This Document Contains Chapters 16 to 18 CHAPTER 16: CONTROL OF GENE EXPRESSION WHERE DOES IT ALL FIT IN? Chapter 16 builds upon the coverage of protein synthesis in Chapter 15 and provides detailed information about gene regulation and applies the concepts of mitosis and gene regulation to explain development in eukaryotic organisms. As in Chapter 15, it is important to stress the differences between prokaryotes and eukaryotes. This information in Chapter 16 is needed to fully understand the principles of biotechnology covered in Chapter 17 and genomics discussed in Chapter 18. SYNOPSIS Prokaryotes and multicellular eukaryotes both control gene expression, but for quite different reasons. Bacteria must exploit the resources of a changing environment. If they do not adapt, they die, but maintaining numerous unused enzymes is metabolically expensive. Multicellular eukaryotes must be protected from those changes. The hallmark of multicellular organisms is homeostasis: maintaining a constant internal environment. To ensure this, genes must be transcribed in a specific order over a specific time frame. Transcriptional control and post-transcriptional control are two primary levels of gene regulation. The former is the more common method. Transcriptional gene control is mediated by influencing the binding of RNA polymerase to the DNA helix. An mRNA transcript cannot be produced if RNA polymerase cannot bind to the promoter. Control to stimulate transcription can also be effected, thus facilitating the binding of polymerase and promoter. The entire DNA helix does not need to unwind for transcription to ensue. Only a small section needs to unwind, just enough to expose the major groove to the structural motif of the correct protein. Nearly all proteins use one of four motifs to bind with their respective DNA region. The most common is the helix-turn-helix motif, two alpha helical regions linked by a short nonhelical region. One of the helices aligns next to the DNA. The other, the recognition helix, physically fits into the DNA major groove. The homeodomain motif is a specialized class of helix-turn-helix that was discovered in homeotic mutants Drosophila. The zinc finger motif uses atoms of zinc to help the protein bind to its DNA. In the leucine zipper motif, two protein subunits create a single Y-shaped DNA-binding site that resembles a partially opened zipper. Prokaryotes alter expression of genes when their environment changes. A common pattern in prokaryotes is that gene products necessary for certain catabolic reactions are only expressed when the substrate is present. Such systems are said to be inducible. Other gene products necessary for anabolic pathways are only expressed when the cell needs to build that particular molecule. These are referred to as repressible systems. Each system involves regulatory proteins that will bind to the DNA and alter genetic expression, either by initiating expression (positive) or suppressing expression (negative). Repressors, regulatory proteins that exhibit negative control, act as OFF switches. These can be seen in both inducible and repressible systems. In the inducible E. coli lac operon, lactose binds to the regulatory protein and prevents it from halting transcription necessary for lactose metabolism. In the repressible trp operon, tryptophan binds to the regulatory protein allowing for the suppression of expression genes necessary for tryptophan synthesis. Activators, regulatory proteins that exhibit positive control, are ON switches to ensure that transcription does not occur unless a specific activating chemical is present. The E. coli catabolite activator protein (CAP) is a good representation of this system. The lac operon of E. coli combines ON and OFF switches to ensure that (1) the lactose degrading enzymes are not produced when glucose is present – there’s no need for it since glucose is a better food source, and (2) they are only produced when lactose is present – there’s no need to make enzymes if their substrate isn’t present. Genetic regulation in eukaryotes is much more complicated than what is seen in prokaryotes. In comparing transcriptional control between eukaryotes and prokaryotes, similarities due exist. Regulatory proteins, called transcription factors, must bind to DNA to regulate transcription. Transcription factors can either be basal transcription factors, proteins necessary for recruitment and proper binding of RNA pol II, or specific transcription factors, proteins that alter expression levels depending on specific signals. Eukaryote gene control greatly depends on the structure of the eukaryotic chromosome. Histones affect gene transcription by physically blocking the promoter with the nucleosome they create. Methylation, once thought to be a primary regulator in vertebrates, helps ensure that once a gene is turned off, it stays off. Post¬transcriptional control is common in eukaryotes. Researchers have found that small RNA molecules seem to interfere with translation directly or the breakdown of the mRNA before translation. The eukaryote primary mRNA transcript is a linear patchwork of coding exons and noncoding introns. The entire sequence is made during transcription, the introns are cut out later. In many cases, the various ways the exons can be spliced back together allows for production of different polypeptides from just one gene. Aside from the importance of gene control, this kind of transcription seems quite wasteful. Only ten percent of all transcribed genes are exons and only half of that ever gets out of the nucleus. It is yet unknown as to whether this is under any kind of selective control. Proteins called translation factors regulate production of polypeptides from the mRNA transcript. Translation repressor proteins can also shut down translation by preventing the attachment of the transcript to a ribosome. Although most mRNA transcripts are very stable, some, like those associated with regulatory proteins and growth factors, are less stable. They possess certain 3’ sequences that make them attractive to mRNA degrading enzymes. This ensures that control by these proteins remains as transitory as it should be. Multicellular eukaryotes depend on cell specialization and have evolved complex developmental processes to ensure that the adult is formed properly. After cleavage of the initial zygote, vertebrate animals go through an involved series of stages of cell movement and tissue formation. Cells can be switched from one developmental path to another via the process of induction. Inducing cells secrete proteins, intercellular signals that determine what kind of tissue a cell will become. The same signal can have different results through variations in concentration. A cell is said to be determined when it has become committed to a particular developmental path. Differentiation is the cell specialization that exists at the end of that path. Therefore, cells can be determined, but not yet differentiated. LEARNING OUTCOMES 16.1 All Organisms Control Expression of Their Genes 1. Identify the point at which control of gene expression usually occurs. 2. Compare strategies for control of gene expression in prokaryotes and eukaryotes. 