Lecture 6 Flashcards
What aspect of development is vulnerable for mutation ?
Regulated inhibition
if you lose control of the cell cycle what is the main process you lose control of ?
Proliferation
Basic premise of the cell cycle
cell cycle is a programmed series of events enabling a cell to duplicate contents and generate 2 daughter cells
Cell cycle progression occurs via changing levels of activity of what ?
Cyclin-dependent protein kinases
Alterations in normal function of what is implicated in cancer prognosis?
of cdk regulators are implicated in cancer prognosis
How many critical experiments laid the foundations of cell cycle phases ??
4
What helps cell counting?
Known doubling time
What is cell cycle order ?
G1 > S >G2 > M
What techniques would you use to test -How do we know how long each phase takes ??
How do we know what stage they’re in ?
Functional in vivo assay or use morphological marker
Pulse chase experiment
take up phosphate – taken up into DNA if cells making DNA amount is therefore a lot
- into sugar phosphate backbone
- if undergoing DNA replication cell will incorporate radioactive phosphate
- then expose to radio film - goes black
What can you do with pulse chase experiment results ??
If you work out proportion of cells on a dish positive for DNA replication
- Number of cells doing something at any one time multiply fraction by doubling time tells you how long the phase lasts for
mitosis in mammalian cells takes how long
roughly an hour
at any one time what percentage of cells is expected to be doing mitosis ??
5%
Halogenated Deoxyuridine mimics what
Thymine
what experiment can you use to test whether DNA replication is taking place ??
Expose cells to DU followed by follow up with microscopy with Abs and detects cells with DNA replication taking place
Place of DNA replication changes as a function in time…
replication machinery stays in pace and DNA moves through it
How would you test for interphase/ Metaphase ?
Tubulin staining by epifluorescence Microscopy
Interphase/metaphase
what phases do drosophila embryos do ?
S and M
Drosophila DNA experiments?
Combo of functional in vivo assays and morphological markers
-DNA stained with dye increases and flies engineered to only have GFP-tubulin
Analysis of DNA content tells us
- How many cells are in particular phase
- How long that phase that last in a particular cell type
stain cells with what ??
Flurophore
amount of fluroscence is proportional to what?
Amount of DNA
FACS
fluorescence activated cell sorting
- Number of cells found plotted against relative amount Of DNA per cell
- Way of funnelling cells in a small channel of liquid – one cell wide passing a pathway of a lazer
- Determine cell size by how long it takes to block that signal
- Detect amount of fluorescence- amount Is proportional to DNA
Make more precise - Make a 3D plot in 2 dimensions
what is more accurate and useful than normal FACs analysis ??
Bivariate FACS analysis more accurate and useful
- addresses the issue of precision > where each phase starts and ends
How and why would you manipulate FACS analysis ??
- interfere with progression >
- Look at effect on the cycle
- E.g cell cycle arrest
- Univariate followed by bivariate FACs to identify phase in which it arrests > see where the arrest has occurred/ what is a drug targeting…
describe cell cycle dissection in yeast?
- Fission yeast and budding yeast
- Perform and length in each phase different between the yeast types
- Light microscopy Identify the stage
- Fission yeast only grows laterally – never grows
what are the two main model systems for cell cycle ??
Budding yeast and fission yeast
Screen for cell cycle mutants
> Stay at particular stage
- Need to generate temperature dependant mutants and lost sof them – not possible in humans
- Cdc mutants – temperature sensitive select mutants that are
- Look for mutants who at high temp don’t grow and low they do at high temp arrest at particular stage in the cycle
- Cells arrest at mutation point
- Possible to introduce library of genes in plasmids small fraction that receive functional gene mutant will be rescued – sequence the gene and identify functional gene
limitation of genetic screens
- dont know what the gene does
Biochemical analysis using animal embryos
- Oocytes in frogs grows without dividing
- Reg rounds’ of divisions once fertilised
- Listen to
- 45 min cell cycle
- Frog egg has another material to make 4000 cell worth stored ahead of time in a big cell
- Take a frog egg in test tube and centrifuge – generate highly conc extract of the cytoplasm
- If you add just dna to the extract it will form a nucleus in vitro
- The whole extract will recapitulate the whole cell cycle in vitro
- E.g metaphase to anaphase transition
