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BIO 2B03 > Module 9 > Flashcards

Module 9 Flashcards

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1
Q

What are the key features of each of the four main cell cycle phases?

A

[G1] Cell growth and gene expression
[S] DNA replication
[G2] Preparation for mitosis
[M] Mitosis and cell division

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2
Q

What happens if cell cycle regulation fails?

A

It can lead to cell death or uncontrolled cell proliferation (e.g., cancer)

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3
Q

What is the G0 phase?

A

A non-dividing, quiescent state that most differentiated cells remain in

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4
Q

What are two possible fates of a stem cell after division?

A

1) Continue to divide
2) Exit the cell cycle and differentiate

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5
Q

What happens if all stem cells stop dividing or over-divide?

A

[no division] loss of ability to replace lost tissues
[excessive division] tumors

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6
Q

True or False: Differentiated cells can typically re-enter the cell cycle

A

False — most lose the ability to divide

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7
Q

What phases make up mitosis (M-phase)?

A

1) Prophase
2) Prometaphase
3) Metaphase
4) Anaphase
5) Telophase, followed by cytokinesis

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8
Q

What happens during Prophase?

A

1) Chromosomes begin condensing
2) Mitotic spindle assembly begins
3) Centrosomes migrate to opposite poles
4) Nuclear envelope breaks down (not visible)
5) Endomembrane system fragments into vesicles (e.g., ER, Golgi)

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9
Q

What happens during Prometaphase?

A

1) Chromosomes fully condensed
2) Spindle fully assembled
3) Kinetochores form at centromeres
4) Spindle microtubules attach to kinetochores
5) Links chromosomes to spindle for movement

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10
Q

What happens during Metaphase?

A

1) Bipolar attachment of sister chromatids to spindle from opposite poles
2) Tension generated by microtubules
3) Chromosomes align at metaphase plate (spindle equator)

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11
Q

What happens during Anaphase?

A

1) Sister chromatids released
2) Chromatids pulled to opposite poles
3) Movement via shortening of kinetochore microtubules
4) Each daughter cell receives one copy of each chromosome

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12
Q

What happens during Telophase?

A

1) Chromosomes decondense
2) Mitotic spindle disassembles
3) Nuclear envelope reforms
4) Endomembrane system reassembles
5) Two new nuclei form at opposite poles

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13
Q

What is Cytokinesis?

A

1) Division of cytoplasm into two cells
2) Cleavage furrow forms
3) Driven by contractile ring (actin + myosin II)
4) Final separation into two genetically identical daughter cells

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14
Q

What are the two major mechanisms regulating the cell cycle?

A

1) Regulated phosphorylation via Cyclin-CDK complexes
2) Regulated degradation via E3 ligase complexes

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15
Q

What happens if a protein is ubiquitinated by an E3 ligase?

A

It is targeted to the 26S proteasome for degradation

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16
Q

What is the role of CDKs in regulation?

A

They phosphorylate target proteins to trigger specific cell cycle events

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17
Q

What are the 4 main Cyclin-CDK complexes in a cell cycle?

A

1) G1 Cyclin-CDK (active in G1)
2) G1/S Cyclin-CDK (promotes entry into S-phase)
3) S-phase Cyclin-CDK (initiates DNA replication)
4) Mitotic Cyclin-CDK (triggers mitosis)

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18
Q

Do all Cyclin-CDK complexes have the same kinase activity?

A

Yes — they have the same basic kinase function, but differ in:

a) Timing of activation
b) Target proteins

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19
Q

What are the three main E3 ligase complexes in the cell cycle?

A

1) SCF
2) APC-Cdc20
3) APC-Cdh1

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20
Q

What is the function of SCF?

A

Releases the cell from G1, allowing it to transition into S phase

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21
Q

What is the function of both APCs? What does it stand for?

A

[APC-Cdc20] regulates transition from metaphase into anaphase
[APC-Cdh1] mediates the exit from mitosis

[abrv] Anaphase Promoting Factor

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22
Q

What are the three major phosphorylation targets of G1 Cyclin-CDK?

A

1) APC-Cdh1 (inactivates it to end M-phase)
2) Transcription factors (activate expression of S-phase genes)
3) S-phase inhibitors (e.g., Sic1 — prepares for DNA replication)

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23
Q

What is the role of SCF in G1?

