Eukaryotic Cell Cycle L13/14 Flashcards
Eukaryotic What needs to be equally partitioned when a cell divides into two daughter cells?
The genetic material
The organelles
In order to divide (go from G1 to S phase), cells need to receive a stimulus. What can that stimulus be?
Growth factors (usually)
Nutrients (sometimes, depending on cell)
What does the quiescence / quiescent stage refer to?
Gap phase, G_0
How long can cells remain dormant for in G_0
Weeks, months or even years for some cell types
Cells will be exposed to growth factors in what situations?
In response to damage, or the need to grow or repair
When cells produce/activate cyclin dependent kinases what happens to them?
Cell re-enters the cell cycle
What phase of the cell cycle is DNA copied?
G1 phase
When does the cell check to make sure the chromosomes have been duplicated properly?
G2 phase
How many centrosomes and copies of DNA are present in a cell in G1 phase?
1
In what phase do we get duplication of centrosomes?
S phase
When do centrosomes move to opposite poles and begin to form the mitotic spindle?
Moving into G2 phase
When do cells duplicate their DNA and organelles?
S phase
When in the cell cycle does the nuclear membrane break down?
G2 - M transition
In what phase does the mitotic spindle fully form?
G2 - M transition
When in the cell cycle do the nuclear membranes of cells reform, and the mitotic spindle collapse?
Telophase
When in the cell cycle do the centrosomes partition
Cytokinesis
What is the difference between biochemists and genetisists approach to investigating something in biology?
Biochemistry: Isolating factors that can recapitulate an activity in a test tube
Genetics: Genetic changes you can make to a cell’s DNA (eg; inactivating a gene) to prevent it from carrying out a process
What happens when you fuse an M phase cell with a cell at any other stage of the cell cycle? What does this show?
Spontaneous condensation of the chromosomes.
There is a soluble mitosis promoting factor in M phase cells, which forces cells into mitosis
What happens when you fuse an S phase cell with a G1 phase, or a cell in G2 phase? What does this show?
Pushes G1 cell into S phase
G2 cells don’t respond
There is a soluble factor promoting S phase entry, but can only affect G1 (temporal control)
What does MPF stand for?
Maturation Promoting Factor (Mitotis promoting factor)
What enzyme controls maturation of oocytes and both meiotic and mitotic divisions?
Maturation Promoting Factor (MPF)
When does MPF activity increase?
When progesterone is added to a G2 arrested cell
Peaks at Meiosis I (then falls)
Peaks at Meiosis II (stays high until falling after fertilisation)
Peaks at every subsequent mitosis
If cyclin B is not destroyed, what happens to cells?
They remain arrested in mitosis
What is cyclin B required for?
Mitotic entry
What is destruction of cyclin B required for?
Exit from mitosis
Why were xenopus (african tree frogs) and sea urchins used for identification of proteins required for G2/M transition?
Their oocytes are:
Relatively large, easy to manipulate
High protein content
Available in high numbers
Naturally synchronised
Rapid synchronous cell divisions following fertilisation
Easier to create cell extracts for biochemical analysis
How was autoradiography used to identify cyclin?
Sea urchin oocytes were fertilised and 35S Methionine was added
The Methionine is only incorporated into newly produced proteins
Protein extracts were taken at different time points postfertilisation and analysed using SDS-PAGE gel
Autoradiography shows new proteins
If cells get stuck at mitosis, what might have happened?
Cyclin B might not have been destroyed
In what organism was cyclin dependent kinase identified?
Yeast
What is schizosaccharomyces pombe?
Fission yeast
What is S. cerevisiae
Budding yeast
How can you tell if S. cerevisiae has arrested in G1?
They will not have a bud
What mutant phenotype for yeast did Lee Hartwell use to identify genes required for cell progression?
Temperature sensitive mutants
What are Yeast TS mutants?
Yeast temperature sensitive mutants, which can not grow at non-permissive temperatures
What are non-permissive temperatures for yeast TS mutants?
Temperatures >35 degrees
The mutants cannot grow / replicate at higher temperatures
What is genetic complementation?
Adding genes back into mutants to see which ones recover lost functions, such as certain resistances to temperature
What are cdc mutants?
Cell division cycle mutants
A plasmid containing which gene was able to rescue TS mutant budding yeast (mutant’s version of gene not working) ?
cdc28 (italics because its a gene)
What does SPF stand for?
S-phase promoting factor
What is SPF required for?
SPF is critical to bypass the G1/S phase transition. (Bypass restriction point [mammals] and START [yeast])
What is MPF required for?
MPF is critical to bypass the G2/M phase transition
What does cdc28 code for?
A cyclin dependent kinase
What is the name of the point cells decide to go from G1 to S phase in mammalian cells and in yeast cells?
Mammalian: Restriction point
Yeast: START
As well as growth factors, what do yeast require to divide?
Sufficient nutrients in their environment
How does the TS mutation for cdc28 affect the cyclin dependant kinase?
Affects protein stability, making it more susceptible to thermal denaturation
Affects kinase’s affinity for cyclin (which it depends on for activation), and at high temperatures they dissociate -> loss of kinase function
How did increasing abundance of G1 cyclin (overexpression) rescue the cdc28 phenotype and lead to identification of the cyclin dependent kinases?
More cyclins meant the kinases were more likely to form complexes and activate, despite their instability (from low affinity)
This allowed complementation, and therefore identification of the presence of cyclin dependent kinases
Compare the variation in kinases and cyclins throughout the cell cycle
Only one kinase is used through the cell cycle, but each phase of the cell cycle is regulated by different cyclins
How can you tell through cell morphology what stage of the cell cycle fission yeast is in?
The length of the cell
Cdc mutants in S. pombe identified arrest at which stage of the cell cycle?
G2/M transition
(They were long cells)
Gene complementation for identifying cell cycle regulators was performed for the _______ using the gene library previously used for the _______ .
S. pombe (Fission yeast)
S. cerevisiae (Budding yeast)
A plasmid containing which gene was able to rescue TS mutant fission yeast (mutant’s version of gene not working) ?
cdc2 (italics because its a gene)
The cyclin dependent kinase controls what in budding yeast and fission yeast? What does this show?
G1/S transition in budding yeast
G2/M transition in budding yeast
G2/M transition in fission yeast
Shows there is a functionally conserved kinase across both yeasts, and potentially in higher eukaryotes
What are the names of the conserved cyclin dependent kinase in budding yeast, fission yeast, and humans?
Budding: Cdc28
Fission: Cdc2
Human: CDK1
What is the phenotype of a cdc2- (knockout for cdc2)?
Cell is unable to enter mitosis
Cell growth continues, resulting in overlarge cells
What happens if cdc2 cannot be phosphorylated?
Cells enter mitosis rapidly (force cells into mitosis (similar to MPF))
Cell growth is insufficient, causing very short cells (wee phenotype)
What is cdc^D?
Constitutively active cdc2 where a negatively charged amino acid is put in phosphorylation site, preventing the kinase from being phosphorylated
What regulates mitotic CDK?
Cyclin binding
Wee 1 kinase (adds phosphates)
Cdc25 phosphatase (removes phosphates)
What affect on mitosis does having a deficit of Cdc25 or excess of Wee1 have?
Prevents mitosis
Elongated cells (due to increased G2)
What affect on mitosis does having an excess of Cdc25 or deficit of Wee1 have?
Inappropriate mitosis
Small cells (decreased G2)