all combined final Flashcards
1
Q
4 phases of cell cycle
A
G1, S, G2, M
2
Q
true or false?
A
TRUE
3
Q
Describe what happens at prophase (3)
A
Prophase
4
Q
What happens at prometaphase?
A
Prometaphase
5
Q
Describe metaphase
A
Metaphase
6
Q
Describe anaphase
A
Anaphase is pulling apart of sister chromatids.
7
Q
Describe telophase (2)
A
•Telophase
8
Q
Describe cytokinesis: (2)
A
•Cytokinesis
9
Q
Just read
A
okay
10
Q
Metaphase is a ___________ phase bc as soon as fully done, anaphase occurs
A
transient
11
Q
What is the key entry point of mitosis and what is it dependent on?
A
The key start point of mitosis is NEB and it is highly dependent on cdk1-cyclin B
12
Q
Describe how cdk1-cyclin B causes NEB?
A
- phosphorylation of nuclear lamins –> disassembly of nuclear lamina –> nuclear envelope breakdown
13
Q
what other activities are dependent on CDK1-cyclin B activity?
A
Assembly of mitotic spindle and capture of chromosomes by spindle microtubules depends on Cdk1-cyclin B activity
14
Q
Therefore, NEB, mitotic spindle assembly, and spindle attachment are all dependent on cdk1-cyclin B. true or false?
A
TRUE
15
Q
What requires cdk1 inactivation? Explain.
A
Spindle elongation and separation of sister chromatids in anaphase require inactivation of Cdk1.
16
Q
Explain how cdk inactivation occurs and what it is necessary for (2):
A
•APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitinates cyclins –> leading to cyclin destruction –> inactivation of Cdks
17
Q
What are the 2 approaches to studying the cell cycle?
A
•Genetic screens in S. cerevisiae and S. pombe –> genetic
18
Q
Describe the genetic screen experiments that were done on yeast cell cycle
A
- large-scale mutagenesis and screen for temp sensitive mutants (cause protein to not fold properly but at lower temps, will fold properly, but increasing temp will prevent folding and give mutant phenotype)
19
Q
Yeast ts-mutants can be isolated that arrest cell division with a morphology corresponding to a specific stage in the cell cycle. true or false?
A
TRUE
20
Q
What does a metaphase arrest result from? (general statement)
A
gene required for anaphase is mutated
21
Q
What does a late anaphase arrest result from? (general statement)
A
gene required for cytokinesis is mutated
22
Q
What specifically were the important results found in the yeast study of arrest?
A
One gene important in pombe - cdc2
23
Q
Describe the procedure that was done to determine the gene that caused the cell cycle arrest in S. pombe:
A
•S. pombe cdc2 = G2/M regulator
24
Q
Describe cloning of the S. cerevisiae cdc28 (G1/S regulator)
A
Genetic screens in S. cerevisiae identified the cdc28 gene, required for G1/S
25
Just read
okay
26
Lil more reading
okay
27
They started just calling cdc28 and cdc2 as what?
CDK1
28
Describe how they determined that CDK1 is a kinase?
•in vitro kinase assay
29
So, describe the experiment that they did that shows the cyclic activity of CDK1:
-synchronize yeast cells (Hydroxyurea block then release)
30
True or false?
TRUE
31
Reading is for champions. Are you a champion?
Yes
32
How are mitosis and meiosis similar?
•entry into meiosis - NEB (aka maturation) - is similar in many respects to entry into mitosis
33
•meiosis II is very similar to a mitotic division (sister chromatids segregate)
homologues
34
Describe how meiosis is regulated:
-prophase --> NEB (progesterone)
35
meiosis is followed by what?
zygote formation and specialized rapid mitotic cell divisions (early embryo)
36
___________ causes nuclear breakdown, meiosis progresses until metaphase II and the ___________ triggers completion of meiosis and you get rapid mitotic divisions of embryogenesis
oocyte
37
Describe meiosis regulation
Oocytes arrest in prophase of meiosis I until Progesterone triggers maturation.
38
Remains in meiosis II until what?
Remains in meiosis II until fertilization
39
Describe the experiment that started the biochemical approach to cell cycle and what important component of regulation it introduced
-Remove cytoplasm from mitotically cycling embryos (eggs that have been released from meiosis II arrest by fertilization or in vitro activation)
40
Describe the experiment that showed cyclic character of MPF
•1977 - Cytoplasm taken from embryos at given times before or after activation - has different abilities to induce maturation in arrested oocytes.
41
He also took cytoplasm from ________ arrested, inject into immature oocyte and could promote maturation
csf
42
Explain how the cyclic nature of MPF was proven to require protein synthesis
•Cycloheximide (blocks protein synthesis) added to dividing embryo leads to arrest before nuclear envelope breakdown (prophase arrest).
43
True or false?
TRUE
44
Describe the identification of cyclin B
35S-Met added to eggs to label all proteins.
