Eukaryotic Cell Cycle L13/14

Question 1

Eukaryotic What needs to be equally partitioned when a cell divides into two daughter cells?

Answer 1

The genetic material

The organelles

Question 2

In order to divide (go from G1 to S phase), cells need to receive a stimulus. What can that stimulus be?

Answer 2

Growth factors (usually)

Nutrients (sometimes, depending on cell)

Question 3

What does the quiescence / quiescent stage refer to?

Answer 3

Gap phase, G_0

Question 4

How long can cells remain dormant for in G_0

Answer 4

Weeks, months or even years for some cell types

Question 5

Cells will be exposed to growth factors in what situations?

Answer 5

In response to damage, or the need to grow or repair

Question 6

When cells produce/activate cyclin dependent kinases what happens to them?

Answer 6

Cell re-enters the cell cycle

Question 7

What phase of the cell cycle is DNA copied?

Answer 7

G1 phase

Question 8

When does the cell check to make sure the chromosomes have been duplicated properly?

Answer 8

G2 phase

Question 9

How many centrosomes and copies of DNA are present in a cell in G1 phase?

Answer 9

1

Question 10

In what phase do we get duplication of centrosomes?

Answer 10

S phase

Question 11

When do centrosomes move to opposite poles and begin to form the mitotic spindle?

Answer 11

Moving into G2 phase

Question 12

When do cells duplicate their DNA and organelles?

Answer 12

S phase

Question 13

When in the cell cycle does the nuclear membrane break down?

Answer 13

G2 - M transition

Question 14

In what phase does the mitotic spindle fully form?

Answer 14

G2 - M transition

Question 15

When in the cell cycle do the nuclear membranes of cells reform, and the mitotic spindle collapse?

Answer 15

Telophase

Question 16

When in the cell cycle do the centrosomes partition

Answer 16

Cytokinesis

Question 17

What is the difference between biochemists and genetisists approach to investigating something in biology?

Answer 17

Biochemistry: Isolating factors that can recapitulate an activity in a test tube

Genetics: Genetic changes you can make to a cell’s DNA (eg; inactivating a gene) to prevent it from carrying out a process

Question 18

What happens when you fuse an M phase cell with a cell at any other stage of the cell cycle? What does this show?

Answer 18

Spontaneous condensation of the chromosomes.

There is a soluble mitosis promoting factor in M phase cells, which forces cells into mitosis

Question 19

What happens when you fuse an S phase cell with a G1 phase, or a cell in G2 phase? What does this show?

Answer 19

Pushes G1 cell into S phase
G2 cells don’t respond

There is a soluble factor promoting S phase entry, but can only affect G1 (temporal control)

Question 20

What does MPF stand for?

Answer 20

Maturation Promoting Factor (Mitotis promoting factor)

Question 21

What enzyme controls maturation of oocytes and both meiotic and mitotic divisions?

Answer 21

Maturation Promoting Factor (MPF)

Question 22

When does MPF activity increase?

Answer 22

When progesterone is added to a G2 arrested cell

Peaks at Meiosis I (then falls)

Peaks at Meiosis II (stays high until falling after fertilisation)

Peaks at every subsequent mitosis

Question 23

If cyclin B is not destroyed, what happens to cells?

Answer 23

They remain arrested in mitosis

Question 24

What is cyclin B required for?

Answer 24

Mitotic entry

Question 25

What is destruction of cyclin B required for?

Answer 25

Exit from mitosis

Question 26

Why were xenopus (african tree frogs) and sea urchins used for identification of proteins required for G2/M transition?

Answer 26

Their oocytes are: Relatively large, easy to manipulate High protein content Available in high numbers Naturally synchronised Rapid synchronous cell divisions following fertilisation Easier to create cell extracts for biochemical analysis

Question 27

How was autoradiography used to identify cyclin?

Answer 27

Sea urchin oocytes were fertilised and 35S Methionine was added The Methionine is only incorporated into newly produced proteins Protein extracts were taken at different time points postfertilisation and analysed using SDS-PAGE gel Autoradiography shows new proteins

Question 28

If cells get stuck at mitosis, what might have happened?

Answer 28

Cyclin B might not have been destroyed

Question 29

In what organism was cyclin dependent kinase identified?

Answer 29

Yeast

Question 30

What is schizosaccharomyces pombe?

Answer 30

Fission yeast

Question 31

What is S. cerevisiae

Answer 31

Budding yeast

Question 32

How can you tell if S. cerevisiae has arrested in G1?

