PLEASE MAKE SURE THAT ALL PAGES ARE NUMBERED and IDENTIFIED!!!!!.

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1 Instructions: The following pages contain twelve questions. You must answer ten out of the twelve questions. Any one question may ask for more than one response so please read each question carefully for specific instructions pertaining to that question. Make sure the graders can read your answers clearly. Please remember as you answer the questions that you have approximately one hour per question. This means that we expect in depth answers unless otherwise noted. Use diagrams to illustrate your answer wherever possible. Although each question is worth the same number of points, not all questions will require the same amount of time. Allocate your time wisely. We recommend that you first make an outline of your answer, rather than just making up your answer as you write. Some questions ask you to propose experiments. Choose the most direct and realistic approaches and explain your experimental rationale as clearly as possible. Be sure to include alternate hypotheses, controls, expected outcomes and possible problems and solutions. Do NOT put your name on any of the question or answer sheets. To keep the exam anonymous, label your pages with the question number (e.g., Question 1) and with a coded name, using the code distributed by Susan. PLEASE MAKE SURE THAT ALL PAGES ARE NUMBERED and IDENTIFIED!!!!!. This is a closed book exam. Absolutely no discussion will be allowed between the students while the exam is in progress. You will be held to an honor code by agreeing to not receive or give aid on this exam. You will have from 8:00 a.m. until 8:00 p.m. to finish the exam. Lunch will be available from 12:00-1:00. No exam materials can leave the room with you during lunchtime or any other time and you may not refer to outside materials during lunch break. The exams will be collected no later than 8:00 p.m. sharp. If you have questions please contact Victor Faundez (Whitehead 446)

2 Page 1 of 13 QUESTION 1 You are interested in studying the assembly of a new type of cytoskeletal polymer present in the unicellular organism Plasmodium emoryi. This new cytoskeletal structure is distinct from actin, microtubules, and intermediate filament-dependent cytoskeletons. You have developed a simple assay that allows you to observe the conversion of the monomer subunits into polymers in vitro. You then discover that a cell extract derived from Plasmodium emoryi contains a unique activity that polymerizes this novel cytoskeletal monomer, one that does NOT require GTP or GDP. A) How would you isolate and identify the protein(s) responsible for the activity? Plasmodium emoryi is a newly discovered species, so its genome has not been sequenced. (3 points) B) Once you ve identified the protein that increases polymer formation, how would you clone the gene that encodes it? Plasmodium emoryi is not amenable to genetic manipulation. (3 points) C) Once you have identified the protein, how would you provide conclusive evidence that it is involved in polymerization in vivo? Remember that Plasmodium emoryi cannot be genetically modified or transfected. Also recall that you have an antibody that recognizes the monomer and polymer forms of this new cytoskeletal component. (2 points) D) What experiment could you perform to demonstrate that the protein you have purified acts as a catalyst to mediate polymerization of the cytoskeletal polymer as opposed to serving as a structural subunit that stabilizes the polymer? (2 points)

3 Page 2 of 13 QUESTION 2 You positionally clone a mammalian gene and make an antibody to a peptide based on its encoded protein, which is designated RIP (Really Interesting Protein). This peptide antibody works well for immunofluorescence and on Western blots. Western blotting reveals that RIP runs as a closely spaced doublet with apparent molecular weights of 65 and 67 kda. You have an interest in determining first why there is a closely spaced doublet recognized by your affinity purified antibody and more importantly how this doublet relates to the in vivo status of RIP. Consequently, you homogenize 10 g of cells and run a concentrated post ribosomal 100,000 xg supernatant on a G-150 gel filtration column. You monitor the fractions coming off this column and you find that about 75% of RIP elutes with the void volume and 25% elutes as a single peak with an apparent molecular weight of 65 kda, both based on size standards on the column and Western blotting of the fractions. The portion of RIP that elutes in the void volume all has an apparent molecular weight of 67 kda as determined by Western blot. A) Why would a portion of RIP elute with the void volume? (1 point) B) How would you determine an accurate molecular weight and composition for the void volume fraction that contains RIP? (3 points) C) Describe two different hypotheses that could explain why there are two RIP bands detected by Western blotting and describe a plausible strategy to test each of your hypotheses. (6 points)

