AQA(B) AS Module 2: Genes and Genetic Engineering Contents

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1 Module 2 - Genetics - page 1 AQA(B) AS Module 2: Genes and Genetic Engineering Contents Specification 2 DNA Nucleotides 4 DNA Structure 6 DNA Function 7 RNA 8 Replication 10 Transcription 12 Translation 14 Mutations 16 The Cell Cycle DNA and Chromosomes 19 The Cell Cycle and Mitosis 21 Asexual Reproduction 23 Sexual Reproduction 29 Genetic Engineering Techniques 33 Applications 50 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 (nmillar@cwcom.net) Head of Biology, Heckmondwike Grammar School, High Street, Heckmondwike WF16 0AH 10/9/00

2 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 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 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.

3 Module 2 - Genetics - page 3 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. The process of DNA replication can be made to occur artificially and repeatedly in a laboratory process called the polymerase chain reaction (PCR). The use of PCR, radioactive labelling and electrophoresis to determine the sequence of nucleotides in DNA. 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.) 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 How animals can be genetically engineered to produce substances useful in treating human diseases, as 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 or Mendelian Genetics, which is the study of heredity at the whole organisms level by looking at how characteristics are inherited. This method was pioneered by Gregor Mendel ( ). It is less fashionable today than molecular genetics, but still has a lot to tell us. This is covered in Module 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.

4 Module 2 - Genetics - page 4 DNA DNA and its close relative RNA are perhaps the most important molecules in biology. They contains 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 have three parts to them: a phosphate group ( PO 2 4- ), which is negatively charged, and gives nucleic acids their acidic properties. a pentose sugar, which has 5 carbon atoms in it. By convention the carbon atoms are numbered as shown (1', 2', etc, read as "one prime", "two phosphate sugar base - O - O P O CH2 prime", etc), to distinguish them from the carbon atoms in the base. 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: O 5 4 C C 3 O C 2 C 1 OH OH N Base: Adenine (A) Cytosine (C) Guanine (G) Thymine (T) Uracil (U) Nucleotide: Adenosine Cytidine Guanosine Thymidine Uridine The bases are usually known by there 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

5 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). This shows a nucleotide triphosphate molecule. - O - O P O - O O P O - O O P O O 5 4 CH2 C C 3 O C C 2 OH OH 1 N Nucleotide Polymerisation 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 the polymerisation, so there is a sugar-phosphate backbone with the bases extending off it. This means that the nucleotides can join together in any order along the chain. Two nucleotides form a dinucleotide, three form a trinucleotide, a few form an oligonucleotide, and many form a polynucleotide. A polynucleotide has a free phosphate group at one 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 the sugar. The terms 3' and 5' are often used to denote the different ends of a DNA molecule.

6 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.)

7 Module 2 - Genetics - page 7 Function of DNA DNA is the genetic material, and genes are made of DNA. DNA therefore has two essential functions: replication and expression. Replication means that the DNA, with all its genes, must be copied every time a cell divides. Expression means that the genes on DNA must control characteristics. A gene was traditionally defined as a factor that controls a particular characteristic (such as flower colour), but a much more precise definition is that a gene is a section of DNA that codes for a particular protein. Characteristics are controlled by genes through the proteins they code for, like this: Expression can be split into two parts: transcription (making RNA) and translation (making proteins). These two functions are summarised in this diagram (called the central dogma of genetics). No one knows exactly how many genes we humans have to control all our characteristics, but the current best estimates are between 60 and 80 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) * no of genes phage λ virus Eschericia coli Bacterium Saccharomyces cerevisiae Yeast Caenorhabditis elegans nematode worm ~ Drosophila melaogaster fruit fly ~ Homo sapiens Human ~ * kbp = kilo base pairs, i.e. thousands of nucleotide monomers. 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!

8 Module 2 - Genetics - page 8 RNA RNA is a nucleic acid like DNA, but with 4 differences: RNA has the sugar ribose instead of deoxyribose RNA has the base uracil instead of thymine RNA is usually single stranded, but can fold into 3-dimentional structures, like proteins. RNA is usually shorter than DNA 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 subunits, small and large, and are assembled in the nucleolus of the 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 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 molecule there is always the base sequence ACC, where the amino acid binds. On the middle loop there is a triplet nucleotide sequence called the anticodon. There are 64 different trna molecules, each 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 adapter with the appropriate anticodon.

9 Module 2 - Genetics - page 9 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 4 3 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. 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 simple 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.

