Darryl R. J. Macer, Ph.D. Eubios Ethics Institute 1990
Our first reaction to the great advances in science, and in the field of genetics, should be admiration (Haring 1975). The more we understand the language and message of the genes in humans and other creatures, the more we can come to understand our history, and understand better our unique position as living creatures. Humanity has gained the ability to adapt our environment to our genes, and is learning how to adapt our genes to the environment. At the same time we appreciate the universality of the genetic code, and the implications this has for our understandings of our biological origins.
Genetics as a science could be considered to have begun with Mendel,'s experiments with the passing of parental characters in peas in the middle of last century. However, the idea that characters or traits were passed from one generation to the next has been known for many millenium. The cross-breeding that produced agriculturally useful crops such as wheat or corn has a long history. So does the breeding of domestic dogs or cats from wild ancestors. The knowledge that interfamilial marriages lead to more handicapped children, and that blue eyed children come from blue-eyed parents is also very old.
Some of the ideas of how human characters were passed on are fascinating to us now, but they were commonly held beliefs for millenia before we were able to understand them. Aristotle thought that the female supplied the "matter" and the male the "motion" that would determine the child's characteristics. There are similar ideas in ancient manuscripts of other cultures also. However, Hippocrates and Galen, and Islamic writers later, thought that because both man and woman produced semen they contributed equally to the process. As it will be discussed in chapter 12, there were ancient eugenic ideas that arose specifically because of, often incomplete, knowledge. In the nineteenth century in Europe, the human genetic traits were thought to be associated with blood. There were exceptions and irregularities, such as why children could have different eye colour from their parents due to recessive alleles which were to be explained by Mendelian genetics. We now know that blue colour is recessive to brown, so that to have blue colour one must have both genes for blue, if there is just one then the eyes should be brown. There were various theories for the transmission of genes, I will not dwell on them but refer to other books (McKie 1988). The principle alternative was Lamarckism, which thought that body characters acquired during life could be passed in the genes from parents to children.
The physical location of the genes has only been determined since the beginning of this century. In 1908 the American geneticist Thomas Morgan identified the genes to be associated with parts of chromosomes. In 1911 they produced the first chromosome maps, and spent many decades working on genetic studies in fruit fly. In the 1940's Avery showed that traits could be passed from one bacteria to another by a chemical called DNA. We will discuss DNA in the next section, and DNA is a widely known word in most languages today. It was the discovery of DNA as the physical substance of genes which could be said to be the start of molecular biology. Before that people thought proteins were the material, because of their complexity. DNA is chemically very simple compared to proteins, and people thought that the genetic store must be complex. It took another decade before all were convinced of the importance of DNA.
The structure of DNA was determined in 1953, by a group of researchers in Britain, including Watson and Crick, for which numerous accounts have been written (Watson 1968). The double helix structure is also commonly associated with the public image of DNA. The majority of the DNA in a cell takes the form of this double helix, though there are other important structural variations that are thought to be important for gene regulation. There are also larger tertiary structures formed by the DNA, and it is also associated with proteins in most cells. The discovery of how the genetic code is translated to protein sequence took another decade, and during this time the science of molecular biology matured. It has since grown so that it now pervades every part of biology, and has been applied to many areas.
Part of the aim of this book is to bring readers up-to-date on advances in genetic technology. It is intended that this is done so that people who have little background in biology can understand enough to get a clear picture of the ideas and capabilities of these techniques, without a need to understand all the details of the procedures. However, a few terms have to be defined and introduced which will aid this comprehension. This is the purpose of this section.
All organisms are constructed of one or multiple cells. The cell is the basic building block of life. Most cells can reproduce themselves, though in higher organisms some cells may be so specialised that they lose this ability, but may still be able to stay alive for the lifetime of the organism. The information for this continued survival, and for the very existence of cells, is contained in the DNA of a cell. It is the DNA which is the focus of genetic studies. What we may consider to be living organisms depends to some extent on our definition of life, a virus has DNA (or RNA) but can only reproduce using another organisms cellular system, so is not independently alive.
A gene is a sequence of nucleotides that function as a coherent unit. Each gene carries the instructions for a specific protein or an RNA molecule. A series of three nucleotides codes for a specific amino acid. DNA carries the information that is required by an organism. This information is translated to proteins in a sequence specific method. A sequence of nuceotides in the DNA is linearly translated into an amino acid sequence. The intermediate messenger in this process is another nucleic acid called RNA. It is not as stable as DNA, so is not used for information storage except in a few viruses. The cycle of information flow is called the central dogma of molecular biology, and is schematically represented in Figure 2-1.
