Final D145 exam winter 2008 3/20/2008 page 1
Your name
This exam is worth 35 points. Take your time, read the questions carefully, and answer them concisely. Some have more than one possible correct answer. Be sure to explain your logic in arriving at the answer you give since explaining your thinking helps me to decide whether a wrong answer still might deserve partial credit. Good luck! Please write legibly only in the space provided. Using a pencil would be a good idea. 1. (10 points) Remember that you are studying genes required for the metastasis of breast cancer cells and identified BCSS1, a gene that is necessary and sufficient for breast cancer stem cells to develop and become metastatic. You are working hard, and thinking just as hard, so your project is moving ahead very rapidly. At the end of your 3rd year, you identified a promoter element that is required for the expression of BCSS1 in breast cancer stem cells and have identified the protein that binds to this BCSS1 enhancer in breast cancer stem cells. Intriguingly, although BCSS1 is only expressed in mammary stem cells, and in breast cancer stem cells, the BCSS1 enhancer binding protein (let's call it mammregulin) is also expressed in liver and brain. This suggests that the proteins that mammregulin interacts with may be the decisive factors in determining whether BCSS1 is expressed in breast cancer stem cells and mammary stem cells. a) (3 points). How will you determine how many proteins bind to the BCSS1 enhancer element in breast cancer stem cells vs. liver cells?
b) (1 point) You decide to use mass spectrometry to identify proteins that bind to mammregulin. What key reagent(s) will you need to accomplish your goal?
c) (3 points) How will you determine whether any proteins you identify in a) interact directly with mammregulin? How will you determine how many interact indirectly with mammregulin?
d) (3 points) How will you determine how many transcripts the mammregulin gene produces and what size they are? How can you determine whether mammregulin mRNA is expressed at higher levels in the liver, brain, mammary stem cells or breast cancer stem cells?
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D145 Final exam winter 2008 Your name 3/20/2008 page 2 2. (10 points) Your next project is to understand how BCSS1 leads to metastasis. Recall that overexpression of BCSS1 is necessary for the formation of breast cancer stem cells that cause carcinoma in situ. BCSS1 overexpression is not sufficient to cause these cells to metastasize. a) (2 points) So far, we don't know much about what sort of protein BCSS1 is. What might be a quick way to learn something about possible functions for this gene?
b) (3 points) It is quite likely that BCSS1 has an endogenous function in mammary stem cells that is disturbed in carcinogenesis. How would you create a mouse model for BCSS1-induced carcinoma in situ? Be sure to state your assumptions and point out any limitations that your approach might have.
c) (3 points) How would you use this mouse model to determine what genes are up-regulated as a result of BCSS1 overexpresssion? What would your controls be?
d) (2 points) How many animals would you need to test in order to be confident whatever that genes you identify are really up-regulated by BCSS1? Explain your reasoning.
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D145 Final exam winter 2008 Your name 3/20/2008 page 3 3. (5 points) Remember the putative mutations in BCSS1 that are necessary for it to cause breast cancer stem cells to give rise to metastatic cancers in the liver? Your initial efforts in this regard to sequence the cDNA did not prove fruitful since you never did find any mutations that would alter the amino acid sequence of the protein. You decided to move on to experiments that are more productive. However, you have a very smart undergraduate working with you to whom you assigned this project. Remarkably, she found that sequences in the region of the gene corresponding to the 3' untranslated region of the mRNA are strongly associated with the metastatic phenotype. How would you determine whether it was a property of the BCSS1 transcript itself that is causing the metastatic phenotype or some other factor such as expression of a microRNA, antisense RNA, presence or absence of a transcription factor binding site in the gene, etc.? Describe the experiments that you would perform to make this determination.
4. (5 points) In order to gain insight into how BCSS1 amplification leads to carcinomas in situ resulting you wish to determine what genes are necessary for the action of BCSS1 to promote the development of cancer cells. What sort of approach could you use to identify genes expressed in breast cancer stem cells that are required for BCSS1 action? Assume that you can obtain sufficient quantities of breast cancer stem cells from your mouse model created in 2 b) for your chosen approach.
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D145 Final exam winter 2008 3/20/2008 page 4
Your name
5. (5 points) One of the proteins you identified in 4 is mammregulin. You hypothesize that there is a positive feedback (feed-forward) loop wherein BCSS1 ampl...