mRNA template. Reverse transcriptase then joins the DNA nucleotides together to produce a fragment of DNA (gene) for insulin production. The DNA strand produced by this technique is known as complementary DNA (cDNA). Double-stranded DNA is produced from this cDNA using DNA nucleotides and the enzyme DNA polymerase. The absence of introns means these fragments can be transcribed by bacteria. 18 2. Using Restriction Endonucleases ‘Processes’ The required gene or fragment of DNA can be removed from the DNA of an organism by using restriction endonuclease enzymes. A gene or a fragment obtained in this way from a eukaryotic organism will contain introns. 3. Using a ‘gene machine’ This enables the production of fragments of DNA without needing pre-existing DNA or mRNA as a template. Often, the amino acid sequence of a protein is used as a template to determine the sequence of DNA nucleotides for a specific gene. This is an automated process where the required nucleotide sequence is programmed into the gene machine. The absence of introns means these fragments can be transcribed by bacteria. Important considerations for Gene Transfer: Introns If the source of a gene being transferred is eukaryotic, and the intended recipient of the fragment is prokaryotic, introns must not be present. Promoter and Terminator Regions These are sections of DNA which must be added to the gene or fragment of DNA for successful transcription of the transferred genes in the recipient cells. Promoter regions – initiate transcription of the gene by promoting the binding of RNA polymerase Terminator region –marks the end of a gene and triggers the release of the mRNA transcribed. Amplification of DNA Fragments of DNA can be amplified (increased in number by replication) Amplification can be achieved by two techniques In vivo – where the copies are made inside a living organism. In vitro – where the copies are made outside a living organism (usually by PCR). 19 Transferring the fragment or gene ‘Processes’ The fragment or gene obtained may be transferred into either bacteria or eukaryotic cells. Vectors Genes are transferred using vectors. In bacteria this is usually a plasmid. Viruses and liposomes (phospholipid sacs) can also be used to transfer foreign genes into cells. Bacteria are widely used to either: Produce a protein coded for by a transferred gene (e.g. human insulin) Clone genes or fragments. This is known as in vivo cloning. The rapid reproduction rate of bacteria enables a transferred gene to be quickly copied so that a large amount of gene product can be obtained. A plasmid is cut using the same restriction endonuclease used to cut the gene. The plasmid DNA and the ‘foreign’ DNA join by base-pairing as they have complementary sticky ends. The enzyme ligase is used to form the phosphodiester bonds. The plasmid with the foreign DNA is referred to as a recombinant plasmid. 20 These plasmid vectors are added to a culture of bacteria, some of which take up the ‘Processes’ recombinant plasmid by a process called transformation. Similar techniques are used to transfer genes into eukaryotic cells. The use of vectors is not guaranteed to work. There are two likely problems: 1. The cells may not take up the vector at all. 2. The cell may take up the vector, but the vector may not contain the gene. e.g. the plasmid may have joined back together without the ‘foreign’ gene/DNA being taken up. Scientists therefore need to check that the gene or fragment has been successfully transferred. This can be done by using marker genes. Marker Genes These genes enable successfully transformed bacteria or eukaryotic cells to be detected and isolated for subsequent culturing. One example of a marker gene is the GFP gene which codes for the production of a green fluorescent protein (GFP). The GFP gene is added to the gene being transferred. Successfully transformed bacteria or eukaryotic cells can then be identified as they fluoresce when viewed with UV light under a microscope. Note: A variety of other marker genes have been developed but you would be given information on these in an exam. You would then need to apply this information to answer the questions set. 21 Evaluating the use of Recombinant DNA Technology There is considerable debate on the risks of using genetically modified (GM) organisms. Some potential humanitarian benefits include: reducing famine and malnutrition by developing GM plants or animals which produce high yields and are resistant to disease producing vaccines and drugs treating genetic diseases by gene therapy (see later notes) Opposition comes from environmentalists and anti-globalisation activists (oppose growth of large multinational companies). They outline potential drawbacks including: possible transfer of foreign genes to non-target organisms, including humans it is an irreversible process with no certainty of economic benefits ethical considerations with regard to permanently altering the genome of animals long term ecological and evolutionary consequences are unknown large companies having a monopoly leading to lack of choice and forcing smaller companies out of business Note: Questions on evaluating genetic modified organisms will require you to use the information provided in the question as part of your evaluation. Answers relating to ‘playing God’ will not be credited. 22  Gene Therapy ‘Processes’ Gene therapy uses recombinant DNA technology for the treatment of genetic diseases. It involves the introduction of functional copies of an allele into an organism which possesses defective alleles of the same gene. Stages involved in this process include: Identifying the gene causing the disease Obtaining and cloning copies of the functional allele Transferring these functional alleles into the patient e.g. by the use of a vector Ensuring that the alleles reach their target cells and function normally. Cystic fibrosis is an inherited disorder that develops in individuals who are homozygous recessive. Carriers are heterozygous and are not affected by the disease. This gene codes for a chloride ion channel protein that controls the movement of chloride ions in and out of cells. Affected individuals secrete large amounts of abnormally, thick and sticky mucus by epithelial cells particularly in the lungs and