1 A clicker-based case study that untangles student thinking about the processes in the central dogma.Karen N. Pelletreau1*, Tessa Andrews2, Norris Armstrong2, Mary A. Bedell2, Farahad Dastoor1, Neta Dean3, Susan Erster3, Cori Fata- Hartley4, Nancy Guild5, Hamish Greig 1, David Hall2, Jennifer K. Knight5, Donna Koslowsky4, Paula P. Lemons6, Jennifer Martin5, Jill McCourt6, John Merrill4, Rosa Moscarella7, Ross Nehm8, Robert Northington1, Brian Olsen1, Luanna Prevost9, Jon Stoltzfus10, Mark Urban-Lurain7, Michelle K. Smith1 Affiliations: 1School of Biology and Ecology, University of Maine. 2Department of Genetics, University of Georgia. 3Department of Biochemistry and Cell Biology, Stony Brook University. 4Department of Microbiology and Molecular Genetics, Michigan State University. 5Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder. 6Department of Biochemistry and Molecular Biology, University of Georgia. 7Collaborative Research in Education, Assessment, and Teaching Environments for the fields of Science, Technology, Engineering and Mathematics (CREATE4STEM), Michigan State University. 8Center for Science and Mathematics Education and Department of Ecology and Evolution, Stony Brook University. 9Department of Integrative Biology, University of South Florida. 10Department of Biochemistry and Molecular Biology, Michigan State University. *Correspondence to: School of Biology and Ecology, 5751 Murray Hall, Orono ME The notes section of this PowerPoint file will contain information about the slide content, clicker question answers and patterns of student response, and suggestions from faculty who have used the activity.

2 What is Duchenne Muscular Dystrophy (DMD)?A diseases that manifests in muscle weakness and wasting Exhibits X-linked recessive inheritance pattern Slides 2-4: A basic background of Duchenne Muscular Dystrophy (DMD) and X-linked inheritance. These slides are optional depending upon the structure of the course, and how much, if at all, the topic as been covered in the class (e.g., some faculty cover this topic earlier in their classes and so do not need to re-introduce; others assign reading materials before class). Here, a few slides with basic information about the disease are provided to put the exercise in context. Suggested Transition: Discuss the impacts the disease has on individuals, optionally show one of the videos so that students better understand the disease phenotype. Muscular Dystrophy Quick Facts One of 9 types of muscular dystrophy. Often occurs in families with no known genetic history. Affects 1 in male births world wide. Progressive degenerative muscle disease with symptoms starting in infancy, wheelchair dependent by adolescence, and survival beyond 30 years is rare. Cause of death is commonly failure of lungs and heart muscle. Dystrophin is the one of the largest known human genes at 2.4Mb. Its official symbol is DMD. To avoid confusion with the disease, we will use DMD to indicate the disease, and write out dystrophin for the gene or protein. The dystrophin protein located in skeletal and cardiac muscle and a small amount in brain and nerves. The dystrophin protein is part of a protein complex that anchors muscle cell cytoskeleton with the extracellular matrix to support and protect muscle fibers as they contract and relax. >1000 possible mutations in the dystrophin gene can result in “muscular dystrophy” (skeletal muscle wasting and atrophy, eventually leading to cardiac weakening). Mutations include large deletions, duplications or point mutations, which can result in non- functional proteins, partially functioning proteins, or no protein accumulation in cells. Two-thirds of the mutations are deletions of one or many exons in the gene. Cytogenetic Location: Xp21.2; 31,119,221-33,339,608 Useful links https://www.mda.org/disease/duchenne-muscular-dystrophy https://www.nlm.nih.gov/medlineplus/ency/article/ htm https://www.genome.gov/ https://www.youtube.com/watch?v=ZrPnmgs4rHM https://www.youtube.com/watch?v=o1uhhpjmzkw

3 Mutations in the dystrophin gene cause Muscular DystrophyThe dystrophin gene: -encodes for the protein dystrophin -is found on the X chromosome Mutations in the dystrophin gene: -result in different alleles -can lead to the expressed phenotype Muscular dystrophy Slides 2-4: A basic background of Duchenne Muscular Dystrophy (DMD) and X-linked inheritance. These slides are optional depending upon the structure of the course, and how much, if at all, the topic as been covered in the class (e.g., some faculty cover this topic earlier in their classes and so do not need to re-introduce; others assign reading materials before class). Here, a few slides with basic information about the disease are provided to put the exercise in context. Suggested Transition: Frame the mechanics of the disease; emphasize that MD is complex and many different mutations exist that can result in the disease. Male affected Female carrier

