7. Your colleague has prepared a cell culture in which cells grow and divide wit

Question

7. Your colleague has prepared a cell culture in which cells grow and divide with a doubling time of roughly 16 hrs. She performs the following experiment (1) Harvest a small number of cells every 4 hrs (2) Isolate the G1/S-, S-, and M-phase cyclin proteins from the cells. (3) Run the cyclin proteins on a gel with results shown below Ohr 12hr 16hr clin G1/S- phase S-phase M-phase A. (2pts) Your colleague is disappointed that the gel result did not show cyclin levels rising and falling during the cell cycle. Why might this be the case? B. (2pts) What additional step can you add to the experiment to be able to show that cyclin levels do change during the cell cycle? C. (2pts) Suppose you run the modified experiment from Part B. Please predict how your gel would look by drawing in bands in the diagram below Ohr 8hr 12hr 16hr Cyclin G1/S phase S-phase M-phase

Explanation / Answer

A) The cyclin levels do not show desired change in concentration because culture contains the random mixture of cells at different stages of cell cycle.

B) To study the changes in levels of cell cycle protein (which are cyclins in this case) with the progression of cell cycle, culture cell first should be synchronized. This is done in order to bring cells at different stages of cell cycle to the same phase. Many physical and chemical methods can be used to "sync" the cells in culture. When all the cells are brought to same phase of cell cycle, it can be followed by growing cells at optimun temerature and harvesting them from culture every 4 hrs. The cyclin proteins harvested at different time periods can then be analyzed for changes in concentration using gel electrophorosis.

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