A

a) Targets phosphorylated S-phase inhibitors for destruction
b) Enables entry into S-phase by activating S-phase-CDK

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24
Q

What are the key targets of G1/S-phase Cyclin-CDK? [2]

A

1) Transcription factors → activate genes for mitosis (e.g., M-phase cyclins)
2) Centrosome duplication proteins

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25
What is the function of S-phase Cyclin-CDK? [3]
1) Activates and assembles pre-replication complexes at origins of replication 2) Ensures each origin “fires” only once per cycle 3) Prevents re-replication of DNA
26
What cellular components are phosphorylated by M-phase Cyclin-CDK? [5]
1) Chromosomal proteins → chromosome condensation 2) Nuclear envelope proteins → breakdown 3) Microtubule-associated proteins (MAPs) → spindle assembly 4) Kinetochore proteins → chromosome-spindle attachment 5) Anaphase-Promoting Complex (APC) → activates degradation machinery
27
What are the two key points in mitosis where protein degradation is required, and what are their associated pathways?
1) Metaphase to Anaphase Transition (MAT): Requires degradation of anaphase inhibitors, Allows separation of sister chromatids, Triggered by APC-Cdc20 activity 2) Mitotic Exit Network (MEN): Requires degradation of mitotic cyclins (e.g., Cyclin B), Allows exit from mitosis and transition into G1, Mediated by APC-Cdh1
28
What is MPF, and how was it discovered?
a) Maturation Promoting Factor (now called Mitosis Promoting Factor, AKA M-phase Cyclin-CDK) b) Discovered in Xenopus laevis eggs c) Found to trigger meiosis and early mitotic divisions
29
What does MPF consist of?
Cyclin B + CDK1
30
How was the first Cyclin identified?
a) Through studies on sea urchin embryos b) Researchers used radiolabeled protein gels to track protein changes over time
31
What was the key observation about the Cyclin protein?
a) Cyclin concentration oscillated during cell cycles b) Correlated with entry and exit from mitosis — every increase in cyclin concentration is followed by an increase in the number of cells undergoing mitosis
32
What is the relationship between Cyclin B levels and mitosis?
When Cyclin B levels rise, cells enter mitosis, When Cyclin B is degraded, cells exit mitosis
33
What three assays were used to monitor cell cycle activity in vitro?
1) MPF Activity (Histone H1 phosphorylation) 2) Cyclin B Concentration (Tracked by antibodies on gel) 3) Sperm Chromosome Behaviour
34
What system did researchers use to study cell cycle regulation in vitro?
1) Xenopus laevis egg extracts (cell-free) 2) Added sperm nuclei to visualize mitotic changes 3) Mimicked embryonic cell cycles outside of cells
35
In the MPF assay, what was observed when untreated Xenopus extracts were used?
1) Cyclin B levels increased, followed by an increase in MPF activity - chromosomes condensed at peak activity (early mitosis) 2) Cyclin B levels decreased, so MPF activity dropped - chromosomes de-condensed (exit from mitosis)
36
In the MPF assay, what was observed when Xenopus extracts treated with RNase were used?
1) Removed mRNA from the extract but kept tRNA and rRNA 2) There was no increase in Cyclin B concentration, no increase in MPF activity, and thus, no mitotic events
37
What does the synchronization between Cyclin B and mitotic behaviour suggest?
Cyclin B concentration directly regulates CDK activity (MPF)
38
Can the mitotic cycles of the MPF assay occur repeatedly in vitro?
Yes, up to 10 rounds of chromosome condensation and de-condensation occur in the extract system
39
What was the purpose of treating the extracts with RNase?
To remove all mRNA, including Cyclin B, testing if Cyclin B is required for MPF activity
40
What happened when wild-type Cyclin B mRNA was added to the RNase-treated extract?
1) Cyclin B protein levels were restored 2) MPF activity resumed 3) Chromosomes condensed and de-condensed cyclically, proving that Cyclin B synthesis is required for mitosis
41
What happened when non-degradable Cyclin B mRNA was added to the RNase-treated extract?
1) Cyclin B levels remained constantly high 2) CDK (MPF) activity stayed elevated 3) Chromosomes entered mitosis but never exited 4) Chromosomes remained condensed, spindle did not disassemble
42
What key conclusion came from these assay experiments?
The synthesis of Cyclin B for CDK (MPF) activation is equally important to the degradation of Cyclin B to exit mitosis
43
What molecular complex degrades Cyclin B?
APC-Cdh1: an E3 ubiquitin ligase
44
How does APC-Cdh1 trigger Cyclin B degradation?
Adds ubiquitin chains to Cyclin B, then Cyclin B is sent to the 26S proteasome
45
What is the result of Cyclin B degradation at the end of mitosis?