45
Describe CSF arrest and CSF extracts:
CSF arrest: after completion of meiosis I, eggs enter meiosis II and arrest in metaphase.
46
Describe the in vitro mitotic cell cycle using CSF extracts and how it showed the necessity of cyclin B for entry into mitosis:
•Production of cycling extracts: cytoplasm from CSF arrested eggs + sperm nuclei + ATP + Ca2+
47
How did the yeast work and cyclin work come together?
•1989 -Affinity purification of Cdk1 from starfish oocytes. Cyclin B copurifies.
48
So they co-purified and MPF was identified as cdk1-cyclin. True or false?
TRUE
49
Name the different cdk-cyclin pairs that are active at different phases of the cell cycle:
G1 CDK --> CDK4-CyclinD or CDK6-cyclinD
50
All Cdk's are ___________ kinases that depend on a cyclin partner for activity and specificity.
Ser/Thr
51
Describe the process of activation of CDKs: (4)
1.cyclin binding
52
So describe the multi-functionality of CDK7
So cdk7 has multiple important roles, it phosphorylates CTD of RNA pol II and phosphorylates cdk1 and cdk2 in order to activate them
53
Describe wee1 kinase
Wee1 kinase is an inactivating kinase, so the phosphorylation from wee1 kinase inactivates cdk, so it needs to be removed from cdk for activation (cdc25 phosphatase does this)
54
Describe the positive feedback loop of CDK1:
Positive feedback loop occurs because active cdk can phosphorylate cdc25 and wee1.
55
Prophase events
•chromosome condensation.
56
Events of prophase all occur bc of cdk1 and then a critical level is reached that allows for __________
segregation
57
in Prophase:
Condensin forms a ring that is thought to loop together parts of the same chromosome to condense it.
58
Explain why interphase chromosomes are decondensed, why prophase are condensed, and describe what is needed for condensation:
•Interphase chromosomes are decondensed - allows for transcription and for DNA replication at S-phase
59
Prometaphase events
•nuclear envelope breakdown (NEB)
60
Describe nuclear envelope breakdown
•NEB is the key initiating event of mitosis.
61
True or false?
TRUE
62
Describe the mitotic spindles (characteristics and types):
They are polar, having a minus end at the centrosome and plus end that goes out
63
What is this?
Astral microtubules
64
What is this?
interpolar microtubules
65
What is this?
kinetochore microtubules
66
Replicated sister chromatids are held together by __________.
cohesins
67
Separation of sister chromatids requires loss of _____________ and pulling of kinetochore microtubules
kinetochore
68
Centromere of chromosome binds to kinetochore proteins which binds to spindle. True or false?
TRUE
69
Describe kinetochore attachment and the shortening of the spindle
Kinetochore attachment:
70
In prometaphase, kinetochore attachments are ____________.
unstable
71
Read over WELL
okay
72
Define the following: monotelic, syntelic, and merotelic:
monotelic - single attachment
73
Describe how bipolarity is ultimately established in mitosis:
Proper segregation requires capture of both sister chromatids by microtubules from the opposite poles (amphitelic attachment).
74
Read over well
okay
75
Why do chromosomes remain at the midzone during metaphase?
In metaphase, chromosomes remain at the midzone because they are attached to each other via cohesins.
76
Why does anaphase occur? What does this require?
Anaphase occurs when cohesin bonds are broken.
77
•Destruction of ____________ and ____________ depends on a ____________ ____________, the ____________
securin
78
What showed that cyclin B gets degraded?
Add 35-S-CycB (radioactive labeled Cyclin B) in the in vitro mitotic cell cycle
79
How did they identify the sequence for degradation (destruction sequence)?
Make deletions from N-terminus --> assay for degradation in cycling extract
80
they got rid of first 90 Aas from N-terminus end resulted in non degradation, so something there was important. true or false?
TRUE
81
What did they notice about the non-degradable cyclin B (delta90CycB)? Explain the significance:
The extract arrests in mitosis (Early anaphase)
82
So, this arrest was not because it was non-functional. It was its presence when it should've been broken down that caused the arrest. True or false?
TRUE
83
What happens if you add both delta90 and normal cyclin B?
The normal cyclin B gets degraded, but arrest still occurs.
84
When a cycling extract has CycBD90 added:
mitotic extract
85
True or false?
TRUE
86
So summarize what happens if you deleted first 90 AA of cyclin B:
-still able to induce entry into metaphase in Xenopus extracts or eggs
87
List the 3 aspects of CDK-cyclinB that have been covered:
•MPF (Cdk1-cyclin B) is necessary for entry into mitosis.
88
How can you determine what sequence within the 90 aa is important for cyclin destruction?
-look for conserved motifs within this region amongst cyclins from different species.
89
After they found the destruction box, indicate what 2 things they needed to find out about the sequence and how they found them out:
•Determine that this sequence is necessary and sufficient to mediate cyclin B destruction.