A typical protein consists of about 300 amino acids. The DNA needed to code for this would be about 1,000 nucleotides long. However, in higher organisms (plants and animals) the gene on the DNA contains an alternating mixture of what are called introns and exons. The total sequence of a gene of this type may also be 1,000 nucleotides, but many are of 10,000 nuceotides in size, and the human dystrophin gene is 2 million nucleotides long! Introns contain sequence that do not code for the protein, and whose function is unknown. Exons are shorter sequences, that code for protein sequences, for example for 60 amino acid units. This process of transcription, the reading of DNA into RNA, and the splicing of the exons in the RNA together to form the messenger RNA to be used for protein synthesis, is represented in figure 2-2.
Modern genetics and molecular biology have led to techniques by which it is possible to find the exact chemical sequence of any gene from any organism. The genotype of an organism is the complete set of genes that they possess. This is determined at the time of conception for multicellular organisms, and is the same in all cells of one individual organism. Every individual of a species possesses a specific genotype, consisting of many genes. The genotype of all cells derived from the same cell will be the same, unless a mutation occurs. For sexually reproducing organisms the genotype of each new individual is different because the genes from two parents are shuffled. The phenotype of an individual is determined by the constant interaction of their genotype and the environment.
The key to modern genetics has been the discovery of DNA. A gene is chemically made of DNA composed of basic building units or bases which are connected in a very long linear string, in a specific sequence. There are only four types of building bases, nucleotides, but because the sequence in any gene (between 400 and 10000 bases) is very long, there are almost an infinite number of possible combinations. The four different nucleotides that make up DNA are adenosine, guanosine, thymidine and cytosine, and are often represented by the letters A, G, T, and C. The two strands of DNA are held together by bonds between the complementary bases: A binds with T, and G binds with C. The information in DNA can be represented as a long sequence of these four letters, and the two strands of the DNA have complementary sequences.
Figure 2-2: Protein Synthesis. Proteins are made on ribosomes, by the reading of a messenger RNA (mRNA) that is transcribed from the DNA. The RNA is spliced in mammalian cells, the function of this is uncertain.
Genetic engineering is now entering its third decade of use. In 1967 an enzyme DNA-ligase was discovered which joined breaks in a DNA chain. It is now widely used to join pieces of DNA. The first artificial gene was made in 1972 by chemical synthesis. There are now automatic DNA synthesisers in most molecular biology laboratories, which are standard equipment used in making probes to be used to screen DNA. Enzymes called restriction endonucleases were found in different bacteria that cut DNA at short, specific sequences of bases. This allows DNA to be chopped into smaller pieces, and methods were used to join the ends of the desired pieces again to other DNA (See figure 2-3). The nucleotide sequence that acts as the recognition signal usually contains the specific nucleotide that the cut is made at, but for some endonucleases the cut may be made at a certain number of nucleotides further along the DNA.
Using these enzymes new pieces of DNA can be incorporated into carriers called vectors as shown in figure 2-4. To allow specific joining of the inserted DNA into the vector, the sticky ends must correspond. If the inserted DNA does not have the correct nucleotide sequence, then short synthetic nucleotide sequences, called linkers, can be added to the ends of the insert DNA before it is joined to the vector.
Enzyme, Bacterial Source
Original DNA Sequence Cut DNA with Sticky Ends
Eco R1, Escherichia coli
5'-G-A-A-T-T-C-3' 5'-G A-A-T-T-C-3'
3'-C-T-T-A-A-G-5' 3'-C-T-T-A-A G-5'
Pst 1, Providencia stuarti
5'-C-T-G-C-A-G-3' 5'-C-T-G-C-A G-3'
3'-G-A-C-G-T-C-5' 3'-G A-C-G-T-C-5'
During 1973-1976 there was a voluntary moratorium imposed by scientists on the practise of introducing foreign DNA into bacteria. The fears were that moving genes widely could have bad consequences, for instance it could cause the spreading in the microbial world of antibiotic resistance, or toxin formation; or that genetic determinants for tumour formation or human infectious diseases would be transferred to bacterial populations, which could then infect human beings. The safety of genetic engineering is a major debate in itself, and is discussed in depth in chapter 8.
Since the decision that such experiments were safe, the technology has been extended to greatly increase the number of different vectors, so that many organisms can be "engineered", and the range of possibilities has also increased with the large number of different genes which have been identified, sequenced and isolated (Marx 1989a). The technological principles are similar for all the manipulations, some details will be given where appropriate in the discussion of some examples. I will not discuss the immeasurable benefit of the techniques themselves for biological and medical research, as these techniques are now the foundation stone of virtually all biochemistry and biological studies.
