Clinical Case Studies Answers to Karp's Cell and Molecular Biology 9th Edition; All Chapters A+ Graded 2025.

This Copy delivers a concise and illustrative narrative that helps students connect key concepts and experimentation of Clinical Case Studies Answers to Karp's Cell and Molecular Biology 9th Edition; All Chapters A+ Graded 2025. Introduction to the Study of Cell and Molecular Biology CASE STUDY: Will antibiotics cure the common cold? Picture the scene: it’s winter, your head aches, your sinuses are clogged, the coughing and sneezing won’t stop. You’ve got a cold (or maybe even the flu). Do you wait it out at home? Or go to your doctor or the medical clinic on campus? Maybe they can give you some antibiotics to clear it up… Colds, the most frequently transmitted infectious diseases in humans, are primarily caused by a group of viruses known as rhinoviruses (from Greek and Latin words for “nose poisons”). Viruses are non-cellular infectious agents that co-opt our cellular machinery to reproduce. The physiological response to a rhinovirus infection involves activation of the immune system, which leads to many symptoms we associate with the “common cold.” But since these reactions may not be specific to the pathogen, it can be hard to know whether rhinovirus or other viruses such as adenovirus or influenza are the culprit. Bacteria, while not the direct cause of cold symptoms, can cause secondary infections that occur during or after the onset of a cold. Questions: 1. Penicillin is an antibiotic that acts by inhibiting the formation of peptidoglycan cross-links in a cell wall. Based on what you know about the nature of viruses and bacteria, will penicillin effectively kill the rhinovirus? 2. People talk about catching a cold by touching surfaces that have been touched by someone else with a cold, such as a door handle or faucet knob. Is this because viruses can colonize and grow on these surfaces? 3. After entering cells, viruses use the host cell machinery to transcribe their viral DNA into RNA or make new copies of their RNA, which will then be translated into proteins that are needed for virus function and replication. There has been a lot of interest and some progress in the development of anti-viral drugs that act to halt the viral replication cycle. Do you think it would effective to target a drug to cellular RNA polymerase to halt viral replication? Why or Why not?Where can I learn more? 1. Palmenberg AC, Spiro D, Kuzmickas R, et al. Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution. Science. 2009;324(5923):55–59. doi:10.1126/science.1165557 2. Common Colds: Protect Yourself and Others [Internet]. Centers for Disease Control and Prevention; [updated 2019 Feb 11]. Available from: CASE STUDY: Defects in Hemoglobin Structure and Function Hemoglobin is the major oxygen carrier that is used to deliver oxygen to our tissues. It is a heterotetrameric protein that is composed of two alpha subunits and two beta subunits. Each subunit has the ability to bind and release oxygen, and its ability to do so is influenced by the structure of the other subunits. Defects in hemoglobin structure or synthesis are collectively termed hemoglobinopathies. This group of diseases results from defects in the synthesis of one of the hemoglobin chains or in defects in the structure of the hemoglobin molecule itself. Patients with defective hemoglobin have characteristic anemia, which leads to pallor, fatigue, and shortness of breath. Other clinical manifestations include reticulocytosis (elevation of the number of young red blood cells), splenomegaly (enlarged spleen), and urobilinuria (excess urobilins, which are breakdown products of hemoglobin, in the urine). Sickle Cell Anemia is a specific type of hemoglobinopathy caused by mutation of a single glutamic acid residue on the surface of hemoglobin to a valine, which results in a change in the surface properties of hemoglobin. This mutant hemoglobin is referred to as HbS. Presence of HbS causes protein aggregation under conditions of deoxygenation. The protein aggregates lead to malformed red blood cells that inhibit capillary flow. Questions: 1. The mutation in hemoglobin is a change from a glutamic acid to a valine. What are the chemical features of these two amino acids that may result in the defects caused by HbS? 2. According to the principles of the hydrophobic effect, where should glutamic acid and valine normally be found in proteins? 3. How then do you think that the mutated valine residue can contribute to the aggregation of hemoglobin molecules in HbS?Where can I learn more? 1. Schechter, A.N., Hemoglobin research and the origins of molecular medicine. Blood, 2008. 112: p. . 2. Berg, J.M, Tymoczko, J. and L. Stryer, Biochemistry, Sixth Edition, 2007. Chapter 7. Hemoglobin: A portrait of a protein in action. W.H. Freeman Publisher.The Chemical Basis of Life CASE STUDY: Protein Conformational Diseases Newly synthesized polypeptides must be properly folded in order to achieve their proper threedimensional protein structure. Defects in the folding process can lead to improper intramolecular and intermolecular interactions including the formation of protein clusters, or aggregates. These aggregates can become large enough to disrupt cell and tissue function. Huntington’s Disease (HD) is a neurological disease that is caused by a mutation in the huntingtin gene. It is an autosomal dominant disorder, meaning that if a person has just one mutant copy of the huntingtin allele, they will be at much higher risk of having the disease and of passing it along to their children. Unlike many genetic diseases, HD does not arise from point mutations in a gene that cause a loss of function. Instead, it is a member of a family of diseases known as the trinucleotide repeat disorders. The huntingtin gene (HTT) contains a repeated section of the sequence CAG, which codes for a long stretch of the amino acid glutamine. This is known as a polyglutamine, or “polyQ” tract. In HD patients, the polyQ tract of the protein huntingtin (HTT) is much longer than in people who are not at risk for the disease. The glutamine side chains do not fold properly and become sticky to each other and to other proteins, which leads to clustering of the protein, precipitous aggregates, and ultimately cellular dysfunction. Major symptoms of the disease, including involuntary movements and dementia, are consistent with neurodegeneration from defective deposits in brain cells. One challenge to the study of studying human neurological diseases is the requirement for good model systems to ask questions about the disease. A useful model can often be found in animal systems, such as the mouse, where genes can either be overexpressed or deleted. The questions below are based on several mouse models that have been developed to look at HD. Questions: 1. Mice that are heterozygous for the HTT null mutation do not display Huntington's disease. (This means that they have only one copy of the gene encoding huntingtin, and it is the normal, wild type HTT allele.) What does this tell you about the mechanism by which the normal HTT protein functions versus the mHTT mutant protein (with expanded polyQ tract at its N-terminus)? 