4 The dystrophin gene is large and can have many different mutations along the DNA.X chromosome Dystrophin gene Slides 2-4: A basic background of Duchenne Muscular Dystrophy (DMD) and X-linked inheritance. These slides are optional depending upon the structure of the course, and how much, if at all, the topic as been covered in the class (e.g., some faculty cover this topic earlier in their classes and so do not need to re-introduce; others assign reading materials before class). Here a few slides with basic information about the disease are provided to put the exercise in context. Dystrophin is huge gene: 2.4 Mb (= 2,400,000bp - It takes 16hrs to be transcribed) For comparison, the median gene size in the human genome is 23,329 bp Suggested Transition: Point out the large size of the gene, and that it can have multiple mutations. The severity of the disease is related to the type and location of the mutation.

5 Case Study: Siblings living with Duchenne Muscular Dystrophy (DMD)Liam: affected with DMD Elijah: unaffected Liam Elijah affected unaffected Slide 5: The case study is presented and the rationale of the activity. Suggestions: Emphasize the fact that the students are exploring an unknown as a scientist would - through a process of reasoning, data interpretation, and use of their knowledge. Suggested Transition: After framing the case study, discuss what information you need to explore why Liam has DMD. This statement could be presented as a question to your students “What do we need to know about these two brothers to understand the inheritance of the disease better?” Your goal: Use the information provided to explore what nucleotide changes could result in Liam developing DMD

6 X chromosome 79 exons encoding 3500 amino acids 1 2 3 4 5 There are five nucleotide differences between Elijah and Liam in the dystrophin gene. locations where DNA differs Slide 6: Chromosome/gene orientation. For these two brothers assume that except for 5 sites indicated, the rest of DNA sequence is identical. Suggestions: Explain to the students the size of the gene, that mutations can occur in >1000 locations but in this case study we will look at five. Emphasize that the gene extends downstream of/ beyond the last nucleotide difference (#5) indicated. Discuss what factors need to be considered to address if a nucleotide difference will result in the disease. Suggested Transition: One option is to ask the question “As a researcher, would you investigate all of these nucleotide differences as possible causes of Duchenne Muscular Dystrophy (DMD) in Liam? How would you prioritize which differences to look at?” A T C G Elijah’s X Liam’s X affected

7 Dystrophin gene structure and the location of the five different nucleotides.affected Gene structure can provide our first clue. Based solely on these labeled features, discuss which of the DNA differences would be more or less likely to result in the muscular dystrophy phenotype? Slide 7: Discussion Prompt 1 (D1) The goal of this slide is to discuss basic gene structure, including the function of introns, exons, and promoters. Once the discussion is done, verbalize that as a researcher you might eliminate difference number 4 as a likely candidate causing Duchenne Muscular Dystrophy (DMD) in Liam. Suggestions: This slide can be used for a point of discussion in your class to address mutations that can occur in introns and can at times result in disease (see reference below). Suggested Transition: Summarize the discussion by stating that the gene structure helps us prioritize how explore the case study. For example you could say “We can tentatively eliminate difference 4 as the most likely cause of DMD, while we explore other nucleotide differences to see if there is a more clear candidate.” Here are several references pertaining to splice site mutations Vanlander AV, Menten B, Smet J, et al. (2015) Two siblings with homozygous pathogenic splice-site variant in mitochondrial asparaginyl-tRNA synthetase (NARS2). Hum Mutat 36: 222–31. Hallmann K, Zsurka G, Moskau-Hartmann S, et al. (2014) A homozygous splice-site mutation in CARS2 is associated with progressive myoclonic epilepsy. Neurology. 83: 2183–87. Agrawal A, Hamvas A, Cole FS, Wamback JA, Coghill C, Harrison K, Nogee LM.(2012) An intronic ABCA3 mutation that is responsible for respiratory disease. Pediatr Res 71(6): Friedman LS, Ostermeyer EA, Szabo CI, Dowd P, Lynch SP, Rowell SE, King M. (1994) Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Gen 8 (4): 399–404. Kananura C, Haug K, Sander T, Runge U, Gu W, Hallmann K, Rebstock J, Hellis A, Steinlein OK. (2002) A splice-site mutation in GABRG2 associated with childhood absence epilepsy and febrile convulsions. Arch Neurol 59 (7): 1137–1141. Parkinson DB, Thakker RV. (1992) A donor splice site mutation in the parathyroid hormone gene is associated with autosomal recessive hypoparathyroidism. Nat Gen 1 (2): 149–152.

