Workshop A1 – Basics of GM Technology
Florianne Koechlin
What is a gene ?
In an official Swiss document I found this definition: “A gene is a section of the DNA, which contains the information for one protein (...). By the way of these proteins genes determine the structure as well as all metabolic processes of an organism.“ This “central dogma of the gene” was (and partly still is) the underlying assumption of genetic engineering. It basically means: Genes are context-independent entities, always encoding the same protein. It’s a onetrack street from gene to protein to everything else; the program of life is in its genes. It's a seductive paradigm, and its beauty is its simplicity. But it has proved wrong in many ways:
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A gene is not an inert, context-independent entity. Surroundings matter. Example: The gene for the protein isomerase occurs in bacteria, yeasts insects and mammals. In the fruit fly isomerase is involved in vision, in mammals it regulates the maturation of immune cells [1]. The gene has different functions in different environments.
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It’s not a “one-way-communication” from gene to protein, the gene being at the beginning of every metabolic pathway. Instead we observe an interactive communication in all directions: genes “talk to” proteins and other genes, proteins “talk to” genes and other proteins. Even the outer environment can have direct impacts on genes and change their expression patterns.
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Specifically, genes can be shut off and turned on by a multitude of different enzymes and small DNA-messenger molecules.
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A gene can encode for many different proteins (not just for one as stated by the central dogma). In a process called ‘alternative splicing’ the genes original nucleotide sequence is split into fragments that are then recombined in different ways to encode for many different proteins, as we can rearrange from the word TIME the words MITE, EMIT and ITEM. A gene that occurs in cells of the inner ear of chicks gives rise to 576 variant proteins. [2]
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The question : ‘What is a gene?’ cannot be answered easily. There are overlapping genes, jumping genes, silent genes, genes with many different functions… the definition of a gene is getting more and more elusive. Genes are part of a dynamic network. The genome is fluid, genes are turned on and off all the time, during development or responding to changes in the environment or fighting off foreign DNA.
Epigenetics – regulation above and beyond the genome
Increasing evidence gives proof that there are complex systems regulating the expression of genes. The totality of this self-controlling regulation network is referred to as the epigenetic network. Richard Strohman, em. Professor from University of Berkeley, writes: "DNA has been called the Book of Life by HGP (Human Genome Project) scientists, but many other biologists consider DNA to be simply a random collection of words from which a meaningful story of life may be assembled. In order to assemble that meaningful story, a living cell uses a second informational system. It is "dynamic" because it regulates changes in products over time, and it is "epigenetic" because it is above genetics in level of organisation. And some of these changed products feed back to DNA to regulate gene expression. The key concept here is that these dynamic-epigenetic networks have a life of their own — they follow network rules not specified by DNA. And we do not fully understand these rules."
Two events might show the importance of epigenetics:
Human genome project: The decoding of the human genome in 2000 was a surprise: The human genome only contains about 30'000 genes, not 120'000 as expected. With 30'000 genes you can not explain the complexity of a human being. You fail to explain why the onion has a genome six times bigger than a human, and why a mouse differs in only 300 genes from a human. There has to be more to human complexity than genes. (New research showed that humans have only 25000 genes.)
Environmental influence on genes: Cloned mice are not only smaller but reluctant to reproduce. Researchers found, that at least 2 genes were turned off in the process of cloning. The offspring of these cloned mice were also too small and they were also sexual muffles, with the same genes turned off. The cloning procedure influenced the genes, not just the cells, but the genes themselves, and these influences were passed through the germ-line to the offspring. As if the genes "remembered" the fact of cloning and passed this experience to further generations. [3] In the meantime more examples of such direct environmental influences on the genes are known, also in plants.
Gene transfers are neither precise nor predictable
The main technique to bring foreign genes into a plants genome is the biolostic method: A gene gun uses .22-caliber ballistics to shoot DNA into cells. This is clearly a “hit or miss” technique. You donot know how many genes integrate into the genomes nor where they go. They break into subtle networks in the cell, might change neighbour-relations or cause reciprocal actions. This makes every gene transfer an inherently unpredictable event.
Furthermore: Getting the target plant to accept the new genes is tricky too. This requires overcoming billions of years of evolutionary resistance that was specifically designed to keep foreign DNA out. This only can be accomplished by building gene-constructs specifically designed to facilitate the process and overcome the cellular defence mechanisms against foreign genes. That is why artificial geneconstructs have to be built: Along with every trait gene, the artificial gene construct also contains genetically engineered vectors and markers, resistance genes, viral promoters made from the cauliflower mosaic virus, genetic switches and other constructs that enable the “transformation” process. [4] This again opens a field of new risk-possibilities.