2. Scientists have generated a “conditional” model for HD in mice. In this study the researchers generated a mouse model where they could selectively induce the expression of an abnormal version of the HTT protein that contains the N-terminus of HTT with a very long polyQ tract (94 CAG repeats). These mice developed neuropathological and behavioral defects associated with HD. Does this result support or refute what you answered in Question 1 about the mechanism by which the HTT protein causes cellular dysfunction?3. One very exciting finding from this conditional mouse model was that if they turned off the expression of the mutant form of the truncated HTT protein, they could halt progression of the disease and even reverse aggregate formation and progressive motor decline. What do these results imply about the mechanism of the function of the abnormal HTT protein, and what are the implications for therapy for this disease? Where can I learn more? 1. Dickey AS, La Spada AR. Therapy development in Huntington disease: From current strategies to emerging opportunities. Am J Med Genet A. 2018;176(4):842–861. doi:10.1002/ajmg.a.38494The Chemical Basis of Life CASE STUDY: Good vs. Bad Fats? There is much information in the popular press touting the virtues of this oil versus that oil, or this margarine versus that margarine. Beyond the obvious marketing strategies used by many companies, is there any scientific basis for why one fat is better or worse for you than another? Furthermore, why do things like French fries and potato chips that taste so good make us fat? If we look into the nutritional information regarding several of the biological building blocks (proteins, carbohydrates, and fats) that we get from our food, one will find that 1 g of protein or carbohydrate contains 4 calories whereas 1 g of fat contains 9 calories. That means we need much less fat to meet our body’s energy requirements than we do for protein or carbohydrates. Fat is readily stored in our body tissues whereas carbohydrates and proteins must be metabolized to be converted into fat. Despite the bad press that fats often receive, they are essential building blocks for our cells and serve multiple physiologically important roles. But what is the difference between fats? This comes down to the structure of the fatty acid side chains and how the fats are processed in our bodies. Fats can be saturated in which their hydrocarbon chains have no double bonds. Unsaturated fats have double bonds along the length. These double bonds create kinks in the side chains, which inhibit the ability of the chains to pack tightly together. This in turn will affect the temperature at which the transition between liquid and solid occurs. Saturated fatty acids are typically solid at room temperature, whereas unsaturated fatty acids are liquid. In some cases, saturated fatty acids may remain solid at human body temperature, as well. In addition saturated fats are readily converted into LDL cholesterol, high levels of which have been associated with heart disease. Questions: 1. You are about to make a delicious batch of chocolate chip cookies. You debate between using butter versus margarine. Given that butter has a lot of saturated fat and margarine has a higher percentage of unsaturated fat, which is the healthier choice and why? 2. The biological properties of molecules are also very important in cooking. If the recipe for those chocolate chip cookies calls for 2 sticks of butter softened at room temperature, will the texture of the cookie dough be the same if you substitute 2 sticks of margarine that has no saturated fat? Why or why not? Provide an explanation based on your knowledge of the structure of the fatty acid side chains. Where can I learn more? 1. 2. Butter vs. Margarine. Harvard Health Publishing. CASE STUDY: Methanol Poisoning Many drugs and toxins act by binding to enzymes, thus altering or inhibiting their function. There are several modes of enzyme inhibition, one of the most common being competitive inhibition, in which a molecule binds to an enzyme’s active site and competes with its normal substrate. These competing molecules are known as antagonists and may also block the activity of receptors on the cell surface. Millions of tons of methanol (CH3OH), the simplest alcohol, are produced each year for a variety of industrial applications and as a fuel source. Methanol is highly toxic to humans. Symptoms of methanol poisoning include upset stomach, dizziness, and vision problems. It can ultimately lead to blindness and death. Methanol toxicity is not due to the methanol itself, but rather to the reactivity of its oxidation products, formaldehyde (CH2O) and formic acid (CH2O2). The conversion from methanol to formaldehyde is carried out by the enzyme alcohol dehydrogenase. Alcohol Formaldehyde dehydrogenase dehydrogenase CH3OH CH2O CH2O2 CO2 + H2O Methanol Formaldehyde Formic acid Metabolic acidosis and tissue injuryQuestions: 1. Despite the development of other pharmacological treatments, the most common therapeutic treatment for methanol poisoning is to put the patient on an IV containing 10% ethanol (C2H5OH). What do you propose the mechanism of action to be for the treatment with ethanol? Please give some explanation of why you came to this conclusion. 2. A curious side effect of ethanol administration is that the patient will become inebriated, but will not have the toxicity associated with methanol poisoning. Why doesn’t ethanol also get broken down into the same toxic metabolites by the alcohol dehydrogenase? 3. There are multiple forms of the enzyme aldehyde dehydrogenase, which are differentially expressed in humans. Some isoforms have a very high Km for their substrate, acetaldehyde. People who expressive this isoform are highly sensitive to the consumption of alcohol and will often show signs of intoxication after only a single drink. Can you provide an explanation for this observation? Where can I learn more? 1. Devlin, T.M. Textbook of biochemistry with clinical correlations. 6th edition. 2006. Chapter 10: 365-412. 2. Kraut JA. Approach to the Treatment of Methanol Intoxication. Am J Kidney Dis. 2016; 68(1) 161- 167. doi: 10.1053/.2016.02.058. Available from 6386(16)30038-5/fulltextBioenergetics, Enzymes, and Metabolism CASE STUDY: Anaerobic Metabolism Humans have evolved in an aerobic (oxygen-rich) world. Much of our metabolism relies on the availability of molecular oxygen, which accepts energy-depleted electrons from the electron transport chain. The high electron affinity of oxygen allows for the oxidation of the important cofactor NADH, which is formed through the reduction of NAD+ during glycolysis and the TCA cycle. This phenomenon is known as aerobic respiration. During vigorous exercise, the cells of human skeletal muscle will get a quick supply of ATP through glycolysis, which produces ATP about 100 times faster than the TCA cycle. As the level of available oxygen decreases, the cells turn to anaerobic glycolysis to regenerate NAD+. In this scenario, the pyruvate formed in glycolysis is converted to lactate by the enzyme lactate dehydrogenase, generating approximately one NAD+ for each molecule of pyruvate hydrolyzed. The electron transport chain is inactive, but ATP can still be produced to power muscle movement. Other organisms use similar reactions to re-oxidize NADH in the absence of oxygen. One wellknown example takes place in brewer’s yeast. Upon digestion of various carbohydrates, pyruvate is produced and then converted to acetaldehyde by the enzyme pyruvate decarboxylase. The acetaldehyde is in turn reduced to ethanol via alcohol dehydrogenase, in a reaction that also generates NAD+. This process has practical uses – for example, beer is made by the breakdown of sugars in different grains to produce ethanol. Questions: 1. One misconception with anaerobic metabolism in human muscle is that the formation of lactate lowers the pH and causes muscle soreness. Below is the metabolic scheme for the conversion of glucose to pyruvate and then to lactate. Based on this reaction, why is it not possible for the lowering of the pH to be from the lactate? Glucose + 2 ADP + 2 Pi + 2 NAD+ 2 pyruvate + 2ATP + 2 NADH + 2 H+ + 2 H2O Pyruvate + NADH + H+ lactate2. Our bodies do not utilize the same enzymes that yeast use for fermentation of pyruvate. If we did, what would happen each time we exercised? 3. A growing body of evidence suggests that lactate, rather than pyruvate, is the major product of glycolysis even under aerobic conditions and can “shuttle” out to other tissues as a metabolic intermediate. Why might the presence of lactate in aerobically respiring tissues have been underappreciated until recently? Where can I learn more? 1. Brooks GA. The Science and Translation of Lactate Shuttle Theory. Cell Metab. 