Lecture 27 Flashcards Preview

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Flashcards in Lecture 27 Deck (26):
1

The isolation of cdc mutants:

- 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

2

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

- 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

3

Cdc mutants (in both pombe and sacromyces):

- 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

4

cdc4:

- Initiation of DNA synthese

5

cdc16:

- mitosis: mircotubules

6

cdc9:

- DNA synthesis, DNA ligase for okozaki fragments

7

cdc28:

- Cyclin dependent kinase
- G1 - S

8

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

- 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

9

Ordering action of cdc functions:

- 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

10

Cloning of cdc genes:

- 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

11

Aspergillus nidulans temperature sensitive mutants:

- 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

12

nim mutants:

- never in mitosis

13

bim mutants:

- blocked in mitosis
- have formed mitotic structures but don't move beyond this

14

nud mutants:

- nuclei have divided but haven't migrated into the hyphae
- nuclear migration defective

15

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

- Nuclear migration and associated machinery

16

Cyclin synthesis and degradation:

- 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

17

The length of G2 determines what?

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

18

Wee 1 encodes a kinase:

- 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

19

Effects of Cdc2 mutations:

- 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

20

Control of G2 to M:

- 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

21

Cellular checkpoints:

- 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

22

Checkpoint mutants:

- 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

23

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

- 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

24

The start check point:

- 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)

25

G1 - S checkpoint details in s. cerevisiae:

- 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

26

CKI:

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