4 Page 3 of 13 QUESTION 3 You are studying the molecular mechanism of regulation of a protein called QualXM. QualXM is involved in triggering entry into mitosis (M phase). Based on your initial data you believe that QualXM is sequestered in the cytoplasm during the G 1, S and G 2 phases and enters the nucleus at the end of G 2 to trigger the G 2 /M transition. You want to determine how the entry of QualXM is regulated. Assume you have a nice immunoprecipitating and immunoblotting antibody to QualXM, cells in S phase expressing QualXM, cells in M phase expressing QualXM, and most any general reagents you might need. Since you have only a limited amount of time, do not go into any detail about methods like cell synchronization, immunoprecipitation, subcellular fractionation or immunofluorescence, etc. Please describe BRIEFLY but clearly how you would have carried out the experiments detailed in A, B, and C to obtain the following results: A) analyze QualXM during S phase and M phase and find out that it is phosphorylated on both serine and threonine in S phase but is only phosphorylated on serine in M phase. (3 points) Given this result, you hypothesize that phosphorylation of QualXM on one or more threonine residues is essential for excluding it from the nucleus. To test this hypothesis you: B) identify the site of threonine phosphorylation on QualXM (assume there is just one site), (2 points), mutate it, (1 point), and C) carry out an experiment that incorporates these mutants to confirm your hypothesis. (4 points) The most critical aspect of this question is that you choose a reasonable approach and method of analysis and that you include proper controls for the methods you use to analyze phosphorylation or its role in regulating subcellular localization.

5 Page 4 of 13 QUESTION 4 Epithelial cells undergo a transition to a more fibroblast like phenotype during certain developmental sequences and during tumor progression. These alterations are referred to as an epithelial to mesenchymal transition (EMT). You are interested in this phenomenon and begin an investigation using a pancreatic epithelial tumor cell line. You hypothesize that the behavior of cells during an EMT is dictated by cell-cell and cell-matrix adhesion. Therefore, you coat plates with either fibronectin or collagen, and you analyze cell morphology and the expression or phosphorylation of proteins shown in the western blot. You obtain the results shown in the figures A and B below. In addition, you find that E-cadherin is markedly down-regulated when cells are plated on collagen type I (not shown). A) Describe the results that you have obtained and indicate any results that are hallmark of an EMT. (2 points) B) What class of cell surface receptors is most likely responsible for mediating the signaling from collagen or from fibronectin? To answer this question, provide a basic description of both of these receptors and the way in which specificity for an extracellular matrix ligand is achieved. (2 points) C) Design two experiments to test whether the increase in N-cadherin levels in cells plated on collagen is relevant to the EMT. Use both gain of function and loss of function approaches, and include controls in each experimental approach. (6 points)

6 Page 5 of 13 QUESTION 5 In animal cells, two prominent microtubule organizing centers (MTOCs) are the centrosome and the basal body. A) Define the organization of the centrosome, illustrating as necessary. (2 points) B) In dividing cells the centrosome duplicates one time. Describe an experiment to determine the stage in the cell cycle (e.g. G 1, S, M, etc.) when the centrosome duplicates. (2 points) C) During duplication of the centrosome, the centrioles duplicate in a semi-conservative manner. What is meant by semi-conservative centriole replication during centrosome duplication? Illustrate as required. (2 points) D) Describe an experiment that tests whether the centrosome nucleates microtubule assembly. (2 points) E) Describe: [a] a fundamental similarity and [b] a fundamental difference between the centrosome and basal body with respect to their roles in nucleating microtubule assembly. (2 points)

7 Page 6 of 13 QUESTION 6 You have isolated two different monoclonal antibodies, MAB-1 and MAB-2. These antibodies react with the same protein (Protein X) which has been implicated in actin assembly. When these antibodies were microinjected into fibroblasts, MAB-1 had no effect on the morphology of the actin cytoskeleton (Figure, middle panel), but MAB-2 strongly inhibited formation of stress fibers (thick bundles of actin filaments). (Figure, right panel) Figure. Fibroblasts were microinjected with control antibody (left), MAB-1 (middle), or MAB-2 (right), fixed and stained with rhodamine-phalloidin to visualize actin filaments. Control antibody and MAB-1 had no effect on stress fibers (thick bundles of actin filaments). However, MAB-2 inhibited formation of stress fibers. A) Stress fibers produce contractile forces in a similar mechanism to muscle contraction. Myosin II (conventional myosin) is also a major component of stress fibers. Describe or illustrate how actin and myosin are organized in stress fibers. The polarity of actin filaments must be clearly described. (3 points) B) MAB-1 and MAB-2 react with the same protein but have different effects on stress fiber formation. Describe one possible explanation for this difference. (1 point) C) You have successfully cloned the gene encoding Protein X. Design experiments to confirm the results of antibody injection and test whether Protein X has a role in stress fiber formation. (6 points)