2018 Apr 3;27(4):757-785. doi: 10.1016/.2018.03.008. Available from 2. Devlin TM. Textbook of biochemistry with clinical correlations. 6th edition. 2006. Chapter 15: 581-636.The Structure and Function of the Plasma Membrane CASE STUDY: Heartburn – Treat it or Prevent it You are going to go out with a bunch of friends to a restaurant with the best pizza in town. Your favorite is the pizza covered with cheese, tomato and lots of garlic and onions. The only problem is that you know that later in the evening you will be suffering from heartburn and indigestion. Decades ago, when your professors were in college, their only choice was some to take some antacids after overeating pizza, but today there are multiple choices of preventative medications that act to stop heartburn before it starts. The stomach is a highly acidic environment that is further acidified after ingestion of food, which triggers a hormone-stimulated cascade. This hormone stimulation results in the activation of a H+/K+ ATPase channel (“proton pump”) that releases additional protons into the stomach cavity. Heartburn occurs when the acid in the stomach undergoes reflux and backs up into the esophagus, giving you that burning feeling in the chest. There are three types of remedies available. Antacids such as calcium carbonate act simply to neutralize stomach acid, thus raising the pH of the stomach juices. This provides temporary relief from heartburn. A second class of compounds, including the drugs Zantac, Pepcid, and Tagamet, act as receptor antagonists on the surface of the gastric cells so that the hormonal stimulation never triggers the H+/K+ ATPase. The third type, which includes Prilosec and Prevacid, directly inhibit the action of the H+/K+ ATPase. Questions: 1. Clinical studies suggest that the proton pump inhibitors are the most effective drugs of the classes described above. Based on the proposed mechanism of action, suggest a plausible reason for why this would be so. 2. Why is it that antacids are effective to take after heartburn starts but that either the receptor antagonists or the proton pump inhibitors are ineffective unless taken before meals? 3. The drugs that are available for heartburn treatment have been extensively tested and are recognized as safe and essential medicines. Nonetheless, in September 2019 samples of Zantac (ranitidine, a histamine receptor antagonist), were pulled from shelves over concern of a cancer risk. What could explain this concern?Where can I learn more? 1. Shin, J.M., et al., Molecular mechanisms in therapy of acid-related diseases. Cell Mol Life Sci, 2008. 65: p. 264-281. 2. [FDA] US Food and Drug Administration. [Internet]. 2019. Zantac (ranitidine): Safety Information - NDMA Found in Samples of Some Ranitidine Medicines. [updated 2019 Sep 6]. Available from: Membrane CASE STUDY: Commonly Prescribed Anti-Depressants Major depression, a persistent feeling of sadness and emotional disengagement, is one of the most common mental illnesses. In 2017, approximately 7% of US adults and 13% of adolescents had at least one depressive episode. A variety of drugs are used to treat major depression, and their widespread use makes the importance of studies looking at their effectiveness and long-term benefits all the more crucial. The neurotransmitter serotonin controls many physiological processes such as mood, sleep, anger, aggression and appetite. The action of serotonin is down-regulated, or diminished, through the activity of transporters on the pre-synaptic neuron that specifically bind and take up serotonin from the synapse. The drugs Prozac and Paxil act at the site of re-uptake by inhibiting the serotonin pumps, allowing more serotonin to bind to receptors on the post-synaptic neuron. These drugs are known as selective serotonin reuptake inhibitors (SSRIs) and are commonly prescribed to treat depression symptoms. Before approval for use in humans, all drugs undergo extensive testing in model organisms such as mice and rats. However, the use of these models to address individual drug sensitivity and combinatorial treatments is limited by the relatively long maturation and gestation process in mammals. Scientists therefore often turn to simpler model organisms, such as the nematode worm C. elegans or fruit fly D. melanogaster, which have short life spans and have a very well-defined neurological system. The following questions are based on a series of experiments carried out in C. elegans looking at the action of Prozac. Questions: 1. In C. elegans, the mod-5 gene encodes the only version of the serotonin reuptake transporter in this organism. Worms with mutations in the mod-5 gene are hypersensitive to treatment with serotonin and have an elevated behavioral response to serotonin exposure (slow response to food). These effects could be altered by treatment of the animals with Prozac. Strikingly, high levels of Prozac also caused a defect in which the animals contracted their noses, regardless of whether they had mutations in the mod-5 gene. How can you interpret this result?2. What do these results imply for the use of Prozac in humans? 3. Can you think of a way that scientists could use a genetic organism such as C. elegans to identify these other targets of Prozac? Where can I learn more? 1. Choy RK, Kemner JM and, Thomas JH. Fluoxetine-resistance genes in Caenorhabditis elegans function in the intestine and may act in drug transport. Genetics. 2006;172:885-892. 2. Dwyer DS, Aamodt E, Cohen B, Buttner EA. Drug elucidation: invertebrate genetics sheds new light on the molecular targets of CNS drugs. Front Pharmacol. 2014;5:177. Published 2014 Jul 28. doi:10.3389/fphar.2014.00177Aerobic Respiration and the Mitochondrion CASE STUDY: Cyanide Poisoning Cyanide is extremely toxic, whether it be administered by the ingestion of the flavorless powder potassium cyanide or by the inhalation of the almond-scented hydrogen cyanide gas. The book and movie Winter of Frozen Dreams detail the story of Barbara Hoffman, a Wisconsin college student who was convicted of murder after poisoning her fiancé and a former lover with cyanide and attempting to cover her tracks. Other famous and fatal instances of acute cyanide poisoning include the suicide of Adolf Hitler and the “Jonestown massacre” in which 900 people died after ingesting a cyanide-laced flavored drink. Cyanide is an electron transport inhibitor, which acts by binding to the Fe3+ heme a3 group on cytochrome oxidase in Complex IV so that electrons cannot be passed to molecular oxygen during aerobic respiration. The electrochemical gradient that provides the energy for ATP synthesis comes to a halt, leading to apoptotic and necrotic cell death. In humans, the end result of acute exposure to high levels of cyanide is death via shutdown of the central nervous system and/or cardiac arrest. Questions: 1. If cyanide poisoning occurs by blocking of electron transport from oxygen, why can’t the effects of poisoning be reversed by the administration of higher levels of oxygen? 2. One treatment for cyanide poisoning is the infusion of sodium thiosulfate. This compound provides exogenous sulfate, which enhances the metabolism of cyanide to thiocyanate, which is less toxic and can be excreted in the urine. If the reaction from cyanide to thiocyanate requires the activity of an enzyme (called rhodanese), what might be a limitation in using sodium thiosulfate as an antidote? 3. Chronic cyanide poisoning is a less pronounced but more common threat to health that is caused by cigarette smoke and work in industries that use cyanide (plastics, metal fabrication). Why would continuous exposure to cyanide be less likely to result in death than acute exposure to high amounts of cyanide? Where can I learn more? 1. Eckstein M. Enhancing public health preparedness for a terrorist attack involving cyanide. J Emerg Med. 2008; 35:59-65. 2. Hater K. Winter of Frozen Dreams. 1992; 337 pgs. 3. [CDC] Centers for Disease Control and Prevention [Internet]. 