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Module 2 - Genetics - page 1 AQA(B) AS Module 2: Genes and Genetic Engineering Contents Specification DNA The Cell Cycle Genetic Engineering Nucleotides DNA Structure DNA Function Replication RNA Transcription The Genetic Code Translation Mutations DNA and Chromosomes The Cell Cycle and Mitosis Asexual Reproduction Sexual Reproduction Techniques Applications 2 4 6 7 8 10 11 13 14 16 19 23 25 30 34 49 These notes may be used freely by A level biology students and teachers, and they may be copied and edited. I would be interested to hear of any comments and corrections. Neil C Millar ([email protected]) Head of Biology, Heckmondwike Grammar School, High Street, Heckmondwike WF16 0AH HGS A-level notes NCM/6/07 Module 2 - Genetics - page 2 Module 2 Specification DNA Structure. DNA is a stable polynucleotide. The double-helix structure of the DNA molecule in terms of: the components of DNA nucleotides; the sugarphosphate backbone; specific base pairing and hydrogen bonding between polynucleotide strands (only simple diagrams of DNA structure are needed; structural formulae are not required). Explain how the structure of DNA is related to its functions. Replication. The semi-conservative mechanism of DNA replication, including the role of DNA polymerase. Transcription. The structure of RNA. The production of mRNA in transcription, and the role of RNA polymerase. Explain how the structure of RNA is related to its functions. Translation. The roles of ribosomes, mRNA and its codons, and tRNA and its anticodons in translation. Genetic Code. How DNA acts as a genetic code by controlling the sequence of amino acids in a polypeptide. Codons for amino acids are triplets of nucleotide bases. Candidates should be able to explain the relationship between genes, proteins and enzymes. Mutations New forms of alleles arise from changes (mutations) in existing alleles. • Gene mutation as the result of a change in the sequence of bases in DNA, to include addition, deletion and substitution. • A change in the sequence of bases in an individual gene may result in a change in the amino acid sequence in the polypeptide. • The resulting change in polypeptide structure may alter the way the protein functions. • As a result of mutation, enzymes may function less efficiently or not at all, causing a metabolic block to occur in a metabolic pathway. Mutations occur naturally at random. High-energy radiation, high-energy particles and some chemicals are mutagenic agents. Reproduction Genes and Chromosomes Genes are sections of DNA which contain coded information that determines the nature and development of organisms. A gene can exist in different forms called alleles, which are positioned, in HGS A-level notes the same relative position (locus) on homologous chromosomes. Mitosis Mitosis increases cell number in growth and tissue repair and in asexual reproduction. During mitosis DNA replicates in the parent cell, which divides to produce two new cells, each containing an exact copy of the DNA of the parent cell. Candidates should be able to name and explain the stages of mitosis and recognise each stage from diagrams and photographs. Asexual Reproduction and Cloning Genetically identical organisms (clones) can be produced by using vegetative propagation, and by the splitting of embryos. Given appropriate information, candidates should be able to explain the principles involved in: • producing crops by vegetative propagation • the cloning of animals by splitting apart the cells of developing embryos. Meiosis During meiosis, cells containing pairs of homologous chromosomes divide to produce gametes containing one chromosome from each homologous pair. In meiosis the number of chromosomes is reduced from the diploid number (2n) to the haploid number (n). (Details of meiosis not required.) Sexual Reproduction and Gametes Sexual reproduction involves gamete formation and fertilisation. In sexual reproduction DNA from one generation is passed to the next by gametes. When gametes fuse at fertilisation to form a zygote the diploid number is restored. This enables a constant chromosome number to be maintained from generation to generation. Differences between male and female gametes in terms of size, number produced and mobility. Sexual Life Cycles Candidates should be able to interpret life cycles of organisms in terms of mitosis, meiosis, fertilisation and chromosome number. Genetic Engineering In genetic engineering, genes are taken from one organism and inserted into another. • The process of DNA replication can be made to occur artificially and repeatedly in a laboratory process called the polymerase chain reaction (PCR). NCM/6/07 Module 2 - Genetics - page 3 • The use of PCR, radioactive labelling and electrophoresis to determine the sequence of nucleotides in DNA. • The use of restriction endonuclease enzymes to extract the relevant section of DNA. • The use of ligase enzyme to join this DNA into the DNA of another organism. • Plasmids are often used as vectors to incorporate selected genes into bacterial cells. • Genetic markers in plasmids, such as genes which confer antibiotic resistance, and replica plating may be used to detect the bacterial cells that contain genetically engineered plasmids. • Rapid reproduction of microorganisms enables a transferred gene to be cloned, producing many copies of the gene. Genetically Modified Microbes Microorganisms are widely used as recipient cells during gene transfer. Bacteria containing a transferred gene can be cultured on a large scale in industrial fermenters. Useful substances produced by using genetically engineered microorganisms include antibiotics, hormones and enzymes. (Details of manufacturing processes not required.) How animals can be genetically engineered to produce substances useful in treating human diseases, as exemplified by genetically engineering sheep to produce alpha-1-antitrypsin which is used to treat emphysema and cystic fibrosis. Gene Therapy and Cystic Fibrosis In gene therapy healthy genes may be cloned and used to replace defective genes. In cystic fibrosis the transmembrane regulator protein, CFTR, is defective. A mutant of the gene that produces CFTR results in CFTR with one missing amino acid. The symptoms of cystic fibrosis related to the malfunctioning of CFTR. Techniques that might possibly be used to introduce healthy CFTR genes into lung epithelial cells include: • use of a harmless virus into which the CFTR gene has been inserted • wrapping the gene in lipid molecules that can pass through the membranes of lung cells. Evaluation of Genetic Engineering Candidates should be able to evaluate the ethical, social and economic issues involved in the use of genetic engineering in medicine and in food production. Genetically Modified Animals Genetics Genetics is the study of heredity (from the Latin genesis = birth). The big question to be answered is: why do organisms look almost, but not exactly, like their parents? There are three branches of modern genetics: 1. Molecular Genetics (or Molecular Biology), which is the study of heredity at the molecular level, and so is mainly concerned with the molecule DNA. It also includes genetic engineering and cloning, and is very trendy. This module is mostly about molecular genetics. 2. Classical, Transmission or Mendelian Genetics, which is the study of heredity at the whole organism level by looking at how characteristics are inherited. This method was pioneered by Gregor Mendel (1822-1884). It is less fashionable today than molecular genetics, but still has a lot to tell us. This is covered in Module 4. 3. Population Genetics, which is the study of genetic differences within and between species, including how species evolve by natural selection. Some of this is also covered in Module 4. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 4 DNA DNA and its close relative RNA are perhaps the most important molecules in biology. They contain the instructions that make every single living organism on the planet, and yet it is only in the past 50 years that we have begun to understand them. DNA stands for deoxyribonucleic acid and RNA for ribonucleic acid, and they are called nucleic acids because they are weak acids, first found in the nuclei of cells. They are polymers, composed of monomers called nucleotides. Nucleotides Nucleotides contain the elements CHONP, and have three parts to them: phosphate sugar base - O O- P O O CHOH O C phosphate 5' 4' or more simply: sugar C1' N C3' C2' OH OH base • a phosphate group (PO 42- ), which is negatively charged, and gives nucleic acids their acidic properties. • a pentose sugar, which has 5 carbon atoms in it. If carbon 2' has a hydroxyl group attached (as shown), then the sugar is ribose, found in RNA. If the carbon 2' just has a hydrogen atom attached instead, then the sugar is deoxyribose, found in DNA. • a nitrogenous base. There are five different bases (and you don't need to know their structures), but they all contain the elements carbon, hydrogen, oxygen and nitrogen. Since there are five bases, there are five different nucleotides: Base: Adenine (A) Nucleotide: Adenosine Cytosine (C) Cytidine Guanine (G) Guanosine Thymine (T) Thymidine Uracil (U) Uridine The bases are usually known by their first letters only, so you don't need to learn the full names. The base thymine is found in DNA only and the base uracil is found in RNA only, so there are only four different bases present at a time in one nucleic acid molecule. The nucleotide above is shown with a single phosphate group, but in fact nucleotides can have one, two or three phosphate groups. So for instance you can have adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP). These nucleotides are very HGS A-level notes NCM/6/07 Module 2 - Genetics - page 5 common in cells and have many roles other than just part of DNA. ATP is used as an energy store (see module 3), while AMP and GTP are used as messenger chemicals (see module 4). Nucleotide Polymerisation sugar-phosphate backbone Nucleotides polymerise by forming phosphodiester bonds between carbon 3' of the sugar and an oxygen atom of the phosphate. This is a condensation polymerisation reaction. The bases do not take part in phosphate the polymerisation, so there is a sugar-phosphate sugar 5' backbone with the bases extending off it. This means base 3' that the nucleotides can join together in any order along 5' the chain. Two nucleotides form a dinucleotide, three form a trinucleotide, a few form an oligonucleotide, phosphodiester bond and many form a polynucleotide. 3' 5' 3' A polynucleotide has a free phosphate group at one OH end, called the 5' end because the phosphate is attached to carbon 5' of the sugar, and a free OH group at the other end, called the 3' end because it's on carbon 3' of 2' new nucleotide joining chain 5' the sugar. The terms 3' and 5' are often used to denote the different ends of a DNA molecule. HGS A-level notes 3' 2' NCM/6/07 Module 2 - Genetics - page 6 Structure of DNA The three-dimensional structure of DNA was discovered in 1953 by Watson and Crick in Cambridge, using experimental data of Wilkins and Franklin in London, for which work they won a Nobel Prize. The main features of the structure are: • DNA is double-stranded, so there are two polynucleotide stands alongside each other. The strands are antiparallel, i.e. they run in opposite directions. • The two strands are wound round each other to form a double helix (not a spiral, despite what some textbooks say). • The two strands are joined together by hydrogen bonds between the bases. The bases therefore form base pairs, which are like rungs of a ladder. • The base pairs are specific. A only binds to T (and T with A), and C only binds to G (and G with C). These are called complementary base pairs (or sometimes Watson-Crick base pairs). This means that whatever the sequence of bases along one strand, the sequence of bases on the other stand must be complementary to it. (Incidentally, complementary, which means matching, is different from complimentary, which means being nice.) 3' 5' C G hydrogen bonds 3' A T G C T A C G C G 5' DNA showing the complementary base pairing between antiparallel strands HGS A-level notes DNA showing the double helix spacefilling model of the double helix NCM/6/07 Module 2 - Genetics - page 7 Function of DNA DNA is the genetic material, and genes are made of DNA. So what do genes (and DNA) do? There are two definitions of a gene: Traditional Definition Modern Definition A gene is an inherited factor that controls a A gene is a section of DNA that codes for a particular characteristic (such as flower colour). particular polypeptide. Surprisingly, these two definitions actually say the same thing, since characteristics are controlled by genes through the proteins they code for, like this: sequences of bases in DNA determines sequence of amino acids in polypeptide determines shape and function of protein (e.g. enzyme) determines characteristics of cell This process of making proteins and so controlling characteristics is called gene expression (because the gene "expresses" itself). Expression can be split into two parts: transcription (making RNA) and translation (making proteins). DNA has one other important function: the DNA, with all its genes, must be copied every time a cell divides by mitosis, so that the daughter cells have identical copies of all the genes. This copying process is called replication. These functions of DNA are summarised in this diagram (called the central dogma of genetics). expression plicatio n transcription RNA translation Protein re No one knows exactly how many genes we humans have to control all our characteristics, but the current best estimate is around 30 thousand. The sum of all the genes in an organism is called the genome, and this table shows the estimated number of genes in different organisms: Species Common name length of DNA (kbp)* virus 48 phage λ Eschericia coli Bacterium 4 639 Saccharomyces cerevisiae Yeast 13 500 Caenorhabditis elegans nematode worm 90 000 Drosophila melaogaster fruit fly 165 000 Homo sapiens Human 3 150 000 * kbp = kilo base pairs, i.e. thousands of nucleotide monomers. no of genes 60 4 000 6 000 ~10 000 ~10 000 ~30 000 Amazingly, genes only seem to comprise about 2% of the DNA in a cell. The majority of the DNA does not form genes and doesn’t seem to do anything. The purpose of this junk DNA remains a mystery! HGS A-level notes NCM/6/07 Module 2 - Genetics - page 8 Replication - DNA Synthesis DNA is copied, or replicated, before every cell division, so that one identical copy can go to each daughter cell. The method of DNA replication is obvious from its structure: the double helix unzips and two new strands are built up by complementary base-pairing onto the two old strands. 