Biotechnology: Principles, Applications, and Social Implications From Protein to Product The techniques used by the biotechnology industry to modify genes and introduce them into transgenic organisms Title Page: Biotechnology: Principles, Applications, and Social Implications Part II: From Protein to Product NOTE: In “Slide Show” format, click on the slide. You will notice that on many pages a series of text boxes or images will appear sequentially on the same slide. This is intended to provide you with a progression of concepts. Remember to study the slide notes to get a better understanding of what is being presented in the slides.

What is Biotechnology? How about some definitions General Definition The application of technology to improve a biological organism Detailed Definition The application of the technology to modify the biological function of an organism by adding genes from another organism The general definition is very broad. Many individuals prefer this definition because they can claim process such as plant breeding or mutagenesis are actually biotechnology. The detailed definition points to the fact that a foreign gene needs to be inserted for a product to be considered a biotech product. Biotechnology involves the modification of a whole range of organisms. This foreign gene can be inserted into plants, animals, fungi, bacteria or viruses. The key for me is that a foreign gene, or an engineered gene from the same species is added back into the organisms. The modified organisms would not have these traits without the intervention of man.

These definitions imply biotechnology is needed because: Nature has a rich source of variation Here we see bean has many seedcoat colors and patterns in nature As we are all aware, most species have an abundance of variation. The photograph of bean seeds is a great illustration of the variation in nature. You can notice not only many different colors, but also many different patterns. A large array of interacting genes are responsible for this variation. But man can always dream of a new use for an organism. These dreams often involve asking the species to do something it does not now normally do. Biotechnology involves added new traits to a species. But we know nature does not have all of the traits we need

But nature does not contain all the genetic variation man desires Fruits with vaccines Grains with improved nutrition Two of the most dramatic examples of man’s dreams of improving the utility of plants is shown here. At the top is the picture of bananas. The goal is to express vaccines in these fruits so that individuals who eat them will receive the vaccine and become immune to the disease. The rice photograph illustrates a recent invention called “Golden Rice.” This strain of rice has been engineered to express enzymes required for the vitamin A pathway that don’t normal exist in this species. By the goal is to provide this as crop as a dietary product that will improve the nutrition and health of those who eat it.

What controls this natural variation? Allelic differences at genes control a specific trait Definitions are needed for this statement: Gene - a piece of DNA that controls the expression of a trait Allele - the alternate forms of a gene Before we can understand who man goes about using biotechnology approaches to modify a species, we must understand basic genetic principles and terminology. All traits are controlled by genes. A gene can have different forms. These forms are called alleles. It is important that you become fluent with these terms and the differences they imply. It is simple as remember that genes have alleles. Or, alleles are alternate forms of a gene.

What is the difference between genes and alleles for Mendel’s Traits? Mendel’s Genes Plant height Seed shape Smooth Wrinkled Allele This slide is intended to help you understand the difference between genes and alleles. Genes control specific traits. Here are two traits of pea that Gregor Mendel, the father of genetics, studied. Plant height is a trait. That trait is controlled by the plant height gene. The plant can be either tall or short. Different alleles of the plant height gene determine if the plant will be tall or short. If you remember from your genetics class, the allele for tall plant height is dominant to the short allele. This means that heterozygous individuals carrying both the tall and short allele will appear to be tall. Using the genetic terminology, this also means that the short allele is recessive to the tall allele. Similarly, seed shape is controlled by a specific gene. The alternate shapes, smooth or wrinkled, are controlled by different allelic forms of the seed shape gene. Similarly, smooth is dominant to the recessive wrinkled phenotype. Tall Short Allele

This Implies a Genetic Continuum A direct relationship exists between the gene, its alleles, and the phenotypes (different forms ) of the trait Alleles must be: similar enough to control the same trait but different enough to create different phenotypes As the previous slide demonstrated, each gene has a specific function. The fact that two alleles control alternate forms of a trait means they must also be very similar to each other, but different enough to produce different phenotypes. In modern terminology, each allele will have very similar DNA sequences and will encode the same protein product. But the sequence of the alleles will differ at some point, and that difference is directly responsible for the various phenotypes generated by the different alleles.

