Question

1 You are a studying the progression of cells through the cell cycle. You are particularly interested in the cyclins E and A, both of which contribute sequentially to the same cell cycle transition. In this case one cyclin initiates the transition and the other completes the process. In these images you see cell extracts from Hela Cells. A) shows the levels of various proteins, while B) shows the amount of DNA in the cells, more DNA shifts this graph to the right, less DNA shifts this graph to the left. Hrs after mitosis Ciz1 Cyclin E Cyclin A 1.2 1.0 0.8 0.6 (4pts) Based on the DNA graphs in B what stages of the cell cycle are the cells undergoing between 0 and 16 hrs. Justify your answer 12 H 0.2 10 Time (Hr) 15 20 Cyclin ACyclin E Ciz1 b. (2pts) Based on the DNA graph and the Western for cyclins A and E what stage of the cell cycle do cyclins A and E generally regulate? (4pts) Given your answer in b, describe what types of proteins or activities are likely to be regulated by these cyclins. Where in the cell would you expect them to be most active? c. d. (3pts) Which cyclin initiates the cell cycle and which one completes the process? How did you know?

Answer 1

I will start answering from question g as I have already answered question a to d .

G.Both mutant 1 and 3 show almost the same level of recovery of protein cyclin E as compared to mutant 1. Site 2 corresponding to mutant 2 shows less recovered band during co-ip indicating that binding is considerably affected compared to other 2 mutants. One needs to perform a densitometry based measurements to get the level of significance.

For cyclin A site 2 and 3 that is point where mutant 2 and 3 is created are important for ciz1 binding as band intensity is significantly reduced for these 2 mutants.

H. To prove this hypothesis , one can make mutants of ciz1, look at its binding to cyclins using co-op. This will help us to know whether binding of cyclins are affected due to these mutations. Next ,one can use fluorescence microscopy to look at the localisation of cyclin-cdk whether they are affected. One can also pull nuclear extracts from these wild type and mutants and see whether the mutants go to nucleus or stay in cytoplasm. Also one can perform flow cytometry to see whether the function of cell cycle is affected or not due to these mutants and compare it to wild type. One can also over express the wild type copy of cyclin E or A or knock them off to see their importance in cell cycle.

Figures should comprise of following results.

A. Co-ip of cyclin E and A with ciz1 mutants. B. Cytosolic and nuclear extracts of wild type and mutant ciz1. C. Fluorescence microscopy of wild type and mutant ciz1 using cyclin E and A antibodies.