1 original DNA double helix enzyme unzips 2 DNA double helix C T A C T A C G G C T G A T T A T A A C C A nucleotides base-pair to old G 4 strands with hydrogen bonds G T A CG GC A T AT 3 free nucleotides 7 two copies of original DNA polymerase enzyme joins nucleotides 5 DNA together with covalent phosphodiester bonds rewinds 6 enzyme DNA into double helix There are two kinds of bonds involved in DNA replication: Weak hydrogen bonds are formed between bases and don't need an enzyme. Strong covalent bonds are formed between adjacent nucleotides in a strand. They are made by the enzyme DNA polymerase. 1. Replication starts at a specific sequence on the DNA molecule called the replication origin. 2. An enzyme unwinds and separates the two strands of DNA, breaking the hydrogen bonds between the base pairs. 3. The new DNA is built up from the four nucleotides (A, C, G and T) that are present in the nucleoplasm. 4. These nucleotides attach themselves to the bases on the old strands by complementary base pairing. Where there is a T base, only an A nucleotide will bind, and so on. 5. The enzyme DNA polymerase joins the new nucleotides to each other by strong covalent phosphodiester bonds, forming the sugar-phosphate backbone. This enzyme is enormously complex and contains 18 subunits. 6. Another enzyme winds the new strands up to form double helices. 7. The two new DNA molecules are identical to the old molecule. Each new DNA molecule contains one "new" strand and one "old" strand. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 9 DNA replication can takes a few hours, and in fact this limits the speed of cell division. One reason bacteria can reproduce so fast is that they have a relatively small amount of DNA. In eukaryotes replication is speeded up by taking place at thousands of sites along the DNA simultaneously. replication forks The Meselson-Stahl Experiment This replication mechanism is sometimes called semi-conservative replication, because each new DNA molecule contains one new strand and one old strand. This need not be the case, and alternative theories suggested that a "photocopy" of the original DNA could be made, leaving the original DNA conserved (conservative replication), or the old DNA molecule could be dispersed randomly in the two copies (dispersive replication). The evidence for the semi-conservative method came from an elegant experiment performed in 1958 by Matthew Meselson and Franklin Stahl. They used the bacterium E. coli together with the technique of density gradient centrifugation, which separates molecules on the basis of their density. 1. Grow bacteria on medium with normal 14 NH4 2. Grow bacteria for many generations on medium 15 with NH4 purify DNA and centrifuge light DNA CsCl solution purify DNA and centrifuge heavy DNA 14 3. Return to NH4 medium for 20 mins (one generation) purify DNA and centrifuge 14 4. Grow on NH4 mediun for 40 mins (two generations) HGS A-level notes These first two steps are a calibration. They show that the method can distinguish between 14 DNA containing N and that 15 containing N. The DNA is visualised under UV light. purify DNA and centrifuge intermediate DNA light DNA intermediate DNA This is the crucial step. The DNA 14 has replicated just once in N medium. The resulting DNA is not heavy or light, but exactly half way between the two. This rules out conservative replication. After two generations the DNA is either light or half-and-half. This rules out dispersive replication. The results are all explained by semiconservative replication. NCM/6/07 Module 2 - Genetics - page 10 RNA RNA is a nucleic acid like DNA, but with 4 differences: • RNA is made of ribose nucleotides instead of deoxyribose nucleotides • RNA has the base uracil instead of thymine • RNA is single stranded (though it can fold into 3-dimentional structures) • RNA is shorter than DNA There are three kinds of RNA, with three different jobs: Messenger RNA (mRNA) mRNA carries the "message" that codes for a particular protein from the nucleus (where the DNA master copy is) to the cytoplasm (where proteins are synthesised). It is single stranded and just long enough to contain one gene only. It has a short lifetime and is degraded soon after it is used. Ribosomal RNA (rRNA) rRNA, together with proteins, form ribosomes, which are the site of mRNA translation and protein synthesis. Ribosomes have two large subunit 25 nm subunits, small and large, and are assembled in the nucleolus of the small subunit nucleus and exported into the cytoplasm. rRNA is coded for by numerous genes in many different chromosomes. Ribosomes free in the cytoplasm make proteins for use in the cell, while those attached to the RER make proteins for export. Transfer RNA (tRNA) tRNA is an “adapter” that matches amino acids to their codon. tRNA amino acid A C C is only about 80 nucleotides long, and it folds up by complementary base pairing to form a looped clover-leaf structure. At one end of the amino acid attachment site molecule there is always the base sequence ACC, where the amino acid binds. On the middle loop there is a triplet nucleotide sequence base pairing called the anticodon. There are 64 different tRNA molecules, each non-paired loops with a different anticodon sequence complementary to the 64 different codons. The amino acids are attached to their tRNA molecule by specific aminoacyl tRNA synthase enzymes. These are highly specific, so that each amino acid is attached to a tRNA anticodon adapter with the appropriate anticodon. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 11 Transcription - RNA Synthesis DNA never leaves the nucleus, but proteins are synthesised in the cytoplasm, so a copy of each gene is made to carry the “message” from the nucleus to the cytoplasm. This copy is mRNA, and the process of copying is called transcription. A C 2 G U RNA polymerase 1 3 4 promoter mRNA 5 mRNA for one gene 6 nuclear pore nuclear envelope cytoplasm rough endoplasmic reticulum ribosomes 1. The start of each gene on DNA is marked by a special sequence of bases called the promoter. 2. The RNA molecule is built up from the four ribose nucleotides (A, C, G and U) in the nucleoplasm. The ribose nucleotides attach themselves to the bases on the DNA by complementary base pairing, just as in DNA replication. However, only one strand of RNA is made. The DNA strand that is copied is called the template strand. The other strand is a complementary copy, called the non-template strand. 3. The new nucleotides are joined to each other by strong covalent phosphodiester bonds by the enzyme RNA polymerase. 4. Only about 8 base pairs remain attached at a time, since the mRNA molecule peels off from the DNA as it is made. A winding enzyme rewinds the DNA. 5. At the end of the gene the transcription stops, so the mRNA molecule is just the length of the gene. 6. The mRNA diffuses out of the nucleus through a nuclear pore into the cytoplasm. There, it attaches to ribosomes for translation. It usually doesn't have far to go to find a ribosome, as many are attached to the rough endoplasmic reticulum, which is contiguous with the nuclear envelope. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 12 Introns and Exons It turns out that genes contain many regions that are not needed as part of the protein code. These are called introns (for interruption sequences), while the parts that are needed are called exons (for expressed sequences). All eukaryotic genes have introns, and they are usually longer than the exons, so genes are often much longer than they really need to be! No one knows what these introns are for, but they need to be removed before the mRNA can be translated into protein. 1 exon 1 exon 2 intron exon 3 intron exon 4 exon 5 intron intron primary transcript introns 2 introns cut out 1 2 3 4 5 exons + introns 3 mature mRNA – just exons 1. The initial mRNA that is transcribed, or primary transcript, is an exact copy of the gene on the DNA, so it contain exons and introns. 2. The introns in the mRNA are cut out and the exons are joined together by enzymes. Some of this joining (or splicing) is done by the RNA intron itself, acting as an RNA enzyme. The recent discovery of these RNA enzymes, or ribozymes, illustrates what a diverse and important molecule RNA is. Other splicing is performed by RNA/protein complexes called snurps. 3. The result is a shorter mature RNA containing only exons. The introns are broken down. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 13 The Genetic Code The sequence of bases on DNA codes for the sequence of amino acids in proteins. But there are 20 different amino acids and only 4 different bases, so the bases are read in groups of three. This gives 43 or 64 combinations, more than enough to code for 20 amino acids. A group of three bases coding for an amino acid is called a codon, and the meaning of each of the 64 codons is called the genetic code. The Genetic Code (mRNA codons) UUU UUC UUA UUG UCU UCC UCA UCG UAU UAC UAA UAG UGU UGC UGA UGG phe leu ser tyr stop cys stop trp CUU CUC CUA CUG CCU CCC CCA CCG CAU CAC CAA CAG CGU CGC CGA CGG leu pro his gln arg AUU AUC AUA AUG ACU ACC ACA ACG AAU AAC AAA AAG AGU AGC AGA AGG ile start/met thr asn lys ser arg GUU GUC GUA GUG GCU GCC GCA GCG GAU GAC GAA GAG GGU GGC GGA GGG val ala asp glu gly There are several interesting points from this code: • The code is degenerate, i.e. there is often more than one codon for an amino acid. • The degeneracy is on the third base of the codon, which is therefore less important than the others. • One codon means "start" i.e. the start of the gene sequence. It is AUG, which also codes for methionine. Thus all proteins start with methionine (although it may be removed later). AUG in the middle of a gene simply codes for methionine. • Three codons mean "stop" i.e. the end of the gene sequence. They do not code for amino acids. • The code is read from the 5' to 3' end of the mRNA, and the protein is made from the N to C terminus ends. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 14 Translation - Protein Synthesis ribosome initiation codon 1. A ribosome attaches to the mRNA at an initiation codon (AUG). The ribosome encloses two CA codons. G U G C A U G C U G U G C mRN A AAC UU codons met 2. The first tRNA molecule with an amino acid attached (met-tRNA) diffuses to the ribosome. Its anticodon CA anticodon attaches to the first mRNA codon by UA C GUG CA UGCUGUGCA AC UU codon complementary base pairing. 3. The next amino acid-tRNA attaches to the met leu adjacent mRNA codon (CUG, leu in this case) by complementary base pairing. CA UA CGA C GUG CA UGCUGUGCA AC UU peptide bond 4. The bond between the amino acid and the tRNA met cut leu is cut and a peptide bond is formed between the two amino acids. These operations are catalysed CA UA CGA C GUG CA UGCUGUGCA AC UU by enzymes in the ribosome called ribozymes. met 5. The ribosome moves along one codon so that a leu cys new amino acid-tRNA can attach. The free tRNA U G GA CA CG CAU GCUGU G CA ACUUA C molecule leaves to collect another amino acid. C The cycle repeats from step 3. 6. The polypeptide chain elongates one amino acid at a time, and peels away from the ribosome, ala cys stop codon val phe folding up into a protein as it goes. This continues for hundreds of amino acids until a stop codon is reached, when the ribosome falls apart, releasing UG A A G C G UC UU CUAGC CA AG GG G the finished protein. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 15 A single piece of mRNA can be translated by many ribosomes simultaneously, so many protein molecules can be made from one mRNA molecule. A group of ribosomes all attached to one piece of mRNA is called a polyribosome, or a polysome. growing polypeptide chain finished protein A mRN Post-Translational Modification In eukaryotes, proteins often need to be altered before they become fully functional. Because this happens after translation, it is called post-translational modification. Modifications are carried out by other enzymes and include: chain cutting, adding methyl or phosphate groups to amino acids, adding sugars (to make glycoproteins) or lipids (to make lipoproteins). Regulation of Gene Expression Not all genes make proteins. Some important genes control the expression of other genes, and so are called control genes. Control genes usually work by regulating transcription, so mRNA is only made in a cell where it is needed and when it is needed. Remember, each cell in your body contains all of your genes, but only a few are actually expressed. For example skin cells could make amylase but don't, and kidney cells could make haemoglobin, but don't. This is because of control genes. Control genes are if anything even more important than structural genes in controlling characteristics. For example control genes control the development of an embryo and determine which cells differentiate into which kind of tissue. They also control the timing of events such as puberty, flowering or ageing. So most characteristics are controlled by many genes working together, and most genes affect many different aspects of a cell’s function. Characteristics are also influenced by non-genetic factors, such as diet and environment. Some genes (called oncogenes) control cell division and growth, and it is a malfunction in these genes that causes cancer. The regulation of gene expression is a highly complex subject, and is still poorly understood. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 16 Mutations Mutations are changes in genes, which are passed on to daughter cells. DNA is a very stable molecule, and it doesn't suddenly change without reason, but bases can change when DNA is being replicated. Normally replication is extremely accurate, and there are even error-checking procedures in place to ensure accuracy, but very occasionally mistakes do occur (such as a T-C base pair). So a mutation is a base-pairing error during DNA replication. A change in a gene could cause a change in the protein made by the gene, and so a change in the cell function: change in base of DNA change in base of mRNA change in mRNA codon different amino acid in protein change in amino acid sequence change in protein structure change in protein function change in cell function Many of the proteins in cells are enzymes, and most changes in enzymes will stop them working (because there are far more ways of making an inactive enzyme than there are of making a working one). When an enzyme stops working, a metabolic block can occur, when a reaction in a cell doesn't happen, so the cell's function is changed. gene P Compound A enzyme P gene Q Compound B enzyme Q Compound C In this example of a metabolic pathway two enzymes (P and Q) are needed to make compound C from compound A. If a mutation occurs in gene P then enzyme P won't be made (or at least will be the wrong shape so won't work), so compound B can't be made. And with no compound B then compound C cannot be made, even if enzyme Q is functional. It's just possible (though unlikely) that a mutation in gene P could make a modified enzyme P that actually worked faster than the original enzyme. This means the metabolic pathway could be faster and the cell's function could be improved. Since mutations change genes, they give rise to new alleles (i.e. different versions of genes). A cell with the original, functional gene has one allele, while a cell with a mutated, non-functional version of the same gene has a different allele. In the above example if compound A was a white pigment in a flower and compound C was a red pigment, then the "red" allele for flower colour would be the original gene P (and functional enzyme P) while the "white" allele for flower colour would be a mutated gene P (and non-functional enzyme P). HGS A-level notes NCM/6/07 Module 2 - Genetics - page 17 So there are three possible phenotypic effects of a mutation: • Most mutations have no phenotypic effect because they don't change the protein or they are not expressed in this cell. These are called silent mutations, and we all have a few of these. • Of the mutations that have a phenotypic effect, most will have a deleterious effect. • Very rarely a mutation can have a beneficial phenotypic effect, such as making an enzyme work faster, or a structural protein stronger, or a receptor protein more sensitive. A small mutation in a control gene can have a very large phenotypic effect, such as developing extra limbs or flowering more often. Although rare, these beneficial mutations are important as they drive evolution. Examples include modified enzymes that make bacteria resistant to antibiotics, cows that produce milk constantly, sweetcorn that tastes sweet and almonds that aren't poisonous. There are three kinds of gene mutation, shown in this diagram: mRNA protein G . . . C G C G U U U C C . . . (part of original gene) arg ser val A G +A SUBSTITUTION C G C A U U U C C arg ser ile DELETION C G C U U U C C A arg pro phe INSERTION C G C A G U U U C arg phe ser Only one amino acid altered. Rest of protein OK. Reading frame altered. Rest of protein wrong. Reading frame altered. Rest of protein wrong. • Substitution mutations only affect one amino acid, so tend to have less severe effects. In fact if the substitution is on the third base of a codon it may have no effect at all, because the third base often doesn't affect the amino acid coded for (e.g. all codons beginning with CC code for proline). However, if a mutation leads to a premature stop codon the protein will be incomplete and certainly non-functional. • Deletion and insertion mutations have more serious effects because they are frame shift mutations i.e. they change the codon reading frame even though they don't change the actual sequence of bases. So all amino acids "downstream" of the mutation are wrong, and the protein is completely wrong and non-functional. However, the effect of a deletion can be cancelled out by a near-by insertion, or by two more deletions, because these will restore the reading frame. A similar argument holds for a substitution. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 18 Mutation Rates and Mutagens Mutations are normally very rare, which is why members of a species all look alike and can interbreed. However the rate of mutations is increased by chemicals or by radiation. These are called mutagenic agents or mutagens, and include: • High-energy ionising radiation such as x-rays, ultraviolet rays, α, β, or γ rays from radioactive sources. This ionises the bases so that they don't form the correct base pairs. Note that lowenergy radiation (such as visible light, microwaves and radio waves) doesn't have enough energy to affect DNA and so is harmless. • Intercalating chemicals such as mustard gas (used in World War 1), which bind to DNA separating the two strands. • Chemicals that react with the DNA bases such as benzene, nitrous acid, and tar in cigarette smoke. • Viruses. Some viruses can change the base sequence in DNA causing genetic disease and cancer. During the Earth's early history there were far more of these mutagens than there are now, so the mutation rate would have been much higher than now, leading to a greater diversity of life. Some of these mutagens are used today in research, to kill microbes or in warfare. They are often carcinogens since a common result of a mutation is cancer. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 19 DNA and Chromosomes The DNA molecule in a single human cell is 1 m long, so is 10 000 times longer than the cell in which it resides (< 100µm). (Since an adult human has about 1014 cells, all the DNA is one human would stretch about 1014 m, which is a thousand times the distance between the Earth and the Sun.) In order to fit into the cell the DNA is cut into shorter lengths and each length is tightly wrapped up with histone proteins to form a complex called chromatin. During most of the life of a cell the chromatin is dispersed throughout the nucleus and cannot be seen with a light microscope. At various times parts of the chromatin will unwind so that genes on the DNA can be transcribed. This allows the proteins that the cell needs to be made. Just before cell division the DNA is replicated, and more histone proteins are synthesised, so there is temporarily twice the normal amount of chromatin. Following replication the chromatin then coils up even tighter to form short fat bundles called chromosomes. These are about 100 000 times shorter than fully stretched DNA, and therefore 100 000 times thicker, so are thick enough to be seen with the light microscope. Each chromosome is roughly X-shaped because it contains two replicated copies of the DNA. The two arms of the X are therefore identical. They are called chromatids, and are joined at the centromere. (Do not confuse the two chromatids with the two strands of DNA.) The complex folding of DNA into chromosomes is shown below. chromosome one chromatid chromatin histone proteins DNA double helix HGS A-level notes centromere micrograph of a single chromosome NCM/6/07 Module 2 - Genetics - page 20 Karyotypes and Homologous Chromosomes If a dividing cell is stained with a special fluorescent dye and examined under a microscope during cell division, the individual chromosomes can be distinguished. They can then be photographed and studied. This is a difficult and skilled procedure, and it often helps if the chromosomes are cut out and arranged in order of size. 1 2 3 4 5 6 7 12 13 14 15 16 17 18 8 19 9 20 10 21 11 22 XY This display is called a karyotype, and it shows several features: • Different species have different number of chromosomes, but all members of the same species have the same number. Humans have 46 (this was not known until 1956), chickens have 78, goldfish have 94, fruit flies have 8, potatoes have 48, onions have 16, and so on. The number of chromosomes does not appear to be related to the number of genes or amount of DNA. • Each chromosome has a characteristic size, shape and banding pattern, which allows it to be identified and numbered. This is always the same within a species. The chromosomes are numbered from largest to smallest. • Chromosomes come in pairs, with the same size, shape and banding pattern, called homologous pairs ("same shaped"). So there are two chromosome number 1s, two chromosome number 2s, etc, and humans really have 23 pairs of chromosomes. • One pair of chromosomes is different in males and females. These are called the sex chromosomes, and are non-homologous in one of the sexes. In humans the sex chromosomes are homologous in females (XX) and non-homologous in males (XY). In other species it is the other way round. The non-sex chromosomes are called autosomes, so humans have 22 pairs of autosomes, and 1 pair of sex chromosomes. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 21 It is important to understand exactly what homologous chromosomes are. We have two copies of each chromosome because we inherit one copy from each parent, so each homologous pair consists of a maternal and paternal version of the same chromosome. Since the homologous chromosomes contain the same genes, this also means we have two copies of each gene (again, one from each parent). This is why we write two letters for each gene in a genetic cross. The two homologous chromosomes may have the same versions (or alleles) of the gene (e.g. AA), or they may have different alleles, because one copy is a mutation (Aa). Sometimes the chromosomes in a cell nucleus are represented by rods called ideograms, although these structures never actually exist round seeds round seeds purple flowers white flowers because the chromatin is usually uncoiled. Each ideogram represents the long coiled DNA molecule in one chromosome. This diagram shows a pair of homologous chromosomes with two genes marked. The plant cell containing these chromosomes is homozygous for the seed shape gene (RR) and heterozygous for the flower colour gene (Pp). maternal chromosome paternal chromosome The only time chromosomes can actually be seen is during cell division. At this point in the cell cycle each chromosome is made of round seeds round seeds purple flowers purple flowers round seeds round seeds two identical chromatids, because each DNA molecule has now been replicated. This diagram shows the same pair of homologous chromosomes during mitosis. The two chromatids in each chromosome contain the same alleles because they're exact replicas white flowers white flowers of each other. But again the two homologous chromosome contain the same genes but different alleles. maternal chromosome paternal chromosome Chromatin DNA + histone complex during interphase Chromosome compact X-shaped form of chromatin formed (and visible) during mitosis Chromatids the two arms of an X-shaped chromosome. The two chromatids are identical since they are formed by DNA replication. Homologous chromosomes two chromosome of the same size and shape, one originating from each parent. They contain the same genes, but different alleles. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 22 Gene Loci Since the DNA molecule extends form one end of a chromosome to the other, and the genes are distributed along the DNA, then each gene has a defined position on a chromosome. This position is called the locus of the gene, and the loci of thousands of human genes are now known. There are on average about 1 000 genes per chromosome, although of course the larger chromosomes have more than this, and the smaller ones have fewer. This diagram shows the loci of a very few example genes in humans: ou ) sf es of ge ne ba s (M o N ng nd r be th om os le ch m ro um en Sample Genes 1 246 2610 Elastase (protease); Amylase; Skeletal muscle actin 2 240 1748 Lactase; Glucagon 3 193 1381 Alkaptonuria 4 191 1024 Huntingtin 5 181 1190 Asthma 6 170 1394 Antibodies; Potassium channel 7 157 1378 CFTR; Trypsin (endopeptidase) 8 143 927 9 132 1076 10 135 983 Red blood cell antigens (blood groups) Smooth muscle actin; Lipase 11 137 1692 Insulin; Haemoglobin 12 132 1268 HOX genes (embryo development) 13 113 Breast cancer; skeletal muscle myosin 496 14 104 1173 AAT 15 99 Cardiac muscle actin; Tay-Sachs disease 16 81 1032 Calcium pump in fast skeletal muscle 17 80 1394 Human Growth Hormone 18 77 Leukemia 19 60 1592 Alzheimers 20 63 710 SCID 21 45 337 Enterokinase (endopeptidase); Down syndrome 22 48 701 906 400 X 148 1141 Rhodopsin (retina photoreceptor); Blood clotting factor VIII Y SRY (sex-determining genes) 59 255 mt 0.02 37 HGS A-level notes Respiration enzymes NCM/6/07 Module 2 - Genetics - page 23 The Cell Cycle Cells are not static structures, but are created and die. The life of a cell is called the cell cycle and has two main phases: 1. Interphase Genes are expressed into proteins, and the cell does its thing. Can last from minutes to years. Towards the end of interphase the DNA, histones and other proteins are replicated, so there is temporarily twice the normal amount of DNA. mi to s i nt er is 2. Mitotic Phase Cell division, or mitosis, takes place. The cell cycle starts again for each daughter cell. phase In different cell types the cell cycle can last from hours to years. For example bacterial cells can divide every 30 minutes under suitable conditions, skin cells divide about every 12 hours on average, liver cells every 2 years, and muscle cells never divide at all after maturing, so remain in the growth phase for decades. • Interphase can be sub-divided into growth and synthesis phases. In the growth phase the cell grows and does whatever it does (e.g. respires, synthesises molecules, secretes hormones, contracts, transmits nerve impulses, etc.). In the synthesis phase DNA and histones are replicated in preparation for mitosis. • The mitotic phase can be sub-divided into four phases (prophase, metaphase, anaphase and telophase). Mitosis is strictly nuclear division, and is followed by cytoplasmic division, or cytokinesis, to complete cell division. Mitosis results in two “daughter cells”, which are genetically identical to each other, and is used for growth and asexual reproduction. The details of each of these phases are shown on the next page. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 24 Cell Division by Mitosis This cell has n = 2; i.e. 2 pairs of homologous chromosomes. Interphase centrioles chromatin nucleolus • no chromosomes visible • DNA, histones and centrioles all replicated nuclear envelope cell membrane Prophase • chromosomes condensed and visible • centrioles at opposite poles of cell • nucleolus disappears Metaphase • nuclear envelope disappears • chromosomes align along equator of cell • spindle fibres (microtubules) connect centrioles to chromosomes Anaphase • centromeres split, allowing chromatids to separate • chromatids move towards poles, centromeres first, pulled by motor proteins "walking" along the microtubule tracks Telophase • spindle fibres disperse • nuclear envelopes form • chromosomes uncoil and become invisible Cytokinesis • In animal cells a ring of actin filaments forms round the equator of the cell, and then tightens to form a cleavage furrow, which splits the cell in two. • In plant cells vesicles move to the equator, line up and fuse to form two membranes called the cell plate. A new cell wall is laid down between the membranes, which fuses with the existing cell wall. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 25 Asexual Reproduction Asexual reproduction is the production of offspring from a single parent using mitosis. The offspring are therefore genetically identical to each other and to their “parent”- in other words they are clones. A clone is defined as a cell or organism (or even a molecule of DNA) that is genetically identical to another cell or organism (or molecule of DNA). Asexual reproduction is very common in nature, and in addition we humans have developed some new, artificial methods. The Latin terms in vivo (“in life”, i.e. in a living organism) and in vitro (“in glass”, i.e. in a test tube) are often used to describe natural and artificial techniques respectively. Cloning (both natural and artificial) is of great commercial importance as brewers, pharmaceutical companies, farmers and plant growers all want to be able to reproduce “good” organisms exactly. Natural methods of asexual reproduction are often quite suitable for some organisms (such as yeast, potatoes and strawberries), but many important plants and animals do not reproduce asexually (such as apples, bananas or sheep), so artificial methods have to be used. Some different methods of asexual reproduction are summarised in this table. Microbes Plants Animals Methods of Asexual Reproduction Natural Methods Artificial Methods binary fission cell culture budding fermenters fragmentation cuttings vegetative propagation grafting parthenogenesis micropropagation budding embryo splitting any invertebrates fragmentation animal only somatic cell cloning parthenogenesis Cloning Plants The natural methods of asexual reproduction used by plants are referred to as vegetative propagation. A bud grows from a vegetative (i.e. not reproductive) part of the plant (usually the stem) and develops into a complete new plant, which eventually becomes detached from the parent plant. There are numerous forms of vegetative reproduction, including: • • • • bulbs (e.g. onion, daffodil) corms (e.g. crocus, gladiolus) rhizomes (e.g. iris, couch grass) stolons (e.g. blackberry, bramble) HGS A-level notes • • • • runners (e.g. strawberry, creeping buttercup) tubers (e.g. potato, dahlia) tap roots (e.g. carrot, turnip) tillers (e.g. grasses) NCM/6/07 Module 2 - Genetics - page 26 Many of these methods are also perenating organs, which means they contain a food store and are used for survival over winter as well as for asexual reproduction. Since vegetative reproduction relies entirely on mitosis, all offspring are clones of the parent. parent plant scale leaves food store clones of parent runner new shoot stem roots Bulb Runners Tubers There are three artificial methods used to clone plants: cuttings, grafting and micropropagation. Cuttings. This is a very old method of cloning plants. Parts of a plant stem (or even leaves) are cut off and simply replanted in wet soil. Each cutting produces roots and grows into a complete new plant, so the original cut plant can be cloned many times. Rooting is helped if the cuttings are dipped in rooting hormone (auxin). Many rooting hormone flowering plants, such as geraniums, African violet and chrysanthemums are reproduced commercially by cuttings. Grafting. This is another ancient technique, used for plant species that cannot grow roots from cuttings. Instead they can often be cloned by grafting a stem cutting (called a scion) onto the lower part of an existing plant (called the rootstock). One rootstock can take several scions, and need not even be the same species as the scion. The resulting hybrid will produce the flowers and fruits of the scion, but scion + root stock binding its size and hardiness will be determined by the rootstock. Careful selection of rootstock species can result in plants that are easier to harvest or can grow in particular soils. Almost all fruit trees, such as apples and pears, are clones of a few popular varieties grafted onto hardy rootstock. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 27 Tissue Culture (or micropropagation). This is a more modern, and very efficient, way of cloning plants. Small samples of plant tissue, called an explant, can be grown on agar plates in the laboratory in much the same way that bacteria can be grown. Any plant tissue can be used for this (e.g. from a leaf). The plant tissue can be separated into individual cells, each of which can grow into a mass of undifferentiated cells called a callus. If the correct plant hormones are added these cells can develop into whole plantlets, which can eventually be planted outside, where they will grow into normal-sized plants. Conditions must be kept sterile to prevent infection by microbes. leaf cells explant shoot stimulating hormones root stimulating hormones plant out callus culture nutrient agar plantlet normal plant Micropropagation is used on a large scale for fruit trees, ornamental plants and plantation crops such as oil palm, date palm, sugar cane and banana. The advantages are: • thousands of clones of a particularly good plant can be made quickly and in a small space • the technique works for plants species that cannot be asexually propagated by other means, such as palms and bananas. • disease-free plants can be grown from a few disease-free cells. In the field, almost all crop plants are infected with viruses. • a single cell can be genetically modified and turned into many identical plants Cloning Animals No vertebrate animal can reproduce naturally by asexual reproduction, and so it has proved very difficult to develop artificial methods of cloning animals. The problem is that in vertebrates (unlike plants and some invertebrates) the differentiation process cannot be reversed. So a skin cell cannot be turned into a liver cell or heart cell. A lot of research is going into finding stem cells – reasonably undifferentiated animal cells that can develop into different tissues. Although some animal cells can be grown in culture, they cannot therefore be grown into complete animals, so tissue culture cannot be used for cloning animals. Two techniques for cloning vertebrates have been developed to circumvent these problems: Embryo Cloning (or Embryo Splitting). The most effective technique for cloning animals is to duplicate embryo cells before they have irreversibly differentiated into tissues. It is difficult and quite expensive, so is only worth it for commercially-important farm animals, such as prize cows, or HGS A-level notes NCM/6/07 Module 2 - Genetics - page 28 genetically engineered animals. A female animal is given a fertility drug (FSH) so that she produces many mature eggs (superovulation). The eggs are then surgically removed from the female’s ovaries. The eggs are fertilised in vitro (IVF) using selected sperm from a prize male. The fertilised eggs (zygotes) are allowed to develop in vitro for a few days until the embryo is at the 16-cell stage. This young embryo can be split into 16 individual cells, which will each develop again into an embryo. (This is similar to the natural process when a young embryo splits to form identical twins.) The identical embryos can then be transplanted into the uterus of surrogate mothers, where they will develop and be born normally. These animals are clones of each other but not of their parents, since the zygote was made by sexual reproduction. Could humans be cloned this way? Almost certainly yes. Human embryos have been split and cloned to the stage of a few cells, for example to make stem cells for treating diseases. These therapeutic cloning experiments with human embryos are permitted, but are very tightly controlled. Growing cloned human babies (reproductive cloning) is not permitted in most countries (including the UK) for ethical reasons. Somatic Cell Cloning (or Nuclear Transfer). The problem with embryo cloning is that you don’t know the characteristics of the animal you are cloning. By selecting good parents you hope it will have good characteristics, but you will not know until the animal has grown. It would be far better to clone a mature animal, whose characteristics you know. Until recently it was thought impossible to grow a new animal from the somatic cells of an existing vertebrate animal. However, techniques have gradually been developed to do this, first with frogs in the 1970s, then with sheep (the famous “Dolly”) in 1996 and more recently with monkeys in 2001. The technique used to create Dolly is similar to embryo cloning, but has one crucial difference. The cells used for Dolly were from the skin of the udder of an adult sheep, so were fully differentiated somatic cells. They were gown in tissue culture for several years before they were used. One cell was fused with a unfertilised egg cell which had had its nucleus removed. This combination of a diploid nucleus in an unfertilised egg cell was a bit like a zygote, and sure enough it developed into an embryo. The embryo was implanted into the uterus of a surrogate mother, and developed into an apparently normal sheep, Dolly. It took 277 attempts to achieve success with Dolly, but once the technique is improved it may be possible to combine this technique with embryo cloning to make many clones of an adult animal. Dolly’s “mother” (identical twin?) was just an ordinary sheep, but HGS A-level notes NCM/6/07 Module 2 - Genetics - page 29 in the future prize animals (or genetically engineered ones) could be cloned in this way. Dolly died in 2003 aged 6 after leading a fairly normal life and giving birth to healthy lambs of her own. Embryo Cloning (embryo splitting) Somatic Cell Cloning (nuclear transfer) A Select prize cow. Give FSH to superovulate Select prize bull B scrape cells from udder tissue Give FSH to superovulate collect eggs collect sperm collect eggs remove nucleus somatic cell in vitro fertilisation (could be grown in culture for years) holding pipette sucking pipette fuse cells with electric current Grow in vitro to 16-cell embryo somatic cell egg cell Grow in vitro to 16-cell embryo split embryo into several "identical twins" implant into surrogate mother grow to 16-cell stage & implant into surrogate mothers C each calf is a clone lamb is a clone of sheep A D HGS A-level notes NCM/6/07 Module 2 - Genetics - page 30 Sexual Reproduction Sexual reproduction is the production of offspring from two parents using gametes. The cells of the offspring therefore have two sets of chromosomes, one from each parent. Sexual reproduction involves two stages: • Meiosis – the special cell division that halves the chromosome number from the normal, diploid number (2n) to the haploid number (n). • Fertilisation – the fusion of two haploid gametes to form a diploid zygote Meiosis Meiosis is a form of cell division. It starts with DNA replication, like mitosis, but then proceeds with two divisions one immediately after the other. Meiosis therefore results in four daughter cells rather than the two cells formed by mitosis. It differs from mitosis in two important aspects: • In meiosis the chromosome number is halved from the diploid number to the haploid number. This is necessary so that the chromosome number remains constant from generation to generation. The halving is done in a particular way: meiosis ensures that each haploid cell has one of each homologous pair of chromosomes. So for example human gametes have 23 chromosomes: one of each homologous pair. Remember that other species have different haploid numbers. meiosis Diploid cell 2 copies of each chromosome 4 haploid cells 1 copy of each chromosome • In meiosis the chromosomes are re-arranged during meiosis to form new combinations of genes. This genetic recombination is vitally important and is a major source of genetic variation. It means for example that of all the millions of sperm produced by a single human male, the probability is that no two will be identical. You don’t need to know the details of meiosis at this stage (that comes in module 4). HGS A-level notes NCM/6/07 Module 2 - Genetics - page 31 Gametes Gametes are the haploid sex cells that will fuse together to form a new diploid individual. Gametes may not be made directly by meiosis, but instead by one or more mitotic divisions of haploid cells. In all plants and animals there are two kinds of gametes – female and male. Female gametes Male gametes Female gametes (ova or eggs in animals, ovules Male gametes are small cells that can move. If in plants) are relatively large cells and they tend they can propel themselves they are called to be stationary. They contain food reserves motile (e.g. animal sperm) but if they can easily (lipids, proteins, carbohydrates) to nourish the be carried by the wind or animals they are called embryo after fertilisation, and they are produced mobile (e.g. plant pollen). They are produced in in fairly small numbers. Human females for very large numbers. Human males for example example release about 500 ova in a lifetime. release about 108 sperm in one ejaculation. Female: Few, Fixed, Food Male: Many, Mobile, Minute It is this difference in gametes that actually defines the sex of an individual. Those individuals that produce small mobile gametes are the males, and those that produce the larger gametes are the females. In some species (such as most flowering plants) the same individual organisms can produce both male and female gametes, so they do not have distinct sexes and are called hermaphrodites. In other species (such as mammals) there are two distinct sexes, each producing their own gametes. These organisms are called unisexual. These diagrams of human gametes illustrate the differences between male and female gametes. 100µm follicle cells jelly coat head membrane cytoplasm midpiece nucleus nucleolus polar body lipid droplets A Human Ovum HGS A-level notes acrosome nucleus mitochondria membrane flagellum tail 10µm A Human Sperm NCM/6/07 Module 2 - Genetics - page 32 Fertilisation Fertilisation is the fusion of two gametes to form a zygote. In humans this takes place near the top of the oviduct. Hundreds of sperm reach the egg and use their flagella to swim through the follicle cells (shown in this photo). When they reach the jelly coat surrounding the ovum they bind to receptors and this stimulates the rupture of the acrosome membrane in the sperms, releasing digestive enzymes, which digest a path through the jelly coat. When a sperm reaches the ovum cell the two membranes fuse and the sperm nucleus enters the cytoplasm of the ovum. This triggers a series of reactions in the ovum (called the cortical reaction) that cause the jelly coat to thicken and harden, preventing any other sperm from entering the ovum. The sperm and egg nuclei then fuse, forming a diploid zygote. In plants fertilisation takes place in the ovary at the base of the carpel. The haploid male nuclei travel down the pollen tube from the pollen grain on the stigma to the ovules in the ovary. In the ovule two fusions between male and female nuclei take place: one forms the zygote (which will become the embryo) while the other forms the endosperm (which will become the food store in the seed). This double fertilisation is unique to flowering plants. The Reason for Sex The reason for sex is variation. For most of the history of life on Earth, organisms have reproduced only by asexual reproduction. Each individual was a genetic copy (or clone) of its “parent”, and the only variation was due to random genetic mutation. The development of sexual reproduction in the eukaryotes around one billion years ago led to much greater variation and diversity of life. Sexual reproduction is slower and more complex than asexual, but it has the great advantage of introducing genetic variation (due to genetic recombination in meiosis and random fertilisation). This variation allows species to adapt to their environment and so to evolve. This variation is clearly such an advantage that practically all species can reproduce sexually. Some organisms can do both, using sexual reproduction for genetic variety and asexual reproduction to survive harsh times. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 33 Sexual Life Cycles The stages of sexual reproduction can be illustrated by a sexual life cycle: diploid cells All sexually-reproducing species have the basic life cycle shown on the right, alternating between diploid and haploid forms. In addition, they will also use mitosis to grow into adult organisms, fertilisation meiosis but the details vary with different organisms. haploid cells In vertebrate animals (including humans), and in flowering plants the dominant, long-lived adult form is diploid, and the haploid gamete cells are only formed briefly. mitosis diploid zygote diploid adult fertilisation meiosis haploid gametes diploid zygote In the fungi kingdom the dominant, long-lived adult form is haploid. Haploid spores undergo mitosis and grow into complete, differentiated adults (including large structures like mushrooms). At some stage two of these haploid cells fuse to form a diploid zygote, fertilisation meiosis haploid adult which immediately undergoes meiosis to re-establish the haploid haploid spores mitosis state and complete the cycle. In some plants and some invertebrate animals the life cycle shows alternation of generations. These organisms have two distinct adult forms; one diploid and the other haploid. In the simpler plants (mosses and liverworts) the haploid form is larger than the diploid form, while in the higher plants (ferns and conifers) and animals the mitosis diploid adult diploid zygote fertilisation meiosis haploid adult haploid spores mitosis diploid form is larger. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 34 Genetic Engineering Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: • inserting a foreign gene from one species into another, forming a transgenic organism • altering an existing gene so that its product is changed • changing gene expression so that it is translated more (overexpressed), or less (deactivated). Techniques of Genetic Engineering Genetic engineering is a very young discipline, and is only possible due to the development of techniques from the 1960s onwards. These techniques have been made possible from our greater understanding of DNA and how it functions, following the discovery of its structure by Watson and Crick in 1953. Although the final goal of genetic engineering is usually the expression of a gene in a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and then cloning it. This table lists the techniques that we shall look at in detail. Technique Purpose 1 PCR To amplify very small samples of DNA 2 Electrophoresis To separate fragments of DNA 3 DNA Sequencing To read the base sequence of a length of DNA Type Analysing DNA 4 Restriction Enzymes To cut DNA at specific points, making small fragments 5 DNA Ligase To join DNA fragments together 6 Plasmids To carry DNA into cells and ensure replication 7 Transformation To deliver a gene into a living cell 8 Genetic Markers To identify cells that have been transformed 9 Replica Plating 10 Fermenters Manipulating DNA Manipulating Cells To make exact copies of bacterial colonies on an agar plate To grow large quantities of a microbe The use of these techniques in a typical genetic engineering project (the manufacture of geneticallyengineered insulin by bacteria) is shown on the next page: HGS A-level notes NCM/6/07 Module 2 - Genetics - page 35 Extract human DNA 6 Extract bacterial plasmids or cut out human gene with Restriction 4 Enzyme or 4 Buy bacterial plasmids Chemistry 1 cut plasmids with same Restriction Enzyme mix together Amplify small DNA sample Sequence human gene 2 Several different products formed Sticky ends anneal. Add DNA ligase to join DNA backbone 5 hybrid plasmid original plasmid circularised human DNA 3 Bacterial cells in culture flask Insert plasmids into bacterial cells 7 Different bacterial cells formed no plasmid hybrid plasmid original plasmid human DNA only killed resistant resistant killed killed resistant 8 Grow on agar plates with antibiotic 1 Identify hybrid colonies 9 Cells Grow on replica agar plates with antibiotic 2 Grow transformed bacteria in culture flask in lab 10 Grow bacteria in industrial-scale fermenter. Genetically-modified bacteria synthesise insulin HGS A-level notes Purify insulin from medium and sell NCM/6/07 Module 2 - Genetics - page 36 1. Polymerase Chain Reaction (PCR) The polymerase chain reaction is a technique used to copy (or amplify) DNA samples as small as a single molecule. It was developed in 1983 by Kary Mullis, for which discovery he won the Nobel Prize in 1993. PCR is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times. The details are shown in this diagram: DNA polymerase 4 nucleotides 1 Original DNA 2 Heat to 95°C Strands separate 5 Repeat Add primers 3 Cool to 40°C Target sequence Primers anneal 4 Heat to 72°C DNA replicated 1. Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme DNA polymerase. 2. Heat to 95°C for two minutes to breaks the hydrogen bonds between the base pairs and separate the two strands of DNA. Normally (in vivo) the DNA double helix would be separated by an enzyme. 3. Add primers to the mixture and cool to 40°C. Primers are short lengths of single-stranded DNA (about 20 bp long) that anneal to complementary sequences on the two DNA strands forming short lengths of double-stranded DNA. The DNA is cooled to 40°C to allow the hydrogen bonds to form. There are two reasons for making short lengths of double-stranded DNA: • The enzyme DNA polymerase requires some existing double stranded DNA to get it started. • Only the DNA between the primer sequences is replicated, so by choosing appropriate primers you can ensure that only a specific target sequence is copied. 4. The DNA polymerase enzyme can now build new stands alongside each old strand to make double-stranded DNA. Each new nucleotide binds to the old strand by complementary base pairing and is joined to the growing chain by a phosphodiester bond. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot HGS A-level notes NCM/6/07 Module 2 - Genetics - page 37 springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72°C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible. 5. Each original DNA molecule has now been replicated to form two molecules. The cycle is repeated from step 2 and each time the number of DNA molecules doubles. This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. After n cycles, there is an amplification factor of 2n. Typically PCR is run for 2030 cycles. 1 cycle 1 molecule 2 cycles 2 molecules 3 cycles 4 molecules 4 cycles 8 molecules 10 cycles 16 molecules 20 cycles 1024 molecules 1 048 576 molecules PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort. The product can be used for further studies, such as cloning, electrophoresis, or gene probes. Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for example in court cases. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 38 2. Electrophoresis This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments. The DNA samples are placed into wells at one end of a thin slab of gel made of agarose or polyacrylamide, and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off. place DNA sample here Direction of movement negative electrode - well gel buffer positive electrode + Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are two common methods for doing this: • The DNA can be stained with a coloured chemical such as azure A (which stains the DNA bands blue), or a fluorescent molecule such as ethidium bromide (which emits coloured light when the finished gel is illuminated with invisible ultraviolet light). • The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as 32 P, then visualised using autoradiography. Ordinary photographic film is placed on top of the finished gel in the dark for a few hours, and the radiation from any radioactive DNA on the gel exposes the film. When the film is developed the position of the DNA shows up as dark bands on the film. This method is extremely sensitive. film after developing In Dark Room photographic film radiation gel invisible DNA spots HGS A-level notes large fragments small fragments NCM/6/07 Module 2 - Genetics - page 39 3. DNA Sequencing This means reading the base sequence of a length of DNA. DNA sequencing is based on a beautifully elegant technique developed by Fred Sanger in Cambridge, and now called Sanger Sequencing. 