MICRB 106- LESSON 8

GMO- Why the Interest?

A big part of the answer lies in agriculture. The world population is steadily increasing and shows no signs of slowing down. With an additional 80 million people per year it's estimated that the world's population could double over the next several decades. The fastest growing regions on earth are also among the least able to provide food. Over time the situation will only get worse. On the other hand, world food production has been fairly stable over the last 30 years. The Green Revolution of the 60's gave an increase in new seed varieties. The use of irrigation and chemical fertilizers allowed for an increase in production that was quite impressive. But the increases are slowing down. This means that, as population increases and food production stays flat, it will be harder and harder to feed the world's population without putting much more land into agricultural production. That's land that will also be needed for housing, transportation and other types of human development. Also some land is simply not useful for growing crops.

Gene Doping

Athletes today have developed a collection of pharmecuticals and other "natural" products to enhance the development of muscle mass. More and stronger muscles gives a performance edge to people who can early millions of dollars each year to basically play a game in front of others. It's good work if you can get it and the competition is severe. (Remember Lance Armstrong?) Therefore a great deal of motivation exists to get better and stronger faster than the other guy. Thus athletes have long tried to take things like steroids to boost muscle mass. However there are severe health complications involved (not to mention an issue of fairness to those who just train really, really hard to get good), so most professional and amateur sports strongly discourage or have banned using such dangerous substances. While some professional sports have in some ways not been too concerned with performance enhancing drugs the International Olympic Committee has been very harsh with anyone found to have taken banned materials. That enforcement is based on testing of body fluids for the presence of anything that looks suspicious. But what would they do if an athlete was able to produce more naturally occurring hormones than another person? They couldn't be accused of doing anything unnatural, that's just the way someone was born after all--right? It would be like banning a player from playing basketball because they were too tall! But what if an athlete was able to alter the genetic structure of their muscles? No drugs are needed so there is nothing to detect. What then? Gene doping is a term used to refer to the abuse of legitimate medical gene therapy treatments that modify a person's genetic makeup. The idea is to modify traits that would enhance sports performance. Muscle repair and growth are ultimately controlled by genes. Erythropoetin is a hormone that controls the production of red blood cells. The more blood cells, the more oxygen, which means muscle won't fatigue as easily. Growth factor hormones control the rate of muscle cell division. One gene of interest in this area is a gene that controls the production of a hormone called Myostatin. Myostatin controls how big a muscle can get. One particular breed of bull has a genetic defect in this gene which allows it to produce very large muscles. This Belgian Blue Bull has a defect in the Myostatin gene and this is the end result. This mutation can occur in humans. Sometimes a child is borne with the same defect. These super-muscular babies have abnormally large muscles in the legs. Since newborns cannot walk, let alone work out, such large muscle development is not normal. So the question becomes, can the gene controlling muscle development in humans be altered in an adult? The answer is yes, eventually. Different genetics labs have been able to achieve this result in mice to produce so called "Schwarzenegger Mice." It's only a matter of time till this is done with humans. Of course this type of terminology is also part of the problem with genetic alteration. It conjures up all sorts of images of giant Terminator-like mice running amuck. So, given this information, should humans alter their genetic makeup for cosmetic reasons? Should athletes be permitted to built the perfect body, not in the gym but in the laboratory?

Engineering Bacteria

Bacteria are widely used in genetic engineering to produce a variety of products useful to society. Everything from medically valuable proteins to useful enzymes that degrade toxic chemical spills is produced by altering the genes in bacteria. Being small with a simple genome they are relatively easy to handle and of course they multiply rapidly so it's possible to produce large quantities very quickly. Another feature of bacteria that make them an excellent tool of genetic engineering deals with a unique genetic structure called a *Plasmid*. Bacteria have a single, circular chromosome. They may or may not also have a round chromosome-like structure called a plasmid. These plasmids are naturally occurring and contain genes that are not necessary for the cell's survival but confer added phenotypes. Antibiotic resistance, pathogenic traits and the ability to spread the plasmid to other cells are all genes the can be found on a plasmid. Some bacteria can even contain multiple plasmids while others don't have any at all. Since these plasmids are relatively small, perhaps only a few thousand base pairs, they are easily mapped. Using electrophoresis and sequencing technology scientists have figured out the exact letter sequence for many plasmids. Since we know what the letters are it becomes an easy matter to search for the letter sequences where a certain restriction enzyme can cut. (image) The image shows a map for a specific plasmid. On it you can see restriction enzyme cut sites for the enzyme EcoR1 or Eco521. This particular plasmid also has a couple of genes for resistance to the antibiotics Ampicillin and Tetracycline. We can cut this plasmid to insert genes in specific places and break the gene for Ampicillin or Tetracycline resistance. The resulting recombinant molecule is then inserted into a bacterial cell and it acquires new phenotypes depending on the gene we insert. This is the basic process of creating a plasmid Vector.