10 Module 2 - Genetics - page 10 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. Replication starts at a specific sequence on the DNA molecule called the replication origin. 2. The enzyme helicase unwinds and unzips DNA, breaking the hydrogen bonds that join the base pairs, and forming two separate strands. 3. The new DNA is built up from the four nucleotides (A, C, G and T) that are abundant 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. A winding enzyme winds the new strands up to form double helices. 7. The two new molecules are identical to the old molecule.

11 Module 2 - Genetics - page 11 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. 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.

12 Module 2 - Genetics - page 12 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. 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 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 stand that is copied is called the template or sense strand because it contains the sequence of bases that codes for a protein. The other strand is just a complementary copy, and is called the non-template or antisense 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. The initial mrna, or primary transcript, contains many regions that are not needed as part of the protein code. These are called introns (for interruption sequences), while the parts that are

13 Module 2 - Genetics - page 13 needed are called exons (for expressed sequences). All eukaryotic genes have introns, and they are usually longer than the exons. 6. The introns are cut out and the exons are spliced together by enzymes. Some of this 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. 7. The result is a shorter mature RNA containing only exons. The introns are broken down. 8. The mrna diffuses out of the nucleus through a nuclear pore into the cytoplasm.

14 Module 2 - Genetics - page 14 Translation - Protein Synthesis 1. A ribosome attaches to the mrna at an initiation codon (AUG). The ribosome encloses two codons. 2. met-trna diffuses to the ribosome and attaches to the mrna initiation codon by complementary base pairing. 3. The next amino acid-trna attaches to the adjacent mrna codon (leu in this case). 4. The bond between the amino acid and the trna is cut and a peptide bond is formed between the two amino acids. This operation is catalysed by an rrna-protein complex called a ribozyme. 5. The ribosome moves along one codon so that a new amino acid-trna can attach. The free trna molecule leaves to collect another amino acid. The cycle repeats from step The polypeptide chain elongates one amino acid at a time, and peels away from the ribosome, 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 the finished protein.

15 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. 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, or adding sugars (to make glycoproteins) or lipids (to make lipoporteins). Regulation of Gene Expression Not all genes make proteins. Some make trna and rrna, which of course are never translated into proteins. Genes that make things are called structural genes (even though they don t just make structural proteins), but many genes don t make anything. Instead they 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. 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) also 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 very poorly understood.

16 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). Changes in DNA can lead to changes in cell function like this: There are basically three kinds of gene mutation, shown in this diagram: The actual effect of a single mutation depends on many factors: A substitution on the third base of a codon may have no effect because the third base is less important (e.g. all codons beginning with CC code for proline). If a single amino acid is changed to a similar one (e.g. both small and uncharged), then the protein structure and function may be unchanged, but if an amino acid is changed to a very different one (e.g. an acidic R group to a basic R group), then the structure and function of the protein will be very different. If the changed amino acid is at the active site of the enzyme then it is more likely to affect enzyme function than if it is part of the supporting structure. Frame shift mutations are far more serious than substitutions because more of the protein is altered (though even a single amino acid change can have a big effect).

17 Module 2 - Genetics - page 17 If a frame-shift mutation is near the end of a gene it will have less effect than if it is near the start of the gene. 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. It a mutation leads to a premature stop codon the protein will be incomplete and non-functional. If the mutation is in a gene that is not expressed in this cell (e.g. the insulin gene in a red blood cell) then it won't matter. If the mutation is in an intron (or the 98% junk DNA) then it probably won't matter. This may even be why so many introns exist. Some proteins are simply more important than others. For instance non-functioning receptor proteins in the tongue may lead to a lack of taste but is not life-threatening, whereas nonfunctioning haemoglobin is fatal. Some cells are more important than others. Mutations in somatic cells (i.e. non-reproductive body cells) will only affect cells that derive from that cell, so will probably have a small local effect like a birthmark (although they can cause widespread effects like diabetes or cancer). Mutations in germ cells (i.e. reproductive cells) will affect every single cell of the resulting organism as well as its offspring. These mutations are one source of genetic variation. As a result of a mutation there are three possible phenotypic effects: Most mutations have no phenotypic effect. 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. Most 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 cell doesn't happen, so the cell's function is changed. An example of this is the genetic disease phenylketonuria (PKU), caused by a mutation in the gene for the enzyme phenylalanine hydroxylase. This causes a metabolic block in the pathway involving the amino acid phenylalanine, which builds up, causing mental retardation. 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.