1. Label 4 test tubes A, T, C and G. Into each test tube add: a sample of the DNA to be sequenced (containing many DNA Radiolabelled DNA 4 polymerase Primer sample nucleotides (TATGACCG) millions of individual molecules) a radioactive primer (so A T C G dideoxy nucleotide that cannot form a phosphodiester bond 1% A* 1% T* 1% C* 1% G* and so stops further synthesis of DNA. Tube A has A T C G A T C G the DNA can be visualised later on the gel), the four DNA nucleotides and the enzyme DNA polymerase. 2. In each test tube add a small amount of a special modified dideoxy A (A*), tube T has dideoxy T (T*), tube C has dideoxy C (C*) and tube G has dideoxy G (G*). The dideoxy nucleotides are present at about 1% of the concentration of the normal nucleotides. 3. Let the DNA polymerase synthesise many copies of the DNA sample. From time to time at random a dideoxy nucleotide will be added to the growing chain and synthesis of that chain will then stop. A range of DNA molecules will be synthesised ranging from full length to very short. The important point is that in tube A, all the fragments will stop at an A nucleotide. In tube T, all the fragments will stop at a T nucleotide , and so on. DNA fragments synthesised in each tube TA* TATGA* TATGACCG 4. The contents of the four tubes are now run side by side on an electrophoresis gel, and the DNA bands are visualised by autoradiography. Since the fragments are now sorted by T* TAT* TATGACCG A TATGAC* TATG* TATGACC* TATGACCG* TATGACCG TATGACCG T C G sequence read in this direction length the sequence can simply be read off the gel starting with the smallest fragment (just one nucleotide) at the bottom and reading upwards. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 40 There is now a modified version of the Sanger method called cycle sequencing, which can be completely automated. The primers are not radiolabelled, but instead the four dideoxy nucleotides are fluorescently labelled, each with a different colour (A* is green, T* is red, C* is blue and G* is yellow). The polymerisation reaction is done in a single tube, using PCR-like cycles to speed up the process. The resulting mixture is separated using capillary electrophoresis, which gives good separation in a single narrow gel. The gel is read by a laser beam and the sequence of colours is converted to a DNA sequence by computer program (like the screenshot below). This technique can sequence an amazing 12 000 bases per minute. Thousands of genes have been sequenced using these methods and the entire genomes of several organisms have also been sequenced. A huge project to sequence the complete 3-billion base sequence of the human genome was recently completed. This information is giving us unprecedented knowledge about ourselves, and is likely to lead to dramatic medical and scientific advances. Once a gene sequence is known the amino acid sequence of the protein that the DNA codes for can also be determined, using the genetic code table. The sequence can also be compared with DNA sequences from other individuals and even other species to work out relationships between individuals or species. These genome sequences represent vast amount of data that must be analysed and compared to existing sequences. Powerful computers, huge databases and intelligent search programs are being developed to deal with this data, which has led to a whole new branch of biology called bioinformatics, and a new way of doing biology without touching a living thing: in silico. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 41 4. Restriction Enzymes These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut phosphodiester bonds in the middle of the polynucleotide chain. Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming sticky ends. A A C T G A A T T C A T A T G T C A T G G T A C sticky ends T G A C T T A A G T A C A C T G T G A C T T A A Restriction Enzyme The cut ends are “sticky” because they have short stretches of single-stranded DNA with complementary sequences. These sticky ends will stick (or anneal) to another sticky end by complementary base pairing (i.e. with weak hydrogen bonds), but only if the sticky ends have both been cut with the same restriction enzyme so that they have complementary sequences. Restriction enzymes have highly specific active sites, and will only cut DNA at specific base sequences, 4-8 base pairs long, called recognition sequences. Restriction enzymes are produced naturally by bacteria as a defence against viruses (they “restrict” viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments. There are thousands of different restriction enzymes known, with over a hundred different recognition sequences. Restriction enzymes are named after the bacteria species they came from, so EcoR1 is from E. coli strain R, and HindIII is from Haemophilis influenzae. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 42 5. DNA Ligase This enzyme repairs broken DNA by joining two A nucleotides in a DNA strand. Ligase is therefore a bit like DNA polymerase. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, A T T C A T G G T A C sticky ends A C T G T G A C T T A A i.e. to join together complementary restriction fragments. Complementary base pairing Two restriction fragments can anneal if they have complementary sticky ends, but only by weak A C T G A A T T C A T G T G A C T T A A G T A C hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete. DNA Ligase Ligase DNA ligase completes the DNA backbone by forming covalent phosphodiester bonds. Restriction enzymes A C T G A A T T C A T G and DNA ligase can therefore be used together to join T G A C T T A A G T A C lengths of DNA from different sources. Ligase 6. Plasmids Plasmids are short circular bits of DNA found naturally in bacterial cells. In genetic engineering plasmids are used as vectors. A vector is a length of DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties: • It is big enough to hold the gene we want (plus a few others), but not too big. • It is circular (or more accurately a closed loop), so that it is less likely to be broken down (particularly in prokaryotic cells where DNA is always circular). • It contains control sequences, such as a replication origin and a transcription promoter, so that the gene will be replicated, expressed, or incorporated into the cell’s normal genome. • It contain marker genes, so that cells containing the vector can be identified. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 43 Plasmids are the most common kind of vector, so we shall look at how they are used in some detail. A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells will readily take up a plasmid. Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase. The R plasmid EcoRI One of the most common plasmids used is the R-plasmid. This BamHI plasmid contains a replication origin, several recognition sequences PstI for different restriction enzymes (with names like PstI and EcoRI), ampicillin resistance gene SalI tetracycline resistance gene replication origin and two marker genes, which confer resistance to different antibiotics (ampicillin and tetracycline). The R plasmid gets its name from these resistance genes. PvuII The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase enzymes. The restriction enzyme used here (PstI) cuts the plasmid in the middle of one of the marker genes (we’ll see why this is useful later). The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA). Several other products are also formed: some plasmids will simply re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join together to form chains or circles. These different products cannot easily be separated, but it doesn’t matter, as the marker genes can be used later to identify the correct hybrid vector. original plasmid Restriction Enzyme hybrid plasmid PstI R-plasmid DNA ligase Restriction Enzyme PstI + circularised DNA Foreign DNA HGS A-level notes NCM/6/07 Module 2 - Genetics - page 44 7. Transformation Transformation means inserting new DNA (usually a plasmid) into a living cell (called a host cell), which is thus genetically modified, or transformed. A transformed cell can replicate and express the genes in the new DNA. DNA is a large molecule that does not readily cross cell membranes, so the membranes must be made permeable in some way. There are different ways of doing this depending on the type of host cell. Transferring into cells in culture • Heat Shock. Cells are incubated with the plasmid in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the plasmid. This works well for bacterial and animal cells. • Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the membrane and allows the plasmid to enter the cell. This is the most efficient method of delivering genes to bacterial cells. • Micro-Injection. A cell is held on a pipette under a microscope and the foreign DNA is injected directly into the nucleus using an incredibly fine micro-pipette. This method is used where there are only a very few cells available, such as fertilised animal egg cells. In the rare successful cases the fertilised egg is implanted into the uterus of a surrogate mother and it will develop into a normal animal, with the DNA incorporated into the chromosomes of every cell. DNA holding pipette zygote injection pipette Transferring into plant cells • Gene Gun. This extraordinary technique fires microscopic gold particles coated with the foreign DNA at the cells using a compressed air gun. It is designed to overcome the problem of the strong cell wall in plant tissue, since the particles can penetrate the cell wall and the cell and nuclear membranes, and deliver the DNA to the nucleus, where it is sometimes expressed. • Plant Tumours. The plasmid is first inserted into a soil bacterium, and then plants are infected with the bacterium. The bacterium inserts its plasmid into the plant cells' chromosomal DNA and causes a "crown gall" tumour. These tumour cells can be cultured in the laboratory and whole HGS A-level notes NCM/6/07 Module 2 - Genetics - page 45 new plants grown from them by micropropagation. Every cell of these plants contains the foreign gene. Transferring into human cells in vivo • Liposomes. Plasmids can be encased in liposomes, which are small membrane vesicles (see module 1). The liposomes fuse with the cell membrane (and sometimes the nuclear membrane too), delivering the DNA into the cell. liposome DNA fuses part of cell membrane • Viruses. The plasmid is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material. Since viruses rely on getting their DNA into host cells for their survival they have evolved many successful methods, and so are an obvious choice for gene delivery. The virus must first be genetically engineered to make it safe, so that it can’t reproduce itself or make toxins. Two viruses are commonly used: 1. Adenoviruses are human viruses that causes respiratory diseases including the common cold. Their genetic material is double-stranded DNA, and they are ideal for delivering genes to living patients in gene therapy. Their DNA is not incorporated into the host’s chromosomes, so it is not replicated, but their genes are expressed. The adenovirus is genetically altered so that its coat proteins are not synthesised, so new virus particles cannot be assembled and the host cell is not killed. 2. Retroviruses are a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA. On infection this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated into every daughter cell. After a certain time, the dormant DNA is switched on, and the genes are expressed in all the host cells. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 46 8. Genetic Markers These are needed to identify cells that have successfully taken up a plasmid and so become transformed. With most of the techniques above less than 1% of the cells actually take up the plasmid, so a marker is needed to distinguish these cells from all the others. We’ll look at how to do this with bacterial host cells, as that’s the most common technique. A common marker, used in the R-plasmid, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid can make this gene product and so are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells, together with cells that have taken up DNA that’s not in a plasmid (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate. bacteria cells (without plasmids) untransformed cells are killed by tetracycline most cells do not take up plasmid bacterial DNA spread thinly on to agar plate containing tetracycline electroporation + plasmid vectors a few cells take up plasmid and become transformed transformed cells survive 9. Replica Plating Replica plating is a simple technique for making an exact copy of an agar plate. A pad of sterile cloth the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar plate, some cells will be deposited and colonies will grow in exactly the same positions on the new plate. This technique has a number of uses, but the most common use in genetic engineering is to help solve another problem in identifying transformed cells. This problem is to distinguish those cells that have taken up a hybrid plasmid (with a foreign gene in it) from those cells that have taken up the normal plasmid. This is where the second marker gene (for resistance to ampicillin) is used. If the foreign gene is inserted into the middle of this marker gene, the marker gene is disrupted and won't make its proper gene product. So cells with the hybrid HGS A-level notes NCM/6/07 Module 2 - Genetics - page 47 plasmid will be killed by ampicillin, while cells with the normal plasmid will be immune to ampicillin. Since this method of identification involves killing the cells we want, we must first make a master agar plate and then make a replica plate of this to test for ampicillin resistance. master (tetracycline) plate replica (ampicillin) plate press to deposit cells press to pick up cells cells stuck to sterile velvet surface identify these colonies grid pattern of colonies makes identification easier missing colonies indicate cells with hybrid vector transfer to another plate Once the colonies of cells containing the correct hybrid plasmid have been identified, the appropriate colonies on the master plate can be selected and grown on another plate. The R-plasmid with its antibiotic-resistance genes dates from the early days of genetic engineering in the 1970s. Scientists are now worried about pathogenic bacteria gaining antibiotic resistance, so have stopped using this technique. In recent years better plasmids with different marker genes have been developed that do not kill the desired cells, and so do not need a replica plate. These new marker genes make an enzyme (actually lactase) that converts a colourless substrate in the agar medium into a blue-coloured product that can easily be seen. So cells with a normal plasmid turn blue on the correct medium, while those with the hybrid plasmid can't make the enzyme and stay white. These white colonies can easily be identified and transferred to another plate. Another marker gene, transferred from jellyfish, makes a green fluorescent protein (GFP). HGS A-level notes NCM/6/07 Module 2 - Genetics - page 48 10. Fermenters Once colonies of transformed bacteria have been identified on an agar plate they can at last be used to make the genetically-engineered product. To do this commercially the bacteria need to be grown in very large quantities in fermenters. Fermenters are so-called because they were developed from the large vessels used in breweries where yeast ferments sugar to alcohol. First bacteria cells are transferred from the agar plate to a liquid medium in a culture flask, where the cells grow for a few days, then the culture flask is used to inoculate a larger laboratory fermenter, where the bacteria grow further. Finally the culture is used to inoculate a huge industrial fermenter. This process is called scaling up: agar plate culture flask (0.5 L) laboratory fermenter (10 L) industrial fermenter (10000 L) All these steps must be done under sterile conditions to ensure that no other microbes contaminate the cultures. In addition the conditions in the fermenters must be very strictly controlled to maximise the growth rate of the bacteria. • Oxygen for respiration is provided by bubbling air through the fermenter and rapid stirring. • Temperature is controlled using a thermostated water jacket. • Pressure increase due the release of gases is controlled using a vent. • pH is controlled by automatically adding acid or alkali. • Sugars and other materials can be added as required by the bacteria. In industrial fermenters these conditions are constantly monitored using probes and controlled by computers. Under these optimal conditions the bacteria can grow very quickly. After a few days growth the culture is run off from the fermentation vessel, and the product is purified from the mixture by a process called downstream processing. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 49 Applications of Genetic Engineering We have now looked at some of the many techniques used by genetic engineers. What can be done with these techniques? By far the most numerous applications are still as research tools, and the techniques above are helping geneticists to understand complex genetic systems. Despite all the hype, genetic engineering still has very few successful commercial applications, although these are increasing each year. The applications so far can usefully be considered in three groups. • Gene Products using genetically modified organisms (usually microbes) to produce chemicals (usually proteins) for medical or industrial applications. • New Phenotypes using gene technology to alter the characteristics of organisms (usually farm animals or crops) • Gene Therapy using gene technology on humans to treat a disease Gene Products The biggest and most successful kind of genetic engineering is the production of gene products. These products are of medical, agricultural or commercial value. This table shows a few of the examples of genetically engineered products that are already available. Product Insulin HGH Enkephalin BST Factor VIII Anti-thrombin Penicillin Vaccines Antibodies AAT α-glucosidase DNAse rennin cellulase PHB Use human hormone used to treat diabetes human growth hormone, used to treat dwarfism human hormone bovine growth hormone, used to increase milk yield of cows human blood clotting factor, used to treat haemophiliacs anti-blood clotting agent used in surgery antibiotic, used to kill bacteria hepatitis B antigen, for vaccination research and clinical use enzyme inhibitor used to treat cystic fibrosis and emphysema enzyme used to treat Pompe’s disease enzyme used to treat CF enzyme used in manufacture of cheese enzyme used in paper production biodegradable plastic Host Organism bacteria /yeast bacteria plants bacteria bacteria goats fungi / bacteria yeast goats / plants sheep rabbits bacteria bacteria /yeast bacteria plants The products are mostly proteins, which are produced directly when a gene is expressed, but they can also be non-protein products produced by genetically-engineered enzymes. The basic idea is to transfer a gene (often human) to another host organism (usually a microbe) so that it will make the HGS A-level notes NCM/6/07 Module 2 - Genetics - page 50 gene product quickly, cheaply and ethically. It is also possible to make “designer proteins” by altering gene sequences, but while this is a useful research tool, there are no commercial applications yet. Since the end-product is just a chemical, in principle any kind of organism could be used to produce it. By far the most common group of host organisms used to make gene products are the bacteria, since they can be grown quickly and the product can be purified from their cells. Unfortunately bacteria cannot not always make human proteins, and recently animals and even plants have also been used to make gene products. In neither case is it appropriate to extract the product from their cells, but in animals the product can be secreted in milk or urine, while in plants the product can be secreted from the roots. This table shows some of the advantages and disadvantages of using different organisms for the production of genetically-engineered gene products. Type of organism Prokaryotes (Bacteria) Eukaryotes Advantages Disadvantages no nucleus so DNA easy to modify; have can’t splice introns; no postplasmids; small genome; genetics well translational modification; small understood; asexual so can be cloned; small gene size and fast growing; easy to grow commercially in fermenters; will use cheap carbohydrate; few ethical problems. can splice introns; can do post-translational Do not have plasmids (except yeast); modifications; can accept large genes often diploid so two copies of genes may need to be inserted; control of expression not well understood. Fungi (yeast, asexual so can be cloned; haploid, so only can’t always make animals’ gene mould) one copy needed; can be grown in vats products Plants photosynthetic so don’t need much feeding; cell walls difficult to penetrate by can be cloned from single cells; products vector; slow growing; multicellular can be secreted from roots or in sap. Animals (pharming) most likely to be able to make human multicellular; slow proteins; products can be secreted in milk or expensive to produce urine growing; We’ll look at some examples in detail. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 51 Human Insulin Insulin is a small protein hormone produced by the pancreas to regulate the blood sugar concentration. In the disease insulin-dependent diabetes the pancreas cells don’t produce enough insulin, causing wasting symptoms and eventually death. The disease can be successfully treated by injection of insulin extracted from the pancreases of slaughtered cows and pigs. However the insulin from these species has a slightly different amino acid sequence from human insulin and this can lead to immune rejection and side effects. The human insulin gene was isolated, cloned and sequenced in the 1970s, and so it became possible to insert this gene into bacteria, who could then produce human insulin in large amounts. Unfortunately it wasn’t that simple. In humans, pancreatic cells first make pro-insulin, which then undergoes post-translational modification to make the final, functional insulin. Furthermore the human insulin gene contain two introns. protein e i transcription e remove introns S translation post-translational modification S S S i e insulin gene primary transcript mRNA mature mRNA pro-insulin insulin Bacterial cells cannot do post-translational modification, nor can they splice out introns. Eventually a synthetic cDNA gene (with no introns) was made and inserted into the bacterium E. coli, which made pro-insulin, and the post-translational conversion to insulin was carried out chemically. expression in bacteria reverse transcriptase S chemical modification S insulin mRNA (no introns) cDNA (no introns) pro-insulin S S insulin This technique was developed by Eli Lilly and Company in 1982 and the product, “humulin” became the first genetically-engineered product approved for medical use. In the 1990s the procedure was improved by using the yeast Saccharomyces cerevisiae instead of E. coli. Yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the production of human insulin. However another company has developed a method of converting pig HGS A-level notes NCM/6/07 Module 2 - Genetics - page 52 insulin into human insulin by chemically changing a few amino acids, and this turns out to be cheaper than the genetic engineering methods. Human Growth Hormone (HGH) HGH is a protein hormone secreted by the pituitary gland, which stimulates tissue growth. Low production of HGH in childhood results in pituitary dwarfism. This can be treated with HGH extracted from dead humans, but as the treatment caused some side effects, such as CreutzfeldtJacod disease (CJD), the treatment was withdrawn. The HGH gene has been cloned and an artificial cDNA gene has been inserted into E. coli. A signal sequence has been added which not only causes the gene to be translated but also causes the protein to be secreted from the cell, which makes purification much easier. This genetically engineered HGH is produced by Genentech and can successfully restore normal height to children with HGH deficiency. Bovine Somatotrophin (BST) BST is a growth hormone produced by cattle. The gene has been cloned in bacteria by the company Monsanto, who now produce large quantities of BST. In the USA cattle are often injected with BST every 2 weeks, resulting in a 10% increase in mass in beef cattle and a 25% increase in milk production in dairy cows. BST was tested in the UK in 1985, but it was not approved and its use is currently banned in the EU. This is partly due to public concerns and partly because there is already overproduction of milk and beef in the EU, so greater production is not necessary. Antibiotics The first antibiotic, penicillin, was discovered in the fungus Penicillium, but most antibiotics now are produced by bacteria, particularly the genus Streptomyces, which produce streptomycin, tetracycline and erythromycin. Attempts are being made to engineer Streptomyces bacteria to produce better antibiotics, or to produce them in larger quantities. A hybrid antibiotic has been made by combining genes from different strains of Streptomyces, but this has proved to be no better than existing antibiotics. So far genetic engineering has only been used successfully to increase the production of existing antibiotics by overexpression of the genes involved. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 53 Rennin Rennin (also known as chymosin) is an enzyme used in the production of cheese. It is produced in the stomach of juvenile mammals (including humans) and it helps the digestion of the milk protein caesin by solidifying it so that is remains longer in the stomach. Traditionally the cheese industry has used rennin obtained from the stomach of young calves when they are slaughtered for veal, but there are moral and practical objections to this source. Now an artificial cDNA gene for rennin has been made from mRNA extracted from calf stomach cells, and this gene has been inserted into a variety of microbes such as the bacterium E. coli and the fungus Aspergillus niger. The rennin extracted from these microbes has been very successful and 90% of all hard cheeses in the UK are made using microbial rennin. Sometimes (though not always) these products are labelled as “vegetarian cheese”. AAT (α α-1-antitrypsin) AAT is a human protein made in the liver and found in the blood. As the name suggests it is an inhibitor of protease enzymes like trypsin and elastase. There is a rare mutation of the AAT gene (a single base substitution) that causes AAT to be inactive, and so the protease enzymes to be uninhibited. The most noticeable effect of this is in the lungs, where elastase digests the elastic tissue of the alveoli, leading to the lung disease emphysema. This condition can be treated by inhaling an aerosol spray containing AAT so that it reaches the alveoli and inhibits the elastase there. AAT for this treatment can be extracted from blood donations, but only in very small amounts. The gene for AAT has been found and cloned, but AAT cannot be produced in bacteria because AAT is a glycoprotein, which means that it needs to have sugars added by post translational modification. This kind of modification can only be carried out by animals, and AAT is now produced by genetically-modified sheep. In order to make the AAT easy to extract, the gene was coupled to a promoter for the milk protein β-lactoglubulin. Since this promoter is only activated in mammary gland cells, the AAT gene will only be expressed in mammary gland cells, and so will be secreted into the sheep's milk. This makes it very easy to harvest and purify without harming the sheep. The first transgenic sheep to produce AAT was called Tracy, and she was produced by PPL Pharmaceuticals in Edinburgh in 1993. This is how Tracy was made: HGS A-level notes NCM/6/07 Module 2 - Genetics - page 54 1. A female sheep is given a fertility drug to stimulate her egg production, and several mature eggs are collected from her ovaries. 