16.2 Regulatory Proteins Control Genes by Interacting with Specific DNA Nucleotide Sequences 1. Describe the common features of DNA binding motifs 16.3 Prokaryotes Regulate Their Genes in Clusters 1. Compare control of enzyme production by induction and repression. 2. Explain how the lac operon is regulated based on the availability of lactose. 3. Explain how glucose affects the production of lactose-utilizing enzymes. 4. Explain how the trp operon is regulated by levels of tryptophan. 16.4 Transcription Factors Control Gene Transcription in Eukaryotes 1. Distinguish between the roles of general and specific transcription factors. 2. Explain how transcription factors can act at a distance from a promoter. 3. Contrast the roles of coactivators and transcription factors. 4. Describe the interactions of the components of the eukaryotic transcription complex. 16.5 Eukaryotic DNA Is Packaged into Chromatin 1. Describe the role of methylation in gene regulation. 2. Describe how alteration of chromatin structure can affect gene expression. 16.6 Eukaryotic Genes Are Also Regulated After Transcription 1. Describe the role of small RNAs in regulating gene expression. 2. Describe how RNA silencing may act to alter chromatin structure. 3. Describe how alternative splicing can produce tissue-specific gene expression. 4. Explain how editing of RNA transcripts can affect gene expression. 5. Describe how gene expression can be regulated at the level of translation. 6. Describe how eukaryotic cells control mRNA degradation. 16.7 Gene Regulation Determines How Cells Will Develop 1. Describe the progressive nature of cell determination. 2. Explain the role of cytoplasmic determinants in determination. 3. Contrast the role of induction with the role of cytoplasmic determinants. COMMON STUDENT MISCONCEPTIONS There is ample evidence in the educational literature that student misconceptions of information will inhibit the learning of concepts related to the misinformation. The following concepts covered in Chapter 16 are commonly the subject of student misconceptions. This information on “bioliteracy” was collected from faculty and the science education literature. • Students have trouble distinguishing chromatin from chromosomes • Students do not fully understand the role of genetics and environment on determining observable variation in organisms • Students think that prokaryotes and eukaryotes have the same DNA structure • Students do not make the connection between environmental or cell signals with gene regulation • Students believe that all genes program for proteins • Students are unfamiliar with the exact nature of regulatory genes • Students believe the eukaryotes have operons • Students believe that all transcription factors are general • Students are unaware of the enzymatic nature of RNA • Students believe that mRNA splicing occurs without variation • Student believe that all gene regulation occurs before or during transcription • Students believe all of development is driven by genes INSTRUCTIONAL STRATEGY PRESENTATION ASSISTANCE Not only did the cell beat us to the punch as far as the assembly line, it also did module building first. Alternative splicing is like building a modular home. The company makes a selection of room modules; the buyer decides which ones to use and where to put them. Do you want two bedrooms or three? Should the dining room or the family room be to the left of the kitchen? It ends up looking like an entirely different house than the one next door. Students must know what each part of the operon does to clearly understand gene regulation and the lac operon. A regulator wouldn’t function properly if it were at the end of the operon any more than a spillway would regulate the flow of water into a mill if it were on the downstream side of the wheel. Don’t let the students confuse exons and introns. The immediate tendency is to associate exon with other words starting with “ex,” where “ex” means out, and assume that an exon is cut out. WRONG! The “ex” in exon derives from expressed, as in expressed sequence. The “in” in intron comes from intervening sequence, that is, the section that is later cut out. (This may be one time that it is beneficial that most students merely memorize words rather than try to understand where they come from.) HIGHER LEVEL ASSESSMENT Higher level assessment measures a student’s ability to use terms and concepts learned from the lecture and the textbook. A complete understanding of biology content provides students with the tools to synthesize new hypotheses and knowledge using the facts they have learned. The following table provides examples of assessing a student’s ability to apply, analyze, synthesize, and evaluate information from Chapter 16. Application • Have students predict the type of gene regulation carried out by chloroplasts and mitochondria. • Have students design an experiment to see if a gene associated with the breakdown of starch is inducible. • Ask students to predict the outcomes of a mutation that prevents the removal exons. Analysis • Have students explain the differences and similarities between prokaryotic and eukaryotic gene regulation. • Ask students to determine the effects of genetic disease that prematurely labels proteins with ubiquitin. • Ask students to predict the possible outcomes if the promoter of a gene develops a frameshift mutation. Synthesis • Ask students to come up with a way to get prokaryotes to regulate eukaryotic enzymes after inserting a gene for that enzyme. • Have students develop a medical use for ubiquitin. • Ask students come up a reason for permanently activating certain inducible genes in agricultural plants. Evaluation • Ask students to evaluate the possible medicinal value of chemicals that inhibit certain transcription factors. • Ask students to determine the safety of drugs that prevent the formation of polyubiquitinated proteins involved in depression. • Ask to evaluate the safety concerns of introducing eukaryotic genes into prokaryotes. VISUAL RESOURCES Palindromes are words that exhibit two-fold rotational symmetry (bob, kook, deed). The phrase “a toyota” is a palindrome as is “a man, a plan, a canal, panama.” Instruct students to search for other examples of palindromes. The scifi film “Gattaca” touches on future (or maybe not so future!) gene technology and the ethical implications of genetic control. Substantial information is available at the movie website http://www.sciflicks.com/gattaca/. IN-CLASS CONCEPTUAL DEMONSTRATIONS A. Stringing Along Gene Regulation Introduction This demonstration provides a tangible model for showing students RNA splicing, methylation, and histone modification. Materials • Thick permanent markers • White Clothesline or thick rope • Yellow twine • Colored modeling clay • Toy car • 1 inch thick slivers of duct tape • Scissors Procedure & Inquiry 1. Review the concepts of eukaryotic gene regulation. 2. Tell the students you will be using rope to represent at the double helix of DNA, a. Use clay to represent the histones and show how DNA can be wrapped tightly around histones to prevent expression. b. Then take the DNA and say you will be adding methyl groups to the DNA strand. Place small chunks of clay on the string to represent methylation. Then say that the car is RNA polymerase and “run” it down the strand. Explain that the “road bumps” inhibit the function of RNA polymerase. 