7.06_2004_exam1 - 7.06 Spring 2004...

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Unformatted text preview: 7.06 Spring 2004 Name___________________________ 7.06 Cell Biology EXAM #1 February 24, 2004 This is an open book exam, and you are allowed access to books, a calculator, and notes but not computers or any other types of electronic devices. Please write your answers in pen (not pencil) to the questions in the space allotted; if you use the back of a sheet make it clear which answer is where. And be sure to put your name on each page in case they become separated! Remember that we will Xerox many exams at random. Good luck! F=2.3 x 10^4 cal/(V*mol) R=1.987 cal/(K*mol) Question 1 (35 pts). You have recently isolated a yeast that grows in industrial wastewater, and find that its optimal growth medium contains glucose, glycine, as well as 150 mM KBr (potassium bromide) and 15 mM KCl. Curious as to how such an organism grows under such strange conditions, you sequence its genome. You find only one gene sequence (i.e. one encoded protein) homologous to the mammalian Ca 2+ and K+/ Na+ pump. Curious as to what ions this pump pumps, you produce large amounts of it, incorporate it into liposomes, and find that the protein is an ATP- powered Cl- and Br- pump. For each ATP molecule hydrolyzed, two Br- ions are pumped outward (from the cytosolic to the exoplasmic face) and two Cl- ions are pumped inward. Furthermore, you examine these yeast cells growing in their preferred medium of 150 mM KBr and 15 mM KCl., and find that their cytosol has a different ionic composition: 15 mM KBr and 150 mM KCl. a, 5 pts) To measure binding of radiolabeled Cl- and Br- to the ATPpowered Cl- and Br- pump, you want to use a detergent to solubilize the 1 of 7 7.06 Spring 2004 Name___________________________ purified protein from your liposomes. Would you use an ionic or nonionic detergent? Explain your answer. b, 5 pts) You find that the potential across the plasma membrane of these yeast cells is 59 millivolts, with the inside of the cell negative to the outside. How would you make this measurement? c, 5 pts) Would you expect that this potential is generated by open Clchannels in the plasma membrane, by open Br- channels in the plasma membrane, or by the ATP- powered Cl- and Br- pump? Explain your answer in a sentence or two. d, 10 pts) Interested in how these cells take up glucose from the culture medium, you find in the plasma membrane a chloride- glucose antiporter – it transports 3 chloride ions in one direction and one glucose in the other. Assuming that this is the sole protein used by the cells to import glucose, calculate the equilibrium concentration of glucose inside the cell if the external concentration is 1 µM (10-6 M). Assume that the temperature is 310 K, the concentrations of K+, Cl-, and Br- in the medium and the cytosol are as stated above, and that the membrane potential is – 59 mV as also stated above. Show all of your work, and in particular show the equations that you use to calculate your answer. e 5 pts) Your yeast cells are haploid and are not known to form diploids (that is, they appear not to have a sexual cycle.) But through exhaustive screening of a mutagenized population of these yeast cells you have isolated a mutant that is defective in its ability to transport glucose efficiently into the cell; this mutant requires 1 mM glucose in the medium for growth whereas the wild- type cell grows in glucose concentrations as low as 1 µM. Suspecting that this strain harbors a mutation in the chloride- glucose antiporter gene, you want to clone the gene. How do you do it, assuming that you can use any plasmids or vectors that already have been used for other yeast strains? Answer in just a few sentences. f, 5 pts) Would you expect to find a chloride- glucose antiporter in any human cell? Why or why not? 2 of 7 7.06 Spring 2004 Name___________________________ Question 2 (15 pts). You find that the purified chloride- glucose antiporter protein is phosphorylated in a test tube following addition of active yeast Protein Kinase A, and you further find that this yeast Protein Kinase A has a regulatory subunit that functions in a manner similar to its homolog in mammalian cells. Furthermore, you find that addition of the yeast hormone tyrosinol to these yeast cells results in activation of adenylyl cyclase and Protein Kinase A and in inhibition of glucose uptake. Thus you hypothesize that phosphorylation of the chloride- glucose antiporter by Protein Kinase A results in inhibition of its function. Determine whether you would expect the following conditions to increase or decrease glucose uptake by the yeast cells, both in the absence of any hormone and in the presence of tyrosinol, and give a brief explanation for your answer. a, 5 pts) A mutation in the Gas subunit that prevents binding of GTP once a bound GDP has dissociated. b, 5 pts) A mutation in the regulatory domains of PK-A that prevents binding to the catalytic subunit. c, 5 pts) A mutation to alanine of the single serine residue in the chlorideglucose antiporter protein that is phosphorylated by Protein Kinase A 3 of 7 7.06 Spring 2004 Name___________________________ Question 3. 