706_F06pset7 - Problem Set 7 1 From B-cells you purify a...

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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. 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. 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 confirm that the N-terminus – not C-terminus – is on the outside of the cell. E. In the course of analyzing the WTP7.06 cDNA by deletion mutagenesis, you remove the first fifty 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 find the mutant protein almost exclu- sively in the nucleus. Provide an explanation as to why the mutant WTP7.06 protein localizes to the
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