L24 Isolation of cdc mutants

Question 1

Fission yeast vs Budding yeast

Answer 1

see onenote

in budding yeast, blur between S and M stages

Question 2

Hartwell

Answer 2

The leap of faith taken by Hartwell was that the cell cycle might depend on a set of genes — cell division cycle or cdc genes — whose function is required at specific timepoints in the cycle. He found evidence for these genes by identifying mutants that arrested the cell cycle at a specific stage.

Question 3

Isolation of cdc mutants - steps

Answer 3

see onenote

Question 4

Characterisation of temp sensitive mutants

Answer 4

see onenote

Because the cell cycle is an essential process, mutagenized yeast cultures were screened for temperature-sensitive mutants that grew and divided normally at the permissive temperature, but arrested cell division with a characteristic phenotype at the restrictive temperature.

cdc mutants - arrested at a specific stage e.g. all arrested at G1 - S 
a ts (temp. sensitive) mutant - random arrest

Question 5

Ts mutant

Answer 5

see onenote

affected in G1 => S

Question 6

cdc examples

Answer 6

see onenote

e. g. cdc4 - initiation of DNA synthesis
e. g. cdc28 - cyclin dependent kinases

Question 7

Shifting temperature effects

Answer 7

e.g. cdc15 - cannot exit mitosis, no cytokinesis when temperature was shifted to 36 celsius

Question 8

Ordering function of cdc mutants

Answer 8

see onenote

Create double mutants at restrictive temperature
CDC28 => cell arrest without buds
CDC7 => cell arrest with buds

CDC28 & CDC7 double mutant => cell arrest without buds
…CDC28 before CDC7

Question 9

Cloning of cdc genes

Answer 9

complementation for growth at non-permissive temp using a library in a shuttle vector

Question 10

Human - yeast cdc cloning by complementation

Answer 10

see onenote slides

complements if there is growth at 36 celsius (normal human temperature)

Question 11

A.nidulans temperature sensitive mutants

Answer 11

see onenote

isolate mutants that can grow at 32 degrees but not at 42 degrees - nud mutants, cannot move nuclei from spore end of mycellium at restrictive temperature

nud mutants - nuclear migration

cloning and characterisation of gene products => molecular machinery of nuclear movement

Question 12

Cyclin synthesis and degradation at single cell level

Answer 12

see onenote

cln2 fused to GFP

• Green being expressed then not, there is a cycle to cyclin expression

Question 13

Check points - 3 checkpoints

Answer 13

see onenote

Check points - checks that everything happens in order sequentially. Fail safe mechanism to make sure everything is okay, also controls size of the cell.

Start checkpoint - is enviro favourable?
G2/M checkpoint - is all DNA replicated?
met-anaphase transition checkpoint - are all chromosomes attached to spindle?

Question 14

wee mutants

Answer 14

see onenote

• Temperature sensitive
• Small at the non-permissive temperature compared to WT, normal in every other regards
• G2 where the growth occurs is shorter

For CDk to be active
Inactive = has two phosphates on Cdk
Active = has one phosphate on Cdk
- Needs one phosphate, cdc25 phosphatase removes one phosphate (2 to 1 phosphate left)
- Inhibition, wee1 kinase phosphorylates tyrosine 15 (2 phosphates on Cdk, inactive)

Question 15

cdc2 mutants

Answer 15

see onenote

cdc2- = always phosphorylated at Tyr15 => always inactive
cdcD = never phosphorylated at Tyr15 => always active

Question 16

Control of G2 to M

Answer 16

see onenote

Question 17

DNA damage checkpoint

Answer 17

see onenote

G2 - M

• Check point not operating => increased mutations due to lack of DNA repair, chromosome aberrations, loss/gain of chromosomes

=> loss/gain of function mutations
=> inappropriate gene expression
=> change in gene dosage

BRCA1 gene involved in DNA repair response

Question 18

Budding yeast checkpoint

Answer 18

see onenote

Question 19

G1 - S checkpoint

Answer 19

see onenote

Operating via a protein that inhibits the complex, not by phosphorylating

sic1, cdk inhibitor protein (CKI, cdk inhibitor)
- prevents initiation of DNA replication until the cell is fully prepared

Question 20

CKI

Answer 20

see onenote

cdk inhibitor protein
- CKI are controlled by phosphorylation followed by ubiquitin dependent proteolysis

Question 21

Meta-anaphase checkpoint

Answer 21

see onenote

spindle assembly checkpoint

• Have all the chromosomes attached properly
• Spindle assembly checkpoint
• May lead to a pause in mitosis in order to get the chromosomes to properly attach
• Risk of chromosome aberrations if the chromosomes do not attach properly

controlled by anaphase promoting complex (APC/C) - results in destruction of cyclins and cdk targets become desphosphorylated