Lecture 27

Question 1

The isolation of cdc mutants:

Answer 1

• Temperature sensitive mutants are used to isolate cdc mutants
• Haploid cells are treated with mutagen
• Diluted and spread on a plate at 22 degrees
• Allow cells to grown into colonies
• Imprint colonies onto two plates
• Grow one plate at 22 degrees
• Grow one plate at 36
• Mutant can grow at 32 degrees, but not at 36 degrees

Question 2

Once temperature sensitive mutants are isolated we have to determine which ones are mutants effected specifically in the cell cycle:

Answer 2

• Shift the temperature from permissive to non-permissive (22 - 36)
• See what stage of the cell cycle the mutants stop growing at
• A temperature sensitive mutant not effected in the cell cycle will randomly arrest at any stage of the cycle

Question 3

Cdc mutants (in both pombe and sacromyces):

Answer 3

• After the temperature has been shifted to the non-permissive temperature (36), all of the cells will arrest at the G1 - S phase
• All blocked at a specific stage
• This is because, after the shift, the temperature sensitive function is inactive, but it is required for the shift from G1 - S

Question 4

cdc4:

Answer 4

• Initiation of DNA synthese

Question 5

cdc16:

Answer 5

• mitosis: mircotubules

Question 6

cdc9:

Answer 6

• DNA synthesis, DNA ligase for okozaki fragments

Question 7

cdc28:

Answer 7

• Cyclin dependent kinase

- G1 - S

Question 8

Look under the microscope to see the effects of the block:

Answer 8

• Cdc8: effects in mitotic and meiotic DNA replication
• Cdc24: bud formation, nuclei keep on dividing, but the cells do not divide
• Cdc10: septin ring of the motherbud neck required fro cytokeneses
• Cdc15: cannot exit mitosis, so there is no cytokineses

Question 9

Ordering action of cdc functions:

Answer 9

• Double mutants allow us to figure out which step is first
• Cdc28 cannot form buds
• Cdc7 cells arrest with buds
• Which of these acts first? Cross them, the double mutant has the phenotype of Cdc28, so Cdc28 functions before Cdc7

Question 10

Cloning of cdc genes:

Answer 10

• By complementation for growth at the non-permissive temperature using a library in a shuttle vector
• Human cDNA library in S.pombe expression vector (no introns)
• Transformed into cdc2 temperature sensitive mutant and selection for growth at 36 degrees
• Complementing colonies contained plasmids expression human cyclin dependent kinase

Question 11

Aspergillus nidulans temperature sensitive mutants:

Answer 11

• Germinating conidia have one nucleus, it is a haploid organism
• Asexual spores can germinate when placed on a medium
• Nuclei divide and go through the hyphae, released as conidia
• Temperature sensitive mutants that can grow at 32 but not at 42
• Screen mutants under the microscope for temperature effects on conidial germination

Question 12

nim mutants:

Answer 12

• never in mitosis

Question 13

bim mutants:

Answer 13

• blocked in mitosis

- have formed mitotic structures but don’t move beyond this

Question 14

nud mutants:

Answer 14

• nuclei have divided but haven’t migrated into the hyphae

- nuclear migration defective

Question 15

nims, bims, nuts, were clones and characterised. What did this study?

Answer 15

• Nuclear migration and associated machinery

Question 16

Cyclin synthesis and degradation:

Answer 16

• Cyclins are degraded throughout the cell cycle
• By fuses Gfp to cyclins in a cell you can measure levels of cyclins throughout the cell cycle
• Cyclins increase for bud formation, then are degraded

Question 17

The length of G2 determines what?

Answer 17

• The cell size

- If G2 is short (like in wee mutants) the cell will be short

Question 18

Wee 1 encodes a kinase:

Answer 18

• Wee-kinase stops the cyclin from becoming active by phosphorylating tyrosine 15 (part of the cdk complex)
• Tr15 is phosphorylated by Wee1, so it cannot operate to go through the cell cycle
• This can be reverse with Cdc25 which chops off the phosphorylation

Question 19

Effects of Cdc2 mutations:

Answer 19

• Cdc2+: WT normal cells
• Cdc2-: no cell division at all, always phosphorylated at tyrosine 15
• CdcD: never phosphorylated at tyrosine 15, so cells are small

Question 20

Control of G2 to M:

Answer 20

• M-cyclin (for entry into mitosis) associates with a Cdk and is phosphorylated (to make it active) by the Cdk-activating kinase
• Wee1, by phosphorylating Tr15, stops it from being active, so it is inactive
• So because it is inactive it progresses through G2
• After a certain length of time a phosphorylation signal activates phosphatase (cdc25) which chops of the Ty15 phosphatase, and the positive feedback signals maintain this situation, so cdc25 is maintained as phosphorylated

Question 21

Cellular checkpoints:

Answer 21

• DNA damage checkpoint at G2-M
• If DNA damage occurs (ds break or block of replication from pyrimidine dimers) cdck activity is inhibited
• The cell can pause at G2 to give time for repairs

Question 22

Checkpoint mutants:

Answer 22

• WT: normal
• Cdc mutants: the cell will be really long, because G2 can’t go into M
• Wee mutants: the cell will be really small at the G2 phase is too short and M is entered too quickly
• Mitotic mutants: such as a septum through the nucleus or the nucleus fragmented etc

Question 23

If erros occur due to failure of check points then damage an result due to:

Answer 23

• Mutations due to lack of DNA repair
• Chromosome aberrations
• Loss/gain of chromosomes (aneuploidy, disomy)
• This can lead to loss/gain of gene function
• Inappropriate gene expression
• Change in gene dosage
• This may result in further accumulation of errors

Question 24

The start check point:

Answer 24

• Between G1 and S
• Now nutrients, Stop in Go phase (not active, just sitting there)
• Pass start and enter cell cycle of happy
• Stop and mate if the partner of opposite mating type is present (mating type pheromones and cell fusion)

Question 25

G1 - S checkpoint details in s. cerevisiae:

Answer 25

• S-phase cyclin complex made up of Sic1 and cdc28 are complex with G1 cyclin and cdc28
• G1 cyclin complex phosphorylates the S cyclin complex,
• Cdc32 and SCF promote the degradation of the Sic1 by ubiquitin dependent proteolysis
• S-phase cyclin triggers S phase entry and DNA replication follows

Question 26

CKI:

Answer 26

• Cycline dependent kinase inhibitor proteins
• Associate with S cyclin and inhibits its activity
• Controlled by phosphorylation followed by ubiquitin dependent proteolysis