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bio 2b03 > module 9 > Flashcards

module 9 Flashcards

(48 cards)

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1
Q

name: steps to mitosis (5)

A
  1. interphase
  2. prophase
  3. prometaphase
  4. metaphase
  5. anaphase
  6. telophase
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2
Q

question: what happens in interphase?

A
  • chromo. replicated into sister chromatids in S phase
  • centrosomes duplicated in G1 and S phase
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3
Q

question: what happens in prophase?

A
  • chromo. begin condensation
  • mitotic spindle begins assembling
  • duplicated centrosomes start to separate to opp. sides
  • nuclear envelope dissolves
  • endomembranes breakdown into small vesicles
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4
Q

question: what happens in prometaphase?

A
  • chromo. attach via centromeres to microtubule spindle
  • kinetochore prot. assemble at centromere of chromo.
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5
Q

question: what happens in metaphase?

A
  • all chromo. attached to spindle from both poles
    ⤷ bipolar attachment
  • chromo. align at middle of spindle
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6
Q

question: what happens in anaphase

A
  • sig. breaks assoc. between sister chromatids
  • chromatids pulled to opp. poles of spindle
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7
Q

question: what happens in telophase?

A
  • cell reverses everything done in prophase
    ⤷ chromo. decondense
    ⤷ spindle disassembles
    ⤷ nuclear envelop reforms
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8
Q

question: what regulates the sequence of events of mitosis?

A
  • 2 classes of prot.
  • CDK (cyclin dep. kinases)
  • E3 ubiquitin ligase complexes
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9
Q

explain: role of CDKs (+ struc.)

A
  • hetero dimeric prot. complex
  • facilitates phosphorylation
  • CDK = regulated by cyclin
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10
Q

explain: role of E3 ligases

A
  • target specific prot. for degradation in proteasome
  • helps degrade cyclin to turn off kinases
  • helps degrade cell cycle inhibitors
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11
Q

name: major classes of cyclin-CDK kinases (4)

A
  1. G1 cyclin-CDK
  2. G1/S-phase cyclin-CDK
  3. S-phase cyclin-CDK
  4. mitotic cyclin-CDK
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12
Q

question: what’s the difference between the types of cyclin-CDK?

A
  • diff. target prot.
  • diff. timing
    ⤷ ex. G1 cyclin-CDK = active in G1
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13
Q

name: main E3 ligase complexes (3)

A
  1. SCF
  2. APC-Cdc20
  3. APC-Cdh1
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14
Q

explain: roles of each E3 ligase complex

A

SCF
- allows transition to S-phase

APC-Cdc20
- regulates transition from meta- to ana-

APC-Cdh1
- mediates exit from mitosis

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15
Q

question: what is happening in G1 phase (explain w/ cyclin-CDK and E3 ligases)

A
  • G1 cyclin-CDK and SCF are involved
  • G1 cyclin-CDK has 3 major targets
    ⤷ phosphorylating prot. at end of mitosis
    ⤷ prepare for DNA rep.
    ⤷ phosphorylate S-phase inhibitors
  • prepares for the next cyclin-CDK
  • phosphorylating S-phase inhibitors makes them a target for SCF
  • activates S-phase cyclin-CDK
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16
Q

question: what is happening in between G1 and M phases (explain w/ cyclin-CDK and E3 ligases)

A
  • G1/S-phase cyclin-CDK and S-phase cyclin-CDK involved
  • G1/S targets transcription factors that regulate exp. of genes coding for mitosis
    ⤷ ex. M-phase cyclin CDK
  • phosphorylating M-phase CDK inhibits it’s activation until cell is ready to start mitosis
  • S-phase cyclin-CDK needed to activate and assemble pre-replication complex
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17
Q

question: what is happening in M-phase (explain w/ cyclin-CDK and E3 ligases)

A
  • M-phase cyclin-CDK involved
  • many phosphorylation targets
    ⤷ chromosomal prot. (for condensation)
    ⤷ nuclear lamins (for envelope breakdown)
    ⤷ MAPs (for assembly of spindle)
    ⤷ kinetochore prot. in centromeres (for assoc. between chromo. and spindle)
    ⤷ APC complex (to prep. cell to go through mitosis phases)
  • regulated degradation
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18
Q

question: what is the regulated degradation that takes place during the M phase?