How BT works

Bt toxin is produced by the bacterium Bacillus thuringensis. The toxin attacks and destroys insects, particularly caterpillars. The insects ingest the toxin and it effectively kills them. This insecticide has been used for decades and has never been shown to cause problems in animals. Even studies looking at possible allergic reactions on humans have not been able to find any cause for concern. Step 1: Insects ingest Bt toxin Step 2: The toxin binds to receptors on the gut and cause the insect to stop eating. Step 3: The toxin then breaks open the lining of the gut and the toxin gets into other tissues. Step 4: The insect dies.

Making a GMO

Early examples of genetic transfer into plants centered on the insertion of a gene from a firefly. The luciferase gene allows an organism to "glow." This was an easily observable trait that was inserted into tobacco plants. The resulting transgenic plant was able to light up by itself, allowing scientists to see the success of the gene transfer. Many other plants have been engineered to contain genes from other plants or even other organisms altogether in order to create traits in a target organism that would not be possible with traditional selective breeding methods. Some goals of this technology are to increase the yield of crops so that more food can be grown using less space. Also, plants can be produced that grow in conditions where they'd normally die out. Drought and frost resistant plants have been developed that are of particular use in countries where the climate makes it hard for many to grow enough food to survive. Transgenic corn and soybean contain genes that make them resistant to herbicides or the insecticide, Bt toxin. The insecticide Bt has been long used to control the destuction of crops by insects. The European corn borer is an insect that's very destructive to corn plants. Now plants can produce their own insecticide without the need for a farmer to spray the field all the time.

Frankenfoods

Genetically modified foods were among the first organisms to be modified for profit. Since everyone needs to eat every day there is a dual interest in genetically modified foods. First, famine and hunger is an ever present condition in many places around the world. Wars and civil unrest in all sorts of countries can be traced to the basic necessity of finding food to feed a family. Agriculture is still practiced in ways unchanged over thousands of years, in many corners of the planet. Obviously it would be more productive to help people grow their own food rather than ship grain thousands of miles from one country with a surplus to a place where people are starving. It's then equally true that if several billion people need to eat every day there exists a captive consumer pool for anyone willing to sell food to people. The profit motive is as huge as the number of people who need to be fed on a daily basis. Biotech companies have worked for many years to produce genetically modified crops that are easier to grow, will grow in harsh environments, or contain more nutrient value. This helps farmers produce more food for more people and everyone gets to make a buck or two in the process. However, there is a strong movement in certain countries that resists this type of genetically modified food crop. These foods are derisively called a Frankenfood to stir up images of a mindless destructive killing organism out to destroy humans. Think of a tomato with a fish growing out it and you'll be on the path to understanding the intensity of the controversy. Actually this is not far from the truth. A gene from a fish acclimated to cold waters has been inserted into a tomato plant. The resulting GMO is able to resist frost and can potentially be grown in northern climates even in the winter time. There is a great deal of zeal behind those opposed to this kind of genetic modification of food. Mostly based on misunderstanding (or, worse, intentionally misleading) science that many web sites spread about partially accurate information designed to scare people into opposing such crops. Of course others use the same imagery to point out the things that can happen if such progress is halted through unjustified criticism. There are risks associated with genetically modified organisms. These are the same risks that the public faces any time a new drug is prescribed at the doctor's office. New risks involve changes to the environment if GMO's were released into wild populations of plants. These are risks that need careful study and evaluation. Weigh the benefits and risks to producing GMO's. Try to carefully weigh any real risks (as opposed to imagined ones) with the possible benefits to humans.

