3. You are working with wild-type cells that have a 16h doubling time. BrdU is a

Question

3. You are working with wild-type cells that have a 16h doubling time. BrdU is a molecule that can be used to measure the amount of DNA inside cells. When added to cell culture, cells will incorporate BrdU into DNA strands during replication as if it were the DNA base thymidine (T). Your general experiment for using BrdU is: 1. Add BrdU to the media and let the cells continue to grow for 16h 2. Harvest cells and label them with a fluorescent antibody against Brdu 3. Measure fluorescence intensity in cells which is proportional to total DNA content 4. Plot a histogram which shows the number of cells with a certain fluorescence intensity Control (Part A) A. (5pts) As a control, you perform the above experiment on normally growing wild-type cells, and find the histogram to the right. Please explain why one peak shows roughly twice the fluorescence intensity as the other, what sets cells are represented by each peak, AND why the high-intensity peak is smaller. relative units) Nocodazole (Part 8) B. (5pts) You perform the general experiment with the following addition: when you add BrdU, you also add the drug taxol, which stabilizes microtubules. You find the histogram to the right. Please describe how the features of this histogram have changed from Part A, and explain why adding taxol could cause these changes referring to mechanisms we have discussed in class. ntensity (relative units) C. (4pts) You construct a cell line with a temperature sensitive mutant of the G1-cyclin. You perform the general experiment with the following additions: when you add BrdU, you shift to higher temperatures, which deactivates the G1-cyclin. Please draw what you predict will be the resulting histogram after you measure BrdU on the figure to the right.F Fluorescence intensity (relative units) (Note: The result from the control is printed as a reference, you should draw your response on the same axes.)

Explanation / Answer

A. we observe two peaks in the above experiment because the histogram is showing the stages of the cells.First peak will be the G1 phase and the secong peak will be the G2 phase.The first peak is double that of second because the cells are more in G1 phase which means that the cells have replicated DNA but not yet divided.

B. taxol arrest the cells undergoing mitosis.Here we can see only one peak because taxol has accumlated all the cells in G1 phase

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