Question

You are given a flask with standard 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 in the dark 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 component of a UV-mediated DNA damage checkpoint. Which FACS plot (see attached figure) best represents how cells in this flask would appear following growth for 30 min at the restrictive 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). #2 #3 #4 #5 #6 LLL 12 2 1 2 relative amount of DNA 1 2

Answer 1

Answer

Plot #2 is best to represent the growth of the cells after 30min in restrictive temperature.

Permissive temperature:- Temperature-sensitive Gene product will be normal.

Restrictive temperature:- Temperature-sensitive gene product will become mutated in other words no functional gene product.

In Flow cytometry, cell cycle analysis study is done using fluorescence-labeled DNA binding dye which binds DNA stoichiometry as the content of the DNA in the cells.

The DNA content in the cell in respective phases

G₀ / G₁ phase = 2n (cells are diploid)

S phase = >2n (As DNA replication has been started some DNA content doubles )

G₂/M Phase = 4n (Cell DNA content is tetraploid (4n) cells are aligning and preparation for Mitosis )

DNA damage Checkpoint genes work on G2 and G2/M phase as the DNA replication occurs where they check for any DNA damage then these checkpoint kinases activate many downstream elements for the repair of the DNA or the cell cycle will be arrested at respective stages.

As cells grow in the restrictive temperature for 30 min so there will be a mutation in the DNA damage checkpoint gene. So there will be unlikely that the cell cycle is the arrested because Checkpoint gene is mutated which is unable to detect any DNA damage. So there will be a normal cell cycle being followed.

In the given condition cell culture is asynchronous in nature. So there will be cells in all the phases of the cell cycle in the next 30 min of the cell cycle,

• cells that were in the G1 phase will enter into the S phase. So the Peak of the S phase will be increased. But the Peak of the G1 phase cells will be decreased.
• Cells that were in the S phase will enter into the G2/M phase then also the peak will increase.
• All the phases of the cell cycle will be present.