18 Module 2 - Genetics - page 18 The kinds of mutations discussed so far are called point or gene mutations because they affect specific points within a gene. There are other kinds of mutation that can affect many genes at once or even whole chromosomes. These chromosome mutations can arise due to mistakes in cell division. A well-known example is Down syndrome (trisonomy 21) where there are three copies of chromosome 21 instead of the normal two. 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. These ionise the bases so that they don't form the correct base pairs. 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.

19 Module 2 - Genetics - page 19 DNA and Chromosomes The DNA molecule in a single human cell is 99 cm long, so is times longer than the cell in which it resides (< 100µm). (Since an adult human has about cells, all the DNA is one human would stretch about 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 times shorter than fully stretched DNA, and therefore times thicker, so are thick enough to be seen under the 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. one chromatid centromere micrograph of a single chromosome Chromatin Chromosome Chromatid DNA + histones at any stage of the cell cycle compact X-shaped form of chromatin formed (and visible) during mitosis single arm of an X-shaped chromosome

20 Module 2 - Genetics - page 20 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 genes per chromosome, although of course the larger chromosomes have more than this, and the smaller ones have fewer. 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. 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. Homologous chromosomes are a result of sexual reproduction, and the homologous pairs are the maternal and paternal versions of the same chromosome, so they have the same sequence of genes 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

21 Module 2 - Genetics - page 21 homologous in females (XX) and non-homologous in males (XY). In other species it is the other way round. The non-sex chromosomes are sometimes called autosomes, so humans have 22 pairs of autosomes, and 1 pair of sex chromosomes. 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 three phases: 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. 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. The growth and synthesis phases are collectively called interphase (i.e. in between 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.

22 Module 2 - Genetics - page 22 Cell Division by Mitosis Interphase chromatin not visible DNA, histones and centrioles all replicated Prophase chromosomes condensed and visible centrioles at opposite poles of cell nucleolus disappears Metaphase Anaphase Telophase Cytokinesis nuclear envelope disappears chromosomes align along equator of cell spindle fibres (microtubules) connect centrioles to chromosomes centromeres split, allowing chromatids to separate chromatids move towards poles, centromeres first, pulled by kinesin motor proteins walking along microtubule the track spindle fibres disperse nuclear envelopes from nucleoli form 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.

23 Module 2 - Genetics - page 23 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. 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. These different methods are summarised in the table. Microbes Plants Animals binary fission budding spores fragmentation Methods of Asexual Reproduction Natural Methods Artificial Methods vegetative propagation parthenogenesis budding fragmentation parthenogenesis invertebra only tes cell culture fermenters cuttings grafting tissue culture embryo splitting somatic cell cloning any animal Natural Methods Binary Fission. This is the simplest and fastest method of asexual reproduction, used by all prokaryotes and by many unicellular protoctists (such as amoeba and paramecium). The nucleus divides by mitosis and then the cell simply splits into two equal-sized daughter cells. Budding. A small copy of the parent develops as an outgrowth, or bud, from the parent, and is eventually released as a independent individual. This method is used by several protoctists, yeasts (fungi) and even by some animals (sponges and cnidarians). Spores. These are simply specialised cells that are released from the parent (usually in very large numbers) to be dispersed. Under suitable conditions each cell can grow into a new individual. They are therefore a bit like seeds, but seeds are strictly used in sexual reproduction. This method is used by most fungi and by the lower plants (mosses and ferns).

24 Module 2 - Genetics - page 24 Fragmentation. This is when an organism spontaneously breaks into two or more fragments, each of which then develops into a new individual. It is used by some simple animals such as sponges, flatworms, ribbon worms and starfish. Do not confuse this with regeneration, where some animals can regenerate a part of their body if it is cut off, but do not reproduce this way (e.g. earthworms, crabs). Vegetative Reproduction. This term describes all the natural methods of asexual reproduction used by plants. 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), runners (e.g. strawberry, creeping buttercup), tubers (e.g. potato, dahlia), tap roots (e.g. carrot, turnip), and tillers (e.g. grasses). 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. Parthenogenesis. This is used by some plants (e.g. the citrus fruits) and some invertebrate animals (e.g. honeybees, aphids, some crustaceans) as an alternative to sexual reproduction. Egg cells simply develop into adult clones without being fertilised. These clones may be haploid, or the chromosomes may replicate to form diploid cells. Artificial Methods Cloning 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, so artificial methods have to be used.