1) MPF (CDK) is inactivated 2) Cell can exit mitosis and re-enter G1 phase
46
What sequence does APC-Cdc20 recognize in Cyclin B for degradation?
The Destruction Box (D-box) near the N-terminus of Cyclin B
47
What amino acids are are required for recognition and ubiquitinylation of Cyclin B?
Arginine (R) [pos 1] Leucine (L) [pos 4] Asparagine (N) or Glutamine (Q) [pos 9]
48
What happens when the D-box sequence is mutated?
1) Cyclin B becomes stable and non-degradable 2) Cannot be recognized by the APC 3) Cell may become arrested in mitosis due to persistent CDK activity
49
T or F: The D-box is both necessary and sufficient for degradation
True
50
What were the results of adding D-box peptide to Xenopus extracts?
[low concentration] slight delay in anaphase [medium] stronger delay [high] complete metaphase arrest
51
What conclusion was drawn from the D-box peptide experiment?
APC must have another essential target besides Cyclin B, an Anaphase Inhibitor (securin)
52
How does APC-Cdc20 trigger sister chromatid separation at anaphase? [pathway: APC, securin, separase, cohesin]
a) After all chromosomes are properly attached to the spindle, APC-Cdc20 is activated b) APC-Cdc20 ubiquitinates securin, targeting it for proteasome degradation c) Securin normally inhibits separase, keeping it inactive but once securin is degraded, separase becomes active d) Active separase cleaves Scc1, a subunit of the cohesin complex e) Cohesin complex (Smc1, Smc3, Scc1) holds sister chromatids together — its cleavage enables their separation f) Result: sister chromatids separate, allowing anaphase to begin
53
How is APC-Cdc20 activated in the first place?
a) One of its subunits is phosphorylated by Cyclin B-CDK early in mitosis b) This primes the APC to become active once Cdc20 binds
54
T or F: The addition of the Cdc20 specificity factor is necessary for the ability of APC to target securin for ubiquitination
True
55
At the end of mitosis, how does APC allow mitotic exit?
a) Replaces Cdc20 as the APC specificity factor b) Targets Cyclin B for degradation c) Inactivates CDK activity d) Allows mitotic exit
56
How is the APC subunit activated and inactivated?
a) Activated by Cyclin B-CDK phosphorylation during mitosis b) Inactivated in G1 by phosphatases in the absence of active Cyclin B-CDK
57
How does the SCF-Sic1 pathway trigger S-phase entry? [pathway]
a) In G1, the S-phase Cyclin-CDK complex is present but inactive because it's bound by an inhibitor protein called Sic1 b) To enter S-phase, the cell must remove Sic1 c) G1 Cyclin-CDK phosphorylates Sic1 at specific residues d) Phosphorylated Sic1 is recognized by the SCF E3 ubiquitin ligase complex e) SCF adds ubiquitin chains to Sic1, making it a target for proteasome degradation f) Once Sic1 is degraded, S-phase Cyclin-CDK is activated g) This activated CDK triggers DNA replication, committing the cell to S-phase
58
Why is SCF-mediated Sic1 degradation critical for cell cycle directionality?
It prevents reversal of the decision to enter S-phase, ensuring the cycle proceeds only in one direction
59
What is a genetic screen?
A method to identify genes in a biological pathway, unbiased since mutations are created randomly (one per cell usually)
60
What is a temperature-sensitive (TS) mutant?
A mutant protein that folds normally at a permissive temp (EX: 24°C) but misfolds at a restrictive temp (EX: 37°C)
61
Why are TS mutants useful in studying essential genes?
They allow conditional inactivation of essential proteins, enabling researchers to observe phenotypes in live cells under different conditions
62
What fission yeast is introduced as the model organism for studying the cell cycle?
S. pombe
63
Describe the elongated and wee phenotypes of S. pombe
[elongated] caused by G2 arrest, cells keep growing but don't divide, results from mutations that delay mitotic entry [wee] caused by premature mitotic entry, cells divide too early (small), results from shortened G2 phase
64
What are the two primary types of cdc mutants?
1) Elongated 2) Wee [cdc] cell division cycle
65
What phenotypes result from Cdc2 mutations?
a) cdc2- (loss of function): elongated phenotype → G2 arrest b) cdc2D (gain of function): wee phenotype → premature mitosis
66
What does Cdc2 control in the cell cycle?
Entry into mitosis, it acts as the mitotic CDK in S. pombe
67
What is the molecular identity of S. pombe MPF?
A Cdc2-Cdc13 heterodimer (Cdc2 = CDK & Cdc13 = mitotic cyclin)
68
T or F: The Cdc2 protein is a 34 kDa protein that has kinase activity and is required for cell division
True
69
What phases of the cell cycle does the Cdc2-Cdc13 complex regulate in S. pombe?