90
What happened to protein A when fused with the destruction box? Explain what was seen on the gel and the meaning.
Protein A fused to destruction box is destroyed over time.
91
get slide 79 of lec15-16 ?
okay
92
Explain the relationship between cdk-cycB and the APC
the cdk cyclin B activates the APC, which targets the cyclin for destruction, thereby inactivating cdk
93
How is the cdk-cycB and APC interaction affected if mitotic extract is used (delta90 cycB or mutation)?
In a situation where cyclin B is incomplete (missing 90 Aas or has a mutation), it is still active in that the cdk can still phosphorylate APC because it can still interact with cyclin B, but it cannot be degraded because destruction box is missing or mutated, so it cannot be ubiquitinated and thus degraded. So, CDK activity keeps up, but APC degradation of cyclin cannot occur despite being active.
94
Summarize the process of Cyclin B cycling and mitosis:
•Cdk1-cycB promotes NEB and other events of mitosis
95
How do we get a cycling of active and unactive APC?
Remember, because you get degraded cyclin as a result of active APC, you no longer have cdk activity, meaning you lose APC activity. This makes the cycle.
96
____________ must be destroyed for late anaphase to proceed
securin
97
When are cohesin complexes assembled?
s-phase
98
Cohesins keep sister chromatids together until ______________ of mitosis
anaphase
99
Describe sister chromatid cohesion:
•During DNA replication (S-phase), cohesin complexes assemble and keep sister chromatids together.
100
_______ is important bc it is the subunit (of cohesion) that is proteolytically cleaved during anaphase - cut in half
scc1
101
Key event of anaphase:
cleavage of scc1 protein (removal of cohesin ring allows for sister chromatid separation)
102
3) delta90 Cyclin B
1) 2 sister chromatids are separated to the daughter nuclei
103
Molecularly, what happens during anaphase to get separation of sister chromatids? Start with sister chromatids being together.
Sister chromatids together bc of cohesin
104
Indicate when APC mutants arrest and the state of the chromosomes:
APC mutants arrest in metaphase with sister chromatids still associated
105
Indicate when stabilized cyclin B arrests and the state of the chromosomes:
•stabilized cyclin B results in arrest with sister chromatids separated (early anaphase - cannot proceed to late anaphase)
106
What happens in securin mutants?
Securin mutants mean that you get active separase when you shouldn't, meaning you get faulty and premature separation (before anaphase)
107
What happens securin D-box mutants?
•D-box mutant of Securin results in failed sister chromatid segregation, just like APC mutants.
108
The main function of APC is to target __________, and the main purpose of __________is to prevent anaphase
securin
109
3) APC/securin double mutant
1) would look just like WT, separated chromatids because separase would prematurely separate before anaphase
110
Explain why APC/securin double mutant gets separation AND arrests in late anaphase:
Separation makes sense because securin mutation means it premature separation as a result of active separase
111
Securin prevents anaphase by protecting ________. APC promotes anaphase by promoting Securin destruction.
scc1
112
3) APC/securin double mutant
1) No because got degraded by separase
113
How did they discover separase?
Took securin protein, fused it to beads and identified proteins that can bind to it (like a Co-IP). They found separase
114
What did separase mutant show?
no separation, looks like APC mutant!
115
true or false?
TRUE
116
Describe what is expected from a separase/securin double mutant
looks like separase mutant (or APC mutant) because securin is only needed to inactivate separase, which is not active bc of the mutation
117
Mutants with stabilized scc1 arrest where?
metaphase
118
Briefly indicate the Securin, Scc1 and Separase interactions
•Prior to anaphase securin binds to and inhibits separase.
119
What keeps the APC inactive?
Spindle assembly checkpoint (SAC)
120
Describe the introductory experiment to the SAC
•Treatment of cells with colchicine or vinblastine (microtubule destabilizing drugs) or with taxol (microtubule stabilizing drug) leads to a mitotic arrest. Cdk activity is high, cyclin B and securin are stable.
121
Describe how the Spindle Assembly Checkpoint works:
•The APC is kept inactive until all kinetochores have made stable connections to spindle microtubules from both poles.
122
As proper attachment occurs, SAC is inactivated at that kinetochore. True or false?
TRUE
123
Mad2 is activated when SAC is activate (it is a part of SAC), promotes the inhibition of what?
cdc20 of APC
124
SAC is recruited to what?
unattached kinetochores
125
How does mad2 work?
•Mad2 component of SAC is recruited to kinetochore, activated, released.
126
Describe the kiss and run model of sac:
Mad2 cycles off of kinetochores as long as it is not occupied
127
true or false?