pancreas. This can have severe consequences. Clinical trials using gene therapy as a treatment for cystic fibrosis have been partially successful in altering the epithelial cells in the lungs of affected individuals. DNA Sequencing Projects This involves techniques to determine the sequence of DNA nucleotide bases in an organism – the genome. Sequencing projects have read the genomes of a wide range of organisms, including humans. Determining the genome of simpler organisms allows the sequences of the proteins that derive from the genetic code (the proteome) of the organism to be determined. This may have many applications, including the identification of potential antigens for use in vaccine production. In more complex organisms the presence of non-coding DNA and of regulatory genes means that knowledge of the genome cannot easily be translated into the proteome. Sequencing methods are continuously updated and have become automated. 23  DNA Probes and Medical Diagnosis ‘Processes’ DNA probes and DNA hybridisation are used to screen individuals for specific alleles. Many human diseases result from mutated genes i.e. alleles. The base sequence of these alleles has been determined by DNA sequencing. PCR can be used to produce a large number of DNA probes specific to disease-causing alleles. Procedure for screening A labelled DNA probe is made that is complementary to the DNA sequence of the allele being investigated. Multiple copies of the labelled DNA probe are made using the Polymerase Chain Reaction. A sample of DNA is obtained from the person or organism being tested. 24  ‘Processes’ The use of labelled DNA probes and DNA hybridisation to locate specific alleles of genes enables patients to be screened for: heritable conditions e.g. sickle cell anaemia, cystic fibrosis, Huntington’s disease individual drug responses – people can respond differently to particular drugs due differences in their alleles. This can lead to personalised medicine – i.e. specific drugs being prescribed for certain individuals for particular diseases. health risks e.g. the BRCA1 gene increases the risk of developing breast cancer by between 50 to 85% Information concerning the presence of identified mutant alleles is used in genetic counselling to: help people understand the probability of them developing a disease. advise prospective parents who may be carriers of disease-causing alleles. to help decide the best course of drug treatment for genetic diseases. 25  Genetic Fingerprinting ‘Processes’ The genome of an organism contains many repetitive, non-coding sequences of nucleotide bases known as variable number tandem repeats (VNTRs). These occur in lots of places in the genome. With the exception of identical twins, the probability of any two individuals having the same number and length of these repetitive sequences is extremely low. However, the more related two organisms are, the more similar their VNTRs will be. Analysis of these VNTRs can therefore be used to determine relatedness between individuals, as well as to match the identity of a DNA sample (for e.g. at a crime scene) to an individual. The Procedure 1. PCR is used to amplify the sample. 2. The amplified DNA is then cut into fragments, using restriction endonucleases. The endonucleases used cut DNA at sites close to, but not within, the VTNRs. This therefore gives a large number of DNA fragments. Samples of DNA from different organisms give some fragments that are the same length, but some that are different. 3. These fragments are separated by gel electrophoresis. 4. The fragments are treated (using alkali) to form single strands. 5. The single strands are transferred, in the positions they have moved to, onto a nylon ‘membrane’. 26  ‘Processes’ 6. Radioactive DNA probes are then added. These are complementary to the repeated sequences, and allow the positions of the fragments to be identified. Lots of different probes are used, each of which binds with the VNTRs by DNA hybridisation. 7. The radioactive probes allow the position of the fragments to be identified when the ‘membrane’ is placed onto an X-ray film i.e. the genetic fingerprint is obtained. The position of the fragments is dependent on the number of nucleotides present. This will correspond to the number of repetitive sequences in each fragment. Fragments with a smaller number of nucleotides travel further. Two genetic fingerprints can be compared. If both fingerprints have a band at the same position on the gel it means that they have the same number of nucleotides and so the same number of repetitive sequences at that place. However, apart from identical twins the genetic fingerprints will not be the same. The use of genetic fingerprinting is important in: Forensic science – compare DNA samples (extracted from blood, hair, semen) from a crime scene with the DNA of suspects. Medical diagnosis – certain diseases involve unique patterns of several alleles and can identified more readily by genetic fingerprinting. Determining genetic relationships and determining genetic variability within a population (more closely related species or organisms have more similar VNTRs) Animal and plant breeding – ensuring genetic diversity is maintained by screening organisms to prevent inbreeding between closely related individuals. 27 Interpreting the results ‘Processes’ If used forensically, the pattern of bands produced from the sample collected at the crime scene will match the pattern produced when a suspect’s DNA is analysed. A DNA sample was obtained from the scene of a robbery – Sample H. Samples were also taken from three suspects – D, E & F They were also taken from the victims – G & J. Which of the suspects is guilty? Control samples are also run to ensure that the experiment is done correctly – B, K & L. Samples – A, C, I and M are DNA Ladders. These are used to judge the sizes of the DNA fragments Genetic fingerprinting can be used to determine how related two individuals are, and is therefore useful in classification and in paternity cases: In these cases, the child’s non-coding sequences (or bands) must have been inherited from one parent or the other. The mother presumably knows the child is hers. The child’s bands that do not correspond with the mother’s must have come from the father. 28