14.1 Cellular Reproduction Flashcards
Basics of the Cell Cycle
Mitotic M Phase
Interphase
•G1 cell checks for mutations and DNA repair mechanisms occur here before S phase when replication
•S synthesis
•G2 pause so cell can grow and prepare
2 checkpoints: G1 to S and G2 to M
3 Categories of Cells
Mitoticly inactive
- Specialized that have lost ability to divide
- Muscle cells, red blood cells
Induced
- Cells that can be induced to divide
- Liver cells, lymphocytes
Mitotically active cells
- Epithelial cells, hematopoietic stem cells
- Always active
2 Cell Fusion Experiments
Expt: Fusion of G1 cells with S-phase cells
Result: G1 nucleus stimulated to undergo replication pushing them into S phase. Something in the cytoplasm of S that’s influencing the G cells
Expt: Fusion of G2 cells with S-phase cells
Result: G2 nuclei not stimulated to undergo replication. S has no effect on G2 cells. Implications that there’s fairly tight control of how many times a cell will go through a replicative cycle. So if its already gone through it wont again. 1 duplication per cycle.
Mitotic Factors Induce chromosomal compaction
•Chromatin of G1 phase nucleus undergoes premature chromosomal compaction to form elongated compacted chromosomes.
•S phase chromatin is also compacted but appears pulverized due to fragile nature of S-phase chromatin
most compact form of a chromo is Mitotic chromo. •But if they’re fused, they start compacting. Therefore something in the M cells that are causing G1 to compact more.
- M-S fused, get pulerized and break up into fragments. S cells are the most loose of cells, so if you try to influence them to compact, it actually fragments the DNA instead.
- G2 nuclei chromatin becomes compacted.
- M-G2 fused. influences them to start packing tighter.
Role of Protein Kinases
• Entry into M-phase initiated by protein kinase
•“Maturation-Promoting Factor (MPF)”
•MPF consists of two subunits
-Catalytic: Kinase
–Regulatory: Cyclin
•Kinase activity dependent on cyclin levels
• kinase are CDK Only active in the presents of cyclin (the regulatory component. cyclin is a protein,)
Discovery of MPF Experiment
Look at maturation in eggs. they have eggs not progestone treated and another group that is progesterone treated.
extract cytoplasm from the eggs at dif times. Extracted from egg that was treated with progestorne. Take cytoplasm and put into egg not treated with progestrone. At 20hrs they get an induction of maturation. Implies that there’s maximun maturation induction levels at about 20hrs.
MPF Activity Cycles in Amphibian Eggs
- Follow up experiment. Effect of fertilization on eggs. MPF is maximal at stages just before clevage.
- MPF activity present in cytoplasm 90 minutes after fertilization
- Activity correlates with cell divisions (cleavages)
Cyclin Protein Levels Fluctuate with Cell Cycle
dividing cells, sea urchin eggs. Ran protein gels, protein extracts at dif time frames in SDS page gel. Looked for proteins that would cycle up and down. Protein A is coincident with cleavage (dashed line). Protein B just keeps going up. Protein A was then referred to as cyclin a protein that cycled the same as cell division.
MPF Activity
- MPF consists of two polypeptides
- 32 kDa and 45 kDa
- 45 kDa protein becomes labeled with with 32P (i.e. cyclin)
- Yeast produce 34 kDa protein with kinase activity (cdc2)
- Antibodies from cdc2 react with amphibian 32 kDa protein
- 32 is kinase activity and 45 is cyclin component
Cyclin-Dependent Kinases
•Cdks first studied in yeast Temperature sensitive mutants identify two stop points -End of G1 -End of G2 •Fission Yeast -cdc2 responsible
Cell Cycle Regulation in Yeast
Two groups of cyclins Cdc2 kinase works with different classes of kinease. The first class of kinase is G1/S that promotes cell to go from G1 to S. Different cyclins can regulate kinase for what it targets, in this case the target is the ORC.
Mitotic cyclase, will change the specificity of the enxyme. One target is lamin proteins which help form the nuclear membrane. Memebrane breaks down due to phosphorylation of the lamin. More than one type of cyclin.
3 Regulation of cdks
Cyclin Concentration
-Differential transcription of cyclins at stages of cell cycle
Controlled proteolysis
- Cyclin concentration regulated by controlled synthesis and degradation
- SCF and APC complexes link cyclins to polyubiquitin chain (degradation signal)
Cdk phosphorylation state
-Kinases CAK and Wee1
Phosphatase Cdc25
cdk Phosphorylation State
1) cdc2K interacts with mitotic cyclin but remains inactive because phosphorylation of a tyrosine residue Tyr15 by Wee1.
2) CAK trasferì a phosphate to Thr161, needed for cdc2K activity. When cell reaches critical size, Cdc25 phosphatase enzyme active and removes inhibitory phosphate of Tye 15. Cdc2K active so drives cell into mitosis.
3) End of mtiosos. phosphate group removed from Thr161 by a phosphatase. Free cyclin degraded. Cell begins another cycle
Cdc2K is effected by 2 kinases CAK and Wee1
Yeast Cell Cycle Mutants
•Wild Type:
Cell through G2 grows and gets bigger then M divides into 2
•Wee1- mutant:
doesn’t become doubly phosphorylated so skips through and doesn’t grow. Very short G2 so very little time to grow. It divides early.
•Cdc25- mutant:
never switches from inactive to active. So continues to grow and will never divide. Turns into a giant cell that will never divide.
Subcellular Localization of Cyclin (Cyclin B1)
- Cyclin B1 accumulates in the nucleus just prior to mitosis
* Phosphorylation of nuclear export signal (NES) blocks export from nucleus