2011. POTASSIUM CYANIDE : Systemic Agent. [updated 2011 May 12]. Available from: CASE STUDY: Mitochondrial Diseases Mitochondria are likely to have evolved through endosymbiosis of bacteria. Over time the mitochondria lost most of their DNA as genes were transferred to the nuclei of the host eukaryotic cells, but retained some genes that code for mitochondria-specific products such as components of the electron transport machinery and tRNAs and rRNAs. Mitochondrial genetic diseases present a challenge to researchers in that they are not transmitted via simple Mendelian inheritance patterns. There are at least two factors that influence mitochondrial DNA replication and segregation. First, mitochondria are maternally inherited because upon fertilization the sperm head penetrates the egg and the paternal mitochondria are selectively degraded. Second, mitochondria are not segregated precisely during cell division. With each cell division, approximately the same number of mitochondria are distributed to the two daughter cells. The exact mitochondrial DNA content varies from cell division to cell division within a tissue and between tissues. This leads to a condition called heteroplasmy, in which tissues can have different mixtures of wild type and mutant mitochondrial DNA. Questions: 1. If a person inherits a mutation in a mitochondrial gene that is required for one of the components of the electron transport chain, how is it possible that the affected individual can live since oxidative phosphorylation is essential for life? 2. If male offspring were affected by mitochondrial DNA mutations and subsequent diseases, do you think the males would be fertile or sterile?Where can I learn more? 1. Reeve AK, Krishnan KJ, Turnbull D. Mitochondrial DNA mutations in disease, aging, and neurodegeneration. Ann New York Acad Sci. 2008;1147: 21-29. 2. van der Bliek AM, Sedensky MM, Morgan PG. Cell Biology of the Mitochondrion [published correction appears in Genetics. 2018 Apr;208(4):1673]. Genetics. 2017;207(3):843–871. doi:10.1534/genetics.117.300262Photosynthesis and the Chloroplast CASE STUDY: Can Plants Get Cancer? When you think about summer, you think about long days and the light and warmth of sunshine. But you might also think about sunscreen, sunglasses, and all those reminders to limit your exposure to direct sunlight. Why? Because you want to ensure that you don’t get skin cancer. Cancers of the skin, such as basal cell carcinoma, are the most commonly occurring types of cancer in humans. High-energy ultraviolet wavelengths of sunlight can directly cause DNA damage in exposed cells, leading to unregulated growth of new skin cells. The American Cancer Society estimates approximately a million cases or more of skin cancer will be diagnosed each year. Plants require direct exposure to high-intensity light more than humans do. Why is it that plants don’t get cancer when they spend their days in the sun without first slathering themselves with sunscreen? The reason is that plant tissues have evolved systems to fine-tune the absorption of energy from sunlight and to release unused energy in a controlled manner. While green chlorophyll pigments are used in photosynthesis, accessory pigments like carotenoids (which are yellow, red or orange) help to protect the plant cells from solar radiation. When sunlight is most intense, both chlorophylls and carotenoids work in concert to harvest solar energy while protecting the plant from DNA damage. Plants can also change the position of light-harvesting complexes to influence the amount of light that is absorbed. Humans created solar protective pigments as well, like the melanin in your skin, but plants are the experts in this area through the evolutionary refinement of photosynthesis. Even though they don’t get cancer the same way we do, plants may harbor tumors and abnormal cell growths due to bacterial, viral or fungal infections. Ionizing radiation including sunlight can cause DNA damage through the formation of thymine dimers, just as it does in our skin cells. Questions: 1. What molecules involved in the process of photosynthesis do you think would be the most likely candidate to act as a good potential sunscreen ingredient?2. Herbal medicines and plant-derived pharmaceuticals are used to treat a variety of diseases and disorders. What does this suggest about other potential mechanisms to prevent and treat skin cancer? Where can I learn more? 1. Stahl W, Sies H. β-Carotene and other carotenoids in protection from sunlight. Am J Clin Nutr. 2012 Nov;96(5):1179S-84S. doi: 10.3945/ajcn.112.034819. 2. Balić A, Mokos M. Do We Utilize Our Knowledge of the Skin Protective Effects of Carotenoids Enough? Antioxidants (Basel). 2019;8(8):259. Published 2019 Jul 31. doi:10.3390/antiox8080259Interactions Between Cells and their Environment CASE STUDY: Collagen Disorders Multiple diseases arise from defects in collagen synthesis and/or assembly. The manifestation of the disease is often a reflection of the type of collagen that is affected, as many different genes encode for collagen and not all genes are expressed in all tissues. Collagen has a unique structure that relies on multiple post-translational modifications and assembly steps. Each polypeptide chain is rich in proline, which occurs much more frequently than in globular proteins. Hydroxylated residues of proline and lysine are also found in collagen chains and contribute to the protein’s stability. The formation of the collagen triple helix is facilitated by the presence of cysteine residues in the C-terminus of the protein that serve as sites of disulfide bond formation. The disulfide bonds bring the three collagen chains into close proximity. Procollagen assemblies contain sequences that will be cleaved by pro-collagen peptidases once collagen is secreted into the extracellular space. The assembly of the collagen fibril occurs in the extracellular space is stabilized by cross-links of the collagen triple helices to other collagen triple helices. These cross-links occur via oxidized lysine residues that are formed by the action of an enzyme called lysine oxidase. Ultimately collagen fibers are assembled from collagen fibrils in the extracellular space. Below are a series of diseases that occur due to defects in collagen synthesis. For each, there is a brief description of the molecular basis of the disease. For each case, describe the collagen assembly step that is affected in the disease state and the effect on collagen structure and function. Questions: 1. Scurvy: Scurvy occurs from a defect in the synthesis of hydroxyproline due to ascorbic acid deficiency. Ascorbic acid is a necessary cofactor for the enzyme prolyl hydroxylase. 2. Osteogenesis Imperfecta (brittle bone disease): This is a group of defects that result from point mutations or exon rearrangements in the coding regions of collagen, or in mutations of chaperone proteins that assist with collagen assembly. 3. In Type VI Ehlers-Danlos Syndrome, there is a decrease in hydroxylysine residues due to a deficiency in the enzyme lysyl hydroxylase.4. In Ehlers-Danlos Type IX, also known as occipital horn syndrome, the lysyl oxidase enzyme is negatively affected by a defect in the intracellular transport of its key cofactor, copper. Where can I learn more? 1. Lu Y, Zhang S, Wang Y, Ren X, Han J. Molecular mechanisms and clinical manifestations of rare genetic disorders associated with type I collagen. Intractable Rare Dis Res. 2019;8(2):98–107. doi:10.5582/irdr.2019.01064 2. Popovich D(1), McAlhany A, Adewumi AO, Barnes MM. Scurvy: forgotten but definitely not gone. J Pediatr Health Care. 2009 Nov-Dec;23(6):405-15. doi:10.1016/.2008.10.008. Epub 2009 Feb 10. 