TRUE
128
Describe what happens in prometaphase, then metaphase, then anaphase as a result of APC/C and SAC activity
In prometaphase, some chromosomes are properly attached while others are still unattached or have monotelic attachments
129
Run through diagram on slide 98 of 15/16
okay
130
slide 100
okay
131
As microtubules gets shortened, it stays attached, causing pull force that drives anaphase B
collar
132
Anaphase is initiated by ___________ mediated destruction of ___________ and ___________
APC/C
133
Describe what initiates anaphase A and B respectively and what they result in:
•Anaphase A - Securin destruction --> loss of cohesion - pulling forces of kinetochore microtubules partially separates sister chromatids
134
What does this describe?
cytokinesis
135
Describe the midbody in cytokinesis:
Cytokinesis continues until a thin bridge connects the two cells. This contains remnants of the central spindle, actin/myosin and matrix material.
136
Important for determining where cytokinesis occurred in the first place.
Interpolar microtubules
137
________________ _________________ release signal that instruct cytokinesis to occur at this site
Interpolar microtubules
138
Them together pulls the membrane together
actin
139
myosin II in contraction of non-muscle cells:
Myosin is a motor protein
140
Motor activity of myosin brings membrane closer together. true or false?
TRUE
141
There are different models for how the contractile ring is established. There is good evidence for one in which the _____________ _____________ (_____________ _____________ ) sets up the contractile ring
central spindle (spindle midzone)
142
How does the central spindle play a key role in contractile ring formation of cytokinesis?
Determining where contraction occurs
143
__________ is essential for the assembly of the actin/myosin contractile ring
RhoA
144
Describe RhoA
RhoA - activated near central spindle (interpolar microtubules)
145
Describe the experiment result that proved RhoA recruits actin and myosin to the contractile ring
WT cell undergoing cytokinesis
146
Describe the latrunculin A experiment (depolymerization of actin):
Drug depolymerizes actin and prevents cytokinesis (no pinching seen)
147
Describe myosin heavy chain (MHC) RNAi knockdown result:
Cell doesn't form cleavage furrow
148
What did the latrunculin A and MHC knockdown experiments conclude?
actin and myosin are both necessary for cytokinesis, but RhoA is in an upstream pathway of actin and myosin because it doesnt depend on them being present for it to be present.
149
What is ECT2?
The GEF for RhoA --> promotes its active GTP-bound state
150
Actin and myosin localize at the site of cleavage, but ECT2 actually localizes to the overlapping interpolar microtubules at the central spindle (spindle midzone). True or false?
TRUE
151
What is the fate of ECT2 RNAi cells?
failure to undergo cytokinesis
152
What is ETC2 necessary for?
RhoA accumulation at the contractile ring (localization of RhoA)
153
Provide the full pathway for mitotic spindle dependent contractile ring assembly:
•CYK-4 associates with the mitotic central spindle
154
Describe CYK-4 role and studies:
CYK-4 is necessary for cytokinesis and for localization of MHC, actin, ECT2 and RhoA.
155
Describe the experiment that answered the following question. "How is RhoA activation regulated so it is active only after mitosis?"
Start with cells synchronized in metaphase at time 0
156
State what they found to be the thing that stops the binding between ect2 and cyk4 and why it makes sense:
Cdk1 phosphorylation stops the binding, which makes sense because in anaphase cdk1 is degraded, which means that the binding can occur in anaphase, allowing for activation of RhoA
157
ECT2 immunoprecipitates CYK-4 in _____________.
anaphase
158
Phosphorylation of ECT2 (pTP) correlates with ability or inability to bind?
inability
159
Therefore Cdk1 phosphorylation of ECT2 prevents recruitment of ECT2 to the spindle midzone. Anaphase cyclin destruction permits ECT2 recruitment. True or false?
TRUE
160
get slide 17 info from lec 17-18
okay
161
Summarize the model for RhoA activation at the contractile ring:
•CYK-4 associates with the mitotic central spindle (interpolar microtubules that don't associate with stuff)
162
In g1 of cell cycle, what complex is formed and what 2 kinases are a result of its formation (provide what the kinases do in the context too)
Prereplicative complex forms (preRC)
163
In order of appearance in the cell cycle, name the CDK/cyclin pairs that pertain to each phase or checkpoint and what they are inhibited by:
•G1: Cdk4/cyclinD (inhibited by INK4)
164
Which cdk cyclin pair needs to be activated for S to occur?
Cdk2/cyclin A
165
Describe what happens in embryonic cells meiosis and mitosis with no G1 phase in terms of cyclin and APC:
No G1:
166
What happens in cells with G1 phase (normal cells) in regards to their cyclin and apc in M and G1:
Cells that have a G1 phase
167
Describe the relationship between the types of APC
apc/c-cdc20 gets phosphorylated to be activated, but then it gets inactivated and APC/C-cdh1 comes along as the first goes down because they are oppositely regulated by phosphorylation
168
What are the reasons for low cdk activity in g1? (3)
- cdh1-APC/C is active, which keeps m-cyclin (cyclinB) level low
169
•________ inhibits Cdk4-cyclinD
p27, p21
170
-•Cells that are terminally ___________ (most cells in our body) arrest permanently in a G1-like state - referred to as ___________
- cdc20
171
The decision whether to enter S phase is coupled to what?
growth
172
•All cells have some mechanism of coupling cell division and growth. Briefly explain this in unicellular and multicellular organisms:
•In unicellular organisms the main determinant of rate of division is nutrient availability for that cell.
