Lecture 27 Flashcards
1
Q
The isolation of cdc mutants:
A
- 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
Q
Once temperature sensitive mutants are isolated we have to determine which ones are mutants effected specifically in the cell cycle:
A
- 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
Q
Cdc mutants (in both pombe and sacromyces):
A
- 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
Q
cdc4:
A
- Initiation of DNA synthese
5
Q
cdc16:
A
- mitosis: mircotubules
6
Q
cdc9:
A
- DNA synthesis, DNA ligase for okozaki fragments
7
Q
cdc28:
A
- Cyclin dependent kinase
- G1 - S
8
Q
Look under the microscope to see the effects of the block:
A
- 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
Q
Ordering action of cdc functions:
A
- 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
Q
Cloning of cdc genes:
A
- 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
Q
Aspergillus nidulans temperature sensitive mutants:
A
- 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
Q
nim mutants:
A
- never in mitosis
13
Q
bim mutants:
A
- blocked in mitosis
- have formed mitotic structures but don’t move beyond this
14
Q
nud mutants:
A
- nuclei have divided but haven’t migrated into the hyphae
- nuclear migration defective
15
Q
nims, bims, nuts, were clones and characterised. What did this study?
A
- Nuclear migration and associated machinery
16
Q
Cyclin synthesis and degradation:
A
- 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
Q
The length of G2 determines what?
A
- The cell size
- If G2 is short (like in wee mutants) the cell will be short
18
Q
Wee 1 encodes a kinase:
A
- 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
Q
Effects of Cdc2 mutations:
A
- 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
Q
Control of G2 to M:
A
- 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
Q
Cellular checkpoints:
A
- 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
Q
Checkpoint mutants:
A
- 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
Q
If erros occur due to failure of check points then damage an result due to:
A
- 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
Q
The start check point:
A
- 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
Q
G1 - S checkpoint details in s. cerevisiae:
A
- 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
Q
CKI:
A
- Cycline dependent kinase inhibitor proteins
- Associate with S cyclin and inhibits its activity
- Controlled by phosphorylation followed by ubiquitin dependent proteolysis
