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The Meaning of Genes...Christchurch School of Medicine Martin Kennedy What is a gene?When Charles Darwin devised his theory of evolution through natural selection in 1859, it required that offspring inherit something from their parents. In 1865 Gregor Mendel described the basic principles of how this inheritance works, which he deduced from his famous observations on pea plants. Both men reached their conclusions with no knowledge of the molecules that underlie these principles, and it took much longer to establish the physical basis of inheritance. Although the term 'gene' was coined in the early 1900s to describe a unit of inheritance, it was not until the 1940s that work on bacteria proved that genes were contained in molecules of DNA (deoxyribonucleic acid). James Watson and Francis Crick described the double helical structure of the DNA molecule in 1953, ushering in an age of molecular understanding of inheritance. In the ensuing years it became clear that all genes are encoded by DNA, which exists as enormously long molecules, packaged into chromosomes - dense bodies found in the nucleus of a cell that are visible through a microscope. A DNA molecule is made up of specific sequences of four different chemical units, called bases, abbreviated A, C, G and T [A = adenine; C = cytosine; G = guanine; T = thymine.]. These sequences contain the information that is inherited, and that help to determine the characteristics of an organism.
How does the information contained in genes get converted to black hair, or red flowers? Very basically, the DNA sequences are copied to molecules of RNA (ribonucleic acid), which can then act to string together proteins according to the original pattern. And proteins are essential to the structure and chemistry of all living cells. As our knowledge of the molecular basis of inheritance has grown, however, our definition of a gene has evolved. Most scientists would now agree that:
What we have learnt in the last few years is that quite a few important genes produce RNA that does not encode proteins, and many important functions have been found for these non-coding RNA molecules. So the molecular view of a gene is a region of DNA that encodes a gene product - either an RNA alone, or an RNA that ultimately encodes a protein. In higher organisms the gene can be spread over large regions of DNA. Some DNA regions or sequences within a gene (exons) are reflected in the final RNA, but they are interspersed with DNA sequences (introns) that are not represented in the final RNA. Although the entire gene is copied into RNA, the RNA from intron regions is deleted by a process called splicing, leaving an RNA molecule (messenger RNA) which can then be used by the cell to produce a specific protein. It is very common for one gene to produce several RNAs that have undergone splicing in different ways, often resulting in the production of more than one protein by a gene. A gene consists of not only of those regions that are copied into RNA, but also several elements or sections of DNA, often located adjacent to the gene, that regulate the copying of the gene into RNA. These control regions, called promoters and enhancers, are the sites where various molecules can bind to switch a gene on or off, or to adjust its level of activity. Why are genes of interest?As we have seen, genes are a set of instructions that guide the structure and chemistry of cells. Humans have about 30,000 genes, although each gene may produce several different RNA or protein products. Scientists strive to understand what genes are and how they work so that they can better appreciate the biology of living things, and how the natural variability of genes contributes to such things as population diversity, evolution, and the occurrence of disease. This knowledge can then be applied in many ways, such as in the detection or diagnosis of disease, for producing new drugs, or in new approaches to the treatment of disease. Genes are a vital component of the hugely complex biological systems that we call life. Although they play a central role in the cell, their expression is moderated by interactions with various other components of the cell, such as proteins, hormones and other substances. The development and biology of an organism is also shaped by complex external influences. These include an organism's physical and social environments, which are in continual interplay with its genetic make-up. What an organism looks like, how it functions, and its role in any ecosystem is a continual interaction between all these influences. The complexity of living systems means that genes do not operate in isolation, and they do not 'control' an organism's life. But they are a vital part of an organism's life, and we can understand biological systems better if we understand the role that genes play. Why carry out genetic modification?Our ability to fully understand the structure and function of individual genes only came once techniques for isolating genes were available. This required removing DNA from the chromosomes of complex organisms (such as humans) and introducing small fragments of the isolated DNA into simple chromosomes, such as those contained in bacterial viruses (phages) or plasmids (small, circular DNA molecules that exist in many bacteria). These recombinant DNA methods, pioneered in the early 1970s, allowed the purification and detailed study of individual genes. Systematic application of recombinant DNA methods over the last 30 years, culminating in the Human Genome Project, has allowed a complete description of the sequence of three billion DNA bases that make up the 23 human chromosomes that contain our genes. Thus a primary motivation for applying genetic modification techniques was to better understand the nature and function of genes. Biotechnological applications of these methods, in which genes are isolated, modified and expressed in genetically modified organisms for medical benefit, also became a very powerful motivating force. How are genes isolated?Some descriptions of genetic investigation seem to imply that scientists work with single genes, but in fact the original DNA must somehow be copied millions of times in order to get enough pure material to work with. This copying can be done in two main ways.
