F lec 15 Flashcards
1
Q
in _________________ all you can see in the cell is a nucleus with amorphous DNA
A
interphase
2
Q
stage of mitosis that is characterized by the condensation of chromosomes, the movement of the centrosomes to opposite poles, and the spindle starts to form
A
prophase
3
Q
the major transition from prophase to prometaphase is _______
A
NEB (nuclear envelope breakdown)
4
Q
the major transition from prophase to _________________ is NEB (nuclear envelope breakdown)
A
prometaphase
5
Q
stage of mitosis characterized by nuclear envelope breakdown (NEB) and the microtubules of the spindle trying to connect with the chromosomes
A
prometaphase
6
Q
stage of mitosis characterized by all of the chromosomes making proper contact with the microtubules of the spindle at the kinetochores (no man left behind)
A
metaphase
7
Q
stage of mitosis characterized by the separation of sister chromatids and the movement of sister chromatids towards spindle poles
A
anaphase
8
Q
stage of mitosis characterized by the formation of the contractile ring at the spindle midzone and the nuclear envelope starts to reform
A
telophase
9
Q
in telophase, the contractile ring forms at the spindle ________________
A
midzone
10
Q
stage of mitosis characterized by cleavage furrow formation and the separation of 2 daughter cells, as well as spindle disassembly
A
cytokinesis
11
Q
____________ ____________ _____________ depends on CDK1-cyclinB activity
A
nuclear envelope breakdown (NEB)
12
Q
nuclear envelope breakdown depends on ________ - ___________ activity
A
CDK1-cyclinB
13
Q
CDK1 and cyclinB activity causes the ________________ of the ____________ ____________, which causes the disassembly of the nuclear lamina and this results in nuclear envelope breakdown
A
phosphorylation, nuclear lamins
14
Q
CDK1 and cyclinB activity causes the phosphorylation of the nuclear lamins, which causes the disassembly of the ____________ _____________ and this results in nuclear envelope breakdown
A
nuclear lamina
15
Q
CDK1 and cyclinB activity causes the phosphorylation of the nuclear lamins, which causes the disassembly of the nuclear lamina and this results in ____________ _____________ ________________
A
nuclear envelope breakdown
16
Q
the assembly of the mitotic spindle and the capture of chromosomes by spindle microtubules depends on __________-_____________ activity
A
CDK1-cyclinB
17
Q
spindle elongation and separation of sister chromatids in anaphase requires the __________________ of __________
A
inactivation, CDK1
18
Q
spindle _______________ and ________________ of sister chromatids in anaphase requires the inactivation of CDK1
A
elongation, separation
19
Q
spindle elongation and the separation of sister chromatids in anaphase requires the inactivation of CDK1, since CDK1 phosphorylates proteins that ________________ ______ _______________ _______________
A
maintain the metaphase state
20
Q
the _______ ubiquitinates cyclins, which leads to cyclin destruction and the inactivation of CDKs, which is essential for anaphase
A
APC/C (anaphase promoting complex/ cyclosome)
21
Q
the APC/C (anaphase promoting complex/ cyclosome) ___________________ ________________, which leads to cyclin destruction and the inactivation of CDKs, which is essential for anaphase
A
ubiquitinates cyclins
22
Q
the APC/C ubiquitinates cyclins, which leads to cyclin destruction and the inactivation of ________, which is essential for ________________
A
CDKs, anaphase
23
Q
CDK inactivation is necessary for _____________ and the the ______________ ________________ ____ _______
A
anaphase, preRC formation in G1
24
Q
the experiments about CDKs that were performed on S. pombe called the yeast “_____________ ____________”
A
fission yeast
25
the experiment about CDKs that were performed on S. cerevisiae called the yeast "_______________ ______________'
budding yeast
26
yeast _____-______________ can be isolated that arrest cell division with a morphology corresponding to a specific stage in the cell cycle
ts-mutants
27
in yeast ts-mutants, at _______ temperatures proteins fold properly and is functional
low
28
in yeast ts-mutants, at _____________ temperatures proteins do not fold properly and are not functional
high
29
in budding yeast (S. cerevisiae), the cdc16 gene mutants caused the cell to arrest in ______________, and therefore the gene was essential for anaphase
metaphase
30
in budding yeast (S. cerevisiae), the _________ gene mutants caused the cell to arrest in metaphase, and therefore the gene was essential for anaphase
