Volume 2: Science
1. Introduction
Background to the science of BSE
DNA, genes and chromosomes
Protein folding and degradation
Infections and immunity
Strain-typing, western blotting and titration

1.13 Readers unfamiliar with biology may find the following paragraphs helpful in understanding the scientific terms used in this volume. We have endeavoured to make the text comprehensible to a general reader, but the spongiform encephalopathies are complex disorders and it is inevitable that scientific terms need to be used. Most such terms are defined in the glossary at the end of the volume, and so we confine the following to a description of some of the elementary biological principles and technology which underpin the science. Those already familiar with the science should move directly to Chapter 2.

DNA, genes and chromosomes

1.14 Living organisms are made of cells. ¹ The cell wall (or cell surface membrane) controls what enters or leaves the cell. Both the cell wall and the contents of the cell comprise chemical components called molecules. In cells, these molecules consist largely of proteins, carbohydrates (which include sugars), fats, minerals and water. All organisms have a set of instructions specifying how to make every cell and every molecule within these cells. These instructions are encoded within each cell in molecules of DNA (deoxyribonucleic acid). In humans, almost all this DNA is contained within a structure known as the cell nucleus and is packaged into chromosomes. Each chromosome is essentially an extremely long molecule of DNA made up of two chains coiled around one another in a double helix (Figure 1.1). The two chains are linked, like a rope ladder, by nitrogenous bases (chemical compounds forming part of the molecule) which pair with bases on the opposite strand, adenine with thymine (A-T) and guanine with cytosine (G-C). Each set of three bases is called a codon. The order of bases along the DNA helix constitutes the genetic code, and will determine the constitution of every protein made by the cell.

Figure 1.1: a - DNA double helix section; b - Schematic representation of a single (coding) strand of DNA

1.15 Proteins perform many vital functions both within cells and outside them. They are an important part of our diet. Normally, they are broken down during digestion. The constituent parts will be used by our cells to make the protein we need. Proteins produced by our cells include keratin, which forms our hair and skin, and collagen, which forms the supporting tissue for muscles, tendons and organs. Other proteins provide us with a range of enzymes, hormones and antibodies. The presence of proteins in the body, how they work and what they do, have an important effect on the phenotype - the physical characteristics (both visible and invisible) of any particular individual. The phenotype is determined both by the organism's genotype (see paragraph 1.30 below) and by its environment.

1.16 Proteins are long chains of amino acids, bound together into a three-dimensional structure with a shape unique to each particular protein. These chains are formed in accordance with instructions in the DNA, and when these instructions are used to create proteins, each codon corresponds to a particular amino acid to be used in building the protein molecule. There are four possibilities for each of the three bases in the codon (A, T, G and C), so there are 4³ or 64 possible different codons. Three of the codons are instructions to stop building, which leaves 61 to code for particular amino acids. Only 20 different amino acids exist, so more than one codon can code for a particular amino acid. The codon for one of the amino acids, methionine, also codes for a start signal. In almost all forms of life the code is the same: whether in a human or in a bacterium, any particular triplet of the four bases codes for the same amino acid. There are enough combinations of amino acids to generate a very large number of different proteins. Proteins are made in parts of the cell outside the nucleus (Figure 1.2), and the relevant segment of the DNA code is conveyed from the nucleus to the sites of protein production by means of messenger ribonucleic acid (mRNA). Messenger RNA is the transcript of the DNA code required for one protein.

Figure 1.2: Protein synthesis

1.17 Segments of DNA, each made up of a thousand or more base pairs, contain the code for each protein. These segments are the genes. In fact, genes tend to be much larger than the coding region as they usually contain several non-coding segments and stretches of DNA involved in regulating gene expression.

1.18 Genes are the essential parts of the chromosomes. The chromosome complement carries the genetic information in about 50,000-80,000 genes; recent studies suggest that the lower number is most likely. In addition to genes, chromosomes contain repeated sequences of bases which are not transcribed and are of uncertain function.

