Comprehensive Lecture Notes on the Cell Cycle, however some of the experimental procedures are all documented well but the findings from the experiments are all present.
Contents can be seen on the first page.
Summary is that it includes:
- Regulation of the G2/M checkpoint
- Regulation...
Cell Cycle
CELL CYCLE.................................................................................................................................................... 1
INTRODUCTION............................................................................................................................................ 3
TRADITIONAL METHODS OF STUDYING THE CELL CYCLE..................................................................................................3
MODEL SYSTEMS FOR STUDYING THE CELL CYCLE..........................................................................................................3
PHASES OF THE CELL CYCLE......................................................................................................................................3
DIFFERENT TYPES OF CELL CYCLES.............................................................................................................................3
CHECKPOINTS IN THE CELL CYCLE..............................................................................................................................4
REGULATORS IN CELL CYCLE....................................................................................................................................4
Dominant M-phase Factor:............................................................................................................................4
Dominant S-phase factor:...............................................................................................................................4
Block to DNA re-replication:...........................................................................................................................5
REGULATION OF THE G2/M CHECKPOINT...................................................................................................... 5
IDENTIFICATION OF MPF........................................................................................................................................5
Discovery of Maturation Promoting Factor (MPF): the dominant M-phase factor.......................................5
Polar Bodies....................................................................................................................................................6
IDENTIFICATION OF MITOTIC CYCLIN..........................................................................................................................6
GENETIC ANALYSIS OF THE CELL CYCLE......................................................................................................................7
Isolation of cell division cycle (cdc) mutants..................................................................................................7
Characterization of cell cycle mutants...........................................................................................................7
Cloning by complementation of cdc2ts, wee1ts, cdc25ts and cdc13ts mutants.................................................7
What did the cloning tell us?.....................................................................................................................................8
Cloning of wee1, cdc25 & cdc13.....................................................................................................................8
MITOTIC PROMOTION FACTOR (MPF) = P34CDC2 & CYCLIN B.........................................................................................9
CELL CYCLE.................................................................................................................................................... 9
Raising antibodies to cdc2..............................................................................................................................9
i. Does the anti-cdc2 antibody cross-react with a protein in the partially purified Xenopus MPF fraction?..9
ii. Do any other proteins in the MPF fraction co- immunoprecipitate with the anti-cdc2 antibody?............9
Mechanism of co-immunoprecipitation assay:.........................................................................................................9
Results:.........................................................................................................................................................10
iii. Does the co-immunoprecipitate have MPF activity?...............................................................................10
Results:.................................................................................................................................................................... 10
DOSAGE EFFECT OF WEE1, CDC25 AND CDC2 ON THE CELL CYCLE................................................................................10
Results:.........................................................................................................................................................11
ROLE OF CYCLINB................................................................................................................................................ 11
In Vitro assay for cell cycle...........................................................................................................................11
Findings:.................................................................................................................................................................. 11
Is the mitotic cyclin B required for MPF activity?.........................................................................................12
Is cyclin B degradation required for exit from the cell cycle?.......................................................................12
Regulation of cyclin B degradation..............................................................................................................12
PHOSPHORYLATION REGULATION:...........................................................................................................................13
Experimental Questions:...............................................................................................................................13
Experimental Tools:......................................................................................................................................13
Experimental procedure:..............................................................................................................................13
1. Does the phosphorylation status of Cdc2 change during the G2/M transition?......................................14
2. Which amino acids are phosphorylated on Cdc2?...................................................................................15
Assay:......................................................................................................................................................................15
Which tyrosine is phosphorylated and dephosphorylated?....................................................................................15
Inhibitory effect of Y15-P:.......................................................................................................................................15
Wee1 & Mik1 phosphorylate cdc2 (cdk1) on Y15.........................................................................................16
Analysis of mik1 and wee1 mutants........................................................................................................................16
Cdc25 dephosphorylation of cdc2(cdk1)......................................................................................................16
Assay:......................................................................................................................................................................16
WHAT ARE THE TARGETS OF ACTIVE MPF?..............................................................................................................17
SUMMARY:.........................................................................................................................................................17
, Cell Cycle
REGULATION OF THE G1/S CHECKPOINT...................................................................................................... 17
CDC2/CDC28 IS REQUIRED FOR START (THIS CHECKPOINT).........................................................................................17