cdc16
31
in budding yeast (S. cerevisiae), the cdc16 mutants caused the cell to arrest in metaphase, and therefore the gene was essential for ____________
anaphase
32
in ____________ yeast, the cdc16 caused the cell to arrest in metaphase, and therefore the gene was essential for anaphase
budding
33
in budding yeast, the __________ gene mutants caused the cell to arrest in late anaphase, and therefore the gene was required for either telophase or cytokinesis
cdc15
34
in budding yeast, the cdc15 gene mutants caused the cell to arrest in __________ _______________, and therefore the gene was required for either telophase or cytokinesis
late anaphase
35
in budding yeast, the cdc15 gene mutants caused the cell to arrest in late anaphase, and therefore the gene was required for either _______________ or ________________
telophase, cytokinesis
36
in _____________ yeast, the cdc15 gene mutants caused the cell to arrest in late anaphase, and therefore the gene was required for either telophase or cytokinesis
budding
37
in fission yeast, ___________ gene mutants arrest in G2
cdc2
38
in fission yeast, cdc2 gene mutants arrest in ________
G2
39
in budding yeast, _________ gene mutants arrest in G1
cdc28
40
cdc28 is required for entry into ___ _____________
S phase
41
in budding yeast, cdc28 gene mutants arrest in ______
G1
42
in order to find where the gene is in the genome for cdc28 or cdc2 or whatever the gene mutation is:
- identify the gene
- start with cdc2 ts mutant
- make cDNA library that in theory contain all genes in the genome
- take each clone from that library and put them into cdc2 ts mutant cells and hope that one of the yeast cells get the cdc2 gene
- plate yeast at restrictive temp where it doesnt grow
- look for the one colony that grows (grows because cdc2 ts mutant was __________________ and therefore ___________________ by the gene clone from the library)
- from that growing colony, purify the plasmid and sequence the gene in that plasmid
complemented, rescued
43
**
in order to find where the gene is in the genome for cdc28 or cdc2 or whatever the gene mutation is:
- identify the gene
- start with cdc2 ts mutant
- make _ that in theory contain all genes in the genome
- take each clone from that library and put them into cdc2 ts mutant cells and hope that one of the yeast cells get the cdc2 gene
- plate yeast at restrictive temp where it doesnt grow
- look for the one colony that grows (grows because cdc2 ts mutant was complemented and therefore rescued by the gene clone from the library)
- from that growing colony, purify the plasmid and sequence the gene in that plasmid**
cDNA library
44
in order to find where the gene is in the genome for cdc28 or cdc2 or whatever the gene mutation is:
- identify the gene
- start with cdc2 ts mutant
- make cDNA library that in theory contain all genes in the genome
- take each clone from that library and put them into cdc2 ts mutant cells and hope that one of the yeast cells get the cdc2 gene
- plate yeast at _______________ temp
- look for the one colony that grows (grows because cdc2 ts mutant was complemented and therefore rescued by the gene clone from the library)
- from that growing colony, purify the plasmid and sequence the gene in that plasmid
restrictive (where it doesnt grow)
45
in fission yeast, cdc2 is considered a _______ regulator
G2/M
46
cdc2 ts mutants at restrictive temperatures will arrest in ______ and the colony will fail to grow
G2
47
cdc2 encodes a _____ (kDa) protein
34 kDa
48
genetic screens in budding yeast identified that the cdc28 gene was required for ________
G1/S
49
at the restrictive temperature, cdc28 mutants arrest in _______
G1
50
cdc28 in budding yeast encodes a _______ (kDa) protein that is 60% identical to cdc2 in fission yeast and both have similarity to known kinases --> therefore cdc28 and cdc2 are functional homologues
34 kDa
51
cdc28 in budding yeast encodes a 34 kDa protein that is 60% identical to cdc2 in fission yeast and both have similarity to known kinases --> therefore cdc28 and cdc2 are functional _________________
homologues
52
cdc28 in budding yeast encodes a 34 kDa protein that is 60% identical to cdc2 in fission yeast and both have similarity to known _____________ --> therefore cdc28 and cdc2 are functional homologues
kinases
53
it was later found that both cdc28 and cdc2 are required for both ________ and __________
G1/S, G2/M
54
