706_F06pset7_key - Problem Set 7 1 From B-cells you purify a plasma membrane protein which you name Want to Pass 7.06(WTP7.06 You raise antibodies

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1 1. From B-cells, you purify a plasma membrane protein which you name “Want to Pass 7.06” (WTP7.06). You raise antibodies against it and use the antibodies to clone a cDNA encoding WTP7.06 from a B-cell library. A crude stretch of the cDNA is shown below: ATG (start codon) TAG (stop codon) open reading frame WTP7.06 A. Given your knowledge of the protein translocation process, annotate the diagram appropriately if WTP7.06 were a single membrane spanning region protein with the N-terminus on the extracellularr side and C-terminus on the cytoplasmic side. ATG (start codon) TAG (stop codon) signal sequence signal peptidase cleavage site internal stop-transfer anchor sequence B. Annotate the diagram appropriate if WTP7.06 were a single membrane spanning protein with its C-terminus on the extracellular side and N-terminus on the cytoplasmic side. ATG (start codon) TAG (stop codon) positive amino acids internal signal-anchor sequence Problem Set 7
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2 D. Based on the actual sequence of the WTP7.06 cDNA, you conclude that the WTP7.06 has the topology described in Part A. Using B-cell line and any other necessary reagents, describe how you would conFrm that the N-terminus – not C-terminus – is on the outside of the cell. You would raise a monoclonal antibodies to each terminus of WTP7.06. You can fluorescently label your antibodies. You would then add these antibodies to intact B-cells. If you tagged N-terminus & C-terminus antibodies with different fluorophores, then you can add them to intact B-cells simultaneously and see which fluorophore remains bound to outer cell membrane after washing. If you tagged them with a same fluorophore, you would need two batches of intact B-cells to which you would add either N-terminus or C-terminus antibody. You should also conFrm that the antibodies are not getting inside the cell. You could break open the cell and show that C-terminus now stains inner face of plasma membrane. You could also digest outer portion of WTP7.06 with a protease, break the cells open, and stain with N- terminus antibody. Alternatively, you could digest outer portion of protein with a protease, break the cells open and IP with each antibody to see which terminus is left. E. In the course of analyzing the WTP7.06 cDNA by deletion mutagenesis, you remove the Frst Ffty codons of the open reading frame (and replace them with a single ATG start codon). To your surprise, when you express this deletion mutant in the B-cell line, you Fnd the mutant protein almost exclu- sively in the nucleus. Provide an explanation as to why the mutant WTP7.06 protein localizes to the nucleus and why the wild-type protein is not normally found there. Again, assume the wild-type pro- tein has the topology described in part A. C. Annotate the diagram appropriate if WTP7.06 spanned the membrane twice such that both the N
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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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706_F06pset7_key - Problem Set 7 1 From B-cells you purify a plasma membrane protein which you name Want to Pass 7.06(WTP7.06 You raise antibodies

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