CASE TEACHING NOTES. Decoding the Flu INTRODUCTION / BACKGROUND CLASSROOM MANAGEMENT
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1 CASE TEACHING NOTES for Decoding the Flu by Norris Armstrong Department of Genetics, University of Georgia, Athens, GA INTRODUCTION / BACKGROUND This case is a clicker case. It is designed to be presented in a class that uses personal response systems, or clickers, in conjunction with a PowerPoint presentation. The case considers how sequences of nucleotides in DNA or RNA store information regarding the sequence of amino acids found in a protein. It asks students to identify the coding region for a gene and to evaluate how mutations (changes) to this sequence might affect the protein that is produced. In the case, students assume the role of an intern working with a branch of the Centers for Disease Control & Prevention (CDC). While at a remote location, most of the team contracts a new strain of flu virus and the intern must help the one unaffected team member determine which strain of flu is most likely causing the illness based on nucleotide sequences from the hemagluttinin gene of the virus. Because the village where they are working lacks computer access, the intern must evaluate the DNA sequences using pencil and paper. Although designed for an introductory biology course for science or non-science majors, the case could be adapted for upper-level courses by including more complex problems or by including aspects of gene expression left out of the case, such as the excision of introns. This case assumes that students already understand the basic structure of RNA/DNA molecules and nucleotides as well as the mechanism of DNA replication. An understanding of the mechanics of transcription and translation would be helpful, but is not required. The case can be completed in two 50-minute classroom sessions or, potentially, one 75-minute class period. Objectives After completing this case, students will be able to: Describe the transfer of information during gene expression from DNA to RNA to protein and the relationship between these sequences. Understand that each chromosome contains a large amount of information and that only a small part of this information is needed to code for a specific protein. Identifying information to make a specific protein requires the use of punctuation at both the DNA level and at the RNA level. Determine the protein coded for by a DNA or RNA molecule, and comment on how changes to the DNA / RNA molecule will affect the protein. Misconceptions Protein expression is a species-specific phenomenon. CLASSROOM MANAGEMENT This case was written for a large lecture section. It has an accompanying handout containing background information on influenza (included at the end of these Teaching Notes). While the information in the handout is not critical to the case, most students will be interested in this topic. It also provides some information on the virus itself relevant to the case. The handout can be distributed in class, posted on a password-protected course management system, or the information in it presented as part of the PowerPoint presentation shown in class. Teaching the Case Slide 1: Title slide with an image of an influenza virus particle. Slide 2: Introduces Jason, a student intern at the National Center for Preparedness, Detection, and Control of Infectious Diseases (NCPDCID), a branch of the CDC. Jason has been allowed to tag along on a medical mission to a rural village in Mexico with some of the CDC s doctors. During their visit, most team members became ill. One of the few people on the team not to get sick, Jason is recruited by the team leader to help analyze the little data they have on the mysterious illness. Page 1
2 Slide 3: Distribute to students the handout, Flu Fact Sheet, included at the end of these Teaching Notes. Slides 4 5: Dr. Phillips tells Jason he will need to examine nucleotide sequences and determine the proteins for which they code. As Jason has not thought much about this process for a while, he will need to review transcription and translation. Slide 6: Points out a problem with examining the information coded on the chromosomes. Because only a small amount of DNA actually codes for protein and each chromosome may have hundreds of different genes, it is necessary to determine where genes start and end. Students who have read the background information on influenza may point out that the virus genome is much smaller (only 14,000 nucleotides). However, not all of these nucleotides code for protein, so the problem still remains. Slide 7: Gives a basic description of the structure of a gene. It does not go into detail regarding size and location of the regulatory region or differences between prokaryotes and eukaryotes, as this is not the emphasis of the case and should be covered in a different class. Slide 8: Describes how the beginning and end of a gene are identified during transcription. The regulatory region contains a nucleotide sequence (promoter) that helps recruit RNA polymerase to the beginning of