Many human proteins are now being commercially manufactured by use of these techniques, including blood clotting factors, interferons, lymphokines, growth hormone, erythropoietin, insulin and various growth factors, which have medical uses. Recombinant DNA techniques are also being used to produce human vaccines. Modified proteins can be made, using genetic engineering to alter the catalytic properties of natural enzymes. Many pharmaceutical products can potentially be made. The medical importance of these recombinant DNA protein products is growing, and the availability of these products makes therapies for a lot of previously untreated or uncured diseases possible. It would not be an overstatement to say that they have and are revolutionising the treatment of disease.
There are also some methods to directly insert DNA into chromosomes, using a natural phenomenon called homologous recombination. This is where matching DNA sequences match up and a break in the DNA occurs allowing insertion of the intermediate piece of DNA. The mechanics are not necessary for this chapter, what may be important is that the only foreign DNA inserted is the new insert, there may not need to be any vector DNA, such as viral sequences, inserted into the DNA. It is possible to replace a chosen nucleotide sequence with a new sequence, between the homologous nucleotide sites, which is precision genetic engineering. The procedure is schematically shown in Figure 2-5.
Figure 2-5: Gene Targeting with Homologous Recombination
Two schemes of targeting are illustrated. Replacement can be made, where a portion of the input sequence interacts with the chromosomal DNA via a double crossover. Information is transferred to the target without other changes. Insertion of a plasmid that carries a portion of the target can result in the insertion of the DNA and partial duplication of the target.
With the recent developments in the treatment and eradication of many infectious diseases as a source of human suffering and death, the effects of genetic disease have been highlighted. Much research in medicine is being conducted in trying to understand, treat and cure some of the four thousand different known genetic diseases. Genetic disease is not usually lethal and some abnormalities have little effect. About 3% of children suffer from some type of genetic disease at birth. Every human possesses a specific genotype, consisting of many units called genes each gene directs the manufacture in our body of a specific component, these components are usually proteins of which the most important class for genetic studies are enzymes.
There are an enormous number of genes in human beings, at least of the order of a hundred thousand different genes, and many may be involved in defining one particular function or character at the phenotypic level. As one may imagine this complex system is in delicate balance, and it only requires a defect in a single gene to disrupt this balance, the effect maybe lethal. Our genes are in long linear strings, called chromosomes. Humans possess 23 different pairs of chromosomes, a total of 46. While every human has the same set of chromosomes and thus types of genes in the same order, each gene has variant types which are called alleles. Alleles differ in their exact sequence of DNA but they perform the same function. Several hundred human genes have been isolated that have been shown to be causally related to specific genetic diseases (Davies & Robson 1987).
DNA Sequence Protein Sequence
Single amino acid change
At conception a sperm cell of the father fuses with an egg cell of the mother, this constitutes a fertilised egg. Each germ cell only has 23 chromosomes, so the fertilised ovum contains a complete set of chromosomes. This is discussed at the beginning of chapter 3 when we consider human embryonic development (Figure 3-1). These chromosomes pair up after fusion and the chromosomes of the complementary pair exchange genes resulting in an interchange of genetic information. Entirely new combinations of genes are thus made, combining different alleles of each gene in a new string, and so forming a new set of alleles in the new genotype.
Only one of each pair of alleles of each gene is needed for the normal function. Some of the alleles may be so different in their sequence from the normal that the protein or enzyme that they produce is nonfunctional. If this is the case then the individual will use the functional allele of the pair and this will normally allow a completely normal life, or phenotype. Sometimes one of the alleles is functional not in producing the normal product but a nonfunctional product: again the individual will probably live normally. But if the individual possesses two nonfunctional, or misfunctional alleles for any gene then the effect will be a genetic disease which varies in seriousness from not being noticeable in one's life to fatality. Normally the defective allele is not used if there is a normal, functional alternative allele, and the allele would be called recessive because of this. People may carry a recessive disease-causing allele without it having any affect on them, but it is possible that it will be passed on to their offspring. In some cases the defective allele is dominant which means even an individual with one normal and one defective gene will suffer from the disease.
Among the 23 pairs of chromosomes there is a pair called the sex chromosomes, called X and Y chromosomes. A female has two X chromosomes, but a male has a mixed pair possessing both one X and a Y chromosome. The genes on the X-chromosome are dominant over those on the Y-chromosome, so if a gene on the X chromosome is defective then it will be expressed and this type of defect is called an X-linked defect. There are about 400 X-linked genetic diseases known (McKusick 1990). However 90% of the known gene mutations are on the other 22 pairs of chromosomes.