8 Dystrophin gene structure✖ affected Difference #2: UCU (ser) Elijah’s mRNA UCA (ser) Slide 8 Discussion Prompt 2 (D2) Use this slide to discuss the first type of mutation encountered in a coding region, and the consequences of a silent mutation. End with eliminating this nucleotide difference as a likely cause of Duchenne Muscular Dystrophy in Liam. Suggestions: Depending on the class, you may need to take time to orient the student to diagram on the slide and the difference between the mRNA and the DNA. Discuss redundancy in the genetic code and silent mutations. Suggested Transition: Emphasize the information at the bottom of the slide. “This type of substitution (a silent mutation) results in the same amino acid in both Elijah’s and Liam’s dystrophin protein – so there will be no affect on protein function. The results from other nucleotide differences are more challenging to predict.” Liam’s mRNA This type of substitution (a silent mutation) results in the same amino acid, and therefore protein. The results from other mutations are more challenging to predict…

9 ✖ ✖ Q1: Is difference #5 a possible cause of DMD in Liam? Yes NoDystrophin gene structure ✖ ✖ affected Difference #5: GAU (asp) Elijah’s mRNA GAG (glu) Slide 9 Clicker question 1 (Q1) Answer A Difference #5 represents a missense mutation: in Elijah (unaffected) the DNA encodes for GAU (aspartate) and in Liam (affected) the DNA encodes for GAG (glutamate). This question is an exploratory question, and most students answer A: “Yes” because, without more information, this difference is a possible cause of the disease. In the next slide, students will learn that aspartate and glutamate are similar in structure. Suggested Transition: After the clicker responses have been discussed, ask students what information would be needed to understand the consequence of this particular missense mutation. Try to lead the students to discuss properties of amino acids. Liam’s mRNA Q1: Is difference #5 a possible cause of DMD in Liam? Yes No

10 What can we deduce about these two amino acids and their possible function?HINT: Your biochemistry colleague reminds you that glutamate and aspartate are both structurally and biochemically similar. Here the two amino acids are highlighted in red. The unique and similar structure is clearly visible on this diagram. Suggestions: Ask students to draw conclusions on the structure and function of these two amino acids based upon the figure. For upper level classes, you can explore why substitutions with similar amino acids may not have significant affects on protein structure and function. Suggested Transition: Wrap up the discussion about amino acid structure and the possibility of this mutation having an impact on phenotype. For example: “We cannot eliminate this mutation as a possible cause of the disease, but researchers may explore other options to see if there is a more definite candidate.” Image modified with permission from: Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson R, Reece JB. (2014). Campbell Biology in Focus. Pearson, Boston.

11 ✖ ✖ ? Dystrophin gene structure affected Difference #1: ? Slide 11 Clicker Question 2 (Q2) Answer: D This question is asking students to examine the promoter region of the gene and to predict how this type of mutation would affect the mRNA levels. The correct answer is D, but often students arrive at C, thinking that a mutation will always reduce transcription. This question can be used to discuss effects of mutations in promoter regions in general, and that a mutation does not always have to down regulate a promoter. Suggested Transitions: Segue into this slide by concluding the last portion of the lesson – for example: “Given the information we have so far, we can not conclude that difference #5 would be the cause of the disease, so we will continue to explore the other differences;” or “difference # 5 may or may not be responsible, we may return to it based on our other findings.” This introduction frames the question mark that appears on this slide above difference #5. Transitioning out of this slide after the discussion with a statement such as “Now you run the experiment, let’s see what the data look like.” References pertaining to promoter mutations and to certain diseases. Wang T, Zang W, Ma Y, Li M, Xuan X, Wang N, Wu R, Li Y, Dong Z, Zhao G. (2012). DNA polymerase beta promoter mutations affect gene transcription, translation and the sensitivity of esophageal cancer cells to cisplatin treatment. Mol Biol Rep 40:1333–1339. Rutter JL, Mitchell TI, Buttice G, Meyers J, Ozelius LG, Brinckerhoff CE. (1998). A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription. Cancer Res 58 : Ludlow LB, Schick BP, Budarf ML, Driscoll DA, Zackai EH, Cohen A, Konkle BA.(1996). Identification of a mutation in a GATA binding site of the platelet glycoprotein Ibβ promoter resulting in the Bernard-Soulier syndrome. J Biol Chem 271: Manco L, Ribeiro ML, Máximo V, Almeida H, Costa A, Freitas O, Barbot J, Abade A, Tamaginini G. (2000). A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency. Br J Haematol 110: Van Wijk R, Van Solinge WW, Nerlov C, Beutler E, Gelbart T, Rijksen G, Nielsen FC. (2003). Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency. Blood 101: Crossley M, Brownlee GG. (1990). Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B. Nature (Lond) 345: Reijnen MJ, Sladek FM, Bertina RM, Reitsma PH. (1992). Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden. Proc Natl Acad Sci U S A 89: Q2: If difference #1 caused DMD, we would predict the mRNA levels in Liam to be __________ the mRNA levels in Elijah? the same as higher than lower than different in some way from