a gene. The ability of RNA polymerase to bind to this site is regulated by other sequences in the regulatory region and enables gene expression to be turned up or down. A different sequence at the end of the coding region causes the RNA polymerase to release the DNA/RNA ending transcription. The case does not discuss specific information regarding the promoter or terminator sequences, but this could be added for more advanced students. Slide 9: Describes the general mechanism of how transcription occurs. A link to an animation for transcription hosted by St. Olaf College is available ( molgenetics/transcription.swf ) as well as a link to DNAi, a free website hosted by Cold Spring Harbor Laboratory ( Animations for transcription at the the DNAi site can be viewed by the following steps: a. Click on Copying the Code. b. Click on putting it together in the upper right side of the window. c. Animations are linked in the lower left corner of the window that opens. i. Transcription links to an animated video of transcription. ii. Interactive links to an interactive animation that walks students through the steps of transcription. Slide 10: Clicker Question #1 asks students to demonstrate that they understand the relationship between DNA and RNA. It also helps the instructor point out that either DNA strand could be used as the template for transcription. The RNA molecule produced must be complementary and anti-parallel to the DNA template. Slide 11: Graphically depicts the correct answer for CQ#1. The instructor should point out that either strand could act as the template strand and contain the information for how to make the protein. The instructor can also note that each RNA sequence is the same (substituting U for T) as the opposing non-template strand. Slide 12: Depicts the problem of how a nucleotide code can be converted into an amino acid code. There are only four nucleotides but 20 different amino acids. Slide 13: Clicker Question #2 asks students to think about how a four-letter code can be used to encode 20 amino acids. Many students will have memorized that the genetic code uses three bases to code for an amino acid, but they will not know why. This question specifically asks the students to suggest how many amino acid combinations could be coded for by combinations of two nucleotides. Formatted as a multiple-choice question, if the clickers being used can accept numeric input, you may wish to re-format this slide to allow for that. Slide 14: Graphically depicts the answer and rationale for the preceding question. Slide 15: Introduces the triplet genetic code. The instructor should point out that many amino acids are coded for by more than one triplet. Optionally, you can point out that codons for the same amino acid tend to be very similar. I would recommend against discussing the 3rd base wobble, as it is a relatively minor detail for introductory-level biology and detracts from the main concepts. The instructor should not point out the start or stop codons. Slides 16 17: Graphically explains how trna and ribosomes read the information stored in the mrna to make proteins. Does not go into detail regarding the ribosomal subunits or A, P, and E ribosomal sites, though these could be included if desired. A link to Page 2
3 another animation hosted by St. Olaf College is provided ( molgenetics/translation.swf) as well as the link to DNAi given earlier in Slide 9 ( index.html). Although this second link is the same, the instructions for viewing the animations for translation are slightly different: a. Click on Reading the Code. b. Click on putting it together in the upper right side of the window. c. Animations are linked in the lower left corner of the window that opens. i. Translation links to an animated video of transcription. ii. Interactive links to an interactive animation that walks students through the steps of translation. Slide 18: Clicker Question #3 asks students to predict what amino acid sequence an RNA fragment from the beginning of an mrna molecule codes for. Many students will immediately break the sequence up into triplets beginning with the first three nucleotides without considering start/stop codons. After the students have answered the question, the instructor should not indicate what the correct answer should be or go over start/stop codons. The next few slides will introduce the start/stop codons and students will have a chance to try this question again. Slides 19 20: Introduces students to the idea of punctuation in the genetic code. Show the slide and wait a few moments. A few students will raise their hands. Ask them why they did so? They will say that the slide told them to. How do they know what the slide says? Some students will eventually point out indicators of a sentence: starts with a capital letter and ends with a period. The letters between these markers spell out a coherent message. In an RNA molecule, only some of the nucleotides code for protein. How do you identify them? Slide 21: Highlights the start and stop codons in the genetic code. Slide 22: Clicker