3. Now, tell the students you will be using rope to represent a pre-mRNA molecule. a. Mark introns with the colored markers b. Ask the students to tell you what happens next and why c. Proceed to cut out the introns while explaining pre-mRNA splicing d. Ask the students to tell you what happens next and why e. Tape the pieces together using the duct tape f. Then explain the addition of the poly A tail by taping the yellow twine to the rope g. Ask the students to explain the function of the poly A tail h. Discuss the mRNA capping process and add a large nub of clay to the end of rope opposite the poly A tail i. Ask the students to explain the function of the capping process j. Explain that the mRNA can now be transported to ribosomes 4. Now use the scissors to chop up the mRNA explaining that mRNA is destroyed in the cytoplasm as a way of regulating gene expression B. Visual Chromosomes Introduction The complexity of information on each human chromosome is amazing to see particularly in context of the gene regulation that takes place during development. The Department of Energy provides a website called “Human Chromosome Launchpad” which has up-to-date cartoon images of the genes on each of the human chromosomes. The website is a useful demonstration tool for hypothesizing about the regulatory mechanisms needed during human development. Materials • Computer with live access to Internet • LCD projector attached to computer • Web browser with bookmark to Human Chromosome Launchpad at: http://www.ornl.gov/sci/techresources/Human_Genome/launchpad/ Procedure & Inquiry 1. Provide students with a brief discussion about the complexity of gene regulation needed for development. 2. Have the students hypothesize about the organization of genes needed to streamline the expression of polygenic and pleiotropic traits. 3. Then go to the Human Chromosome Launchpad website. 4. Click on one of the chromosomes and then click on Images to investigate its details and see an image of the identified genes. The chromosome can be zoomed for viewing using the instructions on the website. Chromosomes can be printed for students to use in group work. 5. Take time to ask the students to review particular features including the assortment of organization of traits of each chromosome. 6. This demonstration can be expanded to a take home activity in which students write a “resume” for a chromosome. LABORATORY IDEAS Lego® My Genes Activity a. Tell students that you would like them to design a model of depicting gene regulation in prokaryotes and eukaryotes. Explain that scientists commonly make tangible models of biological molecules to better understand cellular functions. b. The following materials should be provided to a small group of students: a. Lego® blocks of various colors b. Colored markers c. Scissors d. A roll of cellophane tape e. A roll of Velcro®-type adhesive tape f. Yarn or thick string g. Pop beads of various colors c. Explain to students that the models should show the differences between prokaryotic and eukaryotic gene regulation. The models should also take into account all of the factors involved in controlling genes. d. Have the students explain their models to the class. The students should use their models to compare and contrast the genomic regulation of different cells. e. Then have the class briefly evaluate the various group models for accuracy/ LEARNING THROUGH SERVICE Service learning is a strategy of teaching, learning and reflective assessment that merges the academic curriculum with meaningful community service. As a teaching methodology, it falls under the category of experiential education. It is a way students can carry out volunteer projects in the community for public agencies, nonprofit agencies, civic groups, charitable organizations, and governmental organizations. It encourages critical thinking and reinforces many of the concepts learned in a course. 1. Have students do a presentation on the link between gene regulation and cancer at a college or school health fair. 2. Have students design an educational PowerPoint presentation on gene regulation for high school teachers. 3. Have students tutor middle school or high school biology students studying genetics. 4. Have students design and build an accurate model of operons for a school library or science department. ETYMOLOGY OF KEY TERMS anabolism synthesizing a substance (from the Greek an- up and ballein- to throw) catabolism the breakdown of a substance (from the Greek kata- down and ballein- to throw) micro- small; too small to be seen with the naked eye (from the Greek mikros- small) transcription the process of producing an RNA molecule using the information in a gene (from the Latin trans- across or through and scribere- to write) translation the process of synthesizing a polypeptide using the information in a mRNA molecule(from the Latin trans across or through and latus, past participle of ferre- to carry) CHAPTER 17: BIOTECHNOLOGY WHERE DOES IT ALL FIT IN? Chapter 17 investigates contemporary uses of biotechnology based on the principles of classical and molecular genetics. This chapter gives the instructors many opportunities to ask students critical thinking questions about applications of genetics knowledge. The topics in this chapter also stir much debate in society and should be addressed as part of the coverage. Chapter 17 is the capstone chapter for gene expression coverage from Chapters 15 and 16 of the book should be revisited when covering genomics information in Chapter 18. SYNOPSIS Science has reached the exciting, but potentially dangerous stage, at which we are learning to manipulate the materials of heredity. The first human genes isolated and inserted into bacteria turned these cells into miniature factories producing interferon. Many bacteria possess restriction endonucleases to protect themselves from invading viruses. Scientists use these enzymes to chop up strands of DNA at specific locations. Such specificity assures that a given enzyme will always break up a specific kind of DNA into exactly the same size and number of fragments. These fragments constitute a library of DNA sequence information. Restriction enzyme specificity also assures that all of the fragments possess identical, short sequences called “sticky ends.” Each strand of a sticky end is complementary to the other strand and can be joined to the other ends when treated with a DNA ligase. DNA Fragments, even those from different organisms that have been cut with the same restriction enzyme can be joined enabling the insertion of foreign genes into a plant, animal, or bacterial genome. Bacterial plasmids and viruses are the vehicles by which such genes are inserted into the host DNA, the crux of genetic engineering. There are four steps in this process: cleavage, producing recombinant DNA, cloning, and screening. Cleavage is accomplished using the restriction endonuclease that will produce the desired sticky ends. The fragments are then inserted into the desired vehicle. Unfortunately, very few vehicles actually receive DNA fragments and even fewer get the desired piece. At this point, vehicles not carrying fragments are eliminated, generally by prior association with an antibiotic resistance gene. Each colony of cells is cloned and allowed to multiply, thus replicating