Little is known regarding the kinds and rates of mutations the occur in human beings. Much of our knowledge of genetic disease and mutations comes from the study of mutagens on animals. Animal experiments are used to study the effects of mutagens on DNA. It is likely that most spontaneously occuring mutations are actually induced by external forces, such as ionizing radiation, ultraviolet radiation, viruses and chemicals (OTA 1986). In order to systematically detect a mutated nucleotide in DNA caused by radiation we need much more sensitive methods than are technically available now. The effect of a mutation depends on where they occur in the DNA, it can be harmless or lethal. Mutations that occur in germ cells affect future generations, but mutations that occur in somatic cells may only affect the individual. Somatic mutations play a role in the development of most cancers, being a step in the process. Mutations occuring in one generation, perhaps if due to mutagenic agents, such as radiation from an atomic bomb, or chemical agents, would continue in different ways. Any large chromosomal mutations would probably result in sterility, so would only affect the first generation after. Dominant and X-linked mutations often cause severe disease and interfere with reproduction so would not last many generations. The recessive mutations have the greatest chance of being maintained in the population, none would be eliminated in the first generation, as each individual would only be a carrier, and if only one copy, then no effect. They would be present for generations.
Genetic diseases affect all populations and were apparent before prehistory. The infant mortality rate, the number of babies dying in their first year of life per 1000 live births, in England has decreased much, from 154/1000 in 1900 to 12/1000 in 1980, but the number due to genetic disease has remained similar at about 4.5/1000 (Connor & Ferguson-Smith 1984). In fact because of advances in medical treatment there has been an increase in the number of people living to reproductive age who carry or have genes that are defective, though some sufferers do not reproduce. We all carry about twenty recessive alleles for lethal characteristics, but because these occur at low frequency the incidence of a child being born with two recessive alleles is low (Sikora 1984).
Single gene disorders are the defects of immediate importance when considering the potentials of genetic therapy, although they are only one of many causes of genetic disease. 1% of live births contain known harmful gene defects, some examples are summarised in Table 2-1, and these include dominant, recessive, and X-linked defects. Many gene defects causing genetic disease are due to altered regulatory mechanisms, instead of, or as well as the production of an altered protein. In 0.5% of live births there are chromosomal aberrations, where the number of chromosomes is not 46, one of the most common and well known of these is trisomy 21 or Down's Syndrome (Jones & Bodmer 1974). Many genetic diseases (such as diabetes) are caused by multiple genes, and other diseases such as cancer are the result of environment acting upon an as yet little known genetic base. One type of cancer, retinoblastoma has been found to be caused by a single autosomal dominant mutation, and occurs at a frequency of 0.01% of births (Cavenee & Hansen 1986).
During this last decade we have discovered the mutations responsible for some important single gene diseases, such as Duschenne muscular dystrophy and cystic fibrosis. However, during the next decade there will be attention on very common diseases with complex causes including genetic elements. These include cancer, coronary heart disease, diabetes, high blood pressure, manic depression, schizophrenia, Alzheimer's disease. These are all household words, because their frequency is so high. There may be three or five genes, triggered by environmental factors, acting together. The diseases are complex, but very common. The role of diet, viral infections, smoking and other chemical exposures is unknown. Genetic susceptibility means that a particular gene is only one determinant for developing a complex disorder. In identical twin studies, if one has diabetes or schizophrenia, the other one gets it only 20-50% of the time. There have been conflicting results from gene linkage studies that have studied the disease in different families. For example there was a linkage between schizophrenia to genetic markers on chromosome 5 found by one group, but not in another family study by a different research team.
The genetic mechanism of common gene mutations is still to be determined, and there may be different types. In thalassemia and hemophilia A it is likely that there is a hotspot, where mutation can occur more frequently in the DNA. In some hemoglobinpathies there appears to have been positive selection for some alleles because of heterozygote advantage. There are some genetic diseases that have very common occurrences of mutations, such as beta-thalassemia, hemophilia A, alpha-1-antitrypsin deficiency, phenylketonuria, Gaucher's disease and APRT deficiency while there are other diseases that have rare common mutations such as Lesch-Nyhan disease, ADA deficiency, Duschenne muscular dystrophy, Blood Clotting Factor VIII deficiency and hereditary retinoblastoma. The reasons for different classes depend on the above mechanisms, and is still to be elucidated.
Disease ---------- % Live births
Single Gene Defects 1
Huntington's chorea 0.01
Cystic fibrosis 0.04
Hemophilia A and B 0.01
Progressive Muscular Dystrophy 0.02
Chromosomal Aberrations 0.54
21 Trisomy 0.1
13 Trisomy 0.01
18 Trisomy 0.02
Complex Genetic Traits 2
Congenital Malformations 1.2
Contribution to other diseases ?
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