12 ✖ ✖ ✖ ? Dystrophin gene structure affected Difference #1: Slide 12 Clicker Question 3 (Q3) Answer: B The students observe that there is no change in the amount of mRNA as a result of the promoter mutation and can therefore conclude this difference is likely not a cause of DMD in Liam. The majority of students answer correctly (B). Suggested Transition: Once the student responses are in, you can transition to the next slide by saying that we will explore the last possible nucleotide difference (#3). OMIT for printing: If you print off/provide slides for your students this slide reveals certain answers; you may want to omit from printing/posting before class. Q3: You have now done the experiment and measured the mRNA levels in both Elijah and Liam. If these are your results, would you conclude that difference #1 a likely cause of DMD in Liam? Yes No

13 Slide 13 Suggestions: Use this slide (here and throughout) to mark the transitions between the processes in the central dogma. In this case, the lesson was just addressing a mutation in the promoter region (transcription), so this slide will now re-orient the students to DNA replication for the subsequent questions. Suggested Transition: Now we are going to explore how nucleotide differences may have an effect on the phases of the central dogma beginning with replication.

14 Dystrophin gene structure✖ ✖ ✖ ? affected CAA (gln) UAA (STOP) Difference #3: Elijah’s mRNA Liam’s mRNA Q4: When DNA polymerase reaches the nucleotides encoding the premature stop codon it will… stop when it reaches the first nucleotide encoding the premature stop codon. stop when it reaches the last nucleotide encoding the premature stop codon. not be affected by this base change and will continue to read through the nucleotide difference. Slide 14 Clicker Question 4 (Q4) Answer: C This question is the first of several that address the effect of a premature stop codon on the processes in the central dogma. The majority of students will incorrectly answer that the DNA polymerase will stop prematurely (either A or B). The next slide promotes discussion of the consequences of having DNA polymerase that stopped at every stop codon in the genome. Suggestions: Be sure to explain that nucleotide difference #3 is a premature stop codon. If you would like/if time allows, you can provide the CAA and UAA and ask the students to use a codon table to determine what they encode for. (simply delete the “gln” and “STOP” in the slide above) A reminder, these questions, though simple in their response these are not meant to be quick responses. Remind students that they should be problem solving throughout the activity. To that end, be sure to provide ample time for student thinking and discussion. Suggested Transition: Ask the question “What would happen if DNA polymerase stopped at every stop codon in the genome?”

15 The red boxes along this DNA strand indicate regions where the DNA sequence could code for stop codons. If DNA polymerase recognized these triplet nucleotides as stop codons, what would be the consequence on DNA replication? Slide 15 Discussion Prompt 3 (D3) Use this slide to have the students think about their previous answer and what the outcomes would be. Revisit DNA replication, DNA polymerases, and what is responsible for stopping DNA replication. Suggested Transition: “So let’s revisit this question again given this understanding.”

16 Dystrophin gene structure✖ ✖ ✖ ? affected CAA (gln) UAA (STOP) Difference #3: Elijah’s mRNA Liam’s mRNA Q5: When DNA polymerase reaches the nucleotides encoding the premature stop codon it will… stop when it reaches the first nucleotide encoding the premature stop codon. stop when it reaches the last nucleotide encoding the premature stop codon. not be affected by this base change and will continue to read through the nucleotide difference. Slide 16 Clicker Question 5 (Q5) Answer: C After the previous discussion, students vote again on the effect of a stop codon on DNA polymerase. There is typically a shift towards the correct answer (C). Suggested Transition: Reflect with the students how their answers on this question have changed.

17 Polymerases read only one base at a time,not triplet codons. The DNA polymerase would not be affected by this base change in Liam’s DNA and replication would proceed normally. Slide 17 Use this slide to re-emphasize how DNA polymerases work and why stop codons would have no effect on the process of DNA replication. Suggested Transition: Stress to students that this mutation will not affect DNA replication and DNA polymerase. Now we will explore how this mutation may or may not affect transcription.