8 Page 7 of 13 QUESTION 7 The 4-generation pedigree illustrated below represents a family among whose members 5 individuals are affected by a specific trait (GLO) they glow in the dark. Affected individuals are indicated by filled symbols; unaffected individuals are indicated by open symbols. I 1 2 I I II I I V A) Your goal is to determine whether GLO is a predominantly genetic trait. Assume the pedigree above represents a family of Drosophila melanogaster. How could you answer this question? Describe the essential elements of your experiment, what sort of data you would need to collect, and how you would interpret these data. (5 points) B) Now assume that the pedigree above represents a human family. How could you determine if GLO is a predominantly genetic trait now? Describe the essential elements of your experiment, what sort of data you would need to collect, and how you would interpret these data. (5 points)

9 Page 8 of 13 QUESTION 8 You are interested in a macrophage transmembrane glycoprotein receptor, Zaphod, that binds to a hormone, Bbx. Zaphod is localized to the plasma membrane as well as to punctuate spots within the cells. When you expose cells to Bbx, the amount of Zaphod at the plasma membrane increases. A) Describe two distinct methods to quantify changes in the level of Zaphod at the plasma membrane in response to the hormone treatments. (2.5 points) B) You hypothesize that the punctuate intracellular spots are indicative of localization of Zaphod to specific organelles. How would you identify the intracellular organelles containing Zaphod? Give two specific examples of organelles to be examined and how they would be identified. (2.5 points) C) You want to determine whether the effect of Bbx is an effect on Zaphod mrna levels. How would you test this possibility? (2.5 points) D) After performing the experiments outlined in part "C", you rule out this possibility and further demonstrate that there is no change in the level of Zaphod protein in response to treatment with Bbx. Propose two different models to explain the effect of Bbx on Zaphod that do not require a change in the steady-state level of the Zaphod protein. What experiments would allow you to distinguish between these different models? (2.5 points)

10 Page 9 of 13 QUESTION 9 Cranio-lenticulo-sutural dysplasia (CLSD) is a non-lethal autosomal recessive syndrome characterized by late-closing fontanels, cataracts, facial dysmorphisms and skeletal defects (A). Electron micrographs from patient fibroblasts reveal an extensive enlargement of endoplasmic reticulum cisternea (B). The genetic defect corresponds to an amino acid substitution (F382L) in sec23a, a subunit of the COPII complex. Sequence alignment reveals that this residue is conserved from yeast to humans (C). The COPII cytosolic complex is critical in the formation of vesicles that deliver secretory proteins from the endoplasmic reticulum to the Golgi complex. Saccharomyces cerevisiae mutants lacking SEC23 ( sec23) are not viable. Similarly, yeast Sec23 protein carrying the F382L amino acid substitution fails to rescue the lethal phenotype of the sec23 deletion. However, the F382Y mutation confers a temperature sensitive phenotype when expressed as the only copy of Sec23 in yeast cells. Based on this information: A) Propose two hypotheses that could explain why patients with the F382L amino acid substitution in sec23a are viable whereas the corresponding amino acid substitution in Sec23 is lethal in yeast. (2 points) B) Suggest an explanation for why the F382L amino acid change yields a non-functional protein in yeast whereas the F382Y amino acid change confers some function. (1 point) C) Choose one of your hypotheses (from A) and propose an experimental strategy (with controls) to test the hypothesis. Feel free to use yeast and/or human cells in your strategy if needed. (4 points) D) How would you use the F382Y temperature sensitive yeast mutant to better understand the genetic pathway where SEC23 participates? (3 points)

