Lecture 6

Question 1

What aspect of development is vulnerable for mutation ?

Answer 1

Regulated inhibition

Question 2

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

Answer 2

Proliferation

Question 3

Basic premise of the cell cycle

Answer 3

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

Question 4

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

Answer 4

Cyclin-dependent protein kinases

Question 5

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

Answer 5

of cdk regulators are implicated in cancer prognosis

Question 6

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

Answer 6

4

Question 7

What helps cell counting?

Answer 7

Known doubling time

Question 8

What is cell cycle order ?

Answer 8

G1 > S >G2 > M

Question 9

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 ?

Answer 9

Functional in vivo assay or use morphological marker

Question 10

Pulse chase experiment

Answer 10

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

Question 11

What can you do with pulse chase experiment results ??

Answer 11

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

Question 12

mitosis in mammalian cells takes how long

Answer 12

roughly an hour

Question 13

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

Answer 13

5%

Question 14

Halogenated Deoxyuridine mimics what

Answer 14

Thymine

Question 15

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

Answer 15

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

Question 16

Place of DNA replication changes as a function in time…

Answer 16

replication machinery stays in pace and DNA moves through it

Question 17

How would you test for interphase/ Metaphase ?

Answer 17

Tubulin staining by epifluorescence Microscopy

Interphase/metaphase

Question 18

what phases do drosophila embryos do ?

Answer 18

S and M

Question 19

Drosophila DNA experiments?

Answer 19

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

Question 20

Analysis of DNA content tells us

Answer 20

• How many cells are in particular phase

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

Question 21

stain cells with what ??

Answer 21

Flurophore

Question 22

amount of fluroscence is proportional to what?

Answer 22

Amount of DNA

Question 23

FACS

Answer 23

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

Question 24

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

Answer 24

Bivariate FACS analysis more accurate and useful

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

Question 25

How and why would you manipulate FACS analysis ??

Answer 25

• 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…

Question 26

describe cell cycle dissection in yeast?

Answer 26

• 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

Question 27

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

Answer 27

Budding yeast and fission yeast

Question 28

Screen for cell cycle mutants

Answer 28

> 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

Question 29

limitation of genetic screens

Answer 29

• dont know what the gene does

Question 30

Biochemical analysis using animal embryos

Answer 30

• 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