Allelic Differences for Mendel’s Genes Plant Height Gene Gene: gibberellin 3--hydroxylase Function: adds hydoxyl group to GA20 to make GA1 Role of GA1: regulates cell division and elongation Mutation in short allele: a single nucleotide converts an alanine to threonine in final protein Effect of mutation: mutant protein is 1/20 as active So what about the genes encoding the two traits that Mendel studied. This slide provides the essential information regarding the plant height gene. This gene encodes and important enzyme in the gibberllic acid biosynthetic pathway. If you remember, gibberellic acid is responsible for node elongation in plants. If a plant has a longer nodes, it will be taller than a plant with the same number of nodes, but whose nodes are shorter. The specific gene product is gibberellin 3--hydroxylase. This protein adds a hydroxyl (-OH) group to the gibberellin called GA20 and this process makes the biologically active GA1 gibberellin. The dominant allele contains the normal form of this gene, and the result of its expression will be tall plants. The recessive allele is very similar to the dominant allele. In fact, the only difference is a change in a single nucleotide that results in the change of an alanine to a threonine in the final protein. This may seem like a trivial change, but the result of the change is a 20-fold reduction in the active of the protein encoded by this mutant allele. This comparison highlights the type of information that is gathered from these sequence analysis of different alleles.

Allelic Differences for Mendel’s Seed Shape Gene Gene: strach branching enzyme (SBE) isoform 1 Function: adds branch chains to starch Mutation in short allele: transposon insertion Effect of mutation: no SBE activity; less starch, more sucrose, more water; during maturation seed looses more water and wrinkles So what about the smooth vs. wrinkled phenotypes expressed by the seed shape gene? This gene encodes the strach branching enzyme (SBE) isoform 1. The recessive allele has no activity, and the result in the seed is that it contains much more sucrose than starch. The result of higher sucrose levels is a higher water content. As these seeds dry they lose more water and the seed takes on a wrinkled appearance. Unlike the recessive plant height allele that is the result of a single nucleotide change, the recessive seed shape allele is the result of the insertion of a large piece of DNA into the coding region. This large piece of DNA is a transposable element. This element is similar to the elements that Barbara McClintock discovered in corn.

Central Dogma of Molecular Genetics (The guiding principle that controls trait expression) DNA (gene) RNA Protein Trait (or phenotype) Transcription Translation Plant height Seed shape Now that we understand the relationships between alleles at a gene, it is time to place this understanding in a large context. You should become very fluent with this concept: the Central Dogma of Molecular Genetics. This concept is a unifying principle that describes the manner in which the sequence information in the gene is eventually expressed as a trait (or phenotype). DNA is the biochemical molecule of all genes. DNA contains the genetic code that will eventually be converted into the protein that will control the phenotype expression. But DNA is not directly used for phenotypic expression. Instead, the information in the DNA molecule is used to create an intermediary molecule (RNA). The process to produce RNA is called transcription. RNA is an active molecule in another process called translation that is used to create the protein. The protein itself can have many different functions. It could be an enzyme in a metabolic pathway. Alternatively, it could act as a regulator transcription. Finally, it could serve as a structural component of the cell. Whatever it role is, it will control the final phenotype or the outward appearance of plant.

In General, Plant Biotechnology Techniques Fall Into Two Classes Identify a gene from another species which controls a trait of interest Or modify an existing gene (create a new allele) Gene Manipulation Introduces that gene into an organism Technique called transformation Forms transgenic organisms Gene Introduction Remember our definition of biotechnology??? Here is the detailed one again: The application of the technology to modify the biological function of an organism by adding genes from another organism. As the definition implies, we first need to isolate a gene that will be added to our organism of interest. For example, we may wish to add a gene from a bacteria into a plant species. Another approach would be to isolate a gene from our species of interest, modify that gene to change its function, and then reinsert the modified form back into the species. This is the first major biotechnology step, and it is called gene manipulation. Once we have isolated or modified the gene of interest, the next step is gene introduction. This step involves the addition of the gene to our species of interest. In all cases, this introduction must be accompanied by stable integration of that gene into the genome of the target species. Once the gene is stably integrated, it is passed along to all subsequent generation in the same manner as all other genes in the genome. The technique used to integrate the gene into the species is called transformation, and the modified organism is called a transgenic organism.