the ultimate test to see that cdc28 and cdc2 were the same gene was to take the cdc28 gene and put it in a ts _________ mutant and it ______________
cdc2, rescued
55
they decided to unify the naming of cdc2 and cdc28 and all these homologue kinases and they decided to call them __________
CDK1
56
reason why cdc2 mutants didnt just stop earlier in the pathway:
NOT ON EXAM
57
classic assay for kinase activity to prove cdc28 is a kinase:
identify protein histone H1 that is heavily phosphorylated and assume it would have a substrate that is active CDK
- purify cdc28 by IP
- in test tube, add rad ATP labeled on the gamma phosphate (the last of the 3) because this is the phosphate that gets transferred during phosphorylation
- incubate and hope that cdc28 in the cell extract purification will take ATP and transfer phosphate from ATP to histone H1, making H1 radioactive
- take contents of the incubation and run SDS-PAGE and at the size of H1 the band is radioactive
- if they did this with control with random protein and H1 (not cdc28) then H1 is not radioactive and was therefore not being phosphorylated
- so therefore this confirms cdc28 is a kinase and can phosphorylate H1 in vitro
58
to determine how H1- synchronize yeast cells using __________________ block then release
- take cdc2 from yeast cells in G2 and they have low histone H1 kinase activity
- take cdc2 from yeast cells in M and they have high histone H1 kinase activity
- concluded that cdc2/cdc28 kinase activity varies through the cell cycle, peaking at M phase kinase activity changes throughout the cell cycle:
hydroxyurea
59
it was found that in yeast cells in G2, the cdc2 has _______ histone H1 kinase activity
low
60
it was found that in yeast cells at M phase, the cdc2 has ____________ histone H1 kinase activity
high
61
after looking at H1 kinase activities at different points in the cell cycle, the researchers concluded that the cell cycle is driven by ___________________ in the ___________ _______________ of cdc2/cdc28
oscillations, kinase activity
62
in the biochem experiments involving xenopus, they studied ________________
meiosis (instead of mitosis)
63
mitosis is very similar to the _______________ meiotic division
second
64
upon entry into meiosis, _________ aka ________________ is similar in many respects to entry into mitosis
NEB, maturation
65
the 1st meiotic division differs from mitotic division in that __________________ separate
homologues (instead of the sister chromatids that separate in mitosis)
66
there is no _____ __________ between meiosis I and meiosis II
S phase
67
meiosis II is very similar to mitosis in that ___________ ___________________ segregate
sister chromatids
68
meiosis is regulated in prophase at nuclear envelope breakdown by _________________
progesterone
69
meiosis is regulated in ________________ at _____________ _____________ ____________ by progesterone
prophase, nuclear envelope breakdown (NEB)
70
meiosis is regulated in _________________ until fertilization occurs
metaphase II
71
meiosis is regulated in metaphase II and cannot continue into ________________ until fertilization occurs
anaphase II
72
meiosis is regulated in metaphase II and cannot continue into anaphase II until ________________ occurs
fertilization
73
meiosis is followed by ____________ formation and specialized rapid _________________ _______________ to form the early embryo
zygote, mitotic divisions
74
focusing on frog eggs: Prophase
the oocyte is the developing egg that contains a single nucleus that is in prophase for most of oogenesis
75
progesterone causes ________ aka _________________
NEB, maturation
76
_________________ causes NEB aka maturation
progesterone
77
meiosis arrests in __________________ until fertilization occurs and then after that happens you get rapid mitotic divisions and embryogenesis
metaphase II
78
meiosis arrests in metaphase II until ________________ occurs
fertilization
79
in ________________ of oogenesis, fertilization results in sperm entry to the egg and triggers calcium influx into the oocyte, which triggers the completion of meiosis (which is referred to as egg activation)
metaphase II
80
in metaphase II of oogenesis, ___________________ results in sperm entry to the egg and triggers calcium influx into the oocyte, which triggers the completion of meiosis (which is referred to as egg activation)
fertilization
81