3. Palomo T, Vilaça T, Lazaretti-Castro M. Osteogenesis imperfecta: diagnosis and treatment. Curr Opin Endocrinol Diabetes Obes. 2017 Dec;24(6):381-388. doi: 10.1097/MED..Interactions Between Cells and their Environment CASE STUDY: Proteoglycans: Dietary Supplements or Snake-oil treatments? As our bodies age, cells, tissues, joints, and bones begin to deteriorate, with effects ranging from mild discomfort to chronic pain and lowered quality of life. Osteoarthritis is the most common type of arthritis that affects primarily the elderly. After repeated bending and twisting of the joints, the cartilage begins to wear down. This loss of flexibility and cushioning leads to an increase in joint pain. Cartilage contains mainly collagens and proteoglycans, and studies dating to the 1980s suggested that replacement of these compounds in aging patients would provide the natural building blocks for the cartilage to be renewed. Combinations of the proteoglycans glucosamine hydrochloride (GH) and chondroitin sulfate (GS) are available as dietary supplements. These over-the-counter compounds are not subject to most federal regulations - as long as they do not claim any specific health benefits - and may not undergo the same testing and quality assurance methods as regulated drugs. GH and CS supplements are recommended by physicians and medical organizations to assist with joint health, based on a variety of studies and trials of the compounds’ effects in model organisms and humans alike. Other studies have concluded that these supplements are safe to use but are no more effective than placebo for the relief of pain and inflammation associated with osteoarthritis. Questions: 1. Glucosamine hydrochloride and chondroitin sulfate are usually taken together in supplement formulations. Based on your knowledge of the structure of the extracellular matrix, why are these molecules chosen as supplements to lessen the effects of arthritis? 2. Considering that GH and CS are available over the counter, what reasons can you provide for differing reports of the efficacy of these proteoglycan supplements? 3. One of your elderly relatives comes to you asking about some medical advice they got from a friend. They want to know if they should take a glucosamine and chondroitin supplement for their rheumatoid arthritis. How would you advise them?Where can I learn more? 1. Vangsness Jr C, Spiker W, Erickson J. A review of evidence-based medicine for glucosamine and chondroitin sulfate use in knee osteoarthritis. Arthroscopy 2009;5(1):86–94. 2. Hochberg MC, Martel-Pelletier J, Monfort J, et al. Combined chondroitin sulfate and glucosamine for painful knee osteoarthritis: a multicentre, randomised, double-blind, noninferiority trial versus celecoxib. Ann Rheum Dis. 2016;75(1):37–44. doi:10.1136/annrheumdis- 3. Stellavato A, Restaino OF, Vassallo V, Finamore R, Ruosi C, Cassese E, De Rosa M, Schiraldi C. Comparative Analyses of Pharmaceuticals or Food Supplements Containing Chondroitin Sulfate: Are Their Bioactivities Equivalent? Adv. Ther. 2019 Sep 7. doi: 10.1007/s- 01064-8.Interactions Between Cells and their Environment CASE STUDY: Sticky Cells: Cell Connections, Metastasis and Cancer Cancer is a complicated series of diseases characterized by over-proliferation of cells within a tissue. While in some instances the primary tumor is the ultimate cause of death, in many cases the primary tumor metastasizes, or moves, through the lymph system to invade other tissues. The migration of tumor cells requires changes in the adhesion properties of the cell that allow it to move and invade more readily. There are several molecules involved in cell-to-cell connections and cellular architecture: laminins, matrix metalloproteinases, cadherins, and integrins. While mutations in genes that control the cell cycle or recognize DNA damage are the causes of tumor cell formation, tumor cell spread to other tissues may be caused by mutations in the genes the code for these extracellular proteins. The loss of cellular connections, cellular identification and signaling, and tissue structure may lead to the release of expanding tumor cells into the circulatory and lymphatic systems. Once tumor cells have access to blood and lymph vessels, they can travel to most parts of the body. This spread of tumor cells is called metastasis. Questions: 1. Cancer cells from a person suffering from malignant melanoma are analyzed and found to have an elevated ability to bind to laminin; they also secrete much higher than normal levels of a certain proteolytic enzyme activity. How would these differences from normal, control cells promote metastasis of these cancer cells? 2. In contrast to the situation described in question 1, it has recently been found that nearly 25% of melanomas have mutations in matrix metalloproteinases (MMPs). Why is this contradictory and what does this suggest about the likelihood of MMP inhibitors being effect chemotherapy agents?3. Why would cells that express less E-cadherin be more likely to become malignant and give rise to epithelial cell tumors? 4. If you were able to inject the gene for α5β1 integrin (a fibronectin receptor) into some tumor cells in such a way that it was expressed extremely efficiently, would those cells be more or less invasive? Why? Where can I learn more? 1. Akhavan, A., O. L. Griffith, L. Soroceanu, D. Leonoudakis, M. G. Luciani-Torres, A. Daemen, J. W. Gray and J. L. Muschler ., Loss of Cell-Surface Laminin Anchoring Promotes Tumor Growth and Is Associated with Poor Clinical Outcomes. Cancer Research, 2012. 72(10): p. . 2. Eble, J.A. and J. Haier, Integrins in cancer treatment. Current cancer drug targets, 2006. 6: p. 89- 105. 3. Jeanes, A., C. J. Gottardi and A. S. Yap., Cadherins and cancer: how does cadherin dysfunction promote tumor progression [quest]. Oncogene, 2008. 27(55): p. . 4. Palavalli, L.H., et al., Analysis of the matrix metalloproteinase family reveals that MMP8 is often mutated in melanoma. Nature genetics, 2009. 41: p. 518-520.Cytoplasmic Membrane Systems: Structure, Function, and Membrane Trafficking CASE STUDY: High Cholesterol It is well-established that elevated levels of serum cholesterol (cholesterol that circulates in the blood rather than being taken up into tissues) can place people at an increased risk for cardiovascular disease. Since lipids are not very soluble, large complexes such as low-density-lipoproteins (LDL) and high-density-lipoproteins (HDL) function as cholesterol carriers. LDL Receptor (LDLR) is the cell surface protein that binds to the LDL particles in the serum and internalizes them, thus reducing the levels of serum cholesterol. When LDL uptake into cells is reduced, either by an excess of LDL or a deficit of LDLR, cholesterol builds up in the bloodstream. Over time, the body attempts to cope with the LDL particles in other ways, leading to the symptoms associated with cardiovascular disease. How do people get “high cholesterol”? Genetics plays a role. In familial hypercholesterolemia (FH), patients have a mutation in the gene that encodes the LDLR or in the gene for Apolipoprotein B (ApoB). (ApoB is part of the LDL particle itself). FH is a dominant genetic disorder, so that people who have one mutant copy of either the LDLR or ApoB genes have elevated serum cholesterol and can develop early heart disease in their 30s or 40s. Patients with two mutant copies of the genes have extremely high serum cholesterol and often will develop severe cardiovascular disease will still in their 20s. The content of an individual’s diet is another other major factor for their risk cholesterol-related cardiovascular disease. The most direct correlation is between cholesterol intake and LDL/HDL levels – the more cholesterol someone ingests, the more LDL and HDL are required to carry it through the bloodstream. Other types of lipids, such as fatty acids, are also implicated in LDL metabolism. Saturated fats in diet were long suspected of causing high LDL levels, although this conclusion has been cast in doubt by recent studies. The stronger risk seems to be dietary intake of transunsaturated fats, which have been linked to both elevated levels of LDL and lowered levels of HDL (the “good” cholesterol carrier that is metabolized in the liver). Questions: 1. Given that the effect of specific types of lipids is still under debate, how can someone who is concerned about high cholesterol make good dietary choices? 