Gene Manipulation Starts At the DNA Level The nucleus contains DNA We will discuss gene manipulation and gene introduction separately. The Central Dogma of Molecular Genetics was introduced several slides back. As a remember, it states that the information stored in DNA is transcribed into RNA, the RNA molecule is used in a process called translation to produce translation to produce a protein, and the protein is involved in some process that actually produces the final phenotype of the organism. The dogma implies we need to manipulate the DNA of gene that encodes for the protein if we are going to develop a transgenic organism. The DNA itself is a double-helix molecule that is stored inside the nucleus of the cell. Every cell inside the organism has exactly the same DNA molecule. Source: Access Excellence

DNA Is Packaged Double-stranded DNA is condensed into Chromosomes We normally think of the DNA in the form of a chromosome. Chromosomes are the condensed form of DNA. The simplest form of DNA is the double-stranded molecule. These two strands are complementary to each other. This complementarity is based on the fact that if one strand has an adenine at one nucleotide residue, the complementary strand has a thymine residue at the same location. And if one strand has a guanine at a specific residue, the complemenntary strand has a cytosine residue. This is an important concept because it is the basis of an important screening process called hybridization. The double-strand molecule then undergoes a series of condensation steps to produce the chromosome. Each of this different steps are illustrated here in this slide. It is important to remember that throughout the life-cycle of the cell, DNA is in an uncondensed form. The chromosome only appears during the process of cell division. Chromosomes Source: Access Excellence

Chromosomes Contain Genes All of genes reside on a specific location on the chromosome. Any chromosome will contain thousands of genes. To illustrate this point, the human genome consists of about 35,000 genes which are spread over 22 chromosomes. In contrast, the model plant species Arabidopsis thaliana contains about 30,000 genes on just five chromosomes. This illustrates the point that although plants and animals contain about the same number of genes, the have a different number of genes on their chromosomes. The goal of gene manipulation is to isolate that one gene of interest from among the many genes in the genome. Source: Access Excellence

Genes Are Cloned Based On: Similarity to known genes Homology cloning (mouse clone used to obtain human gene) Protein sequence Complementary genetics (predicting gene sequence from protein) To understand biotechnology, it is important to have a general appreciation of how genes are cloned. These fall into three gene approaches. The first approach is based on the fact that genes between related species have similar DNA sequences. This procedure is called homology cloning. The second procedure is based on the direct relation that exists between the DNA sequence and the final protein sequences. If you have an idea of the protein sequence you can develop a probe to get at the gene. This approach is called complementary genetics. The third procedure relies on experiments that place the gene at a specific genetic location. This procedure is called map-based cloning. Chromosomal location Map-based cloning (using genetic approach)

Homology Cloning Clones transferred to filter Human clone library Mouse probe added to filter Hot-spots are human homologs to mouse gene We will first discuss homology cloning. As mentioned earlier, this procedure is based on the fact that the genes between two closely related species have very similar DNA sequences. To take advantage of this procedure, you need a probe from a species similar to the one you are working on. Let's say you know that someone has cloned a gene from mouse that you are interested in as a human geneticist. You would then contact the person and request a copy of that gene. That will be used as a probe in your experiments. The first step is for you to grow out a clone library. This library contains all of the genes from your species. Therefore a human clone library contains all of the genes in the human genome. The clones are then transferred to a special filter where the DNA for each clone binds. They bind at a spot that is directly analogous to their position on the plate in which they are grown. This relationship is illustrated on this slide. The next step is to add the probe to the library. The probe is normally radioactively labeled. The importance of this will be seen shortly. The probe then will bind by base-pair complementarity to a clone to which it is very similar. This is the hybridization step that was mentioned on an earlier slide. Excess probe is washed off, and the washed filter is then exposed to an x-ray film. The film is then read, and any hot-spot is the location of a clone that is similar to the probe you used. It is hot because the probe was radioactievely-labelled. You then go back to the original plate containing your genes and select the clone containing your gene.

