How can I arrest the budding yeast in a particular cellcycle stage without leading the cell to stress? I want to study mRNA at individual cell cycle stage of budding yeast. For that the cells shouldnot go under any stress, since stress leads the mRNA granules to move to P-bodies. SO please suggest me how to arrest budding yeast to a particular cell cycle stage.
Thank you for your time.
Hi there,
Cell cycle arrest is either the cause or the result of a stress response activation pathway. So I'm afraid you can't avoid any stress response when causing cell cycle arrest. I guess the arrest provoking the least stress response is alpha-factor arrest in G1 (inhibition of cdc28/cln2).
This is not easy. However, as an approach you may get synchronized cell cultures by starving the cells. From there on you can proceed, by launching a new cultivation stage and harvest cells in each stage through carefully monitoring the growth.
Hi again,
As a complement and to go in Manuel's way, alpha factor block and release may also be use to synchronize cells. The other way to proceed in cell synchronization is elutriation (but you need the elutriator!)
I do agree with Dominique's answer. However, elutriation is good to separate budding yeasts form the non-budding ones.Other stages are harder to isolate with this method. If you synchronize cells with the alpha factor you can proceed by launching synchronized cultures and, by harvesting cells in well defined periods you can arrest the harvested cells by immerging them in liquid nitrogen: Then you can store them and analyze the samples whenever you want.
thank you all for your valuable suggestions. Especially thank you Dominique and Manuel for your comments :)
Dear Rafael, I feel using cdc mutants may alter the endogenous mRNA homeostasis to some extent and I dont want that, since I want to see mRNA at physiological level.
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