7.06_2004_exam2key - Name: _ Question 1 (20 points) The...

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Name: _______________________ 7.06 Spring 2004 exam 2 KEY 1 of 9 Question 1 (20 points) The wingless signaling pathway plays an essential role in pattern formation in most if not all metazoans. You therefore decided to collaborate with a micro-array facility to identify the transcriptional targets of this pathway. You send them several cell lines carrying loss of function mutations in known wingless signaling molecules. You ask them to analyze the expression profile using micro-array technology to obtain a clue as to the identity of the mutants. Your collaborator sends you the following primary data (note a filled circle means that the transcript is present, open circle means the transcript is absent): 1 (4 points) Describe how transcripts A, B, C and D are regulated by the wingless signaling pathway. Transcript A: Is not regulated by the wingless pathway Transcripts B and C: are induced by wingless Transcript D: is repressed by the wingless pathway 2 (5 points) Which important component(s) of the wingless pathway could be mutated in mutant 1? Name at least 3 and explain your reasoning. Mutant 1 fails to induce the wingless transcriptional program. It must therefore be defective in a positive regulator of the wingless pathway. It could be defective in frizzled, arrow, disheveled, TCF or β -catenin. 3 (3 points) Which important component of the wingless pathway could be mutated in mutant 2? Name at least 1 and explain your reasoning.
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Name: _______________________ 7.06 Spring 2004 exam 2 KEY 2 of 9 In mutant 2 the wingless pathway is constitutively active. Therefore, it is defective in a negative regulator of the wingless pathway. It could be defective in GSK3 or (optional) the ubiquitin mediated degradation machinery responsible for degrading β -catenin. While you were analyzing your wingless mutants, your collaborator obtained a tumor cell line in which the wingless signaling pathway is constitutively active. 4 (3 points) How could you test biochemically that it is GSK3 that is constitutively active? Outline the experimental detail. I could examine GSK3 kinase activity in the presence or absence of ligand. If GSK3 were constitutively active, GSK3 kinase activity ought to be detected even in the absence of ligand. Experimental design: First, I would immunopreciptiate GSK3 using an antibody bound to beads. I would then incubate the immunoprecipitate with radioactively labeld rATP and substrate ( β -catenin). I would then boil the beads and run the supernatant on a gel and visualize phosphorylated β -catenin by autoradiography. Question 2 (30 points) For your UROP you decide to join a laboratory that studies vulva development in the nematode C. elegans. Your PI suggests that you could start by identifying genes important for vulva development. 1
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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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7.06_2004_exam2key - Name: _ Question 1 (20 points) The...

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