Complementary Genetics 1. Protein sequence is related to gene sequence NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO- ATG GAT-GCT TGG-AGT-AAA C C C G A TCT G C A G 2. The genetic code information is used to design PCR primers Forward primer: 5’-ATGGAT/CGCN-3’ Reverse primer: 5’-T/CTTNC/GT/ACCA-3’ Notes: T/C = a mixture of T and C at this position; N = a mixture of all four nucleotides Reverse primer is the reverse complement of the gene sequence The second cloning procedure is called complementary genetics. And as with that procedure, homology cloning requires a probe from some source. The difference with homology cloning though is that we develop our own probe based on the protein sequence. Probe development is based on two simple facts. The first is that the sequence of the gene in its DNA form will predict the protein sequence. This also implies that if you know the protein sequence you can derive the DNA nucleotide sequence. The second fact is that the placement of an amino acid into a growing protein chain is directed by a sequence of three nucleotides. This fact is illustrated in the slide. You should notice that some amino acids are encoded by one triplet sequence, some by two sequences, and others by three, four or six. As you will see this is a minor issue for complementary genetics approaches. The probe is actually synthesized using a procedure called PCR, or polymerase chain reaction. The DNA synthesis procedure requires DNA primers. One primer is designed to a region in the amino (NH3+) part of the gene. As you can see, the sequence of the forward primer is based on the nucleotide sequence that corresponds to the amino acid sequence. When you look at this sequence you will notice the sequence denoted as “A/T”. This means the primer will actually consist of a mixture primers some with an A at that position and some with a T. The symbol “N” means the primer pool will contain a collection of primers each with one of the four nucleotides at that position. Therefore, this primer pool will consist of 8 (2x4) different primers. The reverse primer is synthesized using the same principles, but it is complementary to the genetic code sequence. (It is complementary because of the fact that DNA consists of two complementary strands. Therefore, the PCR synthesis process is designed to produce both strands.) So, as you can see the first nucleotide is complementary to the A/G bases that encode the lysine (Lys) amino acid, and the primer instead consists of T (thymine) or C (cytosine). As with the forward primer, the reverse primer is actually a pool that consists of 32 [2 (T or C) x 4 (A, T, C or G) x 2 (C or G) x 2 (T or A)] primers.

Complementary Genetics (cont.) 3. Use PCR to amplify gene fragment a. template DNA is melted (94C) 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ b. primers anneal to complementary site in melted DNA (55C) 3’ 5’ 5’ 3’ PCR is a DNA synthesis process. The previous slide described the first step in PCR, the development of the primers. The primers satisfy one of the two requirements of DNA synthesis, a primer from which the new chain grows. The second requirement is a DNA template. To generate a DNA template, the double-stranded DNA molecule must be converted into a single-stranded state. This is done by heating the DNA to a high temperature. Step 3a in the slide shows this process. Once the DNA is single stranded, the temperature is lowered so that the primer can anneal. Once annealed, the temperature is raised to the optimum temperature required by a special DNA polymerase enzyme called Tag polymerase. Once the temperature is reached, the DNA is replicated. As you can see in step 3c, now have two strands of DNA. 3’ 5’ 5’ 3’ c. two copies of the template DNA made (72C)

PCR Animation Denaturation: DNA melts Annealing: Primers bind This is an animation of one step in the PCR process. Take a few minutes and let the animation run through a number of times. It will recycle on its own. This step will show the denaturation (converting the DNA from single- to double-stranded state). The second step is annealing (the binding of the primer to the single-stranded DNA). The final step is extension (the duplication of a strand from the end of the primer). Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated

PCR Again As the previous two slides illustrated, the many feature of the PCR process is the replication of one double-strand DNA molecule into two. But the PCR process does not involve just a single replication cycle. Rather, the step is repeated many times (35-50 times). This repetition leads to an exponential increase in the amount of DNA. At the end, a large amount of DNA is produced that can be used for a number of purposes. One of those purposes, and the one we are interested with here, is the development of a DNA probe. Remember that we started with a protein sequence of interest, and we used that information to design primers to amplify a DNA fragment that would encode that fragment. Therefore at the end of the PCR replication, we have sufficient DNA to use as a probe for library screening.

Complementary Genetics (cont.) 4. Gene fragment used to screen library Clones transferred to filter Human clone library PCR fragment probe added to filter The final step in using complementary genetics for cloning involves screening a library. The steps are exactly the same as we described for homology cloning. The only difference is that we now use the PCR synthesized DNA as our probe. The final result will be the isolation of a DNA clone from the library. That clone will contain DNA sequences that will encode the gene for the protein in which we are interested. Hot-spots are human gene of interest