Answer 32

They will not have a bud

Question 33

What mutant phenotype for yeast did Lee Hartwell use to identify genes required for cell progression?

Answer 33

Temperature sensitive mutants

Question 34

What are Yeast TS mutants?

Answer 34

Yeast temperature sensitive mutants, which can not grow at non-permissive temperatures

Question 35

What are non-permissive temperatures for yeast TS mutants?

Answer 35

Temperatures >35 degrees The mutants cannot grow / replicate at higher temperatures

Question 36

What is genetic complementation?

Answer 36

Adding genes back into mutants to see which ones recover lost functions, such as certain resistances to temperature

Question 37

What are cdc mutants?

Answer 37

Cell division cycle mutants

Question 38

A plasmid containing which gene was able to rescue TS mutant budding yeast (mutant's version of gene not working) ?

Answer 38

_cdc28_ (italics because its a gene)

Question 39

What does SPF stand for?

Answer 39

S-phase promoting factor

Question 40

What is SPF required for?

Answer 40

SPF is critical to bypass the G1/S phase transition. (Bypass restriction point [mammals] and START [yeast])

Question 41

What is MPF required for?

Answer 41

MPF is critical to bypass the G2/M phase transition

Question 42

What does cdc28 code for?

Answer 42

A cyclin dependent kinase

Question 43

What is the name of the point cells decide to go from G1 to S phase in mammalian cells and in yeast cells?

Answer 43

Mammalian: Restriction point Yeast: START

Question 44

As well as growth factors, what do yeast require to divide?

Answer 44

Sufficient nutrients in their environment

Question 45

How does the TS mutation for cdc28 affect the cyclin dependant kinase?

Answer 45

Affects protein stability, making it more susceptible to thermal denaturation Affects kinase's affinity for cyclin (which it depends on for activation), and at high temperatures they dissociate -> loss of kinase function

Question 46

How did increasing abundance of G1 cyclin (overexpression) rescue the cdc28 phenotype and lead to identification of the cyclin dependent kinases?

Answer 46

More cyclins meant the kinases were more likely to form complexes and activate, despite their instability (from low affinity) This allowed complementation, and therefore identification of the presence of cyclin dependent kinases

Question 47

Compare the variation in kinases and cyclins throughout the cell cycle

Answer 47

Only one kinase is used through the cell cycle, but each phase of the cell cycle is regulated by different cyclins

Question 48

How can you tell through cell morphology what stage of the cell cycle fission yeast is in?

Answer 48

The length of the cell

Question 49

Cdc mutants in S. pombe identified arrest at which stage of the cell cycle?

Answer 49

G2/M transition (They were long cells)

Question 50

Gene complementation for identifying cell cycle regulators was performed for the _______ using the gene library previously used for the _______ .

Answer 50

S. pombe (Fission yeast) S. cerevisiae (Budding yeast)

Question 51

A plasmid containing which gene was able to rescue TS mutant fission yeast (mutant's version of gene not working) ?

Answer 51

_cdc2_ (italics because its a gene)

Question 52

The cyclin dependent kinase controls what in budding yeast and fission yeast? What does this show?

Answer 52

G1/S transition in budding yeast G2/M transition in budding yeast G2/M transition in fission yeast Shows there is a functionally conserved kinase across both yeasts, and potentially in higher eukaryotes

Question 53

What are the names of the conserved cyclin dependent kinase in budding yeast, fission yeast, and humans?

Answer 53

Budding: Cdc28 Fission: Cdc2 Human: CDK1

Question 54

What is the phenotype of a cdc2- (knockout for cdc2)?

Answer 54

Cell is unable to enter mitosis Cell growth continues, resulting in overlarge cells

Question 55

What happens if cdc2 cannot be phosphorylated?

Answer 55

Cells enter mitosis rapidly (force cells into mitosis (similar to MPF)) Cell growth is insufficient, causing very short cells (wee phenotype)

Question 56

What is cdc^D?

Answer 56

Constitutively active cdc2 where a negatively charged amino acid is put in phosphorylation site, preventing the kinase from being phosphorylated

Question 57

What regulates mitotic CDK?

Answer 57

Cyclin binding Wee 1 kinase (adds phosphates) Cdc25 phosphatase (removes phosphates)

Question 58

What affect on mitosis does having a deficit of Cdc25 or excess of Wee1 have?

Answer 58

Prevents mitosis Elongated cells (due to increased G2)

Question 59

What affect on mitosis does having an excess of Cdc25 or deficit of Wee1 have?

Answer 59

Inappropriate mitosis Small cells (decreased G2)