There are two possible sources of DNA for the above approaches: DNA from chromosomes (genomic DNA), which is extracted by chemical methods, and complementary DNA (cDNA), which is synthesised using RNA purified from cells as a template to guide the copying. This cDNA doesn't usually exist in nature, because it is a copy of an RNA molecule that has been subjected to splicing, so all of the intron sequences are missing. The cDNA is produced in a test tube using a virus protein that can copy RNA into DNA. There are two possible sources of DNA for the above approaches: DNA from chromosomes (genomic DNA), which is extracted by chemical methods, and complementary DNA (cDNA), which is synthesised using RNA purified from cells as a template to guide the copying. This cDNA doesn't usually exist in nature, because it is a copy of an RNA molecule that has been subjected to splicing, so all of the intron sequences are missing. The cDNA is produced in a test tube using a virus protein that can copy RNA into DNA.
What happens when a gene is moved from one organism to another?After a gene has been isolated, it can be modified before introducing it into another organism. These modifications will usually be carried out by a combination of methods, often involving PCR and the use of bacterial or yeast recombinant clones. Typically, a modification may involve replacing the normal control regions of the isolated gene so that the gene will function in a different organism or in different tissue. For example, if the goal is to express the gene in yeast to make a useful protein, control regions that normally function in yeast may be required. Similarly, if the gene product is to be expressed in cow's milk, control regions that normally function in the mammary gland may be inserted. Most often, a cDNA copy of a gene is used to introduce a gene into another organism. This is because the intron sequences present in the genomic (chromosomal) version of a gene often make it too large to easily manage. The cDNA version is an abbreviated copy that usually contains all the information needed to make a gene product, but which is much simpler to handle than the genomic version. Further alterations are often made to the gene before introducing it into another organism. Once the finalised version of the modified gene is ready it can be introduced into another organism by a wide variety of means, depending on the kind of organism, but in every case the DNA is scooped up by the organism's own cell machinery. For mice, we might inject the purified DNA into fertilised eggs, plants may be infected with bacteria carrying the modified gene, and cells in culture can be encouraged to take up the modified DNA by pulsing them with electricity or treating them with chemicals. The modified DNA will usually be picked up and integrated into the chromosome of the host organism somewhat randomly. Often the modified DNA becomes inserted into the large regions of chromosomal DNA that lie between genes, but it can be integrated within or close to a gene and either disrupt that gene or alter its normal expression. This may have unexpected effects, usually by causing a mutation, which means the transgenic organism will differ unpredictably from a non-transgenic organism. At least two strategies are used to minimise these effects. First, the position at which a recombinant DNA molecule is integrated into the chromosome of a transgenic organism can be established using PCR or other means. This allows an assessment of the impact on nearby genes of the genetic modification, and transgenic organisms produced in an experiment can be screened in this way. Second, methods have been developed for inserting DNA at precise chromosomal locations, less likely to cause unpredictable effects, but these cannot be used in all circumstances. What is produced in the other organism?Once the 'transgene' is introduced into another organism, and assuming it functions (is recognised by the cell as a gene, and RNA is produced from it), the nature of the products will depend on:
If a full and accurate cDNA copy of a gene from any organism is introduced (with appropriate control regions) into a bacterium, a yeast, a plant or an animal, we would expect that it should produce the correct RNA. If the RNA normally encodes a protein, it is likely that the protein will also be produced, and that the chemical make-up of the protein will be very similar in all organisms. The gene may well be expressed in tissues in which it is not normally expressed, although this is often by design (for example, a blood protein in milk). However, proteins can naturally undergo an extraordinary range of modifications once they have been produced, including processing (being broken into pieces by enzymes in the cell), being joined with other proteins and RNA molecules, or coating with sugars and other molecules. These modifications can be specific to the cell, the tissue and the organism in which they are made. For example, a human protein made in yeast should