Map-based Cloning 1. Use genetic techniques to find marker near gene 2. Find cosegregating marker Gene/Marker 3. Discover overlapping clones (or contig) that contains the marker Gene/Marker 4. Find ORFs on contig Gene/Marker The final technique of method of obtaining genes is called map-based cloning. This procedure combines genetic information that locates a gene to a small region of a chromosome. The position of the gene is located to that region by the use of a DNA marker that resides very close to the gene. The first two steps illustrate this point. Typically a marker is discovered at a short distance away from the gene of interest. That marker is then used to discover another marker that is very close that cosegregates with the gene of interest. That cosegregating marker is very close to the gene (closer than the first marker) and is used to isolate a series of overlapping clones or contig. A series of steps is then used to identify ORFs or open reading frames. An ORF is a DNA sequence that has all the characteristics of a gene. Since the DNA marker (and by association the gene of interest) resides on the contig, one of the ORFs will be the gene. We won’t go into the details, but the ORF that is a gene is eventually identified by one of two procedures. Transformation is one approach. As defined earlier, transformation involves the addition of DNA to an organism and changing that organism’s phenotype. For map-based cloning, the ORF is added to a mutant organism, and if the resulting transgenic plant expresses the wild type phenotype then the ORF is the gene you are trying to clone. For example, if you had and ORF that encoded the gene responsible for plant height in pea, that gene could be added to a short pea plant. If the addition of the ORF is the gene for plant height, that transgenic plant would be tall. An alternative approach is to sequence many mutant phenotypes of a specific ORF. That analysis may provide useful information that would allow you to determine a particular ORF is the gene of interest. This is the approach used in human genetics. 5. Prove one ORF is the gene by transformation or mutant analysis Mutant + ORF = Wild type? Yes? ORF = Gene

Gene Manipulation It is now routine to isolate genes But the target gene must be carefully chosen Target gene is chosen based on desired phenotype Function: Glyphosate (RoundUp) resistance EPSP synthase enzyme The development of the transgenic organism uses some gene isolated by the procedures that were just outlined. But it is important that the appropriate gene is used to obtain the specific phenotype you wish to develop. We are going to spend a bit of time concentrating on two important phenotypes: glyphosate (RoundUp) resistance and increased vitamin A content. Each of the phenotypes can be achieved by adding one or several genes to a plant. Increased Vitamin A content Vitamin A biosynthetic pathway enzymes

The RoundUp Ready Story Glyphosate is a broad-spectrum herbicide Active ingredient in RoundUp herbicide Kills all plants it come in contact with Inhibits a key enzyme (EPSP synthase) in an amino acid pathway Plants die because they lack the key amino acids The RoundUp Ready technology is the most visible plant biotechnology product on the market. To better understand plant biotechnology in general, it is important to understand the development of these transgenic organisms. RoundUp is a brand name herbicide manufactured by Monsanto Corp. The active ingredient in this herbicide is glyphosate. The chemical binds to the active site of the EPSP synthase enzyme. This enzyme is a key to the development of a group of amino acids called the aromatic amino acids. When this enzyme is bound by glyphosate, it can not synthesize those amino acids, and the plants die because protein synthesis is severely disrupted. Glyphosate will not bind the to a particular genetically-engineered version of EPSP synthase. Therefore RoundUp Ready crops with this altered enzyme will survive when sprayed with the herbicide. A resistant EPSP synthase gene allows crops to survive spraying

3-Enolpyruvyl shikimic acid-5-phosphate RoundUp Sensitive Plants Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids + Glyphosate X X This slide shows the actual biochemical pathway that we discussed in the previous slide. EPSP synthase synthesizes 3-enolpyruvly shikimic acid-5-phosphate. This is the essential precursor to aromatic amino acids. When plants are sprayed with a glyphosate-containing herbicide, such as RoundUp, this important precursor is not synthesized, and consequently the plant is starved of aromatic amino acids. The result is plant death. Without amino acids, plant dies X X

RoundUp Resistant Plants Shikimic acid + Phosphoenol pyruvate + Glyphosate RoundUp has no effect; enzyme is resistant to herbicide Bacterial EPSP synthase 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) RoundUp Resistant plants have a very simple solution. An engineered version of EPSP synthase, one that was discovered in a bacteria, is introduced into the plant. This enzyme can not be bound by glphosate. Therefore, if a field is sprayed with the herbicide, the introduced version of the gene produces a functional enzyme. The 3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized normally, and the plant produces enough aromatic amino acids to survive. With amino acids, plant lives Aromatic amino acids