2. Some people with healthy diets and no history of cardiovascular disease still have cholesterol accumulation, such as yellow deposits of cholesterol around the eyes called xanthelasma. What might cause this, and what can be done? 3. In addition to genetic predisposition to FH and diet, what other risk factors and conditions are associated with high cholesterol?Where can I learn more? 1. Palacio CH, Harring TR, Nguyen NTT, Goss JA, Mahony CA. Homozygous Familial Hypercholesterolemia: Case Series and Review of the Literature." Case Reports in Transplantation 2011. 2. Forouhi NG, Krauss Ronald M, Taubes Gary, Willett Walter. Dietary fat and cardiometabolic health: evidence, controversies, and consensus for guidance. BMJ 2018; 361. Structure, Function, and Membrane Trafficking CASE STUDY: Unique Sugars Create our Blood Types Ensuring that proteins are targeted to the proper cellular destination is critical in eukaryotic cell function. Not only is this necessary for each protein to be in the proper place to carry out its function but also because certain post-translation modifications take place in the secretory pathway. Improperly processed proteins may be defective in biochemical activity even if they are located in the proper place in the cell. For example, the processing of the A and B antigens for specification of ABO blood types is due to post-translational modification by a glycosyltransferase enzyme. Each antigenic determinant consists of structurally related oligosaccharides present on both glycoproteins and glycolipids that are found on the surface of erythrocytes and other cell types. The presence of different alleles of the ABO glycosyltransferase gene determines which types of modifications occur. A carbohydrate modification known as H antigen is found on all human blood cells and is associated with type O blood. If a person has type A blood, they express the IA allele of the ABO gene, and the resulting enzyme adds N-acetylgalactosamine (GalNac) to the outer galactose residue on the H antigen. If a person has type B blood, they have the IB form of glycosyltransferase, which adds a galactose residue to the outer galactose residue on the O antigen. If a person has type AB blood, it really means that they have a mixture of A and B blood because they produce both forms of the enzyme (the alleles are co-dominant). The consideration of ABO blood types is particularly important when managing blood transfusions. A person can create antibodies against the non-present antigen types. For example, if a person has type A blood, he or she may harbor circulating antibodies to type B antigen. If exposed to type B blood from a donor, the antibody-antigen reaction would lead to a significant immune response as the incompatible blood type is rejected. Recent studies have begun to use blood type groups as a genetic marker for other conditions, as there may be some links between the blood type you inherit and other genes. Questions: 1. If a person had a loss-of-function mutation in the ABO gene, what type(s) of blood group antigen would they produce?2. For the person in question 1, what type of blood could they receive? 3. If the glycosyl transferase enzymes are located in the Golgi complex, how is it that the blood group antigens are found on serum proteins? 4. What methods could be used to make donated blood with type A or type B antigens safe for transfusion into a type O patient? Where can I learn more? 1. Brecher, M. E. and S. N. Hay, ABO Blood Type and Longevity. American Journal of Clinical Pathology, 2011. 135(1): p96-98. 2. Hakomori S. Antigen structure and genetic basis of histo-blood groups A, B and O: their changes associated with human cancer. Biochim Biophys Acta. 1999 Dec 6;1473(1):247-66. 3. Liu QP, Sulzenbacher G, Yuan H, Bennett EP, Pietz G, Saunders K, Spence J, Nudelman E, Levery SB, White T, Neveu JM, Lane WS, Bourne Y, Olsson ML, Henrissat B, Clausen H. Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol. 2007 Apr;25(4):454-64.Cytoplasmic Membrane Systems: Structure, Function, and Membrane Trafficking CASE STUDY: Unique Sugars Create our Blood Types Ensuring that proteins are targeted to the proper cellular destination is critical in eukaryotic cell function. Not only is this necessary for the protein to be in the proper place to carry out its function but also because certain post-translation modifications take place in the secretory pathway. Therefore improperly processed proteins may be defective in their biochemical activity even if they are located in the proper place in the cell. For example, the processing of the A and B antigens for specification of blood types is due to post-translational modification by certain glycosyl transferases. Each antigenic determinant consists of structurally related oligosaccharides present on both glycoproteins and glycolipids that are found on the surface of erythrocytes and other cell types. The presence or absence of the glycosyl transferases determines which types of modifications occur on the O antigen. All people can make the O antigen since it is found on all blood cells. If a person has type A blood, they have the glycosyl transferase enzyme, which adds an N-acetyl galactosamine attached to the outer galactose residue on the O antigen. If a person has type B blood, they have the glycosyl transferase enzyme, which adds a galactose residue to the outer galactose residue on the O antigen. If a person has type AB blood, it really means that they have a mixture of A and B blood because they have both enzymes that modify the O antigen. A person with AB blood with have a mixture of O antigens modified with either N-acetyl galactosamine or galactose. One of the most important aspects of the ABO blood types is when considering donating or receiving blood. A person can create circulating antibodies in their bloodstream to the non-present antigen types. For example, if a person has type A blood, he/she may synthesize circulating antibodies to type B antigen so if he/she received type B blood. The antibody-antigen reaction would lead to a significant immune response. Not only would the incorrect blood type be rejected, but the removal and recycling of those blood cells could lead to greater illness. Recent studies have begun to use blood type groups as a genetic marker for other conditions, as there may be some links between the blood type you inherit and other genes. Questions: 1. If a person had the A gene but it was mutant and non-functional, what type of blood group antigen would they have circulating?2. For the person in question 1, what type of blood could they receive? 3. The glycosyl transferase enzymes are located in the Golgi complex, where are they synthesized and how are they processed? 4. If the glycosyl transferase enzymes are located in the Golgi complex, how is it that the blood group antigens are found on serum proteins? Where can I learn more? 1. Brecher, M. E. and S. N. Hay, ABO Blood Type and Longevity. American Journal of Clinical Pathology, 2011. 135(1): p96-98. 2. Lipshutz Gs, M. S. Z. Q. and et al., ABo blood type–incompatible kidney transplantation and access to organs. Archives of Surgery, 2011. 