not only its own genome but the added fragment as well. The clones are then screened to determine which clonal line contains the desired fragment. Polymerase chain reaction is another new molecular technique that amplifies DNA in an in vitro sample. Frequently the DNA in a sample (of blood for example) is so small that it cannot be analyzed directly. With PCR, the DNA is copied using a microprocessor-controlled thermoregulator. The DNA unzips as the temperature is increased. When it is lowered, polymerase enzymes catalyze the replication of DNA from special primers, making a new strand from each original strand. Thus the amount of DNA is doubled at each cycle – 2 strands to 4 strands to 8 strands to 16 strands and so forth. This method is substantially quicker than cloning the DNA strand via plasmids or viruses. The isolation and amplification of DNA can result in a large number of DNA fragments. Storing and sorting DNA fragments requires a DNA library. Sometimes DNA is not available but an isolated sample of RNA can be converted into a DNA fragment. Reverse transcriptase is used to make a copy of DNA (cDNA) from an RNA molecule. A library of cDNA contains only DNA that is expressed as RNA in a cell. DNA is readily identified using a technique called Southern blotting. Differences in DNA sequences are identified by RFLP analysis. Each individual identified by the RFLP patterns possessed what is referred to as a DNA fingerprint. A process called in vitro mutagenesis in mice produces “knockout mice” in which a known gene is inactivated, allowing a study of the effects of the gene. RNA Interference (RNAi) can achieve similar results as in vitro mutagenesis but the alteration to the genes is not permanent. Biotechnology uses genetic engineering techniques to solve practical problems. The biological community is busy sequencing the entire human genome, certainly an enormous task. DNA fingerprinting has been used to identify and convict numerous criminals. Dozens of commercial applications exist to utilize this revolutionary technology. The most obvious application, pharmaceuticals, however, encounters additional problems of separating the desired product from the rest of the cellular material. Attempts are being made to construct piggyback vaccines, placing genes coding for the exterior of a virulent virus within the harmless vaccinia virus. Agricultural uses range from developing resistance to herbicides, viruses, and insects; to inserting genes for nitrogen fixation and improving growth and plant nutritional value. Society must be informed about these biological processes to ensure our safety and economic well¬being, as well as that of future generations. Lack of sufficient biological knowledge is the source of most of the public’s concern about genetically engineered products. Many assume that BST in milk products may cause human growth problems; they lack the physiological knowledge that this protein is degraded in the stomach like all other proteins. A great many people do not trust governmental safeguards and fear the inadvertent or intentional development of lethal viruses and bacteria. Although there is little scientific need for labeling genetically modified food products, the public has the right to insist upon it. If properly done, labeling should serve to educate consumers as well as inform them. LEARNING OUTCOMES 17.1 Enzymes Can Be Used to Manipulate DNA 1. Explain how restriction endonucleases produce DNA fragments with “sticky ends”. 2. Describe how DNA restriction fragments are joined together. 3. Explain the physical basis for separation of DNA fragments by gel electrophoresis. 4. Describe how transformation allows the construction of transgenic cells. 17.2 Molecular Cloning Allows Propagation of Specific Gene Sequences 1. Describe the use of vectors in molecular cloning. 2. Describe how specific genes can be isolated from a cDNA library 17.3 Analysis of Molecular Clones is an Essential Tool of Modern Biology 1. Explain the Southern Blotting method of identifying genes. 2. Describe how the sequence of a DNA fragment is determined. 3. Demonstrate how the polymerase chain reaction produces large amounts of DNA from a single template. 4. Explain how the yeast system is used to study protein-protein interactions. 17.4 Molecular Clones Can Be Used to Genetically Engineer Cells 1. Describe three applications of cloning technology. 17.5 Applications of Genetic Engineering Include Major Medical Advances 1. Explain how eukaryotic proteins can be produced in bacterial cells. 2. Contrast subunit and DNA vaccines. 3. Evaluate potential problems of gene therapy, and what is being done to counter them. 17.6 Genetically Engineered Plants and Animals Are Revolutionizing Agriculture 1. Compare recombinant technology techniques in plants with those in bacteria. 2. Explain how glyphosate resistance in crop plants confers herbicide tolerance. 3. Explain the operation of stacked GM crops. 4. Describe how researchers have genetically engineered a more nutritious type of rice. 5. Assess whether GM foods are safe to eat, and whether GM crops are harmful to the environment. 6. Explain how human genes can be produced in crop plants and farm animals. COMMON STUDENT MISCONCEPTIONS There is ample evidence in the educational literature that student misconceptions of information will inhibit the learning of concepts related to the misinformation. The following concepts covered in Chapter 17 are commonly the subject of student misconceptions. This information on “bioliteracy” was collected from faculty and the science education literature. • Students believe that all biotechnology is genetic engineering • Students do not fully understand the role of genetics and environment on determining observable variation in organisms • Students do not differentiate the genetic differences between prokaryotes and eukaryotes • Students believe that genetic modification is more unpredictable than selective breeding in determining an organisms characteristics • Students believe that genetically modified organisms are inherently dangerous • Students believe that genetically all modified foods are unsafe or cause allergies • Students believe that gene transfer introduces many characteristics of one organism into another • Students believe the virus vectors used in gene transfer are more dangerous than natural viruses • Students believe it is not possible to introduce the genes of animals into plants • Students believe it is not possible to introduce the genes of plants into animals • Students believe that cloning is an unnatural process • Students believe that genetic modification and cloning introduces unpredictable mutations INSTRUCTIONAL STRATEGY PRESENTATION ASSISTANCE Here we have what’s happening in biology today; where the money is to be made! Discuss the mechanisms of gene technology before discussing its implications. Students have a hard time with the nature of the “sticky ends” resulting from treatment with restriction enzymes. To complicate matters, some blunt cutting enzymes have been discovered as well. It might be helpful to present them with several DNA sequences and show how different restriction enzymes would fragment the sequence. You can then show how identical “sticky ends” can be joined together. Genetic engineering would