L6 Flashcards
(28 cards)
What is the homologue of Cdc28 in S. pombe?
Cdc2
Why have a control point in G2/M?
Timing of replication versus mitosis – want to make sure replication is actually finished
Major site of size control in S. pombe
Proteins involved in the regulation of G2/M in S. cerevisiae
Cyclin-dependent kinase Cdc28 - serine/threonine protein kinase
Clb1, Clb2, Clb3 and Clb4 - B (G2) type cyclins
Proteins involved in the regulation of G2/M in S. pombe
Cyclin-dependent kinase Cdc2 - serine/threonine protein kinase
Cdc13 - B (G2) type cyclin – only uses 1 cyclin
Wee1/Mik1 - tyrosine kinases
Cdc25 - tyrosine phosphatase
Mutations that affect the G2/M transition in S. cerevisiae
Certain temperature sensitive alleles of cdc28 arrest at the non-permissive temperature at G2/M
Deletion of the CLB1, CLB2, CLB3 and CLB4 genes prevents mitosis
CLB2 has the greatest influence on G2/M
Deletion of the CLB2 gene greatly retards G2/M while simultaneous deletion of CLB1, CLB3 and CLB4 genes has little effect
Mutations that affect the G2/M transition in S. pombe
Certain temperature sensitive alleles of cdc2+ arrest at the non-permissive temperature of G2/M
Deletion of the cdc13+ gene prevents mitosis – Cdc13 is essential for getting the cell into M phase
Conditional mutations and overexpression studies of the wee1+, mik1+ and cdc25+ genes revealed that B cyclin association was not all that was required
Studies of the different mutants suggested that the Wee1/Mik1 and Cdc25 proteins worked in opposition to one another and that Cdc2 is regulated by phosphorylation
How does S. pombe divide?
By elongation – forms septum & separates
S. pombe conditional mutations
wee1-ts cdc25-ts cdc2-ts cdc25-ts-wee1-ts mik1- wee1-ts-mik1-
wee1-ts conditional mutation in S. pombe
Ts mutation in wee1 causes the cell to divide at a much smaller size – cell size is linked to this gene
Wee1 is an inhibitor at the boundary
cdc25-ts conditional mutation in S. pombe
Ts mutation in cdc25 (not linked to the cdc25 in cerevisiae) – cell doesn’t undergo mitosis & keeps getting longer
cdc is an activator at the boundary
cdc2-ts conditional mutation in S. pombe
Ts mutation in cdc2 – cell doesn’t enter mitosis
cdc is an activator at the boundary
cdc25-ts-wee1-ts conditional mutation in S. pombe
If you make a double mutant – at the non-permissive temperature it gives the wildtype phenotype
The opposites are blocking & inhibiting (cancel each other out)
Implies the balance between the 2 proteins determines entry from G2 into M phase
mik1- conditional mutation in S. pombe
Mik1- mutation behaves like wee1 & gives a smaller cell size
Also an inhibitor at the boundary
wee1-ts-mik1- conditional mutation in S. pombe
Wee1-ts-mik1- double mutant – cell attempts to divide at an even smaller size leading to mitotic catastrophe
What happens if you overexpress cdc2 or cdc13 in S. pombe?
No effect on the entry into mitosis
This is different to cerevisiae
What happens if you overexpress wee1 in S. pombe?
It inhibits the cell cycle
Cell enters as a much bigger cell
What happens if you overexpress cdc25 in S. pombe?
Cell is driven into M phase a lot quicker
What does the over expression of wee1 and cdc25 in S. pombe tell us?
Reinforces the idea that cdc25 is an activator & wee1 is an inhibitor
Act in opposition to each other
Cell size control in S. pombe
Main control is at G2/M & is controlled by Wee1/Mik1 & Cdc25
Mutations which reduce the size for mitosis increase the G1 time interval
Hence there is a size control at START which is normally cryptic
Cell size control in S. cerevisiae
Cryptic control in pombe at G1 but main one in cerevisiae is at G1
Main control at START and is controlled by Cln3
Mutations which reduce size for START increase the time interval between START and mitosis. Hence there is later cell size control which is normally cryptic
When cln3 cells are smaller, the cell spends longer in G2 as it is not as big as anticipated so it is held in G2 to allow it to grow
How is cell size control different in S. cerevisiae and S. pombe?
The main cell size control for S. cerevisiae and S. pombe is at different points in the cell division cycle
This probably reflects the different life cycles of these yeasts in the wild
S. cerevisiae grows as a diploid in the wild whilst S. pombe grows as a haploid
Why is the fact that S. cerevisiae grows as a diploid in the wild whilst S. pombe grows as a haploid important?
Diploid cells – can use other chromosome to help repair DNA damage
Haploid has no paired chromosome to use as a guide for DNA damage
Diploid has no pressure to go through G1 quickly
Haploid is most susceptible to DNA damage in G1 as it doesn’t have a paired chromosome
As pombe is haploid it wants to spend as little time in G1 to prevent DNA damage – can spend longer in G2
The regulation of Cdc2 activity by phosphorylation in S. pombe
Has 2 phosphorylation sites: tyrosine 15 and threonine 161
Phosphorylation of threonine 161 is required for activity of Cdc2
Phosphorylation of tyrosine 15 inhibits activity of Cdc2
Phosphorylation of tyrosine 15 is dominant to phosphorylation of threonine 161
If they’re both phosphorylated, tyrosine15 is dominant so cdc2 is inhibited
Mik1/Wee1/Cdc25 regulates tyrosine 15 phosphorylation
– Mik1 & Wee1 add phosphates
– Cdc25 removes phosphates
Mutation of tyrosine 15 of Cdc2 to phenylalanine has what affect on S. pombe?
Mutation of the tyrosine15 to F15 looks like a wee1/mik1 double mutant
Enters mitosis at smaller size
Phenylalanine is the most similar structure – mimics the unphosphorylated form of the protein
F = phenylalanine
When we combine the F15 mutation with cdc25 it doesn’t cancel out – site cannot be phosphorylated