146(4): p453-458.The Cytoskeleton and Cell Mobility CASE STUDY: Microtubules and Disease Microtubules (MTs) are highly dynamic polymers, whose physical properties are critical for multiple aspects of cellular function including mitosis, intracellular transport and cell motility. MTs serve as tracks upon which motor proteins can carry cargo to different cellular destinations. In addition, both variable and stable assemblies of MTs comprise cellular structures including the mitotic spindle and the axonemes of cilia and flagella. Defects in multiple proteins that associate with the microtubule cytoskeleton have been correlated with a number of human disorders. Below are summarized a few examples of such conditions, followed by a series of questions to illustrate how scientists use model organisms to probe human disease. The Lis-1 gene and lissencephaly: Lissencephaly is a disease in which neurons do not migrate properly for the development of the cortical layers of the brain during embryonic development. The result is a smooth brain and severe neurological defects. Infants with lissencephaly rarely survive past the first few years of life. A major molecular link to lissencephaly came from the identification of the lis-1 gene that is mutated in classes of patients with lissencephaly. The lis-1 gene product is a regulator of the protein dynein, suggesting that its role in neuronal migration arises from motility defects. It is not yet clear how these defects arise, but one model is that lis-1 causes defects in the orientation of the mitotic spindle within early neuronal cell divisions, which will lead to cellular asymmetry. Another model is that lis-1 regulates dynein motility during migration of the nucleus, which occurs during neuronal migration. Immotile Cilia Syndrome: Defects in axonemal dynein have been associated with immotile cilia syndrome. This results in male infertility because the sperm are unable to move. Patients often have difficulty with respiratory function as well, due to the necessity of respiratory tract cilia to beat properly and to clear the airway of particulates and germs. Ciliopathies: Ciliopathies are collectively considered defects in the formation or function of primary cilia. These diseases are associated with polydactyly, situs inversus, obesity, kidney disease, heart defects, mental retardation, genital abnormalities, retinal degeneration, decreased olfactory discrimination, diabetes, and high blood pressure. One commonality within these diseases is a defect in intraflagellar transport (IFT), the process by which large particles are moved up the axoneme of the primary cilia. Defects in the kinesins that transport IFT particles as well as in several components carried in these particles have been linked to the various ciliopathies.Questions: 1. C. elegans is a nematode that is an excellent model system to probe protein function during early development. Inactivation of the C. elegans lis-1 gene results in a defect in the position of nuclei in certain neuronal cells during development. If you wanted to ask whether lis-1 was interacting in cooperation with dynein, what result might you expect to see in animals with inactivated dynein? 2. If you examined the axonemes of patients with immotile cilia syndrome by electron microscopy, would you expect to see a defect? If so, how might the axonemes be defective? 3. In the nematode worm C. elegans there are a multitude of gene products whose mutation is associated with defects in intraflagellar transport. If mutation in a gene caused a reduction in the rate of motility of IFT particles, what is the likely function of the gene product? Where can I learn more? 1. Prevo B, Scholey JM, Peterman EJG. Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery. FEBS J. 2017 Sep;284(18):. doi: 10.1111/febs.14068 2. DeSantis ME, Cianfrocco MA, Htet ZM, et al. Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein. Cell. 2017 Sep 7;170(6):.e12. doi: 10.1016/.2017.08.037 3. Fassad MR, Shoemark A, Legendre M et al. Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus. Am J Hum Genet. 2018 Dec 6;103(6):984-994. doi: 10.1016/.2018.10.016The Cytoskeleton and Cell Mobility CASE STUDY: Intermediate Filaments and Skin Blisters The intermediate filament (IF) network is a dynamic structure that forms in many cell types. Intermediate filaments are composed of a large number of diverse subunits that can form either homooligomers or hetero-oligomers. Their assembly mechanism involves the initial parallel dimerization of two IF subunits. This dimeric unit then associates in an anti-parallel manner to form a tetramer, which is the basic building block of filament assembly. Keratin proteins are major components of intermediate filaments, and many disorders have been associated with mutations in these proteins. Epidermolysis bullosa (EB) is a group of disorders that are characterized by tearing and blistering of the skin. Patients with EB are also susceptible to secondary infections introduced through damaged skin. Furthermore, EB can affect more than just skin cells. Blood cells and the esophageal mucosa are especially vulnerable in severe cases. Because IF-rich epithelial cells serve as protective barriers, defects in keratin proteins contribute to compromised cellular function and have broad impacts on tissue and organ structure. These defects may be in synthesis, assembly or molecular interactions of keratin-containing intermediate filaments. Questions: 1. Based on the structure of individual keratin subunits, why might a single point mutation in the alphahelical region of the monomer result in a defect in the polymer? 2. One explanation for the molecular basis of epidermolysis bullosa is that keratin mutations inhibit the ability of motor proteins to transport material on intermediate filaments. Is this a plausible model? 3. Though a cure remains elusive, what treatments can you devise that would treat a disease like epidermolysis bullosa at the cellular level?Where can I learn more? 1. Pan X, Hobbs RP, Coulombe PA. The expanding significance of keratin intermediate filaments in normal and diseased epithelia. Curr Opin Cell Biol. 2013 Feb;25(1):47-56. doi: 10.1016/.2012.10.018 2. Epidermolysis bullosa simplex (Web). Genetics and Rare Diseases Information Center, National Institutes of Health. Updated 7/1/19. CASE STUDY: Actin Dynamics and Cell Motility The actin cytoskeleton is employed in the organization of both cell shape and cell movement. Key to both processes is the ability of actin to polymerize at distinct places in the cell and produce force. Cell motility is crucial to the functioning of many cell types, but this movement is not always beneficial. For example, tumor cell migration is a major step in the metastasis, or spread, of cancer. In addition, certain pathogenic bacteria co-opt the cytoskeleton of their host during the infection process and utilize it to propel themselves through cells and into the adjoining cells, thereby propagating the infection. Thus, understanding the basic molecular mechanisms of actin cytoskeleton function has implications in multiple physiological processes. In many experimental studies of cytoskeletal dynamics, the protein components of the cytoskeleton are tagged with a fluorescent marker in order to track their movement. These fluorescently labeled subunits can be illuminated with a laser to lose their light-producing properties (bleaching), or can be injected into cells and illuminated to make them become fluorescent (photoactivation). The latter process is illustrated in the figure below in which photoactivatable monomeric actin (PA-actin) is injected into cell and allowed to equilibrate into the actin cytoskeleton. Laser light is illuminated in a small box near the leading edge of the cell so that only a small number of the actin subunits are labeled. The diagrams illustrate the results of this experiment, in which the researcher follows the edge of the cell as well as the fluorescent bar (green) containing photoactivated actin.Questions: 1. Where did new actin polymerization occur in this cell as it migrated toward the right? 2. Why did you make this conclusion? 3. A similar type of approach can be used to quantify the rate and dynamics of motility of pathogenic bacteria such as Listeria through cells. In this experiment, if a photoactivated mark is placed on the actin filament behind the bacterium, over time the bacterium will move forward, increasing the distance between the bacterium and the photoactivated mark. What do you propose drives the movement of the bacterium and why did you come to this conclusion? Where can I learn more? 1. Theriot, J.A. and T.J. Mitchison, Actin microfilament dynamics in locomoting cells. Nature,1991. 352: p. 126-131. 2. Theriot, J.A., T.J. Mitchison, L.G. Tilney and D.A. Portnoy, The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature, 1992. 