be significantly more difficult without plasmid and viral vectors. Plasmids were presented in the last chapter, viruses were discussed to some extent in the chapter before that. Recall in either case, how the vector is naturally able to insert genetic material into a complete genome, the plasmid into bacteria, the virus into eukaryotes causing some forms of cancer. Science is merely adapting a natural phenomenon to its own benefit. Screening is not only the most difficult part of genetic engineering to do, it is the hardest part to understand. Include the presence of the antibiotic resistance gene at the onset of your discussion. Explain its function at the screening step. The many technical terms associated with gene technology can be confusing; most are associated with genetic engineering in that they are means for identifying the cell with the correct stuff. Probes have been developed for a number of tumor cell lines and Huntington’s disease. The latter is 95% to 98% accurate in determining whether the gene is present. Thus persons with the disease in their family background can be tested long before the onset of the disease itself (most individuals refuse testing or are tested and don’t want to be told the results). Knowledge of test results may impact personal lifestyle and plans for having children as well as insurance and health policies. One merely needs to pick up the science section of the weekly newspaper, or a lay science magazine to see examples of gene technology in action. As a result, it is important to discuss the implications of such research and the necessary scientific and governmental regulations. This is one of the stronger reasons to have some knowledge of biology, to be able to make informed decisions, and to determine if the decisions made by those in power are indeed in the best interest of the populace. Someone will need to make difficult decisions in the not-so-distant future. Just because science can perform certain technological feats doesn’t mean that it should be allowed to do so. Conversely, just because some gene technology is potentially dangerous, doesn’t mean that all related technology should be brought to a halt. It’s your students who will be making the political decisions for the future of the world. HIGHER LEVEL ASSESSMENT Higher level assessment measures a student’s ability to use terms and concepts learned from the lecture and the textbook. A complete understanding of biology content provides students with the tools to synthesize new hypotheses and knowledge using the facts they have learned. The following table provides examples of assessing a student’s ability to apply, analyze, synthesize, and evaluate information from Chapter 17. Application • Have students design a theoretical vector for introducing a bacterial gene into an animal cell. • Have students hypothesize about feasible traits that can be introduced into crops. • Ask students to hypothesize how ligase can be used to make new genes. Analysis • Have students to compare and contrast traditional selective breeding to genetic engineering as a means of producing new agricultural organisms. • Ask students to determine the problems of introducing eukaryotic DNA into prokaryotes using genetic technology. • Ask students to explain why it is possible to damage existing genes when new genes are introduced into genomic DNA. Synthesis • Ask students develop a hypothetical expression vector that would prevent genetically modified crops from reproducing with related wild plants. • Ask students develop ways of using restriction enzymes as a tool for controlling viral diseases. • Ask students come up with a way strategy of using plants to remove hazardous wastes from the soil. Evaluation • Ask students to evaluate the pros and cons of growing genetically modified crops such as Bt corn. • Ask students to assess the value of cloning in agriculture. • Ask student to debate the safety concerns associated with the creation of a new gene. VISUAL RESOURCES Palindromes are words that exhibit two-fold rotational symmetry (bob, kook, deed). The phrase “a toyota” is a palindrome as is “a man, a plan, a canal, panama.” Search the web for thousands of examples, but start here: http://www.cs.rdg.ac.uk/archive/evihcra/ ku.ca.gdr.sc.www//:ptth/. Hopefully you will notice that the URL itself is a palindrome! The scifi film “Gattaca” touches on future (or maybe not so future!) gene technology and the ethical implications of genetic control. Substantial information is available at the movie website http://www.sciflicks.com/gattaca/. IN-CLASS CONCEPTUAL DEMONSTRATIONS A. Virtual Vector Introduction This demonstration provides a tangible model for showing students design elements of an expression vector. Materials • Thick permanent markers • White clothesline or thick rope • Yellow wool or twine • Red wool or twine • Blue wool or twine • Green wool or twine • 1 inch thick slivers of duct tape • Scissors Procedure & Inquiry 1. Review the use of an expression without describing its components . 2. Tell the students you will be using rope to represent a plasmid to be used as an expression vector a. Take a 2” loop of white clothesline or rope and tape it into a loop b. Tell the class that the loop can be a plasmid or a yeast artificial chromosome (YAK). 3. Then ask the class what they would need to make a eukaryotic expression vector. 4. Use the materials in the following manner as the class is making suggestions: a. Use the scissors to represent restriction enzymes for cutting open the plasmid b. Use the duct tape to present ligase bonded regions of the vector c. Use the wool or twine to represent different components of the vector 5. Cut and paste the expression vector based on students comments 6. Then have the class evaluate the accuracy of their vector B. Virtual Biotechnology Concept Map Introduction This fun and fast way to build a concept map engages students in developing a scheme for reviewing all the facts and concepts associated with DNA replication. It helps student select relevant information needed to understand DNA replication. In addition, it helps them incorporate concepts learned in other sections of the book that contribute to an understanding of DNA replication. The simple click and drag animated concept mapping tool should be practiced before using in class. Materials • Computer with live access to Internet • LCD projector attached to computer • Web browser with bookmark to Michigan State University C-Tool: http://ctools.msu.edu/ctools/index.html Procedure & Inquiry 1. Tell students that you would like to do a quick review of the concepts associated with DNA replication. 2. Then go to the Michigan State University C-Tool and add the concept map term “Biotechnology”. Use the “Add” and “Concept Word” feature to place a term on the map background. 