18 NO EFFECT ? ? Slide 18 The lesson now shifts to exploring the effect of a premature stop codon on transcription. Use this slide to summarize what was just learned and re-orient the students to the process of transcription. Suggested Transition: “So we now know that this type of mutation will not affect DNA replication, how do we determine if there is an effect on transcription?”

19 Dystrophin gene structureDifference #3: ? Slide 19 Clicker Question 6 (Q6) Answer C This question asks the students to predict the outcome of a premature stop codon on mRNA size. Roughly half the students incorrectly choose (A), thinking that a premature stop codon will stop transcription and result in a shorter mRNA. Suggested Transition: A high percentage of students may answer this question incorrectly. Once the answers are collected, they are not discussed here. This question is setting up the subsequent question addressing RNA polymerase. Perhaps a simple transition such as “interesting – let’s see what else we can find out” or “let’s look at what is happening at the molecular level during transcription” You may want to wait to reveal the class response to the prediction until after you discuss the results in slide 20. Positive controls would typically be from a known control sample (RNA) to ensure that your procedure has worked and that negative results are reliable. Conversely, negative controls are performed with all reagents but the RNA of interest to ensure that contamination or false positives are not issues. Q6: What do you predict will be the effect of the premature stop codon on mRNA size? It will result in: a shorter mRNA in Liam. a longer mRNA in Liam. the same size mRNA in both Liam and Elijah.

20 Liam’s DNA (affected) coding strand template strand Q7: The above DNA sequence is being transcribed by an RNA polymerase (red square). The premature stop codon mutation in Liam’s DNA is indicated in blue. What will the RNA polymerase do when it reaches the nucleotides encoding the premature stop codon? It will: stop when it reaches the first nucleotide encoding the premature stop codon. stop when it reaches the last nucleotide encoding the premature stop codon. not be affected by this base change and will continue to read through nucleotide difference. Slide 20 Clicker Question 7 (Q7) Answer C This question asks about the effect of a premature stop codon on RNA polymerase. The format used is intentionally similar to that used asking about DNA polymerase (Q4 and 5). Although how DNA polymerases function has already been addressed, many students still answer this question incorrectly and vote that RNA polymerase will stop prematurely (either A or B). Suggested Transition: “Let’s look at the results of the experiment examining mRNA size in Liam and Elijah.”

21 Dystrophin gene structure Difference #3: Slide 21 Suggestions: Present the class clicker responses gathered on slide 19 here and see how their initial prediction compared to the results; or use the results from slide 20 to show how they now understand this concept. Suggested Transition: Reinforce the student understanding that there will be no effect on the mRNA (or transcript) size. Perhaps transition with “let’s see what RNA polymerase would look like” to move to the animation. OMIT for printing: If you print off/provide slides for your students before class this one reveals certain answers and may you want to omit from printing/providing. Here are the results of your experiment. How do the results above compare with your prediction?

22 Elijah’s DNA Liam’s DNA (affected)coding strand Liam’s DNA (affected) coding strand Slide 22 Transcription animation (GIF) Use this animation to solidify understanding of stop codons, RNA polymerase and translation. The animation should play automatically when in presenter mode. If not select the animation, click on “movie setting” and select “play automatically” or hover over the animation until the arrow appears and hit play. Instructions for inserting GIF files (if pre-embedded animations do not work): For MAC -Download any required software updates. -Place the Supporting File S3 in the same folder as your PPT file. From the menu: Select InsertMovie from fileselect the file to insert. (MAC) Once in the file is in the slide, select the movie and a “Format Movie” Tab should appear. Select “Start Automatically” OR FOR PC Select InsertSelect “video” in the “media” section (a small menu will appear)select “video on My PC” to open file browser and insert the GIF Once in the file is inserted, select the video in the slide select the “Playback” tab Use the “Start” drop-down menu to select “automatically”

23 Polymerases read only one base at a time,Elijah’s DNA Liam’s DNA (affected) Polymerases read only one base at a time, not triplet codons. The RNA polymerase would not be affected by this base change in Liam’s DNA and transcription would proceed normally. Slide 23 This slide is a final summary before the lesson moves to translation. Suggested Transition: “Finally, we will explore how a stop codon type of mutation might affect translation.”