2. The eggs are fertilised in vitro. 3. A plasmid is prepared containing the gene for human AAT and the promoter sequence for β-lactoglobulin. Hundreds of copies of this plasmid are microinjected into the nucleus of the fertilised zygotes (see p44). Only a few of the zygotes will be transformed, but at this stage you can’t tell which. 4. The zygotes divide in vitro until the embryos are at the 16-cell stage. 5. The 16-cell embryos are implanted into the uterus of surrogate mother ewes. Only a few implantations result in a successful pregnancy. 6. Test all the offspring from the surrogate mothers for AAT production in their milk. This is the only way to find if the zygote took up the AAT gene so that it can be expressed. About 1 in 20 eggs are successful. 7. Collect milk from the transgenic sheep for the rest of their lives. Their milk contains about 35 g of AAT per litre of milk. 8. Purify the AAT, which is worth about £50 000 per mg. AAT 9. Breed from the transgenic sheep in order to build up a herd of them. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 55 New Phenotypes This means altering the characteristics of organisms by genetic engineering. The organisms are generally commercially-important crops or farm animals, and the object is to improve their quality in some way. This can be seen as a high-tech version of selective breeding, which has been used by humans to alter and improve their crops and animals for at least 10 000 years. Nevertheless GMOs have turned out to be a highly controversial development. None of these new phenotypes is on the specification, but these are some of the most active uses of genetic engineering and are often in the news, so this table gives an idea of what is being done. organism Modification long life tomatoes There are two well-known projects, both affecting the gene for the enzyme polygalactourinase (PG), a pectinase that softens fruits as they ripen. Tomatoes that make less PG ripen more slowly and retain more flavour. The American “Flavr Savr” tomato used antisense technology to silence the gene, while the British Zeneca tomato disrupted the gene. Both were successful and were on sale for a few years, but neither is produced any more. Insect-resistant crops Genes for various powerful protein toxins have been transferred from the bacterium Bacillus thuringiensis to crop plants including maize, rice and potatoes. These Bt toxins are thousands of times more powerful than chemical insecticides, and since they are built-in to the crops, insecticide spraying (which is non-specific and damages the environment) is unnecessary. virus-resistant crops Gene for virus coat protein has been cloned and inserted into tobacco, potato and tomato plants. The coat protein seems to “immunise” the plants, which are much more resistant to viral attack. herbicide resistant crops The gene for resistance to the herbicide BASTA has been transferred from Streptomyces bacteria to tomato, potato, corn, and wheat plants, making them resistant to BASTA. Fields can safely be sprayed with this herbicide, which will kill all weeds, but not the crops. However, this means continued use of agrochemicals, so is controversial. pest-resistant legumes The gene for an enzyme that synthesises a chemical toxic to weevils has been transferred from Bacillus bacteria to The Rhizobium bacteria that live in the root nodules of legume plants. These root nodules are now resistant to attack by weevils. Nitrogen-fixing crops This is a huge project, which aims to transfer the 15-or-so genes required for nitrogen fixation from the nitrogen-fixing bacteria Rhizobium into cereals and other crop plants. These crops would then be able to fix their own atmospheric nitrogen and would not need any fertiliser. However, the process is extremely complex, and the project is nowhere near success. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 56 crop improvement Proteins in some crop plants, including wheat, are often deficient in essential amino acids (which is why vegetarians have to watch their diet so carefully), so the protein genes are being altered to improve their composition for human consumption. mastitis-resistant cattle The gene for the enzyme lactoferrin, which helps to resists the infection that causes the udder disease mastitis, has been introduced to Herman – the first transgenic bull. Herman’s offspring inherit this gene, do not get mastitis and so produce more milk. tick-resistant sheep The gene for the enzyme chitinase, which kills ticks by digesting their exoskeletons, has bee transferred from plants to sheep. These sheep should be immune to tick parasites, and may not need sheep dip. Fast-growing sheep The human growth hormone gene has been transferred to sheep, so that they produce human growth hormone and grow more quickly. However they are more prone to infection and the females are infertile. Fast-growing fish A number of fish species, including salmon, trout and carp, have been given a gene from another fish (the ocean pout) which activates the fish’s own growth hormone gene so that they grow larger and more quickly. Salmon grow to 30 times their normal mass at 10 time the normal rate. environment cleaning microbes Genes for enzymes that digest many different hydrocarbons found in crude oil have been transferred to Pseudomonas bacteria so that they can clean up oil spills. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 57 Gene Therapy Gene therapy is perhaps the most significant, and most controversial kind of genetic engineering. It is also the least well-developed. The idea of gene therapy is to genetically alter humans in order to treat a disease. This could represent the first opportunity to cure incurable diseases. Note that this is quite different from using genetically-engineered microbes to produce a drug, vaccine or hormone to treat a disease by conventional means. Gene therapy means altering the genotype of a tissue or even a whole human. Cystic Fibrosis Cystic fibrosis (CF) is the most common genetic disease in the UK, affecting about 1 in 2500. It is caused by a mutation in the gene for protein called CFTR (Cystic Fibrosis Transmembrane Regulator). The gene is located on chromosome 7, and the usual mutation is a deletion of three bases, removing one amino acid out of 1480 amino acids in the protein. CFTR is a chloride ion channel protein found in the cell membrane of epithelial tissue cells, which line all the open spaces in the human body (such as lungs and gut). The mutation stops the protein channel from working, so chloride ions cannot cross the cell membrane. Normal Bronchiole ClCFTR Cystic Fibrosis Bronchiole Clmutated CFTR less H2O H2O high Ψ low Ψ decreased Ψ runny mucus epithelial cell Alveolar air space increased Ψ alveolus thick sticky mucus epithelial cell Alveolar air space Chloride ions build up inside these epithelial cells, decreasing their water potential (Ψ). Less water therefore diffuses out of these cells to the mucus outside, leaving the mucus drier and more sticky than normal. This sticky mucus block the tubes into which it is secreted, such as the small intestine, pancreatic duct, bile duct, sperm duct, sweat ducts, bronchioles and alveoli. These blockages lead to the symptoms of CF: breathlessness (since the bronchioles are narrowed); lung infections such as bronchitis and pneumonia (since the cilia can't move the thick mucus and so trapped bacteria aren't killed); poor digestion (since the bile and pancreatic ducts are blocked); poor absorption (since thick mucus slows diffusion in the ileum) and infertility (since the sperm ducts are HGS A-level notes NCM/6/07 Module 2 - Genetics - page 58 blocked). Of these symptoms the lung effects are the most serious, causing 95% of deaths. CF is always fatal, though life expectancy has increased from 1 year to about 20 years due to modern treatments. These treatments include physiotherapy many times each day to dislodge mucus from the lungs; antibiotics to fight infections; DNAse and protease drugs to loosen the mucus; enzymes to help food digestion and even a heart-lung transplant. Given these complicated (and ultimately unsuccessful) treatments, CF is a good candidate for gene therapy, and was one of the first diseases to be tackled this way. The gene for CFTR was identified and cloned in 1989. The idea is to deliver copies of this good gene to the epithelial cells of the lung, where they can be incorporated into the nuclear DNA and make functional CFTR chloride channels. If about 10% of the epithelial cells could be genetically modified, this would allow enough chloride ions to be transported to relieve the symptoms of the disease. Note that gene therapy doesn't alter or replace the existing mutated gene, which will still continue to make useless CFTR channels. But in addition, the new gene will make working CFTR channels, which will allow the epithelial cells to function normally. Two methods of delivery are being tried: liposomes and adenoviruses (see p 45), both delivered with an aerosol inhaler, like those used by asthmatics. Clinical trials are currently underway, but as yet no therapy has been shown to be successful. SCID Severe Combined immunodeficiency Disease (SCID) is a rare genetic disease that affects the immune system. It is caused by a mutation in the gene for the enzyme adenosine deaminase (ADA). Without this enzyme white blood cells cannot be made, so sufferers have almost no effective immune system and would quickly contract a fatal infection unless they spend their lives in sterile isolation (SCID is also known as “baby in a bubble syndrome”). Gene therapy has been attempted with a few children in the USA and UK by surgically removing bone marrow cells (which manufacture white blood cells in the body) from the patient, transfecting them with a geneticallyengineered virus containing the ADA gene, and then returning the transformed cells to the patient. The hope is that these transformed cells will multiply in the bone marrow and make white blood cells. The trials are still underway, so the success is unknown. HGS A-level notes NCM/6/07 Module 2 - Genetics - page 59 The Future of Gene Therapy Gene therapy is in its infancy, and is still very much an area of research rather than application. No one has yet been cured by gene therapy, but the potential remains enticing. Gene therapy need not even be limited to treating genetic diseases, but could eventually also help in treating infections and environmental diseases e.g.: • White blood cells have been genetically modified to produce tumour necrosis factor (TNF), a protein that kills cancer cells, making these cells more effective against tumours. • Genes could be targeted directly at cancer cells, causing them to die, or to revert to normal cell division. • White blood cells could be given antisense genes for HIV proteins, so that if the virus infected these cells it couldn’t reproduce. It is important to appreciate the different between somatic cell therapy and germ-line therapy. • Somatic cell therapy means genetically altering specific body (or somatic) cells, such as trachea epithelial cells, bone marrow cells, pancreas cells, or whatever, in order to treat the disease. This therapy may treat the disease in the patient, but any genetic changes will not be passed on the offspring of the patient. • Germ-line therapy means genetically altering those cells (sperm cells, sperm precursor cells, ova, ova precursor cells, zygotes or early embryos) that will pass their genes down the “germ-line” to future generations. Alterations to any of these cells will affect every cell in the resulting human, and in all his or her descendants. Germ-line therapy would be highly effective, but is also potentially dangerous (since the long-term effects of genetic alterations are not known), unethical (since it could easily lead to eugenics) and immoral (since it could involve altering and destroying human embryos). It is currently illegal in the UK and most other countries, and current research is focussing on somatic cell therapy only. All gene therapy trials in the UK must be approved by the Gene Therapy Advisory Committee (GTAC), a government body that reviews the medical and ethical grounds for a trial. Germ-line modification is allowed with animals, and indeed is the basis for producing GMOs. HGS A-level notes NCM/6/07