3. Solicit a few more terms or concepts and then ask the class how the concepts are connected to each other. Use the “Add” and “Linking Line” feature to build a connecting line. 4. Then ask the students to justify the concept linking lines. Use the “Add” and “Linking Word” feature to place student comments on the map. 5. Continue the activity until you feel the students made a comprehensive map. LABORATORY IDEAS Biotechnology research requires computational studies using a DNA database before proceeding with the laboratory work needed to produce a genetically modified organism. This activity introduces students to the use a DNA sequence search engine called BLAST. a. Introduce students to the value of knowing gene sequences before designing expression vectors used to produce genetically modified organisms. Tell them that researchers use electronic on-line databases to search for gene sequences for a particular protein they want to place into an organism. b. Provide students with the following resources: a. Computer with Internet access b. Web browser with a bookmark to BLAST Tutorial (http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html) c. Web browser with a bookmark to BLAST Search (http://www.ncbi.nlm.nih.gov/blast/index.shtml) d. A list of amino acids with their single letter designations e. Codon chart c. Tell the students go the BLAST tutorials on Information Page of the BLAST website. d. Then tell the student to type in the amino acid sequence into the Input box on Part 2 of the PSI-BLAST tutorial at http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/psi1.html: b. Have them press the Search button without making any other changes to the search. c. Then have them discuss with each other the diversity of genes programming that amino acid order in its DNA sequence. e. Now have the students go to the BLAST Search and click on the Protein-protein BLAST (blast) link. f. Instruct them to type in the same amino acid sequence and analyze the results. g. Then have them analyze the results including any information provided in the links from the completed search page. h. Then instruct the students to calculate the approximate DNA sequence for the gene. i. Have the students go to the Nucleotide search and type in the purported DNA sequence. j. Have the students assess the outcomes of their search. They should be able to explain why the search may or may not have found the appropriate gene for the amino acid. LEARNING THROUGH SERVICE Service learning is a strategy of teaching, learning and reflective assessment that merges the academic curriculum with meaningful community service. As a teaching methodology, it falls under the category of experiential education. It is a way students can carry out volunteer projects in the community for public agencies, nonprofit agencies, civic groups, charitable organizations, and governmental organizations. It encourages critical thinking and reinforces many of the concepts learned in a course. 1. Have students do a presentation on biotechnology to a civic group. 2. Have students design an educational PowerPoint presentation of genetic engineering for high school teachers. 3. Have students tutor high school biology students studying genetics. 4. Have students design a series of educational posters about biotechnology for a local school or library. ETYMOLOGY OF KEY TERMS -ase enzyme (modern) bio- life or relating to living organisms (from the Greek bios- mode of life) -de to remove (from the Latin de- from; down; away) di- two; twice (from the Greek di- two) polymer- a chemical compound having many parts (from the Greek polys- many and meros- part) micro- small; too small to be seen with the naked eye (from the Greek mikros- small) prote- of, or relating to, protein; first (from the Greek protos- first) trans- across, through (from the Latin trans- across or through) CHAPTER 18: GENOMICS WHERE DOES IT ALL FIT IN? Chapter 18 blends the earlier coverage of genetics with Chapter 17 to discuss the topic of genomics. As with Chapter 17, Chapter 18 gives the instructors many opportunities to ask students critical thinking questions about applications of genetics knowledge. Chapter 18 builds upon coverage from Chapters 15 and 17. The structure of DNA and protein synthesis should be briefly revisited before covering the genomics information in Chapter 18. Chapter 18 is essential to explain the adaptation of organisms covered later in the textbook SYNOPSIS A genome, all the genetic information of an individual, can be characterized in different ways. In the past, genomes where characterized by genetic maps, or linkage maps, representing the positional relationship of genes on chromosomes. Currently, physical maps are being constructed of many species representing the positional relationship of DNA sequences on a particular chromosome. With the results of the current genome projects, entire genomes are being sequenced and assembled in proper order, similarly to the pieces of a jigsaw puzzle. The participation of large numbers of researchers and the advances in DNA technology, such as automated sequencers, have been crucial to researchers' ability to compile complete or almost complete genome sequences. The study of genomes has several applications. These include determining the minimal genome to support a cell, investigating proteins more fully and the use of comparative genomics to answer evolutionary questions. One of the more surprising findings of the Human Genome project is the actual number of genes a complex organism actually have. Comparing the genomes of several organisms, researchers have found that number of genes does not necessarily correlate with complexity of the organism. With genome sequences now available, researchers are attempting to locate genes within the genome by located coding sequences. By applying the knowledge of gene expression, transcription and translation, researchers can identify regions that appear to code for start and stop codons. These regions are known as open reading frames. Interestingly, evidence seems to indicate that alternative splicing patterns in humans seems to allow for complexity of proteins. The complexity of proteins is due to different patterns of intron splicing following transcription and not from addition of genes. Four classes of protein-encoding genes have been identified in eukaryotic genomes: single-copy genes, segmental duplications, multigene families, and tandem clusters. Also, it seems that a large portion of eukaryotic genomes are actually non-coding regions. The vast amounts of information provided by the genome projects have given rise to new fields of science, particularly genomics. Genomics is generating new interests and building links between genetics, evolution, and development. Comparative genomics is addressing areas such as: How have complex traits evolved? What are the origins of genomic differences? How have developmental mechanisms evolved? Evolutionary histories of species are inscribed in the nucleotides of the DNA molecules in their genomes. Scientists are finding many similarities in DNA sequences in species that had a common ancestor hundreds of millions of years ago. These discoveries are especially interesting because evidence suggests that common or similar genetic sequences have different expressions in different species. Functional genomics is a field that attempts to determine the function of the vast number of proteins an organism can produce. Proteomics, yet another field that has been bolstered by the genome projects, is the study of the proteome, all the proteins coded for by the genome. The information provided by the genome projects has opened many areas of research, both in theoretical science, but also in applied science. Information learned from the genome projects has the potential to improve pharmaceuticals, agriculture and diagnostic tools. But, with this information come many questions. How will society use the information? Will it be used for purposes of screening and possibly discrimination of some individuals? Will it alter the way we view certain behavioral traits? LEARNING OUTCOMES 18.1 The Challenge in Mapping Genomes Is to Order Many Segments 1. Explain how STS sites allow ordering of genomic fragments. 