A
  • ubiquination and degradation at 2 points
  1. anaphase inhibitors = degraded to allow metaphase to anaphase transition (MAT)
  2. mitotic cyclin = degraded to allow cell to exit mitosis via mitotic exit network (MEN)
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19
Q

question: what is MPF?

A
  • first seen in frog eggs
  • mitosis promoting factor
  • a cyclin and CDK heterodimer
  • MPF = M-phase cyclin CDK complex
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20
Q

question: what is cyclin B?

Study These Flashcards
A
  • cycling prot.
  • regulates M-phase cyclin-CDK activity
  • seen from sea urchins
    ⤷ saw that a prot. had varying lvls of conc.
    ⤷ kept going up and down -> cycling
  • saw that increase in cyclin was coordinated w/ increase in number of cells engaging in mitosis
    ⤷ less cyclin = less cells in mitosis
21
Q

explain: in vitro experiment for cyclin

Study These Flashcards
A
  • used assay for MPF activity
    ⤷ looked for phosphorylation of target
  • measured cyclin B conc. on a gel
  • looked for beha. typical for a cell that does mitosis
22
Q

question: what happened in the in vitro experiment when treated w/ and w/out low RNase? what about when adding back in mRNA for cyclin B? what about adding non-degradable cyclin B?

Study These Flashcards
A

WITHOUT RNASE
- cells did mitosis
- cyclin B and MPF matched mitotic events
⤷ increase in cyclin B -> increase in MPF activity
- less cyclin B = less MPF activity = cells did mitosis exiting beha.

WITH RNASE
- removed mRNA but leaves tRNA and rRNA
- no increase in cyclin B
- no increase in MPF activity

ADDING BACK CYLIN B
- mitosis restored the same
- MPF activity returned

NON-DEGRADABLE CYCLIN B
- cyclin B lvls stay high
- cells stay in condensed state and can’t complete mitosis

conclusion = cyclin B is necessary for MPF activity
⤷ and need to be able to remove/degrade cyclin B

23
Q

question: how is cyclin B degraded?

Study These Flashcards
A
  • APC-Cdc20 and APC-Cdh1 target cyclin B
  • ubiquitination and degradation via proteasome
  • degradation begins at anaphase
  • APC-Cdc20 mediates w/ ubiquitinylation
  • degradation actually happens via APC-Cdh1 ubiquitinylation at exit from cell cycle
24
Q

question: how is cyclin B recognized by APC-Cdc20?

