Eukaryotic cell cycle control

Question 1

What is cell cycle control?

Answer 1

~ systems that respond to intracellular stimuli to control cell growth
~ complex signalling pathways
~ regulated by cyclin/CDK activities

Question 2

Function of cyclin-CDKs

Answer 2

ensures DNA replication occurs only once per cell cycle and that chromosomes are equally segregated into daughter cells

Question 3

Cell fusion expeirment

Answer 3

~ M phase-cell + G2 phase-cell = when combined both enter M phase
~ conclusion: M=phase cells have a mitosis promoting factor
~ works with all other phases too

Question 4

Cell fusion experiment #2

Answer 4

~ S phase-cell + G1 phase-cell = 2x S phase cell
~ conclusion: S-phase promoting factor in S-phase cells
~ only works on cells in G1 not G2

Question 5

Which experimental systems were used to study G2/M transition?

Answer 5

~ eggs/embryos of African tree frogs (Xenopus)

~ sea urchins

Question 6

Why were those exp. systems used?

Answer 6

~ relatively large, high protein content
~ readily available in high numbers
~ naturally synchronised
~ can obtain cell extracts from specific points (arrest cell cycle easily)

Question 7

Xenopus Oocyte maturation + identification of MPF

Answer 7

MPF = maturation promoting factor
~ if cytoplasm of egg in metaphase of meiosis II added to G2 arrested oocyte, it undergoes meiosis I
~ proves that MPF exists

Question 8

MPF activity

Answer 8

~ rises and falls in parallel with mitosis

~ high MPF levels arrests oocyte in meiosis until its abundance drops

Question 9

Identification of cyclin as a component of MPF

Answer 9

1. sea urchin oocyte fertilized and 35S Methionine added
2. methionine is only incorporated into newly-made protein
3. protein extracts taken at different time points post fertilisation and analysed by SDS-PAGE
4. autoradiography shows new proteins

Question 10

Cyclin B

Answer 10

~ a constituent of MPF
~ when cyclin B mRNA added = normal mitotic program
~ Cyclin B = required for mitotic entry

Question 11

Effects of a truncated cycle B protein

Answer 11

~ non-degradable by protease and its activity prevents mitotic exit
~ therefore cyclin B (MPF) destruction is needed for mitotic exit