L6

Question 1

What is the homologue of Cdc28 in S. pombe?

Answer 1

Cdc2

Question 2

Why have a control point in G2/M?

Answer 2

Timing of replication versus mitosis – want to make sure replication is actually finished

Major site of size control in S. pombe

Question 3

Proteins involved in the regulation of G2/M in S. cerevisiae

Answer 3

Cyclin-dependent kinase Cdc28 - serine/threonine protein kinase

Clb1, Clb2, Clb3 and Clb4 - B (G2) type cyclins

Question 4

Proteins involved in the regulation of G2/M in S. pombe

Answer 4

Cyclin-dependent kinase Cdc2 - serine/threonine protein kinase

Cdc13 - B (G2) type cyclin – only uses 1 cyclin

Wee1/Mik1 - tyrosine kinases

Cdc25 - tyrosine phosphatase

Question 5

Mutations that affect the G2/M transition in S. cerevisiae

Answer 5

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

Question 6

Mutations that affect the G2/M transition in S. pombe

Answer 6

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

Question 7

How does S. pombe divide?

Answer 7

By elongation – forms septum & separates

Question 8

S. pombe conditional mutations

Answer 8

wee1-ts
cdc25-ts
cdc2-ts
cdc25-ts-wee1-ts
mik1-
wee1-ts-mik1-

Question 9

wee1-ts conditional mutation in S. pombe

Answer 9

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

Question 10

cdc25-ts conditional mutation in S. pombe

Answer 10

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

Question 11

cdc2-ts conditional mutation in S. pombe

Answer 11

Ts mutation in cdc2 – cell doesn’t enter mitosis

cdc is an activator at the boundary

Question 12

cdc25-ts-wee1-ts conditional mutation in S. pombe

Answer 12

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

Question 13

mik1- conditional mutation in S. pombe

Answer 13

Mik1- mutation behaves like wee1 & gives a smaller cell size

Also an inhibitor at the boundary

Question 14

wee1-ts-mik1- conditional mutation in S. pombe

Answer 14

Wee1-ts-mik1- double mutant – cell attempts to divide at an even smaller size leading to mitotic catastrophe

Question 15

What happens if you overexpress cdc2 or cdc13 in S. pombe?

Answer 15

No effect on the entry into mitosis

This is different to cerevisiae

Question 16

What happens if you overexpress wee1 in S. pombe?

Answer 16

It inhibits the cell cycle

Cell enters as a much bigger cell

Question 17

What happens if you overexpress cdc25 in S. pombe?

Answer 17

Cell is driven into M phase a lot quicker

Question 18

What does the over expression of wee1 and cdc25 in S. pombe tell us?

Answer 18

Reinforces the idea that cdc25 is an activator & wee1 is an inhibitor

Act in opposition to each other

Question 19

Cell size control in S. pombe

Answer 19

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

Question 20

Cell size control in S. cerevisiae

Answer 20

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

Question 21

How is cell size control different in S. cerevisiae and S. pombe?

Answer 21

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

Question 22

Why is the fact that S. cerevisiae grows as a diploid in the wild whilst S. pombe grows as a haploid important?

Answer 22

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

Question 23

The regulation of Cdc2 activity by phosphorylation in S. pombe

Answer 23

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

Question 24

Mutation of tyrosine 15 of Cdc2 to phenylalanine has what affect on S. pombe?

Answer 24

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

Question 25

How are Mik1/Wee1 and Cdc25 regulated by size in S. pombe?

Answer 25

Cdc25 is phosphorylated with a timing that mimics dephosphorylation of tyrosine 15 of Cdc2

Wee1 appears to be negatively regulated by the Nim1 kinase
– Nim1 is an inhibitor of Wee1

For example, overproduction of Nim1 suppresses a cdc25ts mutation at the non-permissive temperature
– If we make a Wee1/Cdc25 double mutant they cancel each other out

Question 26

Regulation of Cdc28 in C. cerevisiae by mutating tyrosine 19 to phenylalanine

Answer 26

Tyrosine 15 of Cdc2 in S. pombe is tyrosine 19 of Cdc28 in S. cerevisiae

Mutating tyrosine 19 of Cdc28 to phenylalanine has no effect on cell division, cell size or the length of G2 phase in contrast to S. pombe

Question 27

What is the Wee1 homologue in S. cerevisiae?

Answer 27

Swe1

Overexpression of Swe1 arrests cells in G2

A swe1D overcomes the cell cycle delay of a mih1D

Question 28

What is the Cdc25 homologue in S. cerevisiae?

Answer 28

Mih1

Deletion of the gene delays entry into M phase but does not prevent it