L1 - introduction to cell cycle Flashcards
(28 cards)
What is the cell division cycle made up of?
G1
S
G2
M
Has a driven order
What are GAP phases?
Occur in-between DNA synthesis and mitosis
G1
G2
What is the difference between S. cerevisiae and S. pombe?
Are very different from each other
S. cerevisiae grows by budding
S. pombe grows by elongation
Are both easy to manipulate
Are evolutionary very far apart - ~600 million years
If we can show similarities between the 2, probably means humans have the same similarity
What are permissive conditions?
Conditions under which the mutant strain will grow
What are restrictive or non-permissive conditions?
Conditions under which the mutant strain will not grow
What is a temperature sensitive mutation?
A mutation that shows a phenotype at a temperature at which the wild type grows
What are the types of temperature sensitive mutations?
ts - temperature sensitive
the yeast mutant doesn’t grow at high temperatures (35-37) but will grow at normal permissive temperatures (28-30)
cs - cold sensitive
the yeast mutant doesn’t grow at low temperatures (15-20) but will grow at the normal permissive temperatures (28-30)
Why is temperature important in conditions?
Most likely affects the stability/folding of the mutant protein influencing the function of the protein
Life cycle of S. cerevisiae
Nucleus migrates to a new bud
Formation of the mitotic spindle across the bud neck
Mother cell is bigger than daughter cell when it divides
• creates problem for the cell
• can see exactly where the cell is in the cell cycle
S. CEREVISIAE
Morphology of asynchronous cultures of a wild type strain, a cdc28-ts mutant strain and a noncdc-ts mutant strain growing at the permissive temperature (28 degrees)
Wild type strain - cell number keeps increasing
Cdc28-ts mutant strain - cell size almost doubles before stopping
Noncdc-ts mutant strain - no chance from the wildtype
If the mutation is involved in the cell cycle, all the cells will get stuck in the same place - stop passing a specific place in the cycle
Life cycle of S. pombe
Instead of forming a bud, it elongates - diameter doesn’t change but length does
Nucleus tends to lie in the middle of the cell
Cell divides equally in mitosis
S. POMBE
Morphology of asynchronous cultures of a wild type strain, a cdc28-ts mutant strain and a noncdc-ts mutant strain growing at the permissive temperature (28 degrees)
Wild type strain - cell number keeps increasing
Cdc28-ts mutant strain - cell size almost doubles before stopping
Noncdc-ts mutant strain - same as the wild type
What are Cdc mutations?
Cell division cycle mutations
What do Cdc mutations show?
- Define genes necessary for transit through specific points in the cycle
- The block marks the point beyond which the cells cannot progress
- Block point may not be the same point where the wild type gene normally acts in the cycle
- Mutations isolated are conditional mutations
- Different allele identification may identify several functions of a protein
What is a conditional mutation?
A mutation that has wild-type (or less severe) phenotype under certain “permissive” environmental conditions and a mutant phenotype under certain “restrictive” conditions
phenotype may not be the same as that of the null
• partial functions may leak through or change the function rather than destroy it
Problems with Cdc mutations
Gene pathway redundancy
Mutation is very rare
The Cdc gene has many cell cycle functions
FACS analysis
Fluorescent Activated Cell Sorter
1) Cells are stained with a fluorescent dye which binds DNA
2) Cells are then analysed in the FACS to study the status of the DNA content of the cell population in the cell cycle
3) Fluorescent signal detected by the FACS is directly proportional to the DNA content of the cell
Why is synchrony important in studies of the cell division cycle?
Determining cell cycle fluctuations in protein and RNA synthesis
Examining potential cell cycle protein modifications
Examining protein associations
Examining localisation of proteins
How are cell cultures synchronised?
- Centrifugal elutriation which separates cells bases on cells
• Cells tend to be smaller at the beginning of the cycle so can be centrifuged out and then synchronised - Shifting Cdc-ts mutants to the restrictive temperature then releasing them from the block
- Chemical treatments - eg. hydroxyurea
- In S. cerevisiae, alpha mating factor blocks ‘a’ type haploid cells in G1 phase
Problems with synchronisation
Physiological consequences of perturbing (causing anxiety) the cell cycle
What are the 2 models for the cell cycle regulation?
Sequential steps
Sequential steps and parallel pathways
What happens if you block Cdc-28?
It blocks everything
• Budding
• DNA replication
• Spindle pole body duplication
What happens if you put Cdc-24 to the non-permissive temperature?
It blocks budding
What happens if you put Cdc-7 to the non-permissive temperature?
It blocks DNA replication
