The cell cycle: formative experiments

Question 1

What are the phases of the cell cycle? And where are the check points located within them?

Answer 1

G1= growth in mass
restriction point- check for favourable environmental conditions
S phase= chromosome duplication
checkpoint- check for DNA damage or stalled replication fork
G2 phase= cell growth
checkpoint- check for damaged or unduplicated dna
M= mitosis/ meiosis
checkpoint- check for chromosome attachment to the mitotic spindle

Question 2

What is the pulse chase experiment and what was it used for?

Answer 2

The pulse chase experiment- used to determine which cells are undertaking DNA replication
relies on the uptake of radioactive isotopes into the cells- which is incorporated into their dna
followed by a period of adding unlabelled isotopes- and analysing the displacement of the radioactivity

Question 3

How long does each phase take in mammalian cells?

Answer 3

Mitosis= 1 hour
G1= 10 hours
S=7.5 hours
G2= 3.5 hours

Question 4

What can you use instead of radioactive isotopes to measure DNA replication?

Answer 4

Halogenated derivatives of dioxyuridine- which mimics the base thymidine
can then use antibodies to detect the halogenated DU

Question 5

How else can you visualise mitosis in cells?

Answer 5

Staining for heterochromatin- shows condensed chromosomes

Staining the mitotic spindle- using an antibody against tubulin

Question 6

What are 3 features of Drosophila embryo cell cycles?

Answer 6

They only have S and M phase
each division is very fast- takes only 15 minutes
they don’t form separate cells- all the nuclei are associated with each other

Question 7

Which technique allows you to measure cellular DNA content and thus determine the proportion of cells in each phase?

Answer 7

FACs- fluorescence-activated cell sorting

plot the number of cells found with particular DNA contents

Question 8

How does FACS work

Answer 8

cells pass through the system one cell at a time
a series of lasers pick up the size of the cells via the blocking of the lasers
fluorophores in the cells will fluoresce when the lasers or a specific wavelength excite the cells- a detector detects this fluorescence
the level of fluorescence is proportional to DNA content

Question 9

How can you find exactly where S phase starts and ends?

Answer 9

By doing bivariate FACs
use the pulse chase experiment- halogenated DU to determine when new DNA is being synthesised, along with how much DNA content there is
gives you a 3D plot which makes it much easier to identify the beginning of S phase= new nucleotides being incorporated into the DNA

Question 10

What else is a bivariate FACs useful for?

Answer 10

Determining where a cell cycle arrest is located

Question 11

What are the two model yeast systems?

Answer 11

Fission and budding

Question 12

What are the cell cycle characteristics of budding yeast?

Answer 12

The daughter bud starts to bud in S phase

mitosis is indicated by the organisation of the mitotic spindle

Question 13

What are the cell cycle characteristics of fission yeast?

Answer 13

Yeast only grows in one dimension- laterally

thus can determine the phase of the cell cycle by how long the cell is

Question 14

What sort of yeast mutants were selected for?

Answer 14

Temperature sensitive cell division cycle mutants

Question 15

Why did they have to be temperature sensitive?

Answer 15

If the cells are arrested at one phase then they cannot grow and proliferate, and thus there would be no cells to experiment on

Question 16

How were the mutants temperature sensitive?

Answer 16

At low temperatures they grow

at high temperatures they are arrested in growth- the morphology of the cells tells you where

Question 17

What did they do with the temp sensitive yeast mutants? And why?

Answer 17

Introduces a large library of yeast genes into plasmids, which they then introduced into a population of the mutant cells
They wanted to see which genes would rescue the function of the mutant

Question 18

What are the characteristics of frog eggs?

Answer 18

Oocytes grow as single cells with no division for many months, getting larger
they then go through meiosis to form haploid cells before being deposited
when the eggs are fertilised they go through normal cell division, however because the divisions are so fast- the cells get smaller

Question 19

Why isnt it an issue that the fertilised egg cells get smalller?

Answer 19

The oocyte had enough material- proteins and lipids- to makes 000s of cells worth of DNA
so it is fine that the dividing cells cannot produce their own proteins etc

Question 20

Which experiment was carried out with frog oocytes? What was the conclusion?

Answer 20

They centrifuged the oocytes - releasing the cytosol
they then added chunks of DNA to the mixture
watched as nuclei formed in the test tube
the cell cycle was being recapitulated in vitro
Conclusion= What were the enzymes and proteins in the cytosol which were allowing cell division to occur? - the answer was CYCLINS