173
•In most cells, the decision of when and if to divide is made in ________ of the cell cycle
G1
174
Entry into S-phase involves what sequential activation?
G1-Cdks --> G1/S-Cdks --> S-Cdks
175
rate of cell division must be coupled to rate of cell growth
protein synthesis
176
In S. cerevisiae, nutrients entering the cell activate what pathway? List what this pathway generally promotes (not just in yeast):
TOR kinase
177
Describe Growth and Cell cycle control in budding yeast starting from the accumulation of nutrients in the cell:
•low nutrients --> yeast cell makes less proteins and does not divide.
178
Explain specifically why S phase is so dependent on nutrients levels/growth:
•The G1 cyclin Cln3 is very unstable (constantly subject to ubiquitin-proteasome degradation) AND its translation is inefficient.
179
get slide 31 info from lecture 17-18
okay
180
•Many mitogens are also ____________ ____________.
•In multicellular organisms both growth and the cell cycle are highly regulated.
181
Get info from slide 32 lec 17-18
okay
182
and 33
ight
183
Many mitogens such as _________ and _________activate the __________________ pathway.
PDGF
184
Describe the Ras/MAPK pathway and how it is involved in control of growth and cell cycle
Receptor binding by mitogen (ex. pdgf, egf)--> transphosphorylation
185
Just read
okay
186
Myc and fos promote activity of what?
G1-CDK activity
187
Myc and fos promote G1-Cdk activity. Explain:
•Mitogen signaling leads to transcription of myc and fos.
188
G1-Cdk activity promotes G1/S. Explain:
•Cdk4-cyclin D binds p27, helping to relieve inhibition of Cdk2-cyclin E.
189
Mitogens and Cdk4/cyclin D inhibit p27, helping to relieve inhibition of Cdk2/cyclin E. How does cyclin E get further promoted?
-Cdk4-cyclin D sequesters p27, allowing low level activation of Cdk2-cyclin E
190
What is the no turning back point for S phase?
Mitogens promote activation of the E2F transcription factor
191
get slide 44 info (lect17-18)
okay
192
What is responsible for a rapid increase in levels of G1/S and S-cyclins?
E2F1 and other activator E2Fs
193
•In G0 and G1, E2F1 is kept inactive by binding to ________ .
s-phase
194
G1-Cdks promote S-phase by activating E2F1. Explain this process:
•Mitogens promote Cdk4-cyclin D activity and low levels of Cdk2-cyclin E activity (by sequestering p27).
195
Describe activation of the S-Cdk, Cdk2-cyclin A:
•E2F1 promotes high level transcription of itself, cyclin E and cyclin A.
196
Provide a summary of the G1 to S transition in higher eukaryotes:
•mitogens activate ras/MAPK pathway leading to myc, fos transcription.
197
Describe growth and cell cycle control in yeast vs higher eukaryotes
•In yeast the major cue for growth is nutrient levels and the cue for cell division is growth.
198
Describe how replication is initiated in terms of the molecular recruitment that takes place:
ORC recruits Cdc6
199
What are the roles of CDK2-cyclin A and DDK and what do they result in?
S-Cdk = Cdk2-cyclin A
200
True or false?
TRUE
201
Briefly describe the status of DNA replication in terms of before during and after S phase (3):
•pre-replication complex (preRC) formation in G1
202
Describe the process of BrdU incorporation and what it shows us:
BrdU Incorporation
203
•Pre-RC assembly is inhibited after __________ to ensure that ______________ does not occur.
S-phase
204
What would happen if re-replication wasn't stopped by inhibiting Pre-RC assembly after S-phase?
re-replication would result in tetraploidy - frequently a first step in cancer development.
205
Summarize the activities of the 2 APC complexes in mitosis and G1:
•APC/C Cdc20
206
True or false?
TRUE
207
•S phase initiation requires an ordered sequence of events starting right after completion of mitosis:
-anaphase/early G1(low Cdk activity due to APCCdh1)
208
Pre Rc only forms when there is _________ cdk activity, which is only found in ______ as a result of APC/C _________
low
209
At S and on there is high cdk activity occurs, preventing formation of another pre RC. True or false?
TRUE
210
Get info from slide 59 of 17/18
okay
211
In regards to replication, what happens in G1?
G1 pre-RC assembly (licensing)
212
In regards to replication, what happens in S? (3)
S phase- Initiation of replication
213
In terms of replication, what happens in G2/M?
G2/M- Prevention of re-replication
214
In terms of replication, what happens in anaphase?