1.19 In all organisms, cells multiply by dividing into two. In body cells this is done by a process known as mitosis. Before the onset of mitosis the DNA in the cell synthesises a copy of itself. Accurate replication of DNA must occur if cell division is to take place without error. We give a greatly simplified description of this complex process. Before mitosis occurs, the two strands of the DNA's double helix separate at a number of points (up to 100 per chromosome) and each strand serves as a template upon which the missing partner can be reconstructed by base pairing with free nucleotides (these are bases which are attached to sugar and phosphate groups found in the cells). Replication proceeds in both directions from each initiation point until the two new strands of DNA are complete. At this point, each chromosome appears to be split longitudinally into two 'chromatids', held together at the site of attachment to the mitotic spindle (Figure 1.3). Mitosis now takes place. The nuclear membrane disappears and the two chromatids of each chromosome are drawn apart. The cell membrane reforms between the two groups, thus forming two new 'daughter' cells.

Figure 1.3: Mitosis

1.20 During DNA synthesis the occasional mishap may occur in which the wrong base becomes incorporated into the new DNA strand. This is the simplest way by which a mutation (a change in the genetic material) can arise. A mutation of this kind is called a point mutation. Mutations can also arise through the insertion of one or more bases or longer sequences of DNA into the DNA strand (insertional mutations) or through the deletion of bases (deletion mutations).

1.21 The genes are ordered along the length of the chromosomes, each gene having a fixed position, or locus, in relation to the others (Figure 1.4). Genes at the same locus are called alleles. There may be many alternative pairs of alleles which are normal and not associated with disease. At a particular locus on the chromosome pair, the alleles may be identical and the individual is said to be homozygous at that locus. When the two alleles are different, the individual is heterozygous.

Figure 1.4: Schematic representation of a locus and alleles

1.22 Genetic information is passed from one generation to the next through the formation of eggs and sperm. Generally human body cells have 23 pairs of chromosomes, one member of each pair being contributed by each parent - by the father through the sperm, and by the mother through the egg. Cells with two sets of chromosomes in this way are called 'diploid' cells. But each sperm or egg is a cell which contains only one member of each pair (a total of 23 chromosomes). Because they have only a single copy of each chromosome, they are said to be 'haploid' cells. Fertilisation of an egg by a sperm produces a united diploid cell with the normal complement of 46. As there are two chromosomes of each sort, there are two similar genes of each sort, one from each parent. Chance determines which member of a pair of chromosomes is present in any individual sperm or egg.

1.23 The random assortment of maternal and paternal chromosomes into each gamete (sperm or egg) helps to ensure that no two sperm or eggs are genetically identical. Their uniqueness is further assured by a process of crossing-over between maternal and paternal chromosomes whereby parts of chromosomes are exchanged. This results in 'shuffling' of alleles so that each chromosome in the gamete has a new combination of parental alleles. The mechanism for this occurs during a special type of cell division, termed meiosis, which is used to produce the gametes and reduces the number of chromosomes from the diploid number (46) to the haploid number (23). In meiosis (Figure 1.5) there are two cell divisions although DNA synthesis occurs only once, before the first division. At the first division, the maternal and paternal chromosomes fuse together to form a bivalent, during which crossing-over occurs. This is followed by separation of the two fused chromosomes, which pass into two daughter cells. These daughter cells thus contain 23 chromosomes each of which is composed of two chromatids, which differ from one another as a result of crossing-over. The second meiotic division is like a mitotic division in which the chromatids separate from one another and pass into two separate cells. Each meiotic event thus produces four cells, each with a different combination of alleles due to random assortment of chromosomes and a recombination of maternal and paternal alleles. Meiosis provides two opportunities for mutation to occur, either at the time of DNA synthesis or at crossing-over.

Figure 1.5: Meiosis

1.24 We explained in paragraph 1.21 above that alleles may differ. Why is this so? As described at paragraph 1.20 above, when cells divide mutations may occur. We shall explain below how these mutations can be passed on to children, grandchildren and later generations. A mutation in the distant past may have been inherited to such an extent that a variant of the allele is present in more than 1 per cent of the population. In traditional population genetics, such variations were referred to as 'polymorphisms', and the locus on the chromosome where the gene is found was said to be 'polymorphic'. More recently the term 'polymorphism' has been applied to changes in DNA which do not appear to affect the phenotype.