Question #4 asks students to try the previous question (CQ#3) again. Most students will correctly look for a start codon and determine the amino acid sequence from that point on. Slide 23: Graphically depicts the three reading frames that could be used for the mnra fragment in CQ#3 4. Slide 24: Displays the amino acids coded for by all three reading frames for the mrna fragment. Only reading frame-3 contains a start codon, so would be the best choice. The instructor should point out that only a portion of the mrna and protein sequence was included in this question and that the protein produced would probably be much larger. Since this is just a fragment of the mrna, students might ask if/how it would be possible to determine the correct reading frame if a start codon were not present in the fragment. Reading frames 1 and 2 could be potentially ruled out because these frames contain stop codons that would result in production of very short peptides. Most proteins are hundreds to thousands of amino acids long. Slide 25: Asks the students to apply their new understanding of the genetic code to interpret a fragment DNA sequence from the beginning of the HA gene. The partial DNA and protein sequences are also provided on a handout that is given to the students (included at the end of these Teaching Notes). In addition, the handout includes three more challenging problems that students can work on outside of class. You may want to point out that influenza virus uses RNA for its genome. However, researchers often work with DNA copies (cdna) of the virus s genes and generally store information for genes as DNA sequences. Have the students work together in small groups to determine where the coding region is located in this DNA fragment. The students may ask which fragment they will need to use as the template. Tell the students that they need to figure this out. Walk among the groups as they work through the problem and make sure that they understand what they need to do. Answer their questions, but avoid giving them the answer. Try to ask guiding questions instead. After the students have worked on the problem for a few minutes, ask for a group to volunteer. Slides 26 30: Walks through the answer to the question posed on Slide 25 graphically. The instructor should point out that either strand could be used as the template for transcription. Also, RNA is always read from 5' 3' so one of the mrna strands (#2) must be reversed to be read properly. There are multiple start codons present in the different reading frames for the two potential RNA molecules. Students may ask how it is possible to determine which is the correct one. In this case, they are given the amino acid sequence for the protein that will Page 3
4 help them determine which is the correct reading frame. The case does not discuss how the correct reading frame would be identified if the encoded protein is not known. Information on this could potentially be included in a more advanced class. Slide 31: Introduces the students to the next part of the case in which they must determine how a change in nucleotide sequence can alter the protein that is produced. Slide 32: Clicker Question #5 provides the same nucleotide sequence the students worked on in slides as well as the corresponding sequence from one of the new virus strains. In the interest of time, the students are given the RNA sequence to work with directly. To answer this question, the students will need to do the following: a. Identify the start codon and reading frame for the sequence. This will be the same as in the activity in Slide 25 and the instructor can tell the students this if desired or time is an issue. b. Identify the nucleotide change that has occurred and the codon that will be affected. c. Determine what, if any, change has occurred to the protein that is produced as a result of this nucleotide change. Slide 33: Graphically explains the answer to the question. The new virus strain has an arginine in place of a lysine at the second amino acid in the HA protein. Slides 34 35: Clicker Question #6 is similar to the preceding question (CQ #5). The codon for amino acid seven has been changed to a stop codon. Instead of adding a leucine to the peptide, protein synthesis stops, resulting in a shorter than normal protein. Slides 36 37: Clicker Question #7 is again similar, but this time the codon for amino acid three has been changed but does not alter the amino acid that is incorporated into the protein. Slides 38 39: Clicker Question #8 presents another variation in which a nucleotide has been deleted from the third codon. This does not alter the amino acid for the codon, but results in a frame shift. All of the subsequent codons have been altered, resulting in production of an incorrect protein. One of the new codons codes for a stop, and the protein that is produced is also too short. Slide 40: Clicker Question #9 asks students to decide which mutation is likely to produce an altered yet functional HA protein. Strains #2 and #4 result in abnormally short proteins that will probably not