lecture 7

Question 1

cerveisiae life cycle

Answer 1

• after spindle pole body duplication, bud emergence
• bug dets progressively larger and then chromosome segregation and cytokinesis
• produces a mother celll (regular size) and a much smaller daughter cell that will get larger and grow and then undergo the same process
• spends longer in g1

Question 2

sombe life cycle

Answer 2

fission yeast
- starts with DNA replciation
- rod gets longer and longer
- formation of septum (cytogenesis occurs through this)
- divides into two equal sized cells
- distinct moropholical features at every stage can be used to idnietify what stage of cell cycle its at.
- spends longer in g2 and M phases

Question 3

cdc

Answer 3

cell division cycle mutants, both cerevisae and pombe have temperature sensitive mutants which cause defects in spefici proteins required to progress through the cell cycle.

Question 4

loss of cdc 2 activity in s. pombe

Answer 4

prevents entering into M phase

Question 5

cdc 2

Answer 5

a cyclin dependent kinase (CDK)

Question 6

cdc 2 in cerevisiae = cdc ____ in pombe

Answer 6

28

Question 7

are proteins controlling cell cycle highly conserved between eukaryotic organisms

Answer 7

yes; human CDKs can be used in yeast cells with mutations to gain functional CDKs

Question 8

kinases

Answer 8

• put phosphate gorps on

Question 9

phosphatases

Answer 9

remove phosphate groups from proteins

Question 10

MPF

Answer 10

• heterodimer of CDK an mitotic cyclin (cdc 2)

Question 11

cdc 2 activity

Answer 11

• is not active, phosphorylation activity remains low at G1-S phase and increases towards G2 phase, peaking at the start of mitosis

Question 12

cdc 2 presence/amount

Answer 12

• remains constant

Question 13

what does a western blot of the cdc 2 and cyclin samples show?

Answer 13

• mitotic cyclin bands are greater at one point in cell cycle and disappear and then reappear. (peals at G2,drops off during mitosis and then increases and drops off at that point gain)
• cdc 2 bands remain constant

Question 14

does cyclin have enzymatic activity

Answer 14

• no but it associates with a kinase that does

Question 15

activity of mitosis promoting factor

Answer 15

• peaks at late g2, and drops off
• this correlates with levels of cclin

Question 16

what is the dotted line

Answer 16

it is the level of cyclin (red is the activity of cdc 2)
- beings low in g1 and then increases and peaks in late G2 and then drops off
- having more cyclin present may turn on cdc atvitiy

Question 17

when does MPF form

Answer 17

• late g2, cdc 2 binds cyclin to form and activate MPF

Question 18

why is it that there is a lot of cyclin in s phase but no cdc 2 activity

Answer 18

more than one type of cyclin? there must another level of regulation?

Question 19

when are cyclins presnt

Answer 19

• ## at the cell cycle stage that they trigger and are absent in other cell cycle stages.

Question 20

what are the types of cyclin

Answer 20

G1, G1/S, S and mitotic

Question 21

how are cyclins regulate

Answer 21

• transcription factors that cn turn on at diff phases of the cel cycle
• ubiquitin mediated proteasome dependent degradation

Question 22

what is ub-mediated proteasome-dependent degradation

Answer 22

• degrades the cyclins when no longer needed

Question 23

ub

Answer 23

76 amino acid protein
- found in all eukaryotic clls

Question 24

polyubiquitination

Answer 24

• of a target protein acts as a signal for the targeted protein to be degraded by the proteosome
• proteins are now directed towards “garbage can”
• degradative enzymes isnide
• ate hydrolysis required to move the protein and ub to proteasome for degradation
• all cyclins are destroyed at certain phases of the cell cycle

Question 25

what happens t ub proteins when autophagy doesn't work

Answer 25

- acclamation of UB proteins

Question 26

SCF uniqutuinin ligaes

Answer 26

- target cyclins for destruction by ub at certain phases of the cell cycle

Question 27

is ub degraded in the proteosome

Answer 27

no

Question 28

wee1

Answer 28

kinase phpsorylates CDK, causing it to be activated adds phosphate group at tyrosine pos 15

Question 29

cdc25

Answer 29

phosphatase that removes the inhibitory phosphate group from CDK and promote sits activation

Question 30

what regulates MPF activty

Answer 30

wee1 and cdc25 by addition ore removal of phosphate groups on CDK, removes the phosphate pout on by wee1 kinase, allowing MPF (cyclin idk complex to be fully activated)

Question 31

mutation in wee1 kinase or excess of cdc 25

Answer 31

- phosphate group not added to CDK - increase in mitotyic cyclin during s pace and g2 - premature trigger tino mitosis (cells dot expand and elongate), small cells because of decreased G2 no break from addition of phosphate group - cell enters mitosis too early same phenotype as if there was an excess of cdc 25 (removing the phsppahte group right away)

Question 32

mutation in phpshatase so that it is no longer functional

Answer 32

- phosphate group remains on - cell grows and grows but does not. have signal to enter mitosis - elongated cells that are stuck in g2 and cannot go to mitosis this might also happen if too much/too active wee1 kinase putting on phosphate groups

Question 33

another Layer of regulation after cdc 25 removes phosphate grou

Answer 33

- CAK puts a phosphate group at threonine 161 on CDK

Question 34

what are all the components needed for MPF to be active

Answer 34

- sufficient mitotic cyclin synehtsized - associate it with CDK - inactivation phspph group at tyr 1,5 removal by cdc 25, and then - acting phosphate grou by CAK at thr 161

Question 35

CAK

Answer 35

an activating kinase

Question 36

active MPF rols

Answer 36

- chrom condenation, disassembly of nuclear envelope, interpose microtubule dissamebly and mitotic spindle formation (MAP) - remodeling Golgi, er basically CDK kinase phosphorylation leads to the proomtoion of cell to enter mitosis

Question 37

when does inactivation of MPF

Answer 37

- inactivation at anaphase needed

Question 38

mechanism for inactivating MPF

Answer 38

- rapid mitotic cyclin degradation after polyuiquintainon - requires APC (anaphase promotic complex/cyclosome)

Question 39

APC

Answer 39

ub ligase active at end of mitosis. - sticks on ub molecules onto mitotic cyclin to promote degradation of the cell

Question 40

how do ub ligases (like APC) know where to stick on ub

Answer 40

- destruction box (nine amino acids found in cyclins), helps promote rdestrucftion of proteins by the proteosome, and recognition by ligases -

Question 41

is CDK polyubiqtuianteed ?

Answer 41

NO but cannot have high activity without the mitotic cyclin after anaphase