Introduction - The Basics of Life
The genetic information for all living things is found in DNA (deoxy-ribonucleic acid). This genetic material encodes the information that ultimately shapes what an organism is, controls how it grows and develops, and influences how it behaves. Selective activation and inactivation of genes encoded in DNA enables an organism to produce specific proteins (long chains of amino acids) in particular amounts over time and in response to the internal and external environment of the organism.
The production of protein takes place in two steps. In the first step, called transcription, the permanent DNA message (Figure-1, 1) is copied into a temporary messenger RNA (mRNA, 2) by an enzyme (RNA polymerase). This mRNA message can be read by a complex cellular “machine” called a ribosome (3). In this second step, called “translation,” the ribosome assembles amino acids in an order specified by the mRNA to create a specific protein (4).
The growth, development, and function of an organism is a reflection of gene expression- the timing, pattern, and quantity of RNA and the production of proteins from genes within the organism. Thus, gene regulation is essential to life- from the simplest virus to the most complex mammal.
The transcription of DNA to make messenger RNA is highly controlled by the cell. In order for a higher organism (plant or animal) to function, genes must be turned on and off in coordinated groups in response to a variety of situations. For a plant this may be “abiotic” (non-living) stress such as the rising or setting sun, drought, or heat, “biotic” (living) stress such as insects, viral or bacterial infection, or any of a limitless number of other events. The job of coordinating the function of groups of genes falls to proteins called transcription factors (TxF’s).
How Do Transcription Factors Work?
TxF’s are proteins that regulate gene expression (figure-2). Each gene is preceded by a promoter region that includes a binding site for the RNA polymerase (which will copy DNA to RNA) and a variety of other features, including the “TATA box” (a short segment of repeating thymidine and adenine residues) and one or more enhancer sites, which serve as a binding location for TxF’s.
In order for a messenger RNA to be created, an initiation complex must assemble in the promoter region. This complex consists of over 40 proteins, including the RNA Polymerase, TATA binding protein, and one or more TxF’s.
TxF’s work by binding to DNA at the enhancer site and/or to other proteins in the initiation complex. Through these protein-protein inter-actions, TxF’s are able to control whether RNA polymerase moves forward along the DNA to produce a message. In effect, TxF’s are the “traffic cops” regulating mRNA production.
TxF’s often contain features that help the cell respond to the internal or external environment. Typically, these are binding sites that interact with chemicals within the cell (“ligands”) that modulate the activity of the TxF. For example, TxF’s may bind to hormones, chemicals like glucose, or to other proteins in order to “sense” and respond to the environment.
In order to allow coordinated gene function, a particular TxF may bind to multiple genes, and each gene may be controlled by multiple TxF’s. Further– recall that each TxF is itself a protein, and TxF’s often regulate other TxF’s. TxF’s form complex networks that may control from one to many thousands of genes in response to conditions inside or outside of the cell.
Using TxF's in GM Plants
TxF’s are proteins, and the genes for TxF’s can be inserted into plants in the same manner as other genes, such as the Bt gene for insect resistance or the Roundup Ready gene (CP4-EPSPS).
As with other genes, TxF genes may carry non-specific promoters which allow them to be expressed at all times in all plant tissues, or they may carry tissue selective or other promoters to allow expression only in particular tissues, at particular times, or under particular conditions.
Role of TxF's in plants: Conventional Breeding
Selective plant breeding goes back tens of thousands of years and has produced remarkable results. For example, teosinte, the ancestor of corn, is a short, bushy plant with tiny seeds. Selective breeding has resulted in the tall, large-seeded food plant we use today (Figure-3, Doebley, 1997). Research has now shown that these dramatic changes in plant characteristics are primarily due to selection for changes in key TxF’s.
Further, plants alter TxF and gene expression levels during development and in response to environmental conditions. Humans routinely consume plant materials at various stages of maturity or ripeness, and raised under widely varying conditions and are routinely exposed to foods with varying levels of TxF’s and gene expression.
TxF's in the Diet
TxF’s are essential to the function of all genes in higher organisms, including humans. Thus, TxF’s are in all whole foods derived from plants or animals.
Many TxF’s used in genetically modified crops will be derived from crops used for food and feed and, indeed may simply be additional copies of TxF’s already present in a particular food. TxF’s are conserved within evolutionary development, and TxF’s derived from non-food species will probably be highly similar to those already in the diet.
TxF's: Safety and Health for Humans and Animals
As noted above, TxF’s are present in all foods derived from plants and animals, and have a long history of safe consumption. While TxF’s influence multiple genes, it is important to recognize that humans and animals already consume plants whose TxF’s have been extensively influenced by breeding, and which grow under a wide variety of environmental conditions. Thus, the vast majority of the variability in composition which may be induced via TxF’s is, in most cases, already present within food and feed crops.
Like other proteins introduced using bio-technology, TxF’s undergo safety assessment including acute toxicology studies, bio-informatics analysis to assure that the protein does not resemble known food allergens or toxins, and testing to assure that the protein is rapidly digested.
In order to have an effect within a cell, the TxF would have to survive digestion, be taken up by the body, be transported across the cell membrane, and make its way across the cytoplasm, through the nuclear membrane, and be delivered to the DNA. Once there, the plant TxF’s would need to interact effectively with enhancers and the other proteins of the initiation complex in the consuming organism. There are substantial barriers to the uptake and transfer of proteins, as well as species differences in activity, which make the likelihood of oral activity essentially nil.