18.2 Sequencing Large Genomes Is an Automated Process 1. Explain how we can clone larger fragments of DNA. 1. Differentiate between clone-by-clone sequencing and shotgun sequencing. 18.3 Sequencing the Human Genome Has Revealed Many Surprises 1. Explain the discrepancy between the number of unique mRNAs and unique genes. 2. Describe the four classes of gene copy number on human chromosomes. 3. Describe the six major classes of noncoding DNA on human chromosomes. 4. Describe the evidence that genes have moved between organelle and nuclear genomes. 18.4 Microarrays Allow Comparisons of Genomes of Individuals 1. Describe the different uses of DNA microarrays. 18.5 Comparing Genomes Reveals Evolutionary History 1. Compare the number of genes in the different mammalian genomes. 2. Describe the degree to which genomics is revealing new, previously unknown genes. 3. Explain why some organisms have far more genes than their complexity would seem to indicate. 4. Explain why sequences like the HOX genes are highly conserved within genomes. 5. Compare the rates of genomic change in major groups of organisms. 6. Describe the evidence that genomes continually accumulate genetic differences. 7. Describe the potential functional role of genomic DNA that does not encode proteins. 8. Compare what we know about human and chimp genomes. 18.6 Proteomics Is the Study of All Proteins Encoded by a Genome 1. Distinguish between genomics and proteomics. 18.7 Genomics Has Important Applications 1. Describe how genomics can be used to rapidly identify potential pathogens. 2. Describe how genomics can be used to identify useful genes in crop plants. 3. Describe the issues of intellectual property and privacy raised by genomics. COMMON STUDENT MISCONCEPTIONS There is ample evidence in the educational literature that student misconceptions of information will inhibit the learning of concepts related to the misinformation. The following concepts covered in Chapter 18 are commonly the subject of student misconceptions. This information on “bioliteracy” was collected from faculty and the science education literature. • Students believe that all genes program for proteins • Students do not distinguish between the DNA of prokaryotes and eukaryotes • Students believe that phenotype can be fully by knowing the genotype • Students do not take into account the presence of exon information in genomic DNA • Students do not fully understand the role of genetics and environment on determining observable variation in organisms • Students believe that genes for one characteristic are all located on the same chromosome • Students do not distinguish genomics from proteomics • Students believe that all mutations are deleterious. • Students believe that DNA variation between different organisms is always very high • Students are unfamiliar with the degree of conserved genes between unrelated organisms INSTUCTIONAL STRATEGY The area of genomics is so vast and new that many professional scientists are not completely sure of all the applications or terminology, for that matter, of this field of science. Considering the number of anagrams that have been introduced in previous chapters, the addition of STS, YAC, BAC, etc. can be confusing for many students. I try to reinforce the function of these by explaining the meaning behind the anagram when used. One of the wonderful applications of the area of genomics and recombinant DNA technology in teaching, is you have a chance to demonstrate what we can do with previous biological knowledge. By understanding how genes are expressed, how proteins are encoded in the DNA, researchers have the ability to located possible open reading frames. Also, with the apparent prevalence of alternative splicing in eukaryotes, there is still so much we have to learn. It is an exciting area of science. Applications of the information provided by the genome projects are revealed so often that many students may hear these new "discoveries" on the news. This is one chapter that requires constant updating of information for presentation. Access the genome websites or science news websites for new updates on medical or agricultural applications. HIGHER LEVEL ASSESSMENT Higher level assessment measures a student’s ability to use terms and concepts learned from the lecture and the textbook. A complete understanding of biology content provides students with the tools to synthesize new hypotheses and knowledge using the facts they have learned. The following table provides examples of assessing a student’s ability to apply, analyze, synthesize, and evaluate information from Chapter 18. Application • Have students predict the fragments of DNA produced on a section of the DNA sense strand sequence ACGTCGGATCCGGCCTAGC using the restriction enzyme HaeIII. • Have students explain. • Ask students to hypothesize how unexpected SNPs can effect the action of restriction enzymes on a sequence of DNA. Analysis • Have students to explain how proteomic analysis can give insights into the characteristics of genes that have not yet been sequenced. • Ask students to explain why genomics is not a predictor of how proteins interact in a cell. • Ask students to assess the impact of chemicals that damage DNA on the genomic evolution of an organism. • Ask students to explain how introns interfere with the characterization of a gene based on the mRNA sequence determined by proteomic techniques. Synthesis • Ask students to find a way restriction enzymes can be used to determine the variability of exon base pair sequences in an organism. • Ask students to assess the value of conducting a genomic analysis of a 15,000 year old body found frozen in northern Alaska. • Ask students come up with a strategy in which proteins collected from a frozen ancient plant can be used to build a picture of its genomic information. • Ask students to assess the evolutionary consequences of genetic changes that reduce non-coding DNA in an organism’s genome. Evaluation • Ask students to evaluate the pros and cons of performing SNP analyses on all humans. • Ask students to explain the medical implications of knowing that humans and chimpanzees are 98% similar according to genomic analyses. • Ask student to debate the value of using genomics to determine the probability of a child living to a certain age. VISUAL RESOURCES Palindromes are words that exhibit two-fold rotational symmetry (bob, kook, deed). The phrase “a toyota” is a palindrome as is “a man, a plan, a canal, panama.” Search the web for thousands of examples, but start here: http://www.cs.rdg.ac.uk/archive/evihcra/ku.ca.gdr.sc.www//:ptth/. Hopefully you will notice that the URL itself is a palindrome! The scifi film “Gattaca” touches on future (or maybe not so future!) gene technology and the ethical implications of genetic control. Substantial information is available at the movie website http://www.sciflicks.com/gattaca/. IN-CLASS CONCEPTUAL DEMONSTRATIONS A. Genomic Video streaming Introduction This demonstration provides up-to-date information about genomic applications called gene chips and microarrays. The demonstration uses an animated and narrated video stream to show students how gene chips and microarrays are used in genomics.. Materials • Computer with live access to Internet • Video streaming software preinstralled • LCD projector attached to computer • Web browser with bookmark to Howard Hughs Genomics Videos at http://www.hhmi.org/biointeractive/genomics/video.html. Procedure & Inquiry 1. Review the concept of genomics to the class. 