Electrophoresis

If enough DNA is available for analysis, it is possible to figure out the exact sequence of the letters found in the DNA strand itself. A *gene* is just a set of nucleotides in a very specific sequence. But what are the exact letters that make up a gene? A gene may be a few hundred or even a few thousand nucleotides long. It's entirely possible to sequence a segment of DNA and figure out what the exact sequence of A's and T's and C's and G's really looks like

DNA Fingerprinting

If one looked at the sequence of two humans you would see that more than 99% of the base sequence would be identical. However, that remaining portion that is different would be virtually unique to that individual. While it is not possible with current technology to figure out the complete sequence of a person's DNA it is possible to look for patterns in the DNA to compare two people. In DNA fingerprinting restriction enzymes are used to cut DNA into a collection of different sized pieces. Since two people will have slight variations in their DNA sequence, a restriction enzyme would have cut sites in different places and the DNA would be cut into different sized pieces. If the DNA of two individuals is cut with the same set of enzymes we would generate two piles of DNA in different sizes. Those two sets of DNA fragments can be separated according to size using *Electrophoresis.* In the end, you would expect to see two different banding patterns on the gel. Each pattern would be a nearly unique "fingerprint" made up of a specific set of DNA fragments.

Human Genome Project

In the late 80's the scientific community had developed enough technology to allow for the sequencing of long pieces of DNA. They undertook a project that was thought at the time would consume years and years if not decades of effort to sequence all the several billion letters of the human genome. It turns out that the project took less than a decade due to technological advances in robotics that made what is a very tedious job go much faster. Today we have a general map of the 20,000 or more genes found in humans. What all these genes do is unknown for the most part. Intensive research is ongoing to this day attempting to make sense of all the data generated as part of this project. Some practical applications include the development of a variety of genetic tests that show how some people are predisposed to certain genetic diseases.

Trouble with GMO

Is the manipulation of genes in living organisms new? NO! (not really) If you think about it, humans have been manipulating genes since the dawn of history actually. The process of selective breeding involves allowing pairs of desirable animals, for example to breed offspring that will hopefully have characterists of both of the parents combined. Look at all the breeds of dogs and cats and farm animals seen today. Many of these did not exist even a few hundred years ago. The corn on the cob we enjoy today is derived from indian maize that was quite different when Europeans landed in North America. Many of the items seen in a grocery store were varieties created by selective breeding, we're quite good at that process. *Public Concerns:* Genetically modified organisms are of legitimate concern for a number of reasons. There is a sensitivity about the safety of genetically modified plants, particularly in Europe. The mad cow disease outbreak some years ago has made them particularly sensitive about the food production industry. However, much of what you can read online is simply misplaced fear, plants have been modified but they are not necessarily any more harmful than a new strain of turnip that resulted from selective breeding practices. There has been tremendous amounts of research to ensure safety. There are however a few areas of concern. The biggest one is allergic reactions. People can be allergic to the most mundane things, fatally allergic in some cases. The fear is that if genes are exchanged and someone has an odd allergy problem, it may crop up in foods that are not normally considered a problem. Another issue is the transfer of genes from one plant species to another. There are concerns that such a transfer could have unintended consequences that may result in a loss of biodiversity. These are a couple of issues worthy of discussion and study. *GMO's vs. "Natural" Selective Breeding* So what are the differences between making a genetically modified organism instead of using traditional selective breeding practices. For one, selective breeding is not at all certain to provide success. The whole point of sexual reproduction is to allow for the creation of different outcomes each time. You look and act very different from your siblings yet everyone comes from the same two parents. Selective breeding produces thousands of new phenotype combination. It can take a lot of time and a large dose of luck to stumble upon just the right combination. On the other hand genetic engineering is very, very precise. Rather than mixing up a hundreds of genes and finding the one combination out of thousands of attempts, only one gene, one phenotype, is transferred. Everything else remains exactly the same, but one new desirable trait is conferred. The genes involved are very well understood and their function is well known. Overall, genetic engineering is LESS likely to lead to unknown gene effects.