Anaphase- re-creating conditions for pre-RC formation (licensing)
215
Describe what happens in terms of preRC formation, then inactivation of its components, then how they are eventually promoted again:
pre-RC: ORC recruits Cdc6 and Cdt1 which recruit Mcm helicase.
216
Summarize main events and main activities of the cell cycle:
•G1- low Cdk activity. PreRCs assemble.
217
What are cell cycle checkpoints?
•Delay or arrest in cell cycle progression in response to problems completing a specific step in the cell cycle or in response to other cellular problems
218
How do checkpoints take action? (3)
•Checkpoints may pause the cell cycle and promote repair before continuing. Checkpoints can also induce permanent arrest or apoptosis.
219
True or false?
TRUE
220
What are the important checkpoints? (3)
1. Spindle assembly checkpoint
221
Describe what the spindle assembly checkpoint is and the way it takes place:
•APC/Cdc20 is inhibited in response to incomplete kinetochore/microtubule attachments.
222
Briefly describe G1 DNA damage checkpoint and its significance:
•activated if DNA damage is detected in G1 --> G1 arrest.
223
Briefly describe replicative senescence and its significance:
•Replicative senescence - telomere shortening --> G0 arrest (senescence).
224
What are the three situations that trigger the DNA damage checkpoint?
1. Stalled replication fork
225
What do the 3 DNA damage triggers result in?
They recruit ATR and ATM, 2 kinases that phosphorylate 2 downstream kinases (chk1 and chk2).
226
What are ATR and ATM responsible for (aside from chk1 and chk2 phosphorylation)?
ATM - double strand breaks (leads to permanent cell-cycle arrest or cell death)
227
What do chk1 and chk2 do?
Chk1: production of DNA repair enzymes
228
ATM (and ATR) has two functions at these sites:
1. help recruit repair machinery
229
What did they do to cells to show ATM recruitment?
hit em with ionizing radiation
230
Read over well
okay
231
Wee1 and cdc25 act on what?
cdk1 and cdk2
232
Get info from slide 9 of lecture 19
okay
233
Provide the G1 DNA damage checkpoint pathway:
•ATR and ATM both recognize different types of DNA damage.
234
A very similar checkpoint to DNA damage in g1 can be activated in G2:
•Cdk1 remains phosphorylated by Wee1
235
•In case of more severe damage ATM,ATR, Chk1 and Chk2 phosphorylate and activate what?
p53
236
Describe p53 activity:
•p53 is a transcription factor that induces a permanent cell cycle arrest by activating p21 expression
237
Explain how p53 mediates G1 arrest in response to DNA damage:
In undamaged cells p53 is inactive due to Mdm2 binding
238
Briefly indicate how telomeres are protected and what they are protected from:
•Telomeres Are Protected by telomere binding proteins - sheltrin complex
239
Describe replicative senescence pathway:
•Telomere shortening at each cell division eventually leads to loss of all of the telomere repeats.
240
Why is p53 considered the cellular gatekeeper?
because many issues like DNA damage, telomere shortening and hypoxia lead to its activation, resulting in cell cycle arrest, senescence, or apoptosis
241
What is apoptosis and briefly how does it get triggered?
•Apoptosis is often referred to as programmed cell death or cell suicide.
242
What are these?
Examples of apoptosis as a developmental process
243
•Apoptosis as a response to cell cycle and other cellular checkpoints. True or false?
TRUE
244
What are the big advantages of using c. elegans for apoptosis?
One of the big advantages in c.elegans is that it has a very limited number of cells and because it is see-through, you can follow every single cell during development
245
What information did they get from c. elegans on apoptosis?
Showed that programmed cell death was revealed through cell lineage maps
246
Explain the difference between apoptosis and necrosis:
•Apoptosis involves activation of a cell death pathway while necrosis is a passive process resulting from damage to the cell.
247
•Features of apoptotic cells: (things that happen)
-cytoskeleton collapse, nuclear envelope breaks down, chromatin condenses and breaks into fragments, cell surface blebbing, apoptotic bodies
248
Apoptotic cells identified by incubation in Acridine Orange between digits of hands. Describe how this can be determined experimentally:
Incubating with dna dye called acridine orange binds to dna regardless of alive or dead. But, it cannot enter the membrane of a living cell. The cell membrane loses its integrity in apoptosis, so it allows for acridine orange to enter, so it specifically shows where apoptotic cells are.
249
What would you see on a DNA gel in apoptotic cell?
Chromosomal DNA fragmentation detected on a DNA gel (fragmentation as time goes on)
250
Describe TUNEL labelling:
TUNEL labeling - apoptotic cells have fragmented chromosomes. Label DNA ends with Terminal deoxynucleotidyl Transferase
251
Name and describe the 5 assays for apoptosis:
•Plasma membrane loses integrity
252
What is the state of caspases during normal times in the cell?
Caspases are in our cells at all times in a state in which they need the n terminus to be cleaved to be active (procaspase to caspase)
253
get slide 9 of lecture 20 info
okay
254
True or false?
