A mutant yeast strain stops growing and dividing when shifted from 25 degree C t
ID: 177310 • Letter: A
Question
A mutant yeast strain stops growing and dividing when shifted from 25 degree C to 37 degree C. A population of these yeast growing at 25 degree C was split in two. One sample was left at 25 degree C and the other was shifted to 37 degree C. After 12 hours the cells were fixed, incubated with propidium iodide and analyzed with a FACs. Propidium iodide binds DNA and fluoresces when excited with a laser of the proper wavelength. The results of the experiment are shown in Figure 9 on the right. Exactly 10,000 cells from each sample were analyzed. Explain why the data support an arrest at either the G_2 or spindle assembly checkpoints and not at the G_1 checkpoint. To determine which checkpoint the cells are arrested in you prepare a fraction of the cells at 37 degree C and inject it into a G_2 arrested frog oocyte. The oocyte immediately assembles a matrix spindle. What causes this to happen? Would you conclude from this result that the mutant yeast cells are arrested at the G_2 checkpoint or at the spindle assembly checkpoint? Explain The cartoon in figure 8 on page 13 show mechanism of checkpoint regulation. Describe one gene that may be mutated in yeast cells to produce the phenotype in Figure 9. Explain your choice.Explanation / Answer
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Answer 41: Propidium iodide is a specific fluorescent dye which stains the nucleic acid including RNA and DNA in a cell which can be measured by FACS. According to the question, the cells when shifted to 37 degree celcium temperature show decline in cell survival. Since the G1 phase of cell cycle is involves onset of DNA replication and G2 involves prepraration of cell division, there occurs physiological difference between the two. According to the first graph, it can be relatively seen that the height of first peak is more than double of the second peak. The height of this first peak is nearly similar to that of the FACS analysis peak in the second graph. This states that the DNA in the second set of population has already been doubled and the cell might have started to reach the division phase of cell cycle. Had it been the G1 checkpoint, the FACS peak for the second population would have been nearly half of its present height becuase DNA would not have been completely replicated as it should have been. Thus, these two graphs state that the cells are either halted at the G2 phase or at the spindle assembly of cell division.
Answer 42: When the cytosolic fraction of the yeast cells are injected in an oocyte halted at the spindle assembly checkpoint, the oocyte starts division immediately. This clearly demonstrates the finding in the first part of the question that the yeast cells had indeed passed on the G2 checkpoint and there was a fault in spindle fibre assembly in these cells which prevented these mutant yeast cells to survive under increased temperature.
Since the oocyte might have been given appropriate conditions to divide, all the cytological biomolecules were present in the yeast cytoplasmic extract which made the oocyte divide by forming a normal spindle assembly.
Answer 43: A number of cytosolic/nuclear proteins are involved in regulation of cell cycle control. For example, cyclin dependent kinases remain improtant proteins which prevent entry of cell division into next phase and directly affect spindle assembly. These cyclin dependent kinases are directly involved in modulation of various mobile elements of the cell cycle which are mediated by phosphorylation mediated activation or deactivation. Failure of a cell to regulate the activation/deactivation status of these proteins prevent appropriate spindle assembly or migration of chromosomes to the spindle equator or segregation of chromosomes due to motility defects, hence prevents completion of cell cycle.
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