7.06_2004_PS6 - 7.06 Spring 2004 PS 6 1 of 13 Problem Set...

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7.06 Spring 2004 PS 6 1 of 13 Problem Set 6. Question 1. You are working in a lab that studies hormones and hormone receptors. You are tasked with the job of characterizing a potentially new hormone that is released in response to caffeine and binds its receptor to promote cell proliferation. This amazing hormone stimulates cells to proliferate (in culture) when caffeine is added to the growth media. You have cloned the gene for this hormone, which you call cgh for caffeine growth hormone, and you have antibodies against the protein (CGH). The first thing you’d like to discover is how cgh is mediating its effect. To this end you have generated mutants that do not respond to caffeine stimulation. You now want to figure out where the defects occur in these mutations. You begin by analyzing the first mutant, MutA. MutA is curious because the hormone is produced but seems to be nonfunctional. A western blot for CGH shows that the cells produce CGH, and an Elisa for CGH shows that the cells secrete CGH in response to caffeine stimulation. However, the cells fail to proliferate. Determined to find the defect in MutA, you decide to explore the secretory pathway for defects. You decide to test for glycosylation modification in CGH because you know that the N-linked Man 8 (GlcNAc) 2 of CGH gets modified in the ER by a CGH specific glucotransferase that was recently discovered in your lab . The CGH glucotransferase adds an acetylglucosamine to the Man 8 (GlcNAc) 2 in CGH. From having taken 7.06, you know that the enzyme endoglycosidase D will cleave unmodified Man 8 (GlcNAc) 2 , but once modified the N-linked oligosaccharide becomes resistant to cleavage by endoglycosidase D. The cleaved and uncleaved product can be differentiated by running an SDS PAGE gel of your protein. You decide to isolate CGH form both wild type and MutA cells and test for the acetylglucosamine modification by using endoglycosidase D. The results of this experiment show that CGH from wild type cells are resistant to endoglycosidase D cleavage BUT CGH from MutA cells get cleaved by endoglycosidase D. A) What does this experiment suggest about MutA? Based on the above experiment you now believe there is something wrong with the ER glycosylation modification pathway of CGH. Your first thought is to test for a defective CGH glucotransferase. Luckily since this enzyme was discovered and cloned in your lab, you have antibodies against it. You therefore decide to do an immuno-localization experiment using gold secondary antibodies and electron microscopy (your lab is very rich and happens to have an electron microscope). You prepare cell sections for electron microscopy from both wild type and MutA cells and stain them with antibodies against CGH glucotransferase. What you find is that in wild type cells CGH glucotransferase is
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7.06 Spring 2004 PS 6 2 of 13 found in the golgi, in vesicles, and in the ER. In MutA, however, you only see CGH
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7.06_2004_PS6 - 7.06 Spring 2004 PS 6 1 of 13 Problem Set...

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