function properly. Strain #3 does not alter the protein and it should function the same as the HA protein in the typical flu virus, however recent research indicates that changes in nucleotide sequence that do not change amino acid sequences can affect the function of a gene, presumably by affecting regulation of gene expression. Strain #1 displays only a single amino acid change. Depending on the specific amino acid change that occurs, where in the protein it takes place, and what protein is affected, this could have any range of effects on the function of the protein. Although changes to the active site of enzymes or other critical sites would be most likely to affect protein function, changes to any amino acids that affect protein folding in general can alter the behavior and/or functionality of a protein. As an analogy, ask the students how randomly changing one word in a long paragraph would affect the paragraph. It would depend on each particular situation. At one extreme, the meaning of the paragraph will not be altered at all. In the opposite extreme, the meaning of the paragraph might be significantly altered or destroyed. It is also possible for effects between these two extremes to occur. Slide 41: This slide presents an image of a hemagglutinin molecule. Slide 42: Conclusion of the case. ANSWER KEY Answers to the questions posed in the case study are provided in a separate answer key. The key also includes the answers to the practice problems on the student handout. Those answers are password-protected. To access the answers for this case, go to the key. You will be prompted for a username and password. If you have not yet registered with us, you can see whether you are eligible for an account by reviewing our password policy and then apply online or write to answerkey@ sciencecases.org. REFERENCES Infl uenza Websites Centers for Disease Control and Prevention. (n.d.) Influenza. Accessed July 21, gov/flu/. World Health Organization. (2009) Influenza. Accessed July 21, en/. Kilbourne, E.D. (2006) Influenza Pandemics of the 20th Century. Emerging Infectious Diseases. Page 4
5 12(1): vol12no01/ htm. U.S. Dept. of Health & Human Services: Flu.gov. (n.d.): Pandemics and Pandemic Threats since Accessed July 21, gov/general/historicaloverview.html. Infl uenza Hemaggluttinin DNA sequence Influenza A virus (A/Hiroshima/52/2005(H3N2)) segment 4, complete sequence. ACCESSION EU ( Accessed July 21, Acknowledgements: This material is based upon work supported by the NSF under Grant No. due Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the NSF. Copyright held by the National Center for Case Study Teaching in Science, University at Buffalo, State University of New York. Originally published August 04, Please see our usage guidelines, which outline our policy concerning permissible reproduction of this work. Page 5
6 FLU FACT SHEET The Disease (Centers for Disease Control & Prevention: u/) Contagious respiratory illness caused by influenza viruses. Symptoms include fever, headache, extreme tiredness, sore throat, runny or stuffy nose, muscle aches, and stomach symptoms such as nausea, vomiting, and diarrhea. 5% to 20% of the U.S. population contracts the flu each year. >200,000 people are hospitalized and about 36,000 people die from flu complications such as pneumonia, sinus infections, dehydration, and aggravation of existing medical conditions. Older individuals, young children, and people with certain health conditions are at high risk for serious flu complications. The Virus (Centers for Disease Control & Prevention: u/) Influenza uses RNA instead of DNA as its genetic material. Its genome is 14,000 nucleotides on eight pieces of RNA that code for 11 proteins. Two main influenza viruses (A and B) are responsible for seasonal flu. Influenza A is found in different animals, including ducks, chickens, pigs, whales, horses, and seals. Influenza B viruses circulate widely only among humans. Influenza A is divided into subtypes based on differences in two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). These proteins facilitate viral infection of host cells. In healthy individuals, the immune system learns to recognize the H and N proteins and eventually destroys viruses and infected cells, ending the infection. An individual who has already had the flu can contract the disease again if infected with a different strain of virus their immune system does not recognize. New subtypes of influenza virus are caused by changes in viral proteins. o Antigenic drift refers to small changes that commonly occur and which may allow the virus to temporarily evade the immune system. o Antigenic shift refers to less common, major change in the H and/or N proteins resulting in a new influenza A subtype. Antigenic shift can be promoted by transfer of the flu virus between different species of animals such as pigs, birds, and/or humans. When an antigenic shift occurs, most people have little or no protection against the new virus. If the new virus causes illness and can be transmitted easily from person to person, an influenza pandemic can occur. In April of 2009, a new influenza virus that appears to have originated in pigs was detected in patients in Mexico and the United States. Treatment/Prevention Seasonal flu outbreaks are the result of changes in the particular strains of virus that circulate in populations in a given year. The best way to prevent the flu is by getting a flu vaccination each year. Vaccines train the immune system to recognize and destroy viruses before they can establish an infection and cause illness. Flu Fact Sheet for Decoding the Flu by Norris Armstrong
