Things are not going so well in your graduate research project. You’ve just had

Question

Things are not going so well in your graduate research project. You’ve just had a brief but intense meeting with your research advisor who is not fond of your habit of working late and sleeping late; she thinks it is at the root of your lack of productivity (as she perceives it). A few days later you learn from student colleagues that your advisor is looking to find you. Expecting the axe to fall, you are surprised when you learn that she is excited rather than upset. She has just come across a note you had left on her crowded desk (a few late nights earlier) describing a selection scheme you had crafted for isolating mutants in the ER translocation machinery. You are flabbergasted at her change in mood and her interest in your project.

You quickly settle on the details for the selection, which involves fusing an ER import signal to the N-terminus of the His4 gene product. His4 is a cytosolic enzyme that converts histidinol to histidine. Yeast strains that a defective for an early step in the histidine biosynthetic pathway can grow on added histidinol, if His4 is present. You decide to look for temperature sensitive mutants, which are normal at 24 C but defective at 37 C. Using a strain that expresses His4 with an ER import signal, you select that grow on histidinol at 30 C and screen them for ones that die at 37 C. The first mutant you isolate is in the Sec61 gene, the gene that is later shown to encode a principal component of the translocator through which ribosomes insert nascent proteins across the ER membrane. Your advisor is very impressed with your plan, and she asks you to discuss it at the next meeting of your graduate research committee, where you are asked two questions to be answered here:

(1) Why is it that normal cells with the modified His4 cannot grow on histidinol whereas cells with a defective ER-import apparatus can? (2) Why did the selection scheme set out to find temperature sensitive mutants? Why was the selection applied at an intermediate temperature of 30 C, rather than 24 C or 37 C?

Explanation / Answer

1. Modified His4 (Histidinol dehydrogenase) cannot catabolize the histidol into histidine which is an important essential amino acid for cell growth and protein synthesis in the cytoplasm of normal cells. Defective ER-import apparatus containing cells can grow because they have functional His4 in the cytoplasm that can provide histidine from histidol for regular activities.

2. Cells with mutant Sec1 gene will have a high frequency of leaky growth at 38oC this because temperature sensitive strains with defective protein synthesis and importing to ER membrane. Here, His4 activity could be optimum at 30oC and also normal at 24oC results in the selection of temperature strains could be possible but His4 would not be functional at 37oC that results in cell death. It suggests that temperature sensitivity indicating the availability of histidine into the cytoplasm of yeast on histidinol medium.

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