2. Tell students they will be viewing a genomic strategy called gene chips 3. Show the Gene Chip Manufacturing Video 4. Discuss the possible uses of gene chips with the class. 5. Show the microarrayer in Action Video 6. Discuss the possible uses of microarrays with the class. B. Virtual Electrophoresis Introduction Electrophoresis is one of the fundamental techniques used in the genomic analysis of DNA. This accurate and simple to understand animation helps teach the principles of electrophoresis. Materials • Computer with live access to Internet • LCD projector attached to computer University of Utah Virtual Electrophoresis website • Web browser with bookmark to : http://learn.genetics.utah.edu/units/biotech/gel/ Procedure & Inquiry 1. Provide students with a brief introduction to electrophoresis 2. Then go to the University of Utah Virtual Electrophoresis website. 3. Click on the Forward icon to begin the animation. 4. Take time to ask the students to review particular parts of the animation sequence before proceeding with the next step. 5. At the end of the animation ask the students to explain how electrophoresis is useful in genomic studies. C. Name that Change Game Introduction Arthropods are excellent models for predicting the phenotypic changes that led to their great diversity. This demonstration provides students with the opportunity to hypothesize about the types genomic changes that could have led to the phylogenetic diversity of arthropods. Materials • Computer with live access to Internet • LCD projector attached to computer • Web browser bookmarked to Arthropod Story http://evolution.berkeley.edu/evolibrary/article/3_0_0/arthropodstory Procedure & Inquiry 7. Review the principles of classification with the class. 8. Go through Part 1 (Introducing the Arthropods) and Part 2 (What is an Arthropod) of the on-line lesson. 9. Ask the class to list major features of the arthropods that make them unique from their worm ancestors. Then have the class consider the types of genetic changes needed to achieve those unique differences. 10. Now go to the Cambrian Critters section and ask the students to look at the differences and similarities of the ancestral arthropods compared to the modern ones. 11. Finish up by going through the remaining on-line lessons. 12. Have the students quickly summarize what they learned. LABORATORY IDEAS A. Electrophoresis is one of the earliest tools of genomics analysis. The principles of electrophoresis are not always evident using an actual DNA or protein procedure. Plus, there are many variables that can lead students to poor results making it confusing to make conclusions. This virtual electrophoresis setup provides a user-friendly virtual hands-on laboratory activity for learning the principles of genomics. a. Introduce students to basic principles of electrophoresis as a genomics tool. b. Provide students with the following resources: a. Computer with Internet access b. Web browser bookmarked to http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html. c. New England BioLabs webstie showing restriction enzyme cutting points at http://www.neb.com/nebecomm/products/category1.asp?#2 c. Instruct students to use a drop-down window to select a plasmid to sequence using restriction enzymes and electrophoresis. d. Tell the students to predict the number and relative sizes of the fragments for the particular plasmid when mixed with the restriction enzymes provided in the animation. e. Then have the students load the samples. They should know to load the plasmid each of the different restriction enzymes provided and the molecular weight marker. f. They should then run the gel to completion. g. Have the students record whether their predictions were accurate. h. Have the students predict the molecular weights of the fragments based on the molecular weight marker. i. Have them repeat this procedure for each plasmid. B. Phylogenetic changes due to genomic mutations can be investigating using comparative anatomy approaches. This lab session has students comparing the bones of rodents from owl pellets to human bones on an articulated skeleton. Students will be asked to hypothesize the types of genomic changes involved in the differences seen in the comparable bones. a. Students should be provided with the following materials to perform this open-ended inquiry. a. Owl pellets b. Dissecting probes c. Paper d. Cellophane tape or glue b. Introduce students to the owl pellet by doing a virtual pellet dissection using the Virtual Owl Pellet website at http://www.kidwings.com/owlpellets/virtual/vopfinal.htm. c. Hand out the Owl Pellet Bone Chart found at http://www.kidwings.com/teacher/owlpellets/bonechart.htm. d. Then tell the students that you want them to collect a representative rodent from the owl pellet. They should tape or glue the bones to a sheet of paper e. Have the students compare the rodent bones to the comparable human bones f. The students should be asked to hypothesis the types of genomic changes that would explain differences between the homologous rodent and human bones. LEARNING THROUGH SERVICE Service learning is a strategy of teaching, learning and reflective assessment that merges the academic curriculum with meaningful community service. As a teaching methodology, it falls under the category of experiential education. It is a way students can carry out volunteer projects in the community for public agencies, nonprofit agencies, civic groups, charitable organizations, and governmental organizations. It encourages critical thinking and reinforces many of the concepts learned in a course. 1. Have students organize a human genomics forum for the community. 2. Have students do an educational program for high school students using the Gene Almanac website at http://www.dnalc.org/ddnalc/resources/animations.html. 3. Have students do a simple electrophoresis demonstration for elementary school students. 4. Have students do a genomics display at a local library. ETYMOLOGY OF KEY TERMS -ase enzyme (modern) bio- life or relating to living organisms (from the Greek bios- mode of life) homeo- likeness; resemblance; similarity (from the Greek homoios- like) para- beside; next to (from the Greek para- beside) prote- of, or relating to, protein; first (from the Greek protos- first) retro- backwards (from the Latin retro- back; to the rear) transpose to change in form (from the Latin trans- across or through and ponere- to place) Instructor Manual for Understanding Biology Kenneth Mason, George Johnson, Jonathan Losos, Susan Singer 9780073532295, 9781259592416
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