1.25 The variation between one allele and another may involve loss, gain or replacement of a single base pair (known as a point mutation) or deletion or insertion of a number of contiguous base pairs in the DNA molecule. Whether large or small, these variations in the DNA code may interfere with the structure or function of proteins (in particular because they alter the instructions for protein construction in the cell), and this in turn may lead to particular characteristics in the phenotype of the individual. Many of these characteristics are harmless (such as eye colour) and some may be beneficial. But some are the characteristics of disease, and thus have the potential to cause harm to the individual. Other changes in DNA are neutral: they do not change the function of the protein.

1.26 For some types of inherited characteristic (including diseases) to express themselves it is enough that only one of the offspring's paired chromosomes has the mutant gene. If only one of the paired chromosomes has the mutant gene, the individual is heterozygous for that gene mutation in all his or her cells, and risks passing on the abnormal gene of a characteristic in sperm or egg to half his or her offspring. This form of inheritance is known as autosomal dominant inheritance: autosomal, because the mutation is carried on a non-sex chromosome; and dominant because the characteristic occurs when one or both of the chromosome pair carries the mutant gene. Characteristics which occur only when both chromosomes of the pair carry the mutant gene are termed recessive.

1.27 The discussion in the preceding paragraph requires qualification in relation to sex chromosomes, which in males are not a complete pair. In the case of the sex chromosomes, the female has a true pair of two X chromosomes. She may then be homozygous or heterozygous for particular X chromosome mutations. The male, however, has a 'pair' which do not completely match - one X and one Y chromosome. The Y chromosome is very much smaller than the X. Some mutations of the X chromosome will give rise to particular characteristics (including diseases) in the absence of a normal matching X chromosome to counteract the mutation. These are called sex-linked recessive characteristics. They include colour blindness and haemophilia. Any male who inherits the mutated X chromosome from his mother will express the associated characteristic. A female inheriting the mutated X chromosome from her mother will express the characteristic if it is present in her father as well.

1.28 A mutation of a gene may also arise spontaneously in a body cell and spread locally in the tissue by cell replication. If a mutation arises in a sperm or egg cell, it may be transmitted to offspring derived from the mutated cell. Both in this case, and in the inherited cases described in the previous paragraph, we speak of a 'germ line mutation', because the mutation is present in the 'germ cells' (or gametes) - the egg cell in the female and the sperm cell in the male - which will produce the next generation. A germ line mutation is sometimes described as a 'familial mutation', because it is inherited in families.

1.29 Cells which are not germ cells are called 'somatic' cells. We describe a spontaneous mutation in a somatic cell as a 'somatic mutation'. The characteristic (which may include disease) associated with the mutation will be expressed in the same way, in those cells that are affected, as a familial mutation, because the mutant gene has the same effect. (Thus if a mutant gene causes the diffusion of an abnormal protein through the body, this can be produced both by a somatic and by a familial mutation.) However, a somatic mutation will not be transmitted to offspring because it does not occur in sperm or eggs.

1.30 The complement of genes of an organism is known as its genotype. Expression of the genotype gives rise to an organism's phenotype (see paragraph 1.15 above). 'Genotype' can also be used to refer to the pair of genes specifying a particular characteristic or protein. The total DNA in a single cell is called the 'genome'. The total DNA content of a germ cell (with only a single copy of each chromosome) is sometimes called the 'haploid genome' in contrast to the 'diploid genome' of somatic cells (the non-germ cells which have two sets of chromosomes).

Protein folding and degradation

1.31 The function of a protein depends not only on its amino acid content but also on its three-dimensional structure. Although each protein is produced as a long chain of amino acids, it quickly assumes a specific conformation due to the formation of chemical bonds between its constituent amino acids. The resulting shape of each protein is characteristic of the protein and confers on it the ability to interact with other proteins and molecules. For example, the shape of the protein globin allows it to combine with molecules of iron to form the oxygen-carrying pigment of red blood cells. If a mutation alters the amino acid content of a protein, this may alter the shape of the protein molecule and interfere with its capacity to interact with other proteins, so resulting in loss of function.