TRUE
255
Two classes of caspase:
•Initiator (activator) caspases - cleave other caspases
256
True or false?
TRUE
257
What are the 2 pathways for activation of caspases?
Intrinsic and extrinsic pathway
258
Briefly explain the intrinsic pathway:
-cellular response to various stresses, DNA damage, depletion of survival factors.
259
Briefly explain the extrinsic pathway:
-apoptosis induced via signal from specialized cells - eg cytotoxic T-lymphocytes
260
What does this describe?
Intrinsic pathway
261
What is the intrinsic pathway of apoptosis promoted by? Explain.
•regulated by BH-Domain family of proteins
262
True or false?
TRUE
263
3 important categories of BH-domain proteins. Name and briefly indicate the role of each:
BH123 - forms the mitochondrial pore which releases cytochrome c (and anti-IAPs)
264
Bh3-only are pro-apoptotic because they bind to and inhibit ___________, allowing for ___________ pore formation in mitochondrial membrane
bcl2
265
Read over well
okay
266
Cytochrome C release from the mitochondria is a key event in the ___________ pathway.
Intrinsic
267
Explain what happens once cytochrome C is released from the mitochondria:
Cytochrome C release - binding to Apaf1 --> CARD domain exposed - multimerization (Apoptosome)
268
•________________ activated by cleavage - cleaves ________________ caspases.
BH123
269
What are IAPs?
IAPs are inhibitors of apoptosis, they sit around and are ready to bind to any activated caspase to block it. They can block apoptosis even if initiated properly.
270
What are anti-IAPs? Give an example of one and describe what they do:
Ex. Hid
271
What does this describe?
IAPs
272
What does this describe?
Anti-IAPs
273
Summarize the Intrinsic Pathway of Apoptosis
•aggregation of BH123 proteins - make a pore in mitochondria
274
Describe the regulation of the intrinsic pathway:
•survival factors (anti-apoptotic)
275
add info from slide 28 of lecture 20
okay
276
add info from slide 29 of lecture 20
okay
277
Describe how cell competition leads to apoptosis:
•Cell competition leads to apoptosis. Major mechanism for organizing neural connections.
278
Describe how growth factors can be survival factors:
A growth factor/survival factor binds to a receptor, stimulating a pathway in which Akt kinase is activated, which can phosphorylate Bad (BH3-only protein) to allow for active Bcl2. This Bcl2 can go on to block apoptosis by preventing BH123 aggregation and mitochondrial pore formation
279
Describe how mitogens can be survival factors:
Mitogen binds to activate a receptor that stimulates a pathway that activates MAPK, which can phosphorylate Hid (anti-IAP) to inactivate it, allowing for IAPs to be free to block apoptosis by binding to caspase
280
is p53 anti or pro apoptotic?
pro-apoptotic
281
get info from slide 35 lecture 20
okay
282
get info from slide 36 lecture 20
okay
283
What are the 5 ways that p53 works as a pro-apoptotic transcription factor?
p53 induces BH3-only expression
284
Read over very well
okay
285
What mediates extrinsic apoptosis?
Cytotoxic T-cells binding to virus-infected cells
286
Describe the recognition of virus-infected cells:
MHC proteins + peptide on cell surface
287
True or false?
TRUE
288
True or false?
TRUE
289
get slide 46 info from lec 20
okay
290
What is Fas-mediated Extrinsic Apoptosis?
•FasL - a ligand of the Tumour Necrosis Factor (TNF) family of proteins - integral membrane protein ligands - function as homotrimers.
291
p53 is primary inducer of intrinsic pathway, but also promotes extrinsic by promoting fas receptor expression. true or false?
TRUE
292
get missing info from slide 48 of lec 20
okay
293
Summarize all that happens in apoptosis mediated by CTLs (cytotoxic T-lymphocytes)
•Cytotoxic T cells identify infected cells via T cell receptor binding to foreign peptides presented on MHC receptors.
294
-____________ tumour - cancer that spreads to ____________ sites - via travel of cancer cells through ____________ or ____________ system.
tumour
295
What are carcinomas, sarcomas, leukemias, and lymphomas?
•Carcinomas - arise from epithelial cells - most common and most dangerous cancers in humans.
296
True or false?
TRUE
297
What are the original theories on the origins of cancer? (2)
•Chemicals and other agents that damage DNA (mutagens) as causes of cancer
298
Read over very well
okay
299
Indicate some phenomena we see in normal cells growing in culture (4)
Cells grown in culture form a monolayer:
300
•Chemicals and radiation can transform normal cells into cancer cells in vitro. What are the 4 traits that suggest that they have become cancer cells?
•loss of contact inhibition
301
These 4 traits that indicate cancer cells led to what conclusion?
•These agents cause mutations --> cancer is caused by mutations in our genes
302
Indicate the significance of Rous sarcoma virus (RSV):
First characterized tumour virus. Led to model that cancer is caused by infectious agents.