Careful testing might decrease the probability of risks, but the technique remains basically unpredictable.
Conclusions
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The old paradigm , the central dogma of the gene, has become redundant.
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Enhanced understanding of epigenetics leads to a complete reversion of hierarchy: Genes are not the ones controlling life processes, but rather suppliers of biochemical substances which the cell needs in its respective development state or functional state. Genes are molecules like others too, and the cell signals what is needed and where. It’s not the genes determining the program.
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Gene transfers are basically unpredictable.
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The old paradigm of the gene still is the fundament of the risk assessment philosophy. But when the basic assumptions are wrong, all consequences drawn from these assumptions are obsolete as well. The risk assessment procedures and regulations have to be revised.
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Annex
Problems in producing transgenic plants , as a result of epigenetic regulation of gene expression and basically unprecise gene transfers
By Beatrix Tappeser, Head of Division GMO-Regulation, Biosafety. Federal Agency for Nature Conservation. [5]
Given the complexity of gene regulation it is hardly surprising that producing transgenic plants is far from being a precise process whose effects can be predicted and described. It is the norm for unexpected phenomena to occur when transgenic organisms are created. With plants, unintended changes in plant physiology are produced. These either have nothing to do with the genetic product or are secondary effects of genes which were not known before the genetically engineered change was made. These unexpected phenomena are also called positional and pleiotropic effects.
Positional effects
The function and regulation of a gene is dependent among other matters on its position in the genome. In current transformation methods foreign genes are however introduced randomly into the receptor genome. The integration cannot be controlled with regard to where the transgene is located. At the site of the integration reordering of sequences often occurs. This can happen with both the new gene sequences being integrated and the sequences flanking the receptor genome – usually with unknown consequences.
Pleiotropy
Pleiotropy means the fact that one and the same gene can have different effects. With transgenic organisms changes in several characteristics can arise even if only one gene is introduced. The consequence can be the most varied changes in the cell's entire metabolism. There are for example genes where a pleiotropic effect in "unrelated" morphological features is characteristic. One of the few systematic investigations into positional and pleiotropic effects has been on the Bintje and Escort varieties of potato, which have been transformed by a gene for a virus coat protein for developing viral resistance. PVX (Potato Virus X)-resistant varieties were analysed to examine how similar they were to the original varieties. Only 18 per cent of the Bintje plants, and 82 per cent of the Escort plants, retained matching characteristics. The exact causes of the deviation have yet to be explained. But these findings also show there is a high variation in positional and pleiotropic effects even in the same species.
As has already been described, gene expression is controlled by the interaction of the most varied factors which are part of a complex and dynamic network. It is therefore no surprise that many transgenes are expressed differently than expected, as they are foreign to plants' regulation networks.
The relevant literature is inadequate in its accounts of the unintended changes which can occur in producing transgenic plants because these often lead to unpredictable instability in the desired characteristic. This means that the plants which have poorer "values" are discarded in the laboratory. It is therefore difficult to fully document examples and only a few such can be described.
Roundup Ready soybeans
One popular example is that of Roundup Ready soybeans resistant to herbicides. The lignification of the stem in these transgenic plants is found to be enhanced by up to 20 per cent, probably caused by the new bacterial enzyme, which influences the lignine's metabolism. This change manifestly has an adverse effect on the soybeans when they are under conditions of stress. Heat stress results in lower harvest yields for transgenic plants. The plants' hormonal make-up is also affected by the genetically engineered intervention. The content of diverse phytoestrogens in the soybeans changes by 12-14 per cent.
A Belgian study group furthermore in 2001 published that in integrating foreign DNA into soybeans several rearrangements in the sequence had occurred at a flank region, and that the plant DNA had likewise been rearranged at the point of integration. An abbreviated version of the herbicide- resistance gene, with 254 base pairs, was also found. Then a DNA segment 534 base pairs in length and without sequence homologies to soybeans or other plant DNA was discovered. It is suspected this is part of a vector DNA or other foreign DNA. All this was published five years after the herbicideresistant soybeans were commercially introduced. It is impossible to say if this was not discovered until then or had already been known about by the manufacturing company.
Notes
1) Regine Kollek, „The gene – that obscure object of desire“ in „The Life Industry“, ITP,1996
2) Barry Commoner, „Unraveling the DNA Myth“, Feb.2002, Harpers.
3) Wolf Reik et al., 2001, Science, 293,5532, 1089
4) Claire Hope Cummings, „Trespass“, Worldwatch Institute, 2005
5) Beatrix Tappeser and Ann-Kathrin Hoffmann, 2004, „The old paradigm of genetic engineering is outdated. Some considerations to the central dogma of molecular biology fifty years after the discovery of the structure of DNA"