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7.06_2004_PS8key - 7.06 Spring 2004 PS 8 KEY 1 of 4 Problem...

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7.06 Spring 2004 PS 8 KEY 1 of 4 Problem Set 8 7.06 Cell Biology 1. a) The phosphorylation status of Rb regulates progression into S phase. Describe an experiment to show that the binding of Rb to E2F1 depends on the phosphorylation state of Rb. You could do a co-IP experiment. IP E2F1 with an E2F1 antibody attached to beads. Then do a western using an antibody specific for the non-phosphorylated form of Rb. A band should be observed corresponding to Rb. Repeat the experiment using an antibody to the phosphorylated form of Rb. This time no band should appear. b) The Rb knockout mouse shows ectopic i.e. abnormal S-phase entry during embryogenesis and is not viable. What could explain this and how could you try to prove it? Since there is no Rb protein activity of E2F1 is not regulated and so the cells lack the G1/S checkpoint and keep cycling. One way to confirm this hypothesis would be to create an E2F1 knockout mouse and cross it to the Rb null mouse so that you have double knockouts i.e. Rb-/-, E2F1-/-. This should rescue the ectopic S-phase entry seen in the Rb knockout mouse. c) p53 is another tumor suppressor gene that is often deleted in human cancers. You have three cell lines (A, B, C) that were established from human thymic lymphomas. How would you investigate whether there is functional p53 in these cells? You would take each cell line and grow it under normal conditions and under conditions that induce apoptosis e.g. irradiate the cells. Then you would lyse the cells and do a western blot for a protein whose expression is normally induced by p53 e.g. Bax. If p53 is non-functional in any of these cell lines then you would not see a rise in Bax protein following irradiation. d) Cell lines A and B fail to show a rise in Bax protein levels following irradiation. You carry out a p53 western blot to check whether the defect is up or downstream of p53 induction. Cell line A shows induction of p53 upon irradiation. What could be responsible for the defect in induction of apoptosis in this cell line?
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7.06 Spring 2004 PS 8 KEY 2 of 4 In cell line A p53 expression is increased following irradiation but the protein doesn’t seem to be able to act as a transcription factor for Bax. One possible explanation is that the p53 protein has a mutation in its DNA-binding domain that it uses to bind to the promoters of genes such as Bax.
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