Genetically Modified Organisms (GMO)

Many people are uneasy about the idea of scientists changing the genome of living organisms. No matter how much benefit society may derive from genetically altered plants and animals, some people just don't trust the science behind it. A personal theory is the natural tendency of people to mistrust that which they don't understand. Sometimes through sheer ignorance of science it's impossible to rationally weigh the benefits versus the risks. (Of course Hollywood loves to produce things that make all geneticists look like money hungry loonies ie, Jurassic Park)

Polymerase Chain Reaction

One bit of technology that has increased the speed by which scientists can analyze DNA sequences is the Polymerase Chain Reaction. It's often the case that even when some bit of DNA is found there may only be a small quantity available for study. Often fairly large amounts of DNA are needed for proper analysis. This is true in cases where a small sample of DNA is found at a crime scene or a small bit of tissue is available. A few cells at the end of a hair follicle or a drop of blood provide enough DNA for PCR to amplify and allow analysis. The sample is loaded into a machine containing a special polymerase and all the necessary nucleotides. When it is done there will be thousands of exact copies of the initial sample of DNA.

Restriction Enzymes

One of the first tools necessary for the manipulation of genes in living organisms involves a set of enzymes that cut DNA. *Restriction enzymes*, or restriction endonucleases, are enzymes naturally found in bacteria which use them to chop up DNA for recycling of nucleotides. Unlike some other enzymes, restriction enzymes cut DNA in very specific ways. --First, a given enzyme will cut the DNA at a specific place where it encounters a specific set of letters in the code. Different enzymes cut at different restriction sites. This makes the enzyme a special pair of scissors that will only cut DNA at locations we can choose. --Secondly when the enzymes cut, they make a staggered, rather than a straight clean, cut across both strands. This particular type of cut leaves a small part of single stranded DNA exposed. These *sticky ends* can then bind to another piece of DNA if the single stranded part matches up. Once the DNA is cut with restriction enzymes, new DNA from other sources can be mixed in to create new combinations. If the same enzyme is used, all the pieces of DNA will have the same overlapping sticky ends. This allows DNA from multiple sources to connect together to create a larger piece of DNA. This new combination of multiple strands of DNA is called *Recombinant DNA* since samples from different organisms are recombined into a new molecule. In this way it becomes possible to take a gene from one organism and combine it with the DNA of a different organism. A plant could have a gene from a fish inserted into it. Genes for vitamin production can be inserted into a plant that does not normally make much of that vitamin. One organism can acquire the traits of a different organism and some thing new is created.

Plant Engineering

Producing sufficient food for an ever growing world population continues to be an ongoing struggle. Using gene transfer techniques it is possible to create new varieties of plants to help meet this challenge. So what can plants get in this manner? Plants can be given resistance to drought, insects, and herbicides. Monsanto makes a soy bean that is very resistant to RoundUp which is used to kill weeds. We can make more nutritious plants that contain extra vitamins like Vitamin A (as in golden rice). It's possible to increase the amount of produce made by each plant. The microbe Bacillus thuringiensis produces a toxin (Bt toxin) that kills insects. It has been long used as a spray on fields to kill crop destroying insects. Now it's possible to insert the gene into a corn plant so any insect eating it will die, no more spraying of fields required. There is much research into developing new plants that are easier to grow for a farmer, particularly those in poor countries where famine is a real problem and modern farming practices are rare. Rather than shipping grain to a nation facing drought, a better idea would be to provide seeds of plants that can grow in harsh conditions. These are all goals of plant engineering. The process for inserting desired genes into plants involves using a bacterium ready made for such a task. We can again turn to nature to find a vector delivery system for plants. There is a bacterium called Agrobacterium tumefaciens has a plasmid that naturally inserts DNA into a plant cell. This organism causes plant tumors and is considered a big problem in agriculture. The plasmid can be harnessed to deliver a target gene to plant tissues. In this case we build a vector using this natural bacterial system. All we have to do is insert a gene of interest into the plasmid using the exact procedure as outlined above. Then it's just a matter of growing the plant cells into a mature plant that will now contain our gene of interest. The following animation shows the general process.

