2005_Exam3 - Name: _ 7.06 Cell Biology EXAM #3 April 21,...

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Name: _____________________________ 7.06 Cell Biology EXAM #3 April 21, 2005 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. Please write only on the FRONT SIDE of each sheet. And be sure to put your name on each page in case they become separated! Remember that we will Xerox all of the exams. Good luck! Question 1. 20 pts __________ Question 2. 20 pts __________ Question 3. 25 pts __________ Question 4. 25 pts __________ Question 5. 10 pts __________
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Name: _____________________________ Question 1. (20 points) As mentioned in lecture, there is considerable interest in understanding the mechanism by which type I transmembrane proteins become inserted into the endoplasmic reticulum membrane by means of stop- transfer membrane anchor sequences. A recent paper in Nature probed this by using a “model” type I transmembrane protein, GHR. GHR is made with a cleavable N- terminal ER signal sequence of 22 amino acids. This is followed by a hydrophilic domain of 85 amino acids that contains one consensus site for N- linked glycosylation, Asn – Ala- Ser (NAS). This is followed by a hydrophobic segment – the stop- transfer membrane anchor with the sequence (in one letter code) LIIFGVMAGVIGT IL LISTGI. This is followed by a C-terminal hydrophilic segment that contains one consensus site for N- linked glycosylation, Asn – Gly- Thr (NGT). (a, 4 pts) You generate using recombinant DNA technologies an mRNA that encodes this GHR protein, and translate the protein in a cell- free extract that contains ribosomes and all other proteins and small molecules required for protein synthesis. The reaction also contains microsomes. You observe that GHR protein is produced and translocated as expected. Which consensus sites for N- linked glycosylation will have a sugar chain attached on the GHR protein: the one in the NAS sequence, the one in the NGT sequence, both, or neither? Explain your answer. (b, 4 pts) Will the signal sequence remain at the N- terminus of the GHR protein that was made in the cell-free system from part a) ? Explain your answer.
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Name: _____________________________ (c, 6 pts) You make an mRNA encoding a mutant form of GHR in which the IL (isoleucine – leucine) sequence in the middle of the transmembrane domain is changed to AA (alanine – alanine). Again, you translate this mRNA in the same cell- free extract that also contains microsomes. You now observe two forms of the GHR protein produced – one with one N- linked oligosaccharide, and the other with two. Below is
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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2005_Exam3 - Name: _ 7.06 Cell Biology EXAM #3 April 21,...

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