Shemank Lecture 11 Flashcards
What are the two landmark phases in the eukaryotic cell cycle?
The S-Phase: DNA synthesis phase; the genome is replicated
The M-phase: Mitotic phase: chromosomes are segregated into daughter cells
How can DNA replication be monitored?
DNA replication can be monitored by the incorporation of 3HT (radioactive thymidine).
The percentage of cells in any activity is a measure of what?
The percentage of time spent in that activity
The length of the S phase is calculated how? How about the length of the M phase?
From the percentage of cells labelled with 3HT.
The length of the M phase can be calculated from the percentage of cells seen to be in mitosis or cytokinesis
How do you find the time it takes to complete the G2 phase?
You would see how long it takes the labelled thiamine cells in the S phase to pop up as mitotic cells
What is a fluorescence activated cell sorter?
Cells were stained with propidium iodide that fluoresces when it binds DNA, the amount of fluorescence is directly proportional to the amount of DNA in each cell.
We use a cell sorter and flow cytometer to detect cells, cells drop into tubes and a camera counts them.
Why is there 2x the amount of DNA in the G2 and M phases but way more cells in the G1 phase?
Because the G1 phase went through all of mitosis and divided, but the G2 and M phases didn’t yet so the dna is just doubled in them from the S phase.
Do mitotic cells contain factors that push other cells to compact (enter mitosis)?
Yes
What experiment demonstrates that cells contain factors that stimulate entry into mitosis?
Hela cells from the M phase and PtK2 cells from G1 were fused, the G1 chromosomes impacted prematurely and the m phase cells converted the G1 cells to a m phase like cell.
Hela cells from the M phase and PtK2 cells from S phase were fused, the m phase cells tried compacted but the S phase cells were too fragile and got pulverized.
M phase hela cells and PtK2 cells in G2 and saw the G2 cells were compacted.
so something in m phase cells pushes other to compact and behave as if they are m phase cells.
What factor in M phase cells pushes cells to compact?
The maturation promotion factor- is a protein kinase
What is the maturation promoting factor?
Is what causes compaction of cells and triggers entry into the M phase by activation of a protein kinase. It has two subunits a kinase and a regulatory subunit called cyclin, increased concentration of cyclin activates the kinase.
When cyclin concentration is low what happens to the kinase?
The kinase lacks the cyclin subunit and the maturation promoting factor is inactive
When checking concentration is high what happens to the kinase MPF?
It’s activated and the entry into mitosis is initiated
How is cell cycles regulated in yeast?
At the start of the G2-M transition the MPF kinase (cdc2 kinase) is activated by cyclin which binds to it making a complex, then the cyclin goes down at the end of mitosis, then the Cdc2 kinase begins again at the end of G1 through binding with G1/S cyclins as they increase in the S phase.
what role do other protein kinases play in the activation of cdc2 kinase?
The cdc activation kinase (CAK) phosphorylates a threonine and Wee1 phosphorylates a tyrosine on the Cdc2 kinase subunit for cyclin to bind to it, cdc25 removes an inhibitory phosphate and causes cyclin to be active and the cell gets driven to mitosis