303
Describe the experiment done with RSV and why they did it:
- removed sarcoma from chicken and broke up into small tissue chunks
304
1960's - Infection with RSV leads to: (4)
•loss of contact inhibition
305
What kind of virus is RSV?
Retrovirus --> rna genome, carry reverse transcriptase to make DNA copy, which is integrated into genome
306
Just read
okay
307
In 1974, they discovered how RSV was able to transform normal cells to cancerous. Briefly state what this was:
a single viral gene - src
308
RSV contains the Src gene - and this gene is the one that confers transformation ability to cells. True or false?
TRUE
309
Just read
okay
310
What were the problems with the viral theory for cancer?
1. They had trouble finding the viruses that caused these human cancers
311
True or false?
TRUE
312
What did they later realize about src?
They realized that they all contain src gene whether infected or not, and it looks like a normal euk gene (introns, exons), whereas the viral src gene doesn't have those introns
313
True or false?
TRUE
314
So, what did they conclude about the src gene in RSV?
transforming gene from RSV is a gene that originated in the host (chicken) and got incorporated into the virus at some point. This gene gave the virus a selective advantage and was therefore retained in the viral genome.
315
Ultimately, the understanding of the src gene led to the unification of the viral and genetic origin of cancer theories. Explain:
•Cancer is caused by overexpression of specific genes that originated from our own genome (proto-oncogenes), that are carried in viruses (viral oncogenes).
316
What are these viral oncogenes usually involved in?
A lot of these viral oncogenes are genes that play important roles in growth and cell division.
317
Slow acting vs fast acting tumour viruses: (I don't really understand this concept)
•slower acting tumour viruses may act by inserting near and causing high level expression of host oncogenes. This would only occur by chance after many rounds of cell infection.
318
Slower acting because it requires chance event of landing in the right place for greater expression (Enhancers). True or false?
TRUE
319
What did these discoveries about viral oncogenes suggest about chemical mutagens? How can you prove this?
• Suggested the idea that chemical mutagens cause cancer by converting proto-oncogenes into oncogenes
320
Describe transfection:
Transfection:
321
true or false?
TRUE
322
Explain how transfection can cause cancer in mice from human tumour cells?
•transfect normal mouse cells with DNA from a human tumour
323
How were they able to identify the actual altered gene that caused the transformation of the cells?
•genomic DNA library from these transformed mouse cells
324
True or false?
TRUE
325
Read over well
okay
326
What mutation in the Ras gene could have made it constitutively active?
Mutation in GTPase activity that makes it stuck in GTP bound state
327
Name and describe the 4 ways proto-oncogenes can be converted to oncogene:
1. Deletion or point mutation in coding sequence --> hyperactive protein made in normal amounts
328
What can be used to identify cancer as a result of chromosomal rearrangement?
SKY probe that labels diff colours for diff chromosomes
329
Forms of activation of oncogenes in cancer (5)
•viral copy of oncogene expressed at high levels by viral enhancers.
330
Describe the experiments and subsequent interpretation of them that led to the discovery of tumour suppressor genes:
•Cell fusion experiments: Fuse a cancer cell with a normal cell
331
Describe the 2 forms of retinoblastoma:
Familial and sporadic
332
This led to the idea of the tumour suppressor and what hypothesis?
two-hit hypothesis
333
get explanation of slide 36 (lec 21)
okay
334
Besides genetic changes, how else could loss of heterozygosity occur?
•Loss of heterozygosity can also occur by epigenetic gene silencing - somatically inherited chromatin modifications
335
What are Rb and INK4 considered? Why?
tumour suppressors
336
What is the most important tumour suppressor?
p53
337
Go through slide 39 chart of what happens as a result of p53 mutation
green - breast cancer
338
•Rb identified as the first tumour suppressor gene. True or false?
TRUE
339
What are most familial cancers caused by?
•Most familial cancers are caused by inheritance of recessive mutant alleles of tumour suppressor genes.
340
Why elephants rarely get cancer
•elephants live almost as long as humans - up to 70 years
341
What does this mean?
Cancer requires oncogenes and mutant tumour suppressors
342
Explain Clonal Origins of Cancer:
•Most cancers derive from a single cell that acquires a somatic mutation that helps promote tumour formation.
343
Just read over well
okay
344
Get info from slide 48 (lecture 21)
okay
345
When can genomic rearrangements occur?
As a result of genome instability:
346
Explain genome instability:
•chromosome rearrangements, gene amplification, anneuploidy
347
cancer results from accumulation of mutations that give the cell the following capabilities: (7)
•Self sufficiency in growth signals and/or insensitivity to anti-growth signals - eg.. ras activation, Rb loss
348
Is the order of acquiring these capabilities important? Explain:
No. The order of acquiring these capabilities is not important but once genome instability is acquired, other mutations arise faster