Study These Flashcards
A
  • recognizes short pep. seq. at N-term of cyclin B
  • pep. seq. = destruction box
    ⤷ arginine in pos. 1
    ⤷ leucine in pos. 4
    ⤷ asparagine or glutamine in pos. 9
    ** RxxLxxxxN/Q
  • any mutations to seq. -> undegradable form of cyclin B
  • adding D-box to other prot. will make them degrade the same was as cyclin B
25
question: does APC-Cdc20 have other targets other than cyclin B? if so, what?
- yes - targets an anaphase inhibitor = securin ⤷ ensures the replicated chromatids are secured before anaphase
26
question: how is securin removed?
- activation of APC-Cdc20 -> targets securin - when securin = removed, separase activates - metaphase has a prot. = separase - cleaves one of the cohesin prot. (Scc1) - breaks apart cohesin complex + chromatids = pulled apart
27
explain: role of SCF
- SCF = Skp, Cullin, F-box containing complex - SCF can recog. Sic1 (S-phase inhibitor) ⤷ only recog. when it gets phosphorylated by G1 cyclin-CDK - SCF degrades Sic1 -> activates S-phase CDK -> proceed to S-phase
28
recap: steps of cell cycle
1. early G1 ⤷ DNA pre-replication complex assembles 2. mid/late G1 **G1 cyclin-CDK** ⤷ inactivates APC-Cdh1 ⤷ activates S-phase cyclin-CDK ⤷ phosphorylates S-phase inhibitors 3. S **SCF** ⤷ degrades phosphorylated S-phase cyclin-CDK (Sic1) **S-phase cyclin-CDK** ⤷ activates pre-replicaiton complex 4. G2 **M cyclin-CDK** ⤷ activates early mitotic events 5. M METAPHASE **APC-Cdc20** (in between) ⤷ degrades securin ANAPHASE **APC-Cdh1** (in between) ⤷ degrades mitotic cyclin TELOPHASE AND CYTOKINESIS
29
define: genetic screen
- unbiased search for genes that are involved in a certain mechanism - make mutations in every gene + look at phenotypic effect
30
define: temperature sensitive mutation
- TS mut. - mutated gene codes for a TS prot. - allows researchers to change temp. to turn prot. on and off - ex. folding at permissive temp. vs misfolding at restrictive temps.
31
question: what are the mutations for s.pombe (fission yeast)?
- mut. to cdc2 gene - mut. to cdc13 gene - (TS) mut. to cdc25 - wee1 gene
32
question: what are the mutations from cdc2 in s.pombe?
- mut. in cell cycle regulators - cell division cycle mutants (cdc mutants) - 2 effects on cells ⤷ elongated cell ⤷ wee phenotype - elongated = delayed in G2 -> kept growing instead of going to mitosis - wee = went to mitosis too early -> smaller than normal
33
question: which s.pomble mutation is recessive vs dominant?
- wildtype = cdc2+ - elongated = recessive = cdc2- - wee = dominant = cdcD
34
question: what is the role of the cdc2 prot. in the cell cycle?
- absence of cdc2 = cell fails to divide - cdc2 increased = cell divides too early and too often - cdc2 = key regulator for entry into mitosis - cdc2 = CDK for MPF
35
question: what is the role of cdc13 prot. in cell cycle?
- also a cell cycle regulator - forms heterodimer w/ cdc2 - increases and decreases like cyclin B
36
explain: MPF of s.pombe
- heterodimer of cdc2 and cdc13 - CDK = cdc2 - cyclin = cdc13 - works as M, S, and G phase cyclin-CDK
37
question: what are the mutations from cdc25 in s.pombe?
- loss of func. cdc25 mut. -> elongated phenotype - (gain of func.) cdc25 dominant mut. -> wee
38
question: what is the role of cdc25 in cell cycle?
- lack of cdc25 inhibits entry into M-phase ⤷ meaning its an MPF activator - ex. gain of func. mut. -> early entry to mitosis
39
question: what are the mutations from wee1 in s.pombe?
- loss of func. mut. = wee- -> wee phenotype - gain of func. mut. = weeD -> elongated
40
question: what is the role of wee1 in the cell cycle?
- lack of wee1 causes premature entry to M-phase ⤷ meaning wee1 = inhibitor to MPF
41
question: how do cdc25 and wee1 regulate MPF?
- cdc25 = MPF activator ⤷ encodes a phosphatase that dephosphorylates tyrosine on cdc2 to activate MPF - wee1 = MPF inhibitor ⤷ encodes a tyrosine kinase that phosphorylates tyrosine on cdc2 to inactivate MPF **both affect same tyrosine on cdc2 (tyrosine 15)
42
question: what is the diff. between s.pombe and s.cerevisiae? similarities?
- s.pombe = fission yeast - sp. cerevisiae = budding yeast - budding yeast has formation of daughter cell that buds off mother in G1 - still have same cell cycle regulators - s.pombe's cdc2 = s.cerivisiae's cdc28
43
explain: impact of TS loss of func. mut. in G1 on s.cerevisaie
- mut. disrupts cell cycle causing cell to arrest in G1 - forms daughter bud but cannot enter S-phase - caused by mut. in cdc28
44
define: functional complementation (+ func.)
- technique to ID genes coding for cell cycle regulators - screens through genes to ID one that can fix the mut. and restore wild-type
45
explain: step 1 in functional complementation
- start w/ a TS mut. - permissive T = cells can grow - restrictive T = cells are arrested at G1
46
explain: step 2 in functional complementation
- add genes back in randomly to see which one restores the ability to divide - use genes from cDNA library - see which one makes a change in phenotype
47
question: what is a cDNA library and how is it made and used?
- complementary DNA - cDNA genes = copies of mRNAs of an org. - library made by isolating mRNAs using reverse transcriptase - means library isn't the whole genome ⤷ bits of mRNA expressed at a certain time
48
explain: step 3 in functional complementation
- ID the gene that makes the phenotypic change - go back to cDNA library ⤷ isolate the plasmid and sequence the cDNA - for s.cerevisaie = cdc28 - for s.pombe = cdc2

bio 2b03 (8 decks)

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