week3_key - MCB130 week 3 solutions 1 What is puromycin...

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MCB130 – week 3 solutions 1 1) What is puromycin, what is it used for, and how does it work? It detaches the nascent chain from a ribosome in the middle of translating an mRNA. It can be used to remove the transcripts from purified rough ER microsomes. It will also kill cells because it inhibits protein synthesis. 2) You have isolated microsomes from the ER, and you treat them with puromycin. Is it important to have puromycin inside or outside the microsome? Draw a picture illustrating your answer. Puromycin must be out the outside where the ribosome is incorporating new amino acids bound to tRNAs. See above for diagram. 3) What is conditional mutant? A conditional mutant displays its phenotype only under certain environmental conditions (restrictive conditions), but can grow (somewhat) normally under another set of conditions (permissive conditions). 4) What is density gradient centrifugation? Density gradient centrifugation is a method used to separate cellular components (or other things, I suppose) based of differences in density. Using a sucrose or cesium chloride gradient, different organelles and other parts of a cell can be isolated and separated from the rest of the cell based on where they are located in the tube after centrifugation. 5) You have isolated a temperature-sensitive yeast mutant that shows normal nuclear export at permissive temperatures. However, it shows defective nuclear export at higher temperatures. a) What phenotypes might you observe at higher temperatures?
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