Lecture 6 Flashcards Preview

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Flashcards in Lecture 6 Deck (30)
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1
Q

What aspect of development is vulnerable for mutation ?

A

Regulated inhibition

2
Q

if you lose control of the cell cycle what is the main process you lose control of ?

A

Proliferation

3
Q

Basic premise of the cell cycle

A

cell cycle is a programmed series of events enabling a cell to duplicate contents and generate 2 daughter cells

4
Q

Cell cycle progression occurs via changing levels of activity of what ?

A

Cyclin-dependent protein kinases

5
Q

Alterations in normal function of what is implicated in cancer prognosis?

A

of cdk regulators are implicated in cancer prognosis

6
Q

How many critical experiments laid the foundations of cell cycle phases ??

A

4

7
Q

What helps cell counting?

A

Known doubling time

8
Q

What is cell cycle order ?

A

G1 > S >G2 > M

9
Q

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 ?

A

Functional in vivo assay or use morphological marker

10
Q

Pulse chase experiment

A

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
11
Q

What can you do with pulse chase experiment results ??

A

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

12
Q

mitosis in mammalian cells takes how long

A

roughly an hour

13
Q

at any one time what percentage of cells is expected to be doing mitosis ??

A

5%

14
Q

Halogenated Deoxyuridine mimics what

A

Thymine

15
Q

what experiment can you use to test whether DNA replication is taking place ??

A

Expose cells to DU followed by follow up with microscopy with Abs and detects cells with DNA replication taking place

16
Q

Place of DNA replication changes as a function in time…

A

replication machinery stays in pace and DNA moves through it

17
Q

How would you test for interphase/ Metaphase ?

A

Tubulin staining by epifluorescence Microscopy

Interphase/metaphase

18
Q

what phases do drosophila embryos do ?

A

S and M

19
Q

Drosophila DNA experiments?

A

Combo of functional in vivo assays and morphological markers
-DNA stained with dye increases and flies engineered to only have GFP-tubulin

20
Q

Analysis of DNA content tells us

A
  • How many cells are in particular phase

- How long that phase that last in a particular cell type

21
Q

stain cells with what ??

A

Flurophore

22
Q

amount of fluroscence is proportional to what?

A

Amount of DNA

23
Q

FACS

A

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

24
Q

what is more accurate and useful than normal FACs analysis ??

A

Bivariate FACS analysis more accurate and useful

- addresses the issue of precision > where each phase starts and ends

25
Q

How and why would you manipulate FACS analysis ??

A
  • 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…
26
Q

describe cell cycle dissection in yeast?

A
  • 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
27
Q

what are the two main model systems for cell cycle ??

A

Budding yeast and fission yeast

28
Q

Screen for cell cycle mutants

A

> 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
29
Q

limitation of genetic screens

A
  • dont know what the gene does
30
Q

Biochemical analysis using animal embryos

A
  • 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