Budding Yeast Life Cycle...............................................................................................................................17
IDENTIFICATION OF G1 CYCLINS:.............................................................................................................................18
Genetic/Mutant screen in S. cerevisiae........................................................................................................18
Dosage effects of G1 cyclins.........................................................................................................................18
CELL CYCLE IN S. CEREVISIAE..................................................................................................................................18
IDENTIFICATION OF MAMMALIAN CYCLINS & CDC2 HOMOLOGUES (CDK1, CDK2…CDK5)...................................................18
Cyclin D is required for the G0 to G1 transition in mammalian cells............................................................19
IDENTIFICATION OF MAMMALIAN CYCLIN-DEPENDANT KINASES (CDKS)..........................................................................19
REGULATION OF MAMMALIAN CELL CYCLE...............................................................................................................20
IDENTIFICATION & CHARACTERIZATION OF CDK INHIBITORS..........................................................................................20
Identification of p16 in association with cdk4..............................................................................................20
Assay for cdk4/cyclinD activity.....................................................................................................................20
TUMOUR SUPPRESSORS: RB & P53.............................................................................................................. 21
CDK INHIBITORS P16, P21, P27.............................................................................................................................21
CANCER GENETICS AND THE RETINOBLASTOMA GENE.................................................................................................21
Action of Rb Protein......................................................................................................................................21
P53 – MOST FAMOUS TUMOUR-SUPPRESSOR GENE...................................................................................................22
DNA DAMAGE............................................................................................................................................. 23
DNA DAMAGE RESPONSE GENES...........................................................................................................................24
DNA DAMAGE CONTROL AT G2/M CHECKPOINT.....................................................................................................24
Experimental Evidence:................................................................................................................................24
DNA DAMAGE CONTROL AT G1/S CHECKPOINT.......................................................................................................27
Question: When cells are exposed to gama-irradiation, do Cdc25A levels affect progress through the S-
phase?..........................................................................................................................................................27
P53 Independent Pathways..........................................................................................................................28
P53 Dependant Pathways............................................................................................................................28
CANCER...................................................................................................................................................... 29
........................................................................................................................................................................ 29
PROPERTIES OF CANCER........................................................................................................................................30
Multi-Hit model............................................................................................................................................30
Cancer Phenotype.........................................................................................................................................31
TUMOUR SUPPRESSOR GENES................................................................................................................................31
Properties:....................................................................................................................................................31
PROTO-ONCOGENES AND ONCOGENES....................................................................................................................31
DNA TUMOUR VIRUSES........................................................................................................................................32
CANCER TREATMENT & DIAGNOSIS............................................................................................................. 32
IONIZING RADIATION:...........................................................................................................................................32
CHEMOTHERAPY..................................................................................................................................................33
Mechanisms of action of different types of Drugs in Chemo:......................................................................33
HORMONAL TREATMENTS.....................................................................................................................................34
CANCER DIAGNOSIS.............................................................................................................................................34
COMBINE RADIATION TREATMENT WITH WEE1 INHIBITORS..........................................................................................34
, Cell Cycle
Introduction
Traditional methods of studying the cell cycle
DNA Replication
- 3H-Thymidine incorporation
o Inject autoradiated thymidine into cell then use autoradiography to see if its
been incorporated into the DNA
- Artificial Thymidine analog Bromo-deoxyuridine (BrdU)
o Readily incorporates opposite Adenines.
o If cell is replicating then BrdU will be incorporated
o Fluorescently labelled anti-BrdU antibodies will show up if cell replication has
occurred.
Model systems for studying the cell cycle
- Mammalian cells proliferating in culture
- Genetic analysis in budding yeast
- Animal embryos: Xenopus & sea urchin eggs
Phases of the cell cycle
G1 Phase:
- Immediately after mitosis
S phase:
- DNA Replication
G2 Phase:
- Before mitosis
M Phase:
- Mitosis
G0 Phase:
- After mitosis if cell leaves cycle and stops dividing
Interphase:
- G1 + S + G2
- DNA is diffusely distributed within nucleus
Different types of Cell Cycles
- Embryonic Cell cycles
o Short cycles of constant duration
o Very synchronous
- Growing cell cycles
o Longer cycles of variable duration
, Cell Cycle
- Reversible exit from cell cycle
o Some cells stop growing and leave the cycle until interacted with a growth
factor etc.
Checkpoints in the Cell Cycle
Regulators in Cell Cycle
Dominant M-phase Factor:
- 1970s: Cell fusion experiments showed that there were different regulators at
different stages of the cell cycle
- Active during Mitosis
- Synchronized mammalian cells, mixed cells in presence of virus or chemical agent
that caused their membranes to fuse- formed hybrid cells
- Results:
o G1 fused with M phase cells = chromatin immediately condensed
o G2 fused with M phase cells = chromatin immediately condensed
o S phase fused with M phase cells = chromatin immediately condensed
- Conclusion:
o Dominant M-phase factor
o Mitotic cells must contain a diffusible factors that induce mitosis when
introduced to cells at other stages of the cell cycle
Dominant S-phase factor:
- When G1 and S cells fused
- G1 cells immediately began to replicate their DNA
- Conclusion:
o Factors from S phase cell must induce DNA replication in G1 nucleus – hybrid
cell delays mitosis until replication has completed.
o S-phase factor induces DNA replication
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