24 ? NO EFFECT NO EFFECT Slide 24The lesson now shifts to explore the effect of a premature stop codon on translation. Use this slide to summarize what was just discussed about transcription and re-orient the students to think about translation.

25 Dystrophin gene structure Difference #3: ? Slide 25 Clicker Question (Q8) Answer A This question addresses the impact of a premature stop codon on translation. By this point in the lesson most students have recognized a stop codon will result in a shorter protein and the majority of the students arrive at the correct answer. Suggested Transition: “Let’s see what the results of your experiment look like.” Loading controls ensure the same amount of protein has been loaded in each sample. Q8: What do you predict will be the effect of the premature stop codon on protein size? It will: result in a smaller dystrophin protein in Liam. result in a larger dystrophin protein in Liam. result in the same size protein in both Liam and Elijah.

26 Dystrophin gene structure:Difference #3: Slide 26 Discussion Prompt 4 (D4) Use this slide to discuss why the protein is shorter and what mechanistically is happening. Suggested Transition: “Now we have evidence that a premature stop codon mutation will affect protein size…let’s look at why.” Loading controls ensure the same amount of protein has been loaded in each sample. Here are the results of your experiment. How do these results compare with your prediction? How can you explain the observed difference in the size of the protein?

27 The ribosome does recognize triplet codons.Elijah’s mRNA Liam’s mRNA (affected) The ribosome does recognize triplet codons. The ribosome will stop and fall off when it encounters the premature stop codon that resulted from the base change in Liam’s DNA. As a result, the process of translation will stop, and the protein will be shorter. Slide 27 Translation animation The animation should play automatically when in presenter mode. If not select the animation, click on “movie setting” and select “play automatically” or hover over the animation until the arrow appears and hit play. Instructions for inserting GIF files (if pre-embedded animations do not work): Suggested Transition: “So what specifically recognizes stop codons during translation?” For MAC -Download any required software updates. -Place the Supporting File S3 in the same folder as your PPT file. From the menu: Select InsertMovie from fileselect the file to insert. (MAC) Once in the file is in the slide, select the movie and a “Format Movie” Tab should appear. Select “Start Automatically” OR FOR PC Select InsertSelect “video” in the “media” section (a small menu will appear)select “video on My PC” to open file browser and insert the GIF Once in the file is inserted, select the video in the slide select the “Playback” tab Use the “Start” drop-down menu to select “automatically”

28 What specifically recognizes stop codons during translation?Slide 28 Many faculty use this slide to revisit details of how stop codons work in translation. Suggested Transition: “Let’s summarize what we have covered so far.” Image modified with permission from: Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson R, Reece JB. (2014). Campbell Biology in Focus. Pearson, Boston. Modified from Pearson

29 EARLY TERMINATION NO EFFECT NO EFFECT Slide 29A final summary of what has been learned about how stop codons affect DNA replication, transcription and translation. Suggested Transition: “So we finally evidence to suggest which of these 5 nucleotide differences may result in DMD. The process of working through each of these nucleotide differences their outcomes allows us to better predict the underlying cause of DMD in Liam.”

30 Q9: Which of the five nucleotide differences are mutations? Dystrophin gene structure affected Elijah’s mRNA Liam’s mRNA CAA (gln) UAA (STOP) GAU (asp) GAG (glu) UCU (ser) UCA Q9: Which of the five nucleotide differences are mutations? All five are mutations. The three in the exons (difference #2, #3, #5) The two in exons that change the amino acid sequence (differences #3, #5) The one in the exon that causes DMD (difference #5) Slide 30 Clicker Question (Q9) Answer A The final clicker question addresses the students’ conceptual difficulty that nucleotide differences are only mutations if they are in a coding region or impact phenotype. While many students correctly answer that all of these nucleotide changes are mutations, a subset still think that mutations are restricted to coding regions (answer B) or must change the amino acid sequence (answer C). This question allows for a discussion on what defines a mutation. Suggested Transition: “It is important to understand that all of these nucleotide changes are considered mutations, but in this scenario only one is likely responsible for the disease phenotype.”

31 Liam Elijah affected unaffected Case Study Conclusions: Siblings living with Duchenne Muscular Dystrophy (DMD) UCU (ser) CAA (gln) GAU (asp) Elijah’s mRNA UCA (ser) UAA (STOP) GAG (glu) Liam’s mRNA Slide 31 We can conclude that, in this case, the premature stop codon (nonsense mutation) was the most likely mutation to lead to the expressed phenotype of DMD in Liam because of the effect on translation and the subsequent truncated protein produced.