The Golden Rice Story Vitamin A deficiency is a major health problem Causes blindness Influences severity of diarrhea, measles >100 million children suffer from the problem For many countries, the infrastructure doesn’t exist to deliver vitamin pills The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed. The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β-carotene, a yellow precursor to vitamin A is abundant in the seed. Improved vitamin A content in widely consumed crops an attractive alternative

Lycopene-beta-cyclase -Carotene Pathway in Plants IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Problem: Rice lacks these enzymes Unlike the single-step RoundUp Ready pathway, the β–carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels. Normal Vitamin A “Deficient” Rice

The Golden Rice Solution -Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Vitamin A Pathway is complete and functional Daffodil gene Single bacterial gene; performs both functions In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioniong versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway, was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β-carotene. It is this product that gives "Golden Rice" its characteristic yellow color. Daffodil gene Golden Rice

Metabolic Pathways are Complex and Interrelated Understanding pathways is critical to developing new products The “Golden Rice” story illustrates a key point: it is very important to industry metabolic pathways. These pathways are very important for our understanding of specific products are produced in the organism. Only by understanding this pathways will we be able to create novel new products.

Modifying Pathway Components Can Produce New Products Turn On Vitamin Genes = Relieve Deficiency Modified Lipids = New Industrial Oils This diagram shows in general the interrelationship between the many different pathways. A key point to understand is that the different sub-pathways interconnect. Therefore modify one component of the pathway may affect the production of a product in a separate sub-pathway. Keeping this in mind, we can now envision how to engineer plants so they produce novel products. We have already seen how modifying a vitamin biosynthetic pathway can positively affect vitamin production. We could also improve nutrition in other ways. For example, if we were to focus our attention on the amino acid pathways we could, for example, increase the lysine content in typically lysine-poor grains. Conversely, someday we might be able to improve legumes by introducing the correct genes necessary to enrich the metionine content. We could also envision new products if we modify other pathways. Oils are a key component to both the food and manufacturing industries. A better understanding of the genes in the complex lipid pathway may allow us to produce better industrial oils. Increase amino acids = Improved Nutrition

Trait/Gene Examples Gene Trait RoundUp Ready Bacterial EPSP Golden Rice Complete Pathway Plant Virus Resistance Viral Coat Protein Male Sterility Barnase This slide illustrates the variety of different traits that have been modified in plants. It also shows the particular gene that was introduced into the plant to obtain the specific trait. As you can see, scientists have successfully introduced many different genes and produced many different results. For example, it was discovered that expressing the in the plant a particular protein of a virus, the coat protein, the plant would then become resistant to that virus. This technique has been widely credited with saving the papaya industry in Hawaii, where the papaya ringspot virus nearly eliminated the papaya growing industry. This is a success story that is often overlooked, probably because the problem was to a crop of limited production value. Plant Bacterial Resistance p35 Salt tolerance AtNHX1

Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants It is now time to cover the development of transgenic crops in greater depth. The two major steps are creating a transformation cassette that contains the gene of interest, and then successfully introducing the cassette into the plant.

Transformation Cassettes Contains 1. Gene of interest The coding region and its controlling elements 2. Selectable marker Distinguishes transformed/untransformed plants All transformation cassettes contain three regions. The “gene of interest” region contains the actual gene that is being introduced into the plant. This is the gene that provides the new function to the plant. In this diagram, the region is shown in red. Many plant tissues are treated with the transformation cassette during the transformation step. Not all of these tissues actually receive the cassette. To distinguish those that contain the gene from those that don’t, it is necessary to use a selection process. The selectable marker is a gene that provides the ability to distinguish transformed from non-transformed plants. This is shown by green. The most common method to introduce the transformation cassette is by using the plant pathogen Agrobacterium. For this system to work it is necessary that the cassette contain insertion sequences that are used by the bacteria. These are shown by the gray. 3. Insertion sequences Aids Agrobacterium insertion