7 Flu vaccine contains three influenza viruses: one A (H3N2) virus, one A (H1N1) virus, and one B virus. The flu shot contains inactivated (dead) virus particles. The flu nasal spray contains live, weakened flu viruses that do not cause illness. The viruses used in the flu vaccine change each year based on international surveillance and scientists' estimations of which strains of virus are most likely to circulate in the population in the upcoming year. Some antiviral medications can potentially reduce the severity of flu symptoms. Flu History Influenza epidemics occur yearly in the fall and winter in temperate regions but can circulate throughout the year in some tropical countries. Annual epidemics result in three to five million cases of severe illness worldwide and 250,000 to 500,000 deaths, usually from high-risk groups. Three worldwide pandemics occurred in the 20 th century: 1918, 1957, and These pandemics likely resulted from changes in the hemagglutinin protein associated with a transfer of the influenza virus from animals to humans. In the 1918 influenza pandemic, approximately 2 4 of every 10 people became ill and over 50 million people died from the disease or complications associated with the flu. An unusual aspect of this outbreak was that infection and mortality was highest among adults 20 to 50 years. References Centers for Disease Control and Prevention: World Health Organization: Kilbourne, E.D. Emerging Infectious Diseases. 12(1): 9 14 (2006). U.S. Dept. of Health & Human Services: Flu.gov: Pandemics and Pandemic Threats since pandemicflu.gov/general/historicaloverview.html. Image Source: Flu Fact Sheet for Decoding the Flu by Norris Armstrong
8 Student Handout for Codon Problem Codon Table U Second Base U C A G UUU Phe (F) UCU UAU UGU Cys (C) U Tyr (Y) UUC UCC Ser (S) UAC UGC C UUA Leu (L) UCA UAA UGA Stop A UUG UCG UAG Stop UGG Trp (W) G First base C A CUU CCU CAU His (H) CGU U CUC Leu (L) CCC Pro (P) CAC CGC Arg (R) C CUA CCA CAA Gln (Q) CGA A CUG CCG CAG CGG G AUU ACU AAU Asn (N) AGU Ser (S) U AUC Ile (I) ACC Thr (T) AAC AGC C AUA ACA AAA Lys (K) AGA Arg (R) A AUG Met (M) ACG AAG AGG G Third base G GUU GCU GAU Asp (D) GGU U GUC GCC GAC GGC C Val (V) Ala (A) Gly (G) GUA GCA GAA GGA A Glu (E) GUG GCG GAG GGG G Find and mark the region in the partial Hemagglutinin (HA) gene sequence below that codes for the peptide: Met-Lys-Thr-Ile-Ile-Ala-Leu-Ser-Tyr-Ile 5 GAATGTAGCTCAAAGCAATGATAGTCTTCATGGTTA 3 3 CTTACATCGAGTTTCGTTACTATCAGAAGTACCAAT 5 Practice Problems 1. Find the stretch of DNA in the following sequence that codes for the peptide: Q E G E I V V L V Q N 5 ACAAAAAATGATGAAGGCAATCATTCCTCATAAGATAATAAAAAGTGTATTGAGAGTGA 3 3 TGTTTTTTACTACTTCCGTTAGTAAGGAGTATTCTATTATTTTTCACATAACTCTCACT 5 5 GTGAGCATGAAAAAGATTTAGTATTTAGCAGTGCGGATATGATCCAAGAGGGTGAAATA 3 3 CACTCGTACTTTTTCTAAATCATAAATCGTCACGCCTATACTAGGTTCTCCCACTTTAT 5 5 GTCGTTCTCGTTCAGAATCTTTTGCAGCATAAGTAGTATGTCGATATACTTATCGTTGA 3 3 CAGCAAGAGCAAGTCTTAGAAAACGTCGTATTCATCATACTGCTATATGAATAGCAACT 5 1
9 2. The following gene fragment contains the coding sequence for the beginning of a 394 amino acid. What are the likely first five amino acids for the protein? 5 AAGCTTCAGCGCGAACGACCAACTACCCCGATCATCAGTTATCCTTAAGGTCTCTTTTG 3 3 TTCGAAGTCGCGCTTGCTGGTTGATGGGGCTAGTAGTCAATAGGAATTCCAGAGAAAAC 5 5 TGTGGTGCGTTCCGGTATGGGGGGGGCTGCCGCCAGGTTGGGGGCCGTGATTTTGTTTG 3 3 ACACCACGCAAGGCCATACCCCCCCCGACGGCGGTCCAACCCCCGGCACTAAAACAAAC 5 5 TCGTCATAGTGGGCCTCCAGGGGGTCCGCGGCAAATATGCCTTGACGGACGCCTCTCTC 3 3 AGCAGTATCACCCGGAGGTGCCCCAGGCGCCGTTTATACGGAACTGCCTGCGGAGAGAG 5 3. The DNA sequence below contains the entire coding region for a gene. Below that is the amino acid sequence of the protein coded for by the gene. A cell suffers a mutation that results in an altered protein being produced (shown below). Describe the location and type of mutation that has occurred. DNA sequence: 5 AACAGCATAAACACAAAATCCATCAAAAATGTCGATGAAATATCTGATGTTGTTGTTCG 3 3 TTGTCGTATTTGTGTTTTAGGTAGTTTTTACAGCTACTTTATAGACTACAACAACAAGC 5 5 CTGCTATGATAATTAGATCATTCGCCAATAGTGGTAACGCTATCGAAACGACATTGTCA 3 3 GACGATACTATTAATCTAGTAAGCGGTTATCACCATTGCGATAGCTTTGCTGTAACAGT 5 5 AGAATTACAAACACTACAACAGATATTCCAGCTATCTGATTATGCGGTCCAGAGGGAAA 3 3 TCTTAATGTTTGTGATGTTGTCTATAAGGTCGATAGACTAATACGCCAGGTCTCCCTTT 5 Original Protein Sequence: M L L F A A M I I R S F A N S G N A I E T T L S R I T N T T T D I P A I Mutated Protein Sequence: M L L F A A M I I R S F A N S G N A N R N D I V K N Y K H Y N R Y S S Y L I M R S R G K 2
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