Problems with BT

So if Bt is so safe why not put the gene into everything? There are two issues that come up with Bt corn, for example. One is the fact that Bt corn is not approved for consumption in humans by the FDA. One particular corn variety, StarLink, produced by Aventis Crop Sciences, contains a protein known as Cry9C. This protein is not easily digested so it could, possibly, be an allergen in humans, but this has not been tested. However, this particular corn variety is approved for use in animal feed so it's widely grown for that purpose. In 2003 a number of taco shells produced by Taco Bell were being sold in grocery stores. They tested positive for the Cry9C protein. Somehow corn being grown for cows found its way into a factory that made taco shells for consumers. This caused great controversy and the corn variety is no longer widely used. Another possible issue with the use of Bt plants is the effect on other insects species that are not causing a problem. Insecticide is insecticide after all. It won't kill just the "bad" insects. Bt corn can produce the pesticide in its pollen. There are concerns that the pollen may interact with other plants and confer this insecticide gene to another, unintended plant species. Of concern is the transfer of the Bt gene to milkweed. The monarch butterfly feeds on milkweed and the concern is that this transfer of pesticide gene from corn to milkweed would inadvertently kill off the monarch butterfly population. Since everyone loves the monarch butterfly (think the Bambi of insects...) no one wants to risk using the Bt corn. New research suggests, however, that in the field, the impact on monarch butterfly populations has been negligible. Still it's an argument that is often put forth by opponents of genetically modified crops.

History of Gene Manipulation

Society has been exposed to fantastical visions of creatures like Frankenstein or mutant, killer animals created in a lab. There is a visceral reaction among people that it's unnatural to play with life in this way. Some accuse science of trying to play god. This sometimes common view, while not accurate, permeates the thinking of a good many people. The reality tends to be much less exotic. After all, genetic manipulation has been occurring for centuries. Humans have been interested in creating new varieties of plants and animals for thousands of years. Almost all domesticated animals in their current form today exist due to a centuries long breeding program. The same can be said about the grains, fruits, and vegetables we eat every day. Agriculture of the past several millennia would not have developed without the use of specialized varieties of domesticated (genetically manipulated) plants. One could argue that modern civilization would not exist unless our ancestors had developed effective farming techniques including those that rely on selective breeding programs. Exactly what was happening was not understood by people even a hundred years ago. Everyone knew that children looked a bit like their parents and animals with desired traits could be produced if parents of certain traits would have offspring. Selective breeding programs were considered something of a science even though the methods were crude and the results did not always turn out as expected. Today scientists have a much better understanding of genetics thanks to pioneers like Gregor Mendel for his work on plants and the team of Watson and Crick for their description of DNA itself. We have come a long way and have an excellent understanding of what genes are and how to analyze and manipulate them. Scientists have developed tools and technologies that were unimaginable even 30 or 40 years ago.

Plasmid Vector

The creation of a Plasmid Vector involves several steps. A vector is a vehicle we create that will deliver a gene to a target cell. Since bacteria are accustomed to having plasmids inside the cell, these make an ideal vehicle to transfer a gene from another source into a bacterial cell. --The first step involves acquiring an appropriate plasmid from a bacterial cell. These are so widely used they can be ordered in a tube from a scientific supply company. --The next step involves cutting the plasmid open using a specific restriction enzyme. Since the plasmid is mapped we have a very high degree of certainty about where this cut will be. Additionally, we use the same enzyme to cut DNA from another source to get the gene we're interested in inserting into the bacterial cell. As an example, we could be trying to insert the gene for Insulin into a bacterial cell so we can grow large numbers of bacteria and get them to produce Insulin for diabetics. --Once we have a number of cut plasmids and a pile of cut DNA from another source (the target gene) we can mix the pieces together and allow them to form recombinant DNA. The key here is that we use the same enzyme on all samples. Because the restriction enzymes cut so specifically, every single piece of DNA, no matter the source, will have the exact same sticky ends. Because of this the pieces can stick to each other in all sorts of combinations. Some plasmids will reform into their original circular shape just by reconnecting their ends together. The enzyme *Ligase* helps seal the ends together Other plasmids will reform but will have new pieces of DNA inserted. Some of these pieces will not contain the gene we want. Others could contain the gene of interest. It becomes necessary to find the one of interest among all of the ones that are not useful. That happens in a later step. --Next a simple process of passing an electrical current through the cells opens up breaks in the membrane and allows the plasmids to pass into the bacteria cells. Some bacteria will now have plasmids inside, others will not. Some of those will contain useless DNA inserts but hopefully at least one will contain the gene of interest. Finding just one cell with the plasmid containing the gene of interest is all that's needed to grow billions of identical cells. But how to find that one cell? --The last step involves looking at a number of bacteria on Petri dishes to find the one with the target gene. This is done by looking for phenotype changes since the plasmid we used had genes that would change the survivability of bacteria in the presence of antibiotics. Remember, the one plasmid shown had two genes for antibiotic resistance but cut sites in the middle of these genes. If we cut the plasmid in the right place and inserted a new gene there, the gene for antibiotic resistance is destroyed. All we need to do then is find a cell resistant to one antibiotic but not the other. By using Petri plates containing combinations of antibiotics one can figure out which cells contain the gene of interest. Other plasmids can be used that contain antibiotic resistance genes and genes that cause a color change if the bacteria are given different sugars as food. Whatever the mechanism, the change in phenotype can be discerned eventually. This is a long process but again the goal is to find that one cell with the correct gene. From it billions of cells can be grown to produce the product of the gene we inserted. In this way it becomes easy to mass produce the product for commercial use.