in metaphase II of oogenesis, fertilization results in ____________ entry to the egg and triggers calcium influx into the oocyte, which triggers the completion of meiosis (which is referred to as egg activation)
sperm
82
in metaphase II of oogenesis, fertilization results in sperm entry to the egg and triggers ____________ ___________ into the oocyte, which triggers the completion of meiosis (which is referred to as egg activation)
calcium influx
83
in metaphase II of oogenesis, fertilization results in sperm entry to the egg and triggers calcium influx into the oocyte, which triggers the completion of meiosis (which is referred to as _______ _______________)
egg activation
84
egg activation allows meiosis to continue and then mitotic divisions ensue from there that are ____________ and ___________________
rapid, synchronized
85
experiment to identify maturation promoting factor (what causes the NEB in response to progesterone):
Maturation Promoting Factor (MPF)
86
to determine the activity of MPF:
- cytoplasm taken from embryos at given times before or after activation has different ability to induce maturation in arrested oocytes
- found that there was Period of MPF activity (Low then high then low again)
87
researchers saw that MPF activity was high in ________________
mitosis
88
researchers saw that MPF activity was low in _______________
interphase
89
it was seen that if you take cytoplasm from a ______ ______________ or ______________ _____________ this could promote maturation
CSF oocyte, dividing embryos
90
experiment to see that MPF levels fluctuate:
- cycloheximide (blocks protein synthesis) is added to a dividing embryo and this leads to arrest before NEB (prophase arrest)
- extracts from these embryos lack MPF activity
- therefore protein synth is required in each cell cycle for MPF activity to function
JUST READ
91
MPF activity, which is required for entry into meiosis: ___________Upon entry into mitosis
present
92
MPF activity, which is required for entry into meiosis:_____________at the completion of mitosis
inactive
93
MPF activity, which is required for entry into meiosis: requires new _____________ _____________ every cycle
protein synthesis
94
identification of cyclin B experiment:
- added 35S-methionine (radioactive) to eggs to label all proteins
- identified a protein that cycles and called it a cyclin
- later came to be cyclin B
95
Experiment to try and simulate fertilization by sperm: - take oocytes in CSF ________________ arrest and take extracts from these oocytes
- combine these extracts with a source of DNA, an energy source, and add calcium to the CSF extract
- the idea is that sperm entry triggers calcium influx so they thought they could simulate this by just adding calcium
- they end up being able to cycle just like in a regular cell (slide 36-37)
metaphase II
96
experiment to simulate fertilization in test tube:
- as soon as they added calcium, ________ occurred and the ______________ _____________ forms inside the epindorph tube and the DNA attaches to the mitotic spindle
NEB, mitotic spindle
97
researchers did a ___________________ and found that cyclin B and CDK1 co-purified together
co-immunoprecipitation
98
researchers did a co-immunoprecipitation and found that _____________ and _________ co-purified together
cyclin B, CDK1
99
it was later found that if you took this copurified complex of CDK1 and cyclin B alone it could induce ________ in a xenopus oocyte
NEB
100
the complex of CDK1 and cyclin B alone became known as _______
MPF (maturation promoting factor)
101
all CDKs are ___________ or ____________ _______________ that depend on a cyclin partner for activity and specificity
serine, threonine kinases
102
First Step --> cyclin binding
Second Step --> phosphorylation of T-loop domain of CDK by CAK-CDK7
Third Step --> dephosphorylation of 2 specific residues by an inhibitory phosphatase called cdc25
- these 2 sites have different activity on the CDK
- Wee1 is the kinase that puts this inhibitory phosphate on the CDK and inhibits the CDK
- cdc25 removes the inhibitory phosphate and activates the CDK
Fourth Step --> release from CDK inhibitors like p27 that can bind to CDK and cyclin and prevent the CDK from phosphorylating its substrates
READ IT
103
once you activate a CDK, this starts a ___________ ____________ _____________ since the CDK then phosphorylates Wee1 and cdc25
- this inactivates wee1 to lead to even more CDK activation
- this further activates cdc25 and you get even more removal of the inhibitory phosphate
positive feedback loop