Gene of Interest Promoter Region Coding Region TP Promoter Region Controls when, where and how much the gene is expressed ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development) Transit Peptide Targets protein to correct organelle ex.: RbCS (RUBISCO small subunit; choloroplast target All of these components of the transformation cassette contain multiple components. In addition to the coding region that encodes the protein product, the gene of interest region also contains two important controlling regions. The promoter region resides just before the coding region and determines when, where, and to what degree the gene of interest will be expressed. In general, two types of promoter regions are used. A constitutive promoter turns the gene on in all tissues at all times. In general, this leads to a relatively high level of gene expression. The most often used constitutive promoter controls the expression of the 35S RNA of the cauliflower mosaic virus. It is abbreviated as CaMV35S promoter. Other promoters direct a very specific expression pattern. For example, the glutelin 1 promoter directs that the expression of the glutelin storage protein at a specific time of seed development. It also ensures the protein is only expressed in the rice endosperm. If the gene of interest is preceded by the CaMV35S promoter, it will be expressed in all tissues at all times. Conversely, the expression of the target gene could be limited to the endosperm if it is controlled by the glutelin 1 promoter. Some, but not all genes, encode protein that function in the plant organelles. These organelles are the chloroplast and the mitochondria. For example, photosynthesis, and part of the carbon and lipid metabolism pathways are carried out in the organelles. To ensure these protein are delivered to the appropriate organelle, a transit peptide is required. This is a short amino acid sequence that is found directly before the coding region. This sequence is recognized by proteins in the outer membranes of the appropriate organelle. This recognition process leads to the import of the protein into the organelle. Therefore, if you are gene of interest functions in the organelle, an appropriate transit peptide must be included in the transformation cassette Coding Region Encodes protein product ex.: EPSP -carotene genes

Selectable Marker Promoter Region Normally constitutive Coding Region ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter Coding Region Gene that breaks down a toxic compound; non-transgenic plants die ex.: nptII [kanamycin (bacterial antibiotic) resistance] aphIV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance] As stated above, the selectable marker is a gene that encodes a protein product. For it to be expressed, it also needs a promoter region. It is typical to use the constitutive CaMV35S RNA promoter. The gene it controls encodes a protein that enables a transformed plant to survive in the presence of normally toxic compound. The most often used selective agents are kanamycin and hygromyin, two bacterial antibiotics, and the herbicide glufosinate. The protein encoded by the selectable marker genes generally renders these selective agents harmless to the transgenic plant.

X Effect of Selectable Marker Non-transgenic = Lacks Kan or Bar Gene Plant dies in presence of selective compound X Transgenic = Has Kan or Bar Gene Plant grows in presence of selective compound This slide shows the effect of the selectable marker.

Insertion Sequences Required for proper gene insertions TL TR Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA Required for proper gene insertions The insertion sequences straddle the gene-of-interest coding region and the selectable marker. These are use by Agrobacteria to create a DNA molecule that is sent out of the bacteria into the plant where it is eventually inserted into the nucleus of a cell in the recipient plant tissue. If the cell follows the proper developmental pathway that leads to a new plant, every cell in that plant will contain the sequences in between the insertion sequences.

Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette) aphIV 35S Gt1 psy 35S rbcS crtl TL TR T-DNA Border Hygromycin Resistance Phytoene Synthase Phytoene Desaturase Insertion Sequence Selectable Marker Gene of Interest Gene of Interest This slide demonstrates one of the transformation cassettes used to develop “Golden Rice” was developed. Slowly click through this slide, and you will see each of the components of the cassette.

Delivering the Gene to the Plant Transformation cassettes are developed in the lab They are then introduced into a plant Two major delivery methods Agrobacterium Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens. The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun. Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants. Tissue culture required to generate transgenic plants Gene Gun

A Requirement for Transgenic Development plant grows to maturity Plant Tissue Culture A Requirement for Transgenic Development Callus grows This slide shows the basic steps of plant tissue culture. Some plant part is placed is on a defined culture media. That media induces the the tissue to develop callus. Callus is an undifferentiated mass of cells. These cells then grow into plant shoots, which are later rooted. The small seedling will then grown into a mature, seed-producing plant. When developing transgenic plants, the transformation cassette is introduced into that plant part that can be induced to grow new plants. A plant part Is cultured Shoots develop Shoots are rooted; plant grows to maturity

A natural DNA delivery system Agrobacterium A natural DNA delivery system A plant pathogen found in nature Infects many plant species Delivers DNA that encodes for plant hormones DNA incorporates into plant chromosome Hormone genes expressed and galls form at infection site In many ways, Agrobacterium, has been the most successful method of delivering DNA into plants. It is a naturally occurring plant pathogen. It inserts DNA into the nucleus of a plant cell. That DNA contain genes that encode hormones and food products the bacteria uses to support its own growth. Here you can see the gall growth on the plant tissue. This is the natural result of the infection. Gall on stem Gall on leaf

The Galls Can Be Huge This photograph shows exactly how large the gall can grow.

