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Question 3 A genetic screen undertaken in budding yeast has identified a gene whose product encodes a protein kinase that controls the transition of cells from G2 into mitosis. By sequence comparison, you identify a human homologue of the yeast protein, which you name PK1. You now wish to explore the regulation and function of PK1 in cultured human cells. Answer the following questions explaining the methodologies you would use. a) You wish to study PK1 activity in synchronous populations of cells that are in different stages of the cell cycle. Describe how you might firstly treat the cells with a named drug in order to synchronise them in a specific stage of the cell cycle. Include a brief explanation of how this drug acts and in which stage of the cell cycle the cells would be arrested. Having treated multiple plates of cells with the drug, you then wash out the drug and incubate each of the plates for different periods of time (from 0-24 hours), as shown in the table below, so that each will have progressed to a different point in the cell cycle. 1 7 8 9 10 11 12 13 14 16 18 20 22 24 Plate number Incubation time (h) b) 0 c) 2 2 3 4 4 6 5 6 8 10 Cell extracts are prepared for each plate of cells. These are analysed by SDS-PAGE and Western blotting using an antibody that only detects the active (phosphorylated) form of PK1. 12 At which stage of the cell cycle would you expect the strongest signal for active PK1 to be observed? Briefly explain why this would be the case. Draw a graph with labelled axes to show the relative activity level of PK1 that you might expect to observe across the different samples, ranging from 0 to 100%. Also mark on your graph the approximate positions of interphase and mitosis. Assume that one full cell cycle takes 24 hours. N.B. Bear in mind the starting point of your experiment after release from the particular cell cycle block that you chose in (a).

Question 3 A genetic screen undertaken in budding yeast has identified a gene whose product encodes a protein kinase that controls the transition of cells from G2 into mitosis. By sequence comparison, you identify a human homologue of the yeast protein, which you name PK1. You now wish to explore the regulation and function of PK1 in cultured human cells. Answer the following questions explaining the methodologies you would use. a) You wish to study PK1 activity in synchronous populations of cells that are in different stages of the cell cycle. Describe how you might firstly treat the cells with a named drug in order to synchronise them in a specific stage of the cell cycle. Include a brief explanation of how this drug acts and in which stage of the cell cycle the cells would be arrested. Having treated multiple plates of cells with the drug, you then wash out the drug and incubate each of the plates for different periods of time (from 0-24 hours), as shown in the table below, so that each will have progressed to a different point in the cell cycle. 1 7 8 9 10 11 12 13 14 16 18 20 22 24 Plate number Incubation time (h) b) 0 c) 2 2 3 4 4 6 5 6 8 10 Cell extracts are prepared for each plate of cells. These are analysed by SDS-PAGE and Western blotting using an antibody that only detects the active (phosphorylated) form of PK1. 12 At which stage of the cell cycle would you expect the strongest signal for active PK1 to be observed? Briefly explain why this would be the case. Draw a graph with labelled axes to show the relative activity level of PK1 that you might expect to observe across the different samples, ranging from 0 to 100%. Also mark on your graph the approximate positions of interphase and mitosis. Assume that one full cell cycle takes 24 hours. N.B. Bear in mind the starting point of your experiment after release from the particular cell cycle block that you chose in (a).

Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Question 3 A genetic screen undertaken in budding yeast has identified a gene whose product encodes a protein kinase that controls the transition of cells from G2 into mitosis. By sequence comparison, you identify a human homologue of the yeast protein, which you name PK1. You now wish to explore the regulation and function of PK1 in cultured human cells. Answer the following questions explaining the methodologies you would use. a) You wish to study PK1 activity in synchronous populations of cells that are in different stages of the cell cycle. Having treated multiple plates of cells with the drug, you then wash out the drug and incubate each of the plates for different periods of time (from 0-24 hours), as shown in the table below, so that each will have progressed to a different point in the cell cycle. 1 Plate number Incubation 0 time (h) b) Describe how you might firstly treat the cells with a named drug in order to synchronise them in a specific stage of the cell cycle. Include a brief explanation of how this drug acts and in which stage of the cell cycle the cells would be arrested. c) d) e) 2 2 3 4 4 6 5 6 7 8 9 610 8 10 12 14 16 Cell extracts are prepared for each plate of cells. These are analysed by SDS-PAGE and Western blotting using an antibody that only detects the active (phosphorylated) form of PK1. 10 11 12 13 18 20 22 24 At which stage of the cell cycle would you expect the strongest signal for active PK1 to be observed? Briefly explain why this would be the case. Draw a graph with labelled axes to show the relative activity level of PK1 that you might expect to observe across the different samples, ranging from 0 to 100%. Also mark on your graph the approximate positions of interphase and mitosis. Assume that one full cell cycle takes 24 hours. N.B. Bear in mind the starting point of your experiment after release from the particular cell cycle block that you chose in (a). The activity of many mitotic kinases is regulated by cyclins which are present at different levels throughout the cell cycle. With the aid of a diagram, explain how cyclin degradation is regulated. State two other processes that could lead to changes in protein levels of cell cycle regulators.
Transcribed Image Text:Question 3 A genetic screen undertaken in budding yeast has identified a gene whose product encodes a protein kinase that controls the transition of cells from G2 into mitosis. By sequence comparison, you identify a human homologue of the yeast protein, which you name PK1. You now wish to explore the regulation and function of PK1 in cultured human cells. Answer the following questions explaining the methodologies you would use. a) You wish to study PK1 activity in synchronous populations of cells that are in different stages of the cell cycle. Having treated multiple plates of cells with the drug, you then wash out the drug and incubate each of the plates for different periods of time (from 0-24 hours), as shown in the table below, so that each will have progressed to a different point in the cell cycle. 1 Plate number Incubation 0 time (h) b) Describe how you might firstly treat the cells with a named drug in order to synchronise them in a specific stage of the cell cycle. Include a brief explanation of how this drug acts and in which stage of the cell cycle the cells would be arrested. c) d) e) 2 2 3 4 4 6 5 6 7 8 9 610 8 10 12 14 16 Cell extracts are prepared for each plate of cells. These are analysed by SDS-PAGE and Western blotting using an antibody that only detects the active (phosphorylated) form of PK1. 10 11 12 13 18 20 22 24 At which stage of the cell cycle would you expect the strongest signal for active PK1 to be observed? Briefly explain why this would be the case. Draw a graph with labelled axes to show the relative activity level of PK1 that you might expect to observe across the different samples, ranging from 0 to 100%. Also mark on your graph the approximate positions of interphase and mitosis. Assume that one full cell cycle takes 24 hours. N.B. Bear in mind the starting point of your experiment after release from the particular cell cycle block that you chose in (a). The activity of many mitotic kinases is regulated by cyclins which are present at different levels throughout the cell cycle. With the aid of a diagram, explain how cyclin degradation is regulated. State two other processes that could lead to changes in protein levels of cell cycle regulators.
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