Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Cell Biology > Module 9 - Cell Cycle Regulation > Flashcards

Module 9 - Cell Cycle Regulation Flashcards

You may prefer our related Brainscape-certified flashcards:
AP® Biology flashcards Biology 101 flashcards
1
Q

cell cycle phases

A

cell cycle could be divided into 4 events: G1, S-phase, G2, mitosis

  • cell cycle must be highly regulated so that there is an alteration between DNA replication (s-phase) and cell division (M-phase)
  • these events must also be coordinated with the growth of. the cell
  • failure in the precise regulation of the events of the cell cycle can either lead to cell death, or overproliferation of cells
  • in multicellular organisms, loss of cell division means that the cells cannot be replaced and tissues cannot. be repaired
  • unregulated cell division is manifested as cancer
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

G1

A
  • first gap phase

- cell is actively growing, engaged in gene expression and the synthesis of new proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

S-phase

A

when the cell is ready to divide, it will replicate the genome entirely in S-phase (DNA synthesis phase)
-now a single chromosome contains 2 identical copies called replicated sister chromatids

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

G2

A

at the completion of s-phase, cells enter into a second gap phase G2, and when the cell is ready, will enter into mitosis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

G0

A
  • most cells will leave this cycle of active division and enter into a quiescent state where division. does not occur called G0
  • may be a short period of inactivity before re-entering into the cycle or the cell may begin the stepwise process of differentiating to become a specific type of cell
  • once differentiation has occurred, the cell is no longer capable of cell divison
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

cell divison vs differentiation

A
  • balance between cell divison and differentiation is important
  • if a population of SC stops dividing and all enter into Go the body loses its ability to regenerate and replace lost tissues
  • if the SC divide too much without differentiating, the body forms a tumor
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Mitosis

A

divided into phases

1) interphase (G1-S-G2)
2) prophase
3) prometaphase
4) metaphase
5) anaphase
6) telphase
7) cytokinesis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

interphase

A

the cell prepares for mitosis
-the chromosomes, a combination of DNA and associated proteins, are replicated in S-phase and the centrosomes are duplicated in G1 and S-phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

prophase

A

the chromosomes begin the process of condensation

  • assembly of. the mitotic spindle also begins as the duplicated centrosomes separated to opposite sides of the cell
  • ther changes that are not visible include the dissolution of the nuclear envelope and the breakdown of the endomembrane of the cell into small vesicles
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

prometaphase

A
  • the chromosomes are maximally condensed and are in the process of attaching via their centromeres to the fully. formed bipolar microtubule spindle
  • kinetochore proteins assemble at the centromeres to mediate the association with the plus ends of spindle microtubules
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

metaphase

A
  • characterized by the attachment of every chromosome to the spindlemicrotubules from both poles of the mitotic. spindle
  • this is called bipolar attachment
  • the tension that results from the forces pulling a duplicated. chromosome simultaneously towards both poles causes chromosomes to aggregate in the middle of the mitotic spindle, referred to as the equator of the spindle
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

anaphase

A

at the movement at which all chromosomes have achieved a bipolar attachment, a signal releases. the association between replicated sister chromatids and the sister chromatids are now pulled to opposite poles of. the spindle
-this process of sister chromatid separation defines anaphase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

telophase

A
  • once sister chromatids separation has occurred, the cell begins to reverse all the cellular changes that occurred in prophase
  • chromosomes decondensed, the spindle disassembles, and the nuclear envelope and endomembrane system reassembles
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

cytokinesis

A

mitosis is completed by separating the 2 cells, each containing an exact replicate of. the nucleus, through the process of cytokinesis that pinches off the cell membranes between the 2 cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

regulation of the cell cycle

A

regulated phosphrylation = cyclin CDK complexes

regulated degradation = E3 ubiquitin ligase complexes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

cyclin - CDK complexes

A

heterodimeric protein complexes
-the kinase activity of the complex is regulated through association with a protein called a cyclin
-an activated kinase will initiate various cellular processes through the phosphorylation of target proteins
4 classes of cyclin-CDK complexes activated in different parts of the cell cycle
1) G1-cyclin-CDK = active in G1, but leads to. the activation of the s-phase-cyclin-CDK
2) G1/S-phase-cyclin-CDK
3) S-phase-cyclin-CDK = required to transit. the cell into s-phase
4) mitotic-cyclin-CDK = will phosphorylate a collection of proteins required for all the cellular changes that occur in prophase
-all have the same structure and same kinase activity, but differ in target proteins and timing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

E3 ligase complexes

A

also required to regulate cell cycle events by targeting specific proteins for degradation in the proteasome
-in this way, cyclin’s could be degraded to turn off kinases of cell cycle inhibitors could be degraded when checkpoints are passed in the cycle
-3 distinct E3-ligase complexes
1) SCF complex releases the cell from G1 and allows. the transition into S-phase
The anaphase-promoting complex (APC) is an E3 ligase complex that has different target proteins depending upon the association with alternate accessory proteins called cdc20 and cdh1
2) APC-cdc2 = regulates the transition from metaphase into anaphase
3) APC-cdh1 = mediates exit from mitosis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

G1 cyclin-CDK and SCF E3 ligase

A

G1 cyclin-CDK will act in G1 to prepare the cell for DNA synthesis
3 major targets (1) by phosphorylation proteins such as the APC-cdh1 at. the end of mitosis, it signals. that mitosis is complete, (2)the cell must prepare for DNA replication. G1-cyclin-CDK targets TFs for phosphorylation. This modification activates the TFs and leads to the concerted expression of s-phase proteins. including nucleotide syntheses, replication. factors and DNA polymerase and more (3) G1-cyclin CDK will phosphorylate s-phase. inhibitors. These proteins bind to and prevent activation of the s-phase cyclin CDK. Phosphorylation of the inhibitor make sit a target for ubiquitinylation and subsequent degradation.
-therefore G1-cyclin-CDK