still retain the same sequence of sub-units (amino acids), but it will have quite a different pattern of sugars attached. If the same protein was made in bacteria, it would lack these sugars altogether. These modifications can be crucial to the correct functioning of a protein, so it is common for genes expressed in divergent species to not work properly. This is much less of a problem when transferring genes between closely related species, such as human and mouse. However, some human genes have been made to work very well in organisms as different as yeasts and fruit flies. So, when a gene is transferred into another organism, it should still make the same RNA and protein it did in its host organism, but the final nature of the protein, and the way it functions, may be somewhat different due to these specific and important modifications. What does it mean for one gene to be identical to another?We mentioned above that mice and humans are closely related species. At face value this seems absurd, but we are talking here in genetic terms. So what does it mean to be genetically similar, or even the same? Scientists often examine similarity between genes from different species by measuring the degree of identity between their DNA sequences. Essentially the same complement of genes occurs in all organisms, reflecting the evolutionary relatedness of species (we all derive from a common ancestor). Simple organisms may have relatively few genes, but these genes will look similar in their DNA sequence to those in higher organisms. It is possible to trace the evolutionary origins of the genes in human chromosomes by comparing them with genes of more and more distantly related species. For example, almost all genes in the human genome can be clearly discerned in the mouse genome, often with relatively minor differences in DNA sequence or gene structure. Therefore, scientists often talk of the mouse and other organisms as having the 'same' genes as occur in humans, but rarely (if ever) would the genes be identical. Differences in the sequence of human DNA occur at a rate of about 1 in every 1200 bases, meaning that human genomes differ by about 2.5 million bases between individuals. This means that when one gene is examined in many individuals it is common to see several different forms of the gene. So, even the 'same' human genes will not necessarily be identical. A gene introduced into a transgenic organism may be similar to that of the host organism or the organism from which it was derived, but it is unlikely to be identical to either. During the process of developing the transgenic organism, the sequence of the gene may have been modified, and it will most likely have been coupled with a control region that does not come from the original gene. Therefore, in most situations it would be more correct to refer to 'equivalent genes' rather than the same genes. There are several levels of equivalence.
Perhaps the ultimate test of equivalence comes, ironically, from transgenic experiments in which human genes are used to replace the equivalent gene in a mouse, a fruit fly, or a yeast. These types of experiments have demonstrated that many genes are functionally interchangeable between species. What do scientists mean by 'human genes'?Scientists use the term 'human genes' in several ways, depending on the context in which it is used. The common use refers to those genes naturally found on human chromosomes, but in other circumstances the term may be applied to:
If we move a human gene into some other organism, does this make the transgenic organism somehow more like a human?Some people are worried that if a 'human gene' occurs in a food product, say, then in some sense they are eating something human, or more like a human. This worry is unfounded. First, as we have seen, humans have many of the same genes as other organisms, and even if the gene is unique to humans the actual DNA will not have been physically derived from a human, but will have been replicated in one of several ways. Second, 'human genes' in another organism result in proteins that are often different from human proteins, due to a variety of modifications brought about by the very different physical environment. Finally, because an organism reflects the activity and interaction of:
adding one or a few human genes into this immensely complex system is not going to 'make the organism more human', just as eating a steak does not make me more cow. Every organism is already adapted to cope with a considerable degree of genetic variability, as shown by the genetic diversity inherent in every population of living things. Adding new genes to some extent simply mimics this natural diversity. The addition of large numbers of human genes into another organism would most probably severely disrupt the biology of the transgenic organism, with disabling consequences. An organism affected in this way would still not be in any way human (or even human-like).
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