GMO: In the Beginning

The first genetically modified food was the tomato. The FDA approved the Flavr Savr tomato in 1994 but the company that produced it was not able to make it work financially. The idea was to create a tomato that ripened on the vine but would stay firm and would thus have a longer shelf life. Another company tried to place a gene from arctic flounder into a tomato in an effort to make the plant resistant to frost. Again, this tomato plant was not a commercial success even though it was used by opposition groups to demonize the technology. In recent years a tomato has been developed that is resistant to salt. Normally tomato plants would not be able to grow in soils that contain much salt. This product would allow farmers to use the estimated 25 million acres of farmland that's lost each year when it becomes too salty to use any longer. *Golden Rice:* Golden rice was created by Peter Beyer and Ingo Potrykus who published their results in the journal Science in 2000. A gene for beta carotene production was transferred from a daffodil into a rice plant. This is a precursor for the production of Vitamin A and Vitamin A deficiency affects a huge number of women and children around the world each year. The WHO estimates 140-250 million children under the age of 5 are affected by this vitamin deficiency. Since people eat more rice worldwide than any other foodstuff, the idea was to enrich the rice with another, less common nutrient. In this way the need to ship bottles of Fintstones vitamins is reduced for those in need of basic nutrition. Most people in the world don't get to eat as well as we do here in the US. More recently a new variety (Golden Rice 2) has been introduced that has 20X the beta carotene of the original golden rice. Opponents of golden rice have argued that this is nothing more than a temporary fix for the underlying problem of world hunger, poverty, and malnutrition. Those affected by Vitamin A deficiency are equally at risk of deficiency in other vitamins as well. One genetically engineered crop won't fix all of the problem. They also argue that the introduction of a crop of rice like this will naturally lead to decreased production of other varieties of rice. Thus we'll see a decrease in biodiversity. Do these concerns negate the practical value of Vitamin A enhanced rice?

Human Engineering

The permanent correction of a genetic condition has long been considered the holy grail of modern medicine. The entire pharmaceutical industry is built around the development of drugs that alter human physiology that is not functioning properly. We treat the symptoms but can't truly cure the disease. One can manage high blood pressure but can't make it vanish. Gene therapy involves the insertion of genes that correct the function of mutated ones. Diabetes is a disease where something is wrong with the insulin protein and sugar metabolism cannot be controlled. If an intact, functional gene could be inserted into the cells of the body, each cell would produce the proper protein and the diabetes condition would disappear, permanently. The trick is to get the proper gene into the proper cell. A vector is needed. In bacteria, a plasmid could be constructed and inserted into a cell. Eucaryotic cells will not function with a plasmid so another approach is needed. Viruses function by attaching to host cells and inserting their genetic material. They then take over the host cell and use it to manufacture more viruses. If the viral DNA is removed and the proper human gene is inserted, the virus will then infect a target cell and deliver the functional gene. The following animation shows the process of treating a disease like Cystic Fibrosis. While still experimental, these procedures are highly regarded as having the potential to dramatically change the way medicine treats and cures diseases.

Genetic Engineering (Biotechnology)

Using recombinant DNA technology it is possible to create transgenic organisms and treat genetic diseases in humans. There are three basic areas of genetic manipulation of living organisms, *bacteria, plants and animals, including humans.*