Question

You are given a flask with liquid media containing a population of S. pombe cells that have a temperature sensitive mutation of a gene that causes complete loss of function of the gene product at the restrictive temperature. The flask had been incubating at the permissive temperature before it was given to you, and the cells in the flask were replicating asynchronously with respect to the stage of the cell cycle. The mutated gene in this strain of yeast encodes an essential subunit of a DNA polymerase required for DNA replication, but not DNA repair. (Assume that the cell only has one DNA polymerase that performs DNA replication and that cells paused in DNA replication can restart and complete this process). Which FACS plot (see attached figure) best represents how cells in this flask would appear following growth after 2 hours in culture at the restrictive temperature followed by 60 min in culture at the permissive temperature? (Note, assume that FACS plot #1 shows analysis of a population of S. pombe replicating asynchronously (i.e. each cell within a population is at a random stage of the cell cycle) in culture. In the rich media assume the yeast have a cell cycle time of 100 min, with G1=50 min; S = 25 min; G2 = 15 min and M = 10 min). #1 #2 #4 #6 ces in Gand Mhes Sphase i 2 1 2 rative amount of DNA percell 1 2

Answer 1

Ans:

Plot #3

G1 phase takes 50 min to complete it. It reaches S phase next. Since the essential subunit of DNA polymerase in mutated, its function is lost. So it has to restart again. So, the G1 phase again it takes 50 mins more, making 100 min in total. By the time it reaches again S phase, DNA polymerase is mutated and again it goes for restart. Hence, the number of cells get accumulated in G1 phase.

Now after the third time in the G1 phase for 50 mins, i.e., after 150 min, cells can synthesize their DNA, because after 2 hours it is transferred to permissive temperature.