11 Page 10 of 13 QUESTION 10 Bacillus anthracis secretes a toxin that is the major virulence and disease-causing agent in mammals. The toxin binds to cells, is internalized, and finally it translocates into the cytoplasm where the toxin exerts its toxic activity. Chemical mutagenesis of CHO cells followed by selection of cells in the presence of toxin leads to the isolation of a cell clone that is resistant to toxin (CHO-B(-)). This phenotype is evident even when CHO-B(-) cells are challenged with higher toxin concentrations. A binding assay that examines specific binding of fluorescently labeled recombinant anthrax toxin to the resistant clone is indistinguishable of background binding (red line, (CHO-B(-)) as determined by flow cytometry. In contrast, cells sensitive to the toxin bind substantial levels of the toxin (CHO-B(+), gray shaded area) Figure 1. Flow cytometry determination of Anthrax toxin binding in CHO cells. X axis depict fluorescence intensity of bound anthrax toxin. Y axis depicts cell count A) Other bacterial toxins use cellular receptors to gain entrance to cells. You hypothesize that the anthrax toxin uses a receptor-dependent mechanism to enter cells. How would you identify such a receptor? Describe in detail one experimental strategy to isolate the receptor. (2 points) B) What type of mutations in the receptor could account for the toxin resistance in CHO-B(-) cells? Describe two possible mutations and the underlying mechanism that eliminates toxin binding. (3 points) C) In step A you identified/isolated a molecule that you suspect is the receptor for the toxin. How would you prove that this molecule is the receptor? Describe two experiments and the corresponding controls to test the hypothesis that the molecule that you isolated/identified is the receptor. (5 points)

12 Page 11 of 13 QUESTION 11 You are studying a receptor tyrosine kinase and want to investigate the nature of the autophosphorylation of this protein. To do this, you construct three forms of the receptor in an expression vector: 1) a normal form with an active kinase domain and one tyrosine that is phosphorylated upon activation, 2) a kinase-dead mutant that is larger than wildtype and carries an inactivating mutation in the kinase domain, and 3) a truncated form that has an active kinase domain but is missing the tyrosine residue (see Figure below). You express these forms in a cell line that lacks expression of this receptor (that is, there is no endogenous receptor at the plasma membrane in these cells). To test whether cis or trans phosphorylation occurs, you express the forms singly and in pairs (see lanes on gel below). You then treat these cells with ligand and radioactive ATP ( 32 P on the γ-p) and then immunoprecipitate the receptor proteins from a cell extract. You analyze expression of the receptors by immunoblotting with an antibody that recognizes all three forms of the receptor and you detect phosphorylation by autoradiography. Your expression results are shown in the first blot below. A) Draw the expected autoradiography results if the receptor autophosphorylates in trans. (3 points) B) Draw the expected autoradiography results if the receptor autophosphorylates in cis. (3 points) C) Would your results for A and B change if you inadvertently purchased ATP that was labeled with 32 P on the α-p? Why or why not? (2 points) D) Briefly describe one mechanism for how phosphorylation of a tyrosine residue within a receptor tyrosine kinase can lead to downstream signaling events. (2 points)

13 Page 12 of 13 QUESTION 12 Although the tumor suppressor gene (TSG) protein that you are studying is present in normal cells and readily detectable be Western blotting, TSG protein is not detectable by Western blotting in any of the six breast cancer cell lines that you test. The mrna transcript for TSG also can not be detected in the cancer cell lines. A) Design an experiment to determine if decreased TSG expression in the tumor cell lines is due to transcriptional or post-transcriptional regulation. (3 points) B) From the experiment in A, you find that TSG transcription is decreased in the tumor cells. Factor Y is the only known trans-activator for TSG, and you show by western that there is plenty of Factor Y present in the tumor cells. You suspect that Factor Y is unable to bind to the sole, wellcharacterized TSG promoter in the tumor cells. Design an in vivo experiment to demonstrate that this is, indeed, the case. (3 points) C) You show in experiment B that Factor Y can NOT bind the TSG promoter in the tumor cells. You sequence the Factor Y gene in the tumor lines and find no mutation in any of them. Someone suggests that you test whether Trichostatin A, an HDAC inhibitor, affects TSG expression in these cell lines, and you observe a significant increase in TSG expression in the presence of the drug. Propose why Trichostatin A might alter TSG transcription. (1 point) Design an experiment and describe the results that would verify your hypothesis. (3 points)