25 pts The hormone TGFß1 inhibits the proliferation of many types of body cells, and loss or inactivation of a TGFß receptor or a component of its downstream signaling pathway frequently occurs in human cancers. As an oncologist, you are studying a particularly aggressive type of AML leukemia, and have cloned lines of pure leukemia lymphocyte cells from each of five patients. Each of the cell lines contains a mutation in some component of the TGFß signaling pathway, but you do not know whether the cells themselves are sensitive or resistant to the growth- inhibitory effects of TGFß: i) Patient A has a deletion in one Smad3 allele and a point mutation in the other Smad 3 gene such that the nuclear localization sequence (NLS) is nonfunctional. ii) Patient B has a deletion in one Smad3 allele and a frame shift mutation in the other Smad 3 gene such that the carboxyl- terminal 9 amino acids, including the three serine residues that are phosphorylated by the Type I TGFß receptor, are deleted. iii) Patient C has a deletion in one Smad4 allele and a mutation in the other Smad 4 gene such that the mutant Smad4 protein cannot bind to Smad3. iiii) Patient D has a deletion in one allele encoding the Type I TGFß receptor, and a mutation in the second allele such that the Type I receptor cannot become phosphorylated by the Type II receptor. (v) Patient E has a deletion in one Smad4 allele and a mutation in the other Smad 4 gene such that the mutant Smad4 protein cannot bind to the Ski protein 4 of 7 7.06 Spring 2004 i) Name___________________________ a, 10 pts) Explain, for each of the five patients, whether or not the indicated mutations would cause the leukemic cells to become resistant to growth inhibition by added TGFß, and for each case explain your answer in one or two sentences. b, 5 pts) How could you determine the location – nucleus or cytosol – of Smad3 in living cultured leukemia cells? c, 10 pts) For each of the indicated cell lines, explain whether, in the presence of added TGFß, you would expect the Smad3 protein and the Smad4 protein to be localized to the nucleus or to the cytosol. Use N for nucleus and C for cytosol, and explain each of your answers. Smad3 Smad4 Wild-type (nonleukemic) lymphocytes Patient A lymphocytes Patient B lymphocytes Patient C lymphocytes Patient D lymphocytes Patient E lymphocytes Question 4, 15 pts. You are a brilliant gastroenterologist who has just finished analyzing a collection of mouse mutants, isolated at the Jackson laboratories in Bar Harbor, Maine because they are underweight. You find that two strains have an unusually high stomach pH of 4 rather than the normal pH of 1. The stomach lumen acidification is achieved by secretion of HCl by parietal cells in response to endocrine signals. The H+/K+- ATPase pump, together with Cl- and K+ channels and a Cl-/ HCO3- antiporter, are responsible for HCl secretion into the stomach lumen. a. 5 pts) You suspect that in one of your mouse lines the Cl-/ HCO3antiporter has a mutation that causes it to become localized to the apical plasma membrane rather than its normal site in the basolateral plasma membrane. Describe, step- by- step, a simple experiment you could to determine where in the plasma membrane this antiporter is located. 5 of 7 Name___________________________ 7.06 Spring 2004 b. 5 pts) Why would mis-localization of the Cl-/ HCO3- antiporter to the apical plasma membrane result in an elevation of the pH of the stomach lumen? c. 5 pts) In a second line of mutant mice you determine that there is a mutation in the Claudin1 gene such that the number of tight junctions that normally interconnect the epithelial cells lining the stomach is reduced from 6 to 2 per cell pair. Explain why a mutation in this protein would result in an elevation of the pH of the stomach lumen? Question 5, 10 pts. You are interested in signaling by the newly isolated hormones BMP6, BMP7, and BMP8. Knowing nothing about the nature of the cell surface receptors for these hormones, you conduct a binding experiment with 35S labeled BMP6, and determine that your lung cell line contains 50,000 surface receptors with a dissociation constant of 10-11M. You cannot radiolabel either BMP 7 or BMP8, so you decide to carry out a competition assay. The binding reaction contains 2 x 10-12 M 35S labeled BMP6 and the indicated molar concentrations of unlabeled BMP 7 or BMP8, and you measure the fraction of 35S labeled BMP6 bound to the cell surface, relative to the amount bound in the presence of no added unlabeled hormone: 100 80 B MP8 60 B MP7 40 20 10-12 10-11 10-10 10-9 10-8 10-7 unlabeled hormone (Molar) 6 of 7 7.06 Spring 2004 Name___________________________ a. 5 pts. ) Which hormone – BMP6 or BMP7, binds with a higher affinity to cell surface receptors? Explain your answer. b. 5 pts) Does the data indicate that there are more than one type of cell surface receptor that binds BMP6? Explain your answer. 7 of 7 ...
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This note was uploaded on 01/23/2012 for the course LSM lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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