25 Module 2 - Genetics - page 25 Cell Culture. Microbes (bacteria and some fungi) can be cloned very easily in the lab using their normal asexual reproduction. Microbial cells can be isolated and identified by growing them on a solid medium in an agar plate, and selected strains can then be grown up on a small scale in a liquid medium in a culture flask. Fermenters. In biotechnology, fermenters are vessels used for growing microbes on a large scale. Fermenters must be stirred, aerated and thermostated, materials can added or removed during the fermentation, and the environmental conditions (such as ph, O 2, pressure and temperature) must be constantly monitored using probes. This will ensure the maximum growth rate of the microbes. 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 plant can be cloned many times. Rooting is helped if the cuttings are dipped in rooting hormone (auxin). Many flowering plants, such as geraniums, pelargoniums, 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 its size will be determined by the rootstock. Almost all fruit trees, such as apples and pears are clones of a few popular varieties grafted onto hardy rootstock.

26 Module 2 - Genetics - page 26 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 now be grown on agar plates in the laboratory in much the same way that bacteria can be grown. Initially the explant had to be meristem tissue (i.e. undifferentiated buds), but the technique has improved so that any tissue can now be used (e.g. from a leaf). The plant tissue can be separated into individual cells, each which can grow into a mass of cells called a callus, and 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. Micropropagation is used on a large scale for fruit trees, ornamental plants and plantation crops such as oils palm, data 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 disease-free plants can be grown from a few disease-free cells. In the field, almost all crop plants are infected with viruses. the technique works for plants species that cannot be asexually propagated by other means, such as palms and bananas. a single cell can be genetically modified and turned into many identical plants Although some animal cells can be grown in culture, they cannot be grown into complete animals, so tissue culture cannot be used for cloning animals. 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 genetically engineered animals. A female animal is fed 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.)

27 Module 2 - Genetics - page 27 The identical embryos can then be transplanted into the uterus of surrogate mothers, where they will develop and be born normally. Could humans be cloned this way? Almost certainly yes. A human embryo was split and cloned to the stage of a few cells in the USA in 1993, just to show that it is possible. However experiments with human embryos are now banned 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 animal (in contrast to plants). However, techniques have gradually been developed to do this, first with frogs in the 1970s, and most recently with sheep (the famous Dolly ) in The technique used to create Dolly is similar to embryo cloning, but has one crucial difference. The cell used for Dolly was from the skin of the udder, so was a fully differentiated somatic cell. This 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 hundreds of attempts to achieve success with Dolly, but once the technique is improved it will be possible to combine this technique with embryo cloning to make many clones of an adult animal. Dolly s mother was just an ordinary sheep, but in the future prize animals (or genetically engineered ones) could be cloned in this way.

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29 Module 2 - Genetics - page 29 Sexual Reproduction Sexual reproduction is the production of offspring from two parent using gametes. The cells of the offspring have two sets of chromosomes (one from each parent), so are diploid. Sexual reproduction involves two stages: Meiosis- the special cell division that makes haploid gametes Fertilisation- the fusion of two gametes to form a diploid zygote These two stages of sexual reproduction can be illustrated by a sexual life cycle: 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, but the details vary with different organisms. In the animal kingdom (including humans), and in flowering plants the dominant, long-lived adult form is diploid, and the haploid gamete cells are only formed briefly. 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, which immediately undergoes meiosis to reestablish the haploid state and complete the cycle. In the plant kingdom the life cycle shows alternation of generations. Plants 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) the diploid form is larger.

30 Module 2 - Genetics - page 30 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: The chromosome number is halved from the diploid number (2n) to the haploid number (n). This is necessary so that the chromosome number remains constant from generation to generation. Haploid cells have one copy of each chromosome, while diploid cells have homologous pairs of each chromosome. 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). Gametes The usual purpose of meiosis is to form gametes- the sex cells that will fuse together to form a new diploid individual. In some algae and fungi the gametes are roughly the same size. This is called isogamy. There are no male and female sexes, but there can be + and - strains, who reproduce together. In all plants and animals the gametes are different sizes. This is called heterogamy. The larger gametes tend to be stationary and contain food reserves (lipids, proteins, carbohydrates) to nourish the embryo after fertilisation. These are the female gametes (ova or eggs in animals, ovules in plants), and they are produced in fairly small numbers. Human females for example release about 500 ova in a lifetime. The smaller gametes can move. If they can propel themselves they are called motile (e.g. animal sperm) but if they can easily be carried by the wind or animals they are called mobile (e.g. plant pollen). These are the male gametes, and they are produced in very large numbers. Human males for example release about 10 8 sperm in one ejaculation. 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

31 Module 2 - Genetics - page 31 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 are called unisexual. These diagrams of human gametes illustrate the differences between male and female. 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 tails 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 make 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