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Flashcards in Lecture 27 Deck (26):

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


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


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



- Initiation of DNA synthese



- mitosis: mircotubules



- DNA synthesis, DNA ligase for okozaki fragments



- Cyclin dependent kinase
- G1 - S


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


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


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


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


nim mutants:

- never in mitosis


bim mutants:

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


nud mutants:

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


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

- Nuclear migration and associated machinery


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


The length of G2 determines what?

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


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


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


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


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


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


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


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)


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



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