Natural Infection Process Is Complex The interaction between the bacteria, Agrobacterium, and the host plant is very complex. It has been studied in great depth and many of the major details of the interaction have been described. It is this understanding that allowed other scientists to convert Agrobacterium into a plant transformation vector that delivers the cassette. It works this way. An Agrobacterium strain in which the phytohormones have been removed is used. The transformation cassette is then introduced into that Agrobacterium strain. The source tissue for plant transformation is infected with the strain. Then the steps described previously are applied. The tissue is grown in the presence of the selective agent. That tissue culture media also contains the hormones necessary to allow the plant tissue develop new plants. Because of the selection pressure placed on that tissue, those new shoots will contain the important genes of interest found in the transformation cassette. For simplicity sake, many details are being left out. But these are many steps.

But Nature’s Agrobacterium Has Problems Infected tissues cannot be regenerated (via tissue culture) into new plants Why? Phytohormone balance incorrect regeneration Solution? Transferred DNA (T-DNA) modified by Removing phytohormone genes Early on it was known that tissues infected with Agrobacterium could not be coaxed to regenerate new plants. Soon it was realized that the plant hormone balance was not correct. To over come this effect, the genes encoding the phytohormones were removed. Once removed, plant tissues infected with the modified Agrobacterium could produce the regenerated plant. With this realization, it was a simple step to envision how to deliver genes of interest into a plant: include these genes in the cassette. Retaining essential transfer sequences Adding cloning site for gene of interest

The Gene Gun DNA vector is coated onto gold or tungsten particles Particles are accelerated at high speeds by the gun Particles enter plant tissue DNA enters the nucleus and incorporates into chromosome The second method currently in use is the gene gun. The principle is very simple. The transformation cassette DNA is coated onto a particle. That particle is then accelerated (using ballistics or an air stream). The particle then enters the plant cell. At that point, the transformation cassette DNA is eluted off the particle, and by a process that is not known, the DNA becomes integrated into the nucleus of the cell. The same basic principles guiding plant transformation with Agrobacterium is used with the gene gun. A tissue source that is capable of being manipulated to produce new plants is treated; in this case it is shot with particles containing the transformation cassette. The tissue is then placed under selection, and those shoots that develop contain the gene of interest. Integration process unknown

Transformation Steps Prepare tissue for transformation Introduce DNA Leaf, germinating seed, immature embryos Tissue must be capable of developing into normal plants Introduce DNA Agrobacterium or gene gun Culture plant tissue Develop shoots Root the shoots This slide summarizes the steps necessary for plant transformation. Field test the plants Multiple sites, multiple years

The Lab Steps And this slide illustrates those steps.

CBF transcription factors Lab Testing The Transgenics Insect Resistance Cold Tolerance This and the next slide illustrate the types of traits that can be obtain using genetic engineering of plants. Notice the particular types of genes that were used to obtain these traits. Some genes encode a protein that directly provides the trait. This is illustrated by the gene that encodes the Bt-toxin protein that is harmful to plant insect pests. Other genes encode protein that regulate the expression of a trait. Cold tolerance can be added to a crop by introducing gene that encode a transcription factor. These factors interact with other genes to turn on their expression. Transgene= Bt-toxin protein Transgene= CBF transcription factors

Mercuric ion reductase More Modern Examples Salt Tolerant Mercury Resistance More recently, such varied traits as salt tolerance and mercury resistance have been introduced into plants transferring genes for specific proteins. Transgene= Glyoxylase I Transgene= Mercuric ion reductase

The Next Test Is The Field Herbicide Resistance Non-transgenics The last step in plant genetic engineering is field testing. This slide shows a field that contains herbicide resistant and tolerant plants. Transgenics

Final Test Consumer Acceptance RoundUp Ready Corn Before After What is needed is for the public to accept these crops. Examples such as these, were a corn crop is freed of weed pressure make a compelling case for acceptance of these new agricultural products. But, it should be noted that these traits are all producer orientated. Before After

The Public Controversy Should we develop transgenics? Should we release transgenics? Are transgenics safe? Are transgenics a threat to non-transgenic production systems? The public, in general, is interested in the consumer perspective. Here are the general questions that drive the controversy. Are transgenics a threat to natural eco-systems?