L6 - the cell cycle Flashcards
What is the cell cycle?
A programmed series of events enabling a cell to duplicate it’s contents and generate 2 daughter cells
How do we know the length of the cell cycle?
The time it takes for a cell to double (24hrs)
How do we know the length of S phase?
Incubate cells with 32P, taken up by cells undergoing DNA synthesis - can calc the % of cells doing DNA synth which is representative of the length of S phase (35% - 7.5 hrs)
How do we know the length of M phase
Morphological assays allow us to visualise cells in M (1hr) phase
What does the analysis of a cell’s DNA content tell us?
Whether the cells are proliferating or not. G2/M phase has double the amount of DNA as in G1. S phase has a lower DNA content than G1 but higher than G2
Why is yeast a good model system for the cell cycle?
Easily identifiable morphological features to see which phase of the cell cycle the yeast is in. Allows us to study effects of mutations on the cell cycle
What are cyclins?
Proteins that are expressed at different levels during the cell cycle. They bind to CDKs to activate them
How were cyclins discovered?
Tim Hunt. Looked at protein synthesis in sea urchins, saw proteins that appeared and then were degraded just before mitosis. Cyclins - synthesised cyclically
What are the major cyclin/CDK complexes?
G1: D/CDK4/6
G1/S: E/CDK2
S: A/CDK1/2
M: B/CDK1
What are the main activators of CDKs?
Cyclin
What are the main inhibitors of CDKs?
CKIs. Act to inhibit CDKs by phosphorylation of adj threonine/tyrosine residues
What induces the expression of CKIs?
Anti-proliferative signals, DNA-damage signals
What is the role of mitogens?
Inhibit CKIs - block inhibition of CDKs
What is the role of Rb in the cell cycle?
It regulates the restriction point. RP is the place where cells decide to undergo mitosis or enter G0. In the presence of mitogens, RB will allow cells to enter mitosis. Rb is a pt that binds and inhibits E2F (a principle TF for all genes, req for entry into S-phase).
Which cyclin/CDK complex phosphorylates Rb and what does the phos do?
Cyclin D CDK4/6. The phosphorylation has an inhibitory role on Rb - allowing entry into CC
How are yeast used as an experimental model?
Mutate yeast and screen for mutants that disrupt the cell cycle (can be easily visualise). Grow yeast at different temperatures to identify mutations that are temperature sensitive. Cells arrest at execution point - this is when the mutated protein is needed. Screen libraries of DNA plasmids to see when plasmids rescue the phenotype - the screen to identify the protein
How were xenopus oocytes used to study the cell cycle?
Early stages of cell cycle in oocytes - cells get smaller, cc is fast = no time to synth new pt. This means eggs have enough pt to make 4000 cells worth of DNA/nuc/metabolites. Take egg cyto and add DNA - recap CC in vitro
What is MPF and how was it identified?
Maturation promoting factor. Oocytes are arrested in G2 of CC - inject cyto of egg (metaphase) into oocyte - premature maturation. MPF identified as the factor that induces maturation.
What is the importance of Cdc25 and Wee1?
Both regulate the activity of Cdc2 in yeast. Cdc2 drives entry into mitosis (Cdk1 in humans). Cdc25 mutants are longer, Cdc25 positively regulates Cdc2 - driving mitosis. Wee1 mutants are shorter, enter mitosis early, Wee1 normally inhibits the function of Cdc2, stopping mitosis.
What are the main CKIs?
p16 (INK4A) p15 (INK4B) p18 (INK4C) P19 (INK4D) All inhibit D-CDK4/6 - stop entry into cell cycle p27 (Kip1) p57 (Kip2) p21 (Cip2) Inhibit the other cyclins/CDK complexes stopping progression through the cell cycle
What is the role of TGF-b?
Produced by cells to activate INK4 family of CKIs. This blocks entry into cell cycle - no division - contact inhibition