357: p. 257-260. 3. Carlier, M.F. and D. Pantaloni, Control of actin assembly dynamics in cell motility. The Journal of biological chemistry, 2007. 282: p. .The Nature of the Gene and the Genome CASE STUDY: Flying Models for Human Genetic Disease The trinucleotide repeat disorders are a series of genetic diseases that are characterized by an expansion of trinucleotides within the coding sequence of the gene. One favored model is that the expanded trinucleotides encode for a long stretch of a particular amino acid, which results in protein aggregation leading to neurological dysfunction. (See the Chapter 2 case study on Huntington’s Disease for an example.) Spinocerebellar Ataxia type 1 (SCA1) is a neurological disease that is characterized by a progressive loss of cerebellar neurons. SCA1 is caused by an expanded CAG repeat in the Ataxin-1 (ATXN1) gene. Like Huntington’s Disease, SCA1 is autosomal dominant with an average age of onset in the late 30s. One difficulty in studying any human disease is to develop good model systems to probe the molecular pathways of the defect and to begin to identify potential drug targets for treatment of the disease. The fruit fly Drosophila melanogaster has proven to be an excellent model system for the study of neuronal function. The nervous system of the fruit fly is relatively simple compared to humans, and yet many biomedically important proteins such as Ataxin-1 are found in both species. The continued presence of this protein across evolutionary time suggests that Ataxin-1 has a universal role in neuronal function. An additional advantage of the fruit fly as a model organism is that it is possible to carry out genetic screens, in which researchers can look for enhancers or suppressors of gene function to identify other molecular components that function in a given pathway. To generate a model of SCA1, scientists directed the expression of an expanded CAG repeat of the Ataxin-1 gene in Drosophila (atx-1) and then examined the consequences of overexpression or downregulation of a number of other genes. They also conducted a study in which huntingtin (htt) rather than ataxin-1 contained expanded CAG repeats. Below are a series of questions that deal with these studies. Questions: 1. The researchers found that overexpression or repression of some modifier genes in flies resulted in a reduction in neuronal defects that were caused by overexpression of the CAG repeat of either Atx-1 or huntingtin. What do these results suggest about the molecular mechanisms through which the expanded CAG repeats of Atx-1 might function?2. The researchers also found that overexpression or inhibition of some modifier genes in flies caused different effects on the symptoms caused by overexpression of the CAG repeat of htt or ataxin-1 in flies. This meant that overexpression of some modifier genes enhanced the effects of the CAG repeat of htt while those same modifier genes suppressed the effects of the CAG repeat of ataxin 1. What do these results suggest about the molecular mechanisms by which expanded CAG on htt and ataxin-1 act? 3. When combined together, what do the apparent contradictory results stated in questions 1 and 2 suggest toward our understanding of potential targets for therapy? Where can I learn more? 1. Branco J, Al-Ramahi I, Ukani L, Pérez AM, Fernandez-Funez P, Rincón-Limas D, Botas J. Comparative analysis of genetic modifiers in Drosophila points to common and distinct mechanisms of pathogenesis among polyglutamine diseases. Hum Mol Genet. 2008 Feb 1;17(3):376-90. Epub 2007 Nov 5. DOI: 10.1093/hmg/ddm315 2. Kuiper EF, de Mattos EP, Jardim LB, Kampinga HH, Bergink S. Chaperones in Polyglutamine Aggregation: Beyond the Q-Stretch. Front Neurosci. 2017;11:145. Published 2017 Mar 23. doi:10.3389/fnins.2017.00145The Nature of the Gene and the Genome CASE STUDY: Chromosomal Translocations and Leukemia The organization of the genetic material within chromosomes is essential to proper gene regulation. Disruption of this organization can result in improper expression of genes and thus improper levels of proteins in cells. Rearrangements of the genetic material can result in loss of information and thus the formation of mutant proteins. Either of these deleterious conditions can lead to a disease state. Chronic myelogenous leukemia (CML) is a type of blood cancer in which the white blood cells known as granulocytes are produced in excess. CML is associated with chromosomal translocations between the long arms of chromosomes 9 and 22, resulting in a der (a derivative chromosome, number 9) and an abnormal 22 called the Philadelphia chromosome (see figure below). The translocation creates a gene fusion between the bcr gene (normally found on chromosome 22) with the C-abl gene (normally found on chromosome 9). This new gene fusion called bcr-abl is found on the Philadelphia chromosome, which results from the translocation. The function of the normal bcr gene is not clear, but the C-abl gene is a tyrosine kinase (an enzyme that puts phosphates from ATP onto tyrosine residues of other proteins) that is normally involved in cell differentiation and cell division. The gene translocation, which occurs in a bone marrow stem cell, creates the bcr-abl fusion in the blood cell lineage. The translocation results in constitutive expression of the transgene, which promotes cell division. Questions: 1. While only bone marrow transplants are considered curative treatments for CML, drugs such as imatinib (sold as Gleevac) normalize blood cell counts and induce full remission in a large number of CML patients. Gleevac competes with ATP binding in the active site of the bcr-abl transgene. Why would this drug be effective in the treatment of CML? 2. While Gleevac and related compounds are highly effective in most CML cases, researchers are stilllooking for improved treatments and better patient outcomes. What questions might they be asking in their research studies? 3. Why would FISH (fluorescence in situ hybridization) be an especially powerful technique to identify this disease? Where can I learn more? 1. Druker, B.J., Translation of the Philadelphia chromosome into therapy for CML. Blood, 2008. 112: p. . 2. Muller, B.A., Imatinib and its successors--how modern chemistry has changed drug development. Current pharmaceutical design, 2009. 15: p. 120-133. 3. American Cancer Society, About Chronic Myeloid Leukemia. [Internet] 2018. Available from DNA to RNA to Protein CASE STUDY: Transcription-linked DNA Repair The major control point in cellular protein levels comes from the regulation of mRNA. Protein translation from mRNA is comparatively less regulated, so the amount of protein in the cell correlates with the amount of message present. mRNA levels are achieved by a balance of synthesis and degradation. As a result, the machinery of transcription plays a critical role in a cell’s molecular identity. TFIIH is a transcription factor that is part of the pre-initiation complex for RNA Polymerase II in eukaryotic cells. It is a multi-subunit enzyme complex with multiple enzymatic activities. One subunit of TFIIH is a helicase that helps unwind the DNA at the transcription start site. Other TFIIH subunits have protein kinase activity that phosphorylate the carboxy- terminal domain of RNA Pol II and contribute to the enhanced processivity of the polymerase (its ability to read long stretches of DNA before dissociating from the template). In addition to its functions in transcription initiation, TFIIH is also involved in transcription-linked DNA repair. The RNA polymerase stalls when it encounters damaged DNA. TFIIH can then re-associate with the DNA template and stimulate nucleotide excision repair. This provides a mechanism by which the cell can constantly monitor the fidelity of its genome and prevent the production of aberrant proteins. Questions: 1. Among the ten subunits of the TFIIH complex are the XPB and XPD proteins, which are involved in the DNA excision repair mechanism. People with mutati