In contrast to Xenopus, it regulates not only the M-phase but S-phase and G-phase as well, it's the sole CDK-cyclin complex in this organism
70
What is the function of the Cdc25 protein in the cell cycle?
A phosphatase and an activator of MPF, which promotes entry into mitosis by dephosphorylating Tyr15 on Cdc2 of MPF
71
What phenotype is observed with a loss-of-function and gain-of-function mutation in cdc25?
[loss of function] elongated cells → delayed mitosis [gain of function] wee phenotype → premature mitosis
72
What phenotype is observed with a loss-of-function and gain-of-function mutation in wee1?
[loss of function] wee phenotype → premature mitosis [gain of function] elongated cells → delayed mitosis
73
What is the function of the Wee1 protein in the cell cycle?
A tyrosine kinase and an inhibitor of MPF, which inhibits mitosis by phosphorylating Tyr15 on Cdc2 to keep MPF inactive
74
Is MPF active or inactive when Tyr15 on Cdc2 is phosphorylated or dephosphorylated?
[phosphorylated] MPF inactive [dephosphorylated] MPF active
75
In fission yeast (S. pombe), what steps are required to activate MPF?
1) Cyclin (Cdc13) binds to Cdc2 → forms inactive MPF 2) Wee1 phosphorylates Tyr15 → MPF remains inactive 3) CAK phosphorylates Thr161 → partially activates MPF 4) Cdc25 removes phosphate from Tyr15 → MPF is fully activated
76
What is the role of CAK in MPF activation?
1) Phosphorylates Thr161 on Cdc2 2) Adds activating phosphorylation 3) MPF remains inactive until Tyr15 is also dephosphorylated by Cdc25
77
Can cells divide without Wee1 and Cdc25?
Yes, but division is slow and inefficient, these regulators are not essential but optimize cell cycle timing
78
How does S. cerevisiae differ from S. pombe in cell division?
1) Forms a bud in G1 before entering S-phase 2) Asymmetrical division instead of binary fission
79
Are the same cell cycle regulators used in both S. pombe and S. cerevisiae?
Yes, CDKs, cyclins, Wee1, Cdc25 all have conserved functions and mechanisms
80
What happens when budding yeast cells carry a cdc29 temperature sensitive mutation?
a) Cells arrest in G1 at restrictive temperature b) Form daughter buds but fail to enter the S-phase
81
What is the function of Cdc28 in S. cerevisiae?
a) Homologue of Cdc2 (S. pombe) b) Sole CDK in budding yeast c) Required for progression through all phases of the cell cycle
82
What is functional complementation?
A genetic technique used to identify the function of a gene, which involves introducing a wild-type gene copy to a mutant cell to restore the normal phenotype
83
What is the first step in a functional complementation experiment?
a) Use a TS mutant that grows at 25°C (permissive) but not at 35°C (restrictive) b) Confirm the mutation causes G1 arrest at the restrictive temperature
84
What is the second step in a functional complementation experiment?
a) Introduce a cDNA library into the mutant cells b) Grow transformed cells at the restrictive temperature c) If a cDNA restores normal growth at the restrictive temperature, it's likely the wild-type version of the mutated gene and has rescued the phenotype
85
What indicates successful functional complementation in a TS screen?
a) If a mutant cell divides normally at restrictive temp after receiving a cDNA b) This implies the cDNA encodes the wild-type version of the mutated gene
86
What is the third step in a functional complementation experiment?
a) Isolate the cDNA that rescued the mutant phenotype b) Extract the plasmid that holds the cDNA c) Confirm the identity of the gene (Gene Z) responsible for restoring normal function
87
What was gene Z in the S. cerevisiae complementation experiment?
Cdc28, the budding yeast CDK
88
What does it mean that S. pombe cdc2 can rescue a S. cerevisiae cdc28 mutant?
It demonstrates functional conservation of CDK genes and shows cell cycle regulation is conserved across eukaryotes
89
T or F: The Cdc28 gene in budding yeast and the Cdc2 gene in fission yeast code for two different proteins, CDK1 and CDK 2 respectively.
False - they both code for CDK1
90
What are the G1/S and M cyclins in S. cerevisiae?
[G1/S] Cln1, Cln2, Cln3 (combine with Cdc28 to form S-phase promoting factor [SPF]) [M] Clb1, Clb2 (combine with Cdc28 to form MPF) - similar to Cyclin B
91
Are CDK-cyclin regulatory principles conserved across eukaryotes?
Yes, from yeast to vertebrates, all use CDK-cyclin pairs with similar functions and timing
92
What differs between yeast and vertebrate CDK regulation?
[yeast] 1 CDK + multiple cyclins [vertebrates] multiple CDKs + multiple cyclins
93
What are the two techniques used to identify genes?
1) Functional Complementation in S. cerevisiae 2) Genetic Screens in S. pombe

BIO 2B03 (10 decks)

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