In fact, common sense and long experience tell us that TxF’s will have no systemic effects in humans. Consumption of TxF’s in food has no known effect on cell. (Although cells do respond to food consumption, this is normal physiology and is mediated by TxF’s in the consuming organism). Further, if it was common for dietary TxF’s to readily migrate among cells or spread systemically, cellular function and organization would deteriorate into chaos.
TxF's: The Environment
TxF’s from human, plants, and animals already exist in the environment. Most proteins are readily degraded in the environment and are poorly taken up by other organisms. Bacteria generally cannot utilize TxF’s from higher organisms for gene regulation, and will not be affected by the use of TxF’s in plants.
Assessing the Safety of Polygenic Traits
The current generation of commercial genetically modified plants primarily involves insertion of genes for single proteins having insecticidal or enzymatic function. Such genes are expected to have little effect on broad gene expression. In contrast, TxF’s are expected to modulate expression of a large number of genes, and it is important to consider how one might assess the safety of such a plant in regards to gene expression.
As noted, plant gene expression changes markedly as a plant develops in response to abiotic and biotic stressors. Thus, gene expression patterns will differ radically depending upon, for example, maturity at time of harvest and weather conditions before and during harvest, time of day, etc. The reality, however, is that gene expression levels per-se are not relevant to crop efficiency and safety – what matters is the resulting performance and food composition.
Both agronomic performance and food composition are extensively assessed for bioengineered crops. Composition assessment entails extensive evaluation of nutrient (protein, fiber, sugars, vitamins, etc.) and anti-nutrient (phytates, trypsin inhibitors, lectins, etc.) properties as well as assessment for any known plant toxins naturally present in food varieties. Because TxF’s are regulatory factors with no synthetic or other metabolic function, it is highly unlikely that they will produce novel toxins.
The safety assessment of feed and food derived from plants with genetic modifications to modulate endogenous gene expression, including the use of TXF’s, has recently been reviewed (Kier and Petrick, 2008).
What is the role of "-omics" in TxF safety assessment?
Various techniques, broadly referred to as “omics” now exist to simultaneously measure the expression levels of large numbers of genes (genomics), proteins (“proteomics”) or inter-mediary metabolites (“metabolomics”). TxF’s are expected to alter gene expression and may affect downstream expression levels of proteins and metabolites. However, it is important to recall that protein production and metabolic function are also extensively regulated at stages beyond the production of messenger RNA. These homeostatic mechanisms tend to moderate the impact of changes in gene expression. Further, gene function can change rapidly– over minutes to hours, while final food composition in a crop may develop over weeks to months. Thus, gene expression results are expected to be a poor predictor of food composition and safety.
Gene function does fluctuate quite significantly in response to normal environmental events including time of day, weather conditions, etc. This makes “normal” ranges of gene expression very difficult to define, and in fact such “normal” levels are likely to be very broad for most genes.
Given these circumstances, there is little if any safety information that can be derived from genomic or proteomic technologies.
Although metabolomics can detect the presence of a variety of metabolites, accurate measurements are not typically achievable. Most methods are restricted to metabolites of high abundance, and many metabolites have no nutritional significance. Further, databases detailing the normal ranges for nutritionally important metabolites are currently absent of very limited. Compositional and nutritional analyses should logically be focused on important nutritional, anti-nutritional, and toxic components - precisely the type of analysis that is currently performed for biotech feed and food.
"In short, the "omics" technologies offer tremendous data acquisition capabilities but are currently of limited value in safety assessment (Kier and Petrick, 2008). Appropriate assessments will continue to rely on traditional agronomic performance, plant morphology, and targeted compositional analysis pending development and under-standing of extensive metabolomic databases.
- Tjian, R., 1995. Molecular machines that control genes. Scientific American 272, 54-61.
- Carroll, S.B., 2000. Endless forms: the evolution of gene regulation and morphological diversity. Cell 101, 577-580.
- Doebley, J., Stec, A., Hubbard, L., 1997. The evolution of apical dominance in maize. Nature 386, 485-488.
- Doebley, J., Lukens, L., 1998. Transcriptional regulators and the evolution of plant form. The Plant Cell 10, 1075-1082.
- Martinez, E., 2002. Multi-protein complexes in eukaryotic gene transcription. Plant Molecular Biology 50, 925-947.
- Tautz, D., 2000. Evolution of transcriptional regulation. Current Opinion in Genetics and Development 10, 575-579.
- Kier LD, Petrick JS. Safety assessment considerations for food and feed derived from plants with genetic modifications that modulate endogenous gene expression and pathways. Food and Chemical Toxicology 46:2591–2605, 2008.
- Daniel A. Goldstein, M.D. is a Medical Toxicologist and Director of Medical Sciences and Outreach for Monsanto.
- David Songstad, Ph.D. is a Plant Biologist in Scientific Affairs, Monsanto.
- Eric Sachs, Ph.D. is a plant scientist and Lead for Scientific Affairs, Monsanto.
- Jay Petrick, Ph.D. is a Monsanto scientist investigating the safety aspects of Transcription Factors