1.32 Animal and plant cells are constantly making proteins. They are also constantly unmaking ('degrading') them. The time lapse between making and degrading a particular protein is generally no more than a matter of weeks, and may be a few minutes. A short turnover time has the advantage of allowing a rapid response to changes in the cell environment. A common way in which proteins are degraded is by 'enzyme digestion'. This is a process in which an enzyme (itself a protein) uses water to split the protein into amino acids or groups of amino acids called peptides. The enzymes which perform this function are called 'proteinases' or 'proteases'.

1.33 The degrading of a protein by an enzyme is also dependent on the protein having its normal three-dimensional structure. A change in shape may make the protein resistant to degradation. As we shall see in relation to the pathogenesis of TSEs, alteration in the three-dimensional structure of the prion protein is associated with resistance to degradation, and results in the accumulation of insoluble aggregates with the consequent destruction of nerve cells which is the hallmark of such diseases.

1.34 One of the ways in which the function of a protein can be determined is by studying the effect on the phenotype when the gene for that protein is absent or defective. Opportunities for this occur, for example, in the investigation of patients with genetic disease due to a single gene mutation. Studies can also be undertaken in experimental mice in which the gene in question is removed, altered or replaced by the techniques of gene modification. The replacement of a mouse gene by a synthetic gene or by a gene from another species produces what is referred to as a transgenic mouse (Figure 1.6). Genetically modified mice in which a mouse gene has been deleted (knockout mice) have provided important evidence about the function of the protein coded by the gene in the study of human diseases. Other experiments with transgenic mice using a construct containing a mutant human gene can also help in understanding the effects of the mutation.

Figure 1.6: Generation of transgenic mice

Infections and immunity

1.35 The origin and development of a disease is called 'pathogenesis'. Micro-organisms (organisms invisible to the human eye) and certain other substances causing disease are called 'pathogens', and anything of this kind which produces disease is said to be 'pathogenic'. Infectious diseases are caused by bacteria, viruses and fungi among other pathogens. The pathogen may be transmitted by inhalation (eg, pneumonia), by ingestion (eg, food poisoning) or by direct contact in particular ways (eg, HIV, the precursor of AIDS). It may also be transmitted in the womb (eg, congenital rubella).

1.36 We have mentioned earlier the suggestion that the TSE agent might be a virus. viruses are composed of nucleic acid (either DNA or RNA) and viral protein (Figure 1.7). They can only propagate within the living cells of a host. 'Slow viruses, for example HIV, have evolved a relationship within the host's cells which allows them time to proliferate before being attacked by the host's defence mechanisms. Nonetheless, the presence of slow viruses can be identified by tests which utilise the immune system (as described in the next paragraph).

Figure 1.7: Schematic diagram of the human immunodeficiency virus (HIV) as a model for virus structure

1.37 Infections with pathogens evoke a defensive response from the host through the host's immune system. During early development of the immune system, humans establish the means to attack foreign proteins. In order to do so, however, they must first establish tolerance to their own proteins, so that the immune system does not attack them. The system reacts to the foreign proteins of the invading pathogen by mobilising the white cells of the blood (macrophages) to engulf and destroy the invader. This process is associated with inflammation (ie, redness, tenderness), rise in temperature and swelling, due to increased blood supply at the point of infection. Other white cells (plasma cells) in the lymph nodes and other tissues of the lymphoreticular system (LRS) - spleen, thymus, tonsils, appendix, etc - respond by generating antibodies to the foreign proteins (antigens). The antibody response is the second-line response to a primary infection and may take some days to develop. Reinfection with the same pathogen is countered more quickly as the plasma cells are already programmed to produce the appropriate antibody. The antibody response is the basis for the use of vaccination in the prevention of infectious disease.

1.38 In exceptional circumstances an individual will develop an immune response to its own tissues. The exception occurs as a result of the entry into the body of a foreign protein which in part mimics one of the host's own proteins. The vector of the mimicking protein is usually a micro-organism. The antibodies produced by the host against the foreign protein then cross-react with the host protein, which may lead to inflammation and all the other signs of an immune response. This class of disease is referred to as autoimmune disease. One of the best known examples in humans is rheumatic carditis, in which antibodies produced by the host against a bacterium (Group A Streptococcus) may cross-react with protein in the internal lining of the heart and heart valves leading to chronic heart failure.

Strain-typing, western blotting and titration

1.39 The identity of a particular micro-organism can be determined by one or other of a variety of laboratory tests. The morphology (form and structure) of a bacterium can be investigated using a light microscope, but an electron microscope is required to visualise a virus. Bacteria may be further characterised by their ability to grow in specially defined culture media, or by their sensitivity or resistance to antibacterial agents. Many diagnostic tests depend on the use of specific antibodies raised in experimental animals to kill the micro-organism. More recently, analysis of nucleic acid sequences within the micro-organism has been exploited to provide the most sensitive means of determining its particular type. Such analysis may also help in investigating the source of an outbreak of disease, for example E. coli 0157 infection. In this case '0157' can be described as a 'strain' of E. coli, meaning a genetically distinct type of E. coli bacteria.

1.40 Inoculation of experimental animals was used in the past to identify pathogens. Thus, the bacillus of tuberculosis was identified by transmission to guinea pigs. This procedure is no longer required as it has been replaced by the alternative methods described above. However, TSEs are the exception, and bioassay in experimental host animals, particularly mice, remains the principal means for the detection of TSE infectious agents. Different inbred lines of mice, variously sensitive or resistant to the TSE agent, are used and by this means a number of different strains of scrapie and CJD can be distinguished. The types of TSE agent are identified by the length of the incubation period (the time between inoculation and onset of the disease) and by the pattern of disease produced in the brain of the experimental host. This process is called strain-typing. Confusingly, the lines of experimental host animals are also called 'strains', but the object of strain-typing is to identify strains of the infective agent.

1.41 One commonly used technique for the identification of specific antigens within pathogens, and especially TSE agents, is known as western blotting (Figure 1.8). This technique distinguishes proteins on the basis of their size and depends on the availability of antibodies against the protein of interest. Proteins of different size can be separated from one another by the rate at which they move across a gel under the influence of an electric field (electrophoresis); smaller proteins move faster than larger proteins. The position of each protein can be detected in the gel by having an antibody to the protein labelled with a dye. When the position of the protein of interest is compared with the position of proteins of known size, an estimate can be made of the size of the test protein. The detection is easier if the proteins are transferred from the gel to a filter prior to detection with the antibody. This part of the procedure is known as blotting.

1.42 As particular antigenic proteins may differ in size in different varieties of the same pathogen, western blotting can be a useful adjunct in strain-typing (Figure 1.9).

Figure 1.8: Western blotting: the process

Figure 1.9: Western blotting: examples

1.43 Titration is a process used in chemistry to ascertain the amount of a substance in a solution by measuring the volume of a standard reagent required to react with it. In the absence of alternative methods of identification of the TSE agent, titration can only be conducted by a technique using experimental animals, usually mice (Figure 1.10). A known mass of tissue to be assayed is ground up in salt solution to produce a fine suspension to a 1 per cent concentration. A series of dilutions containing 0.1 per cent, 0.01 per cent and so on, is made and a standard amount of each is injected into groups of mice. At very high dilutions it may be that no animals succumb to disease. At very low dilutions (ie, higher concentrations) they may all do so. The dilution at which 50 per cent of the animals acquire the disease is the unit of infective dose (ID₅₀). The unit of lethal dose (LD₅₀) is that which kills 50 per cent of the animals within their normal lifespan. The degree of infectivity of the original sample can be expressed in ID₅₀ units per gram from the dilution (titre) of the original sample. So, for example, if a 0.001 per cent dilution of infective material results in the death of 50 per cent of animals, the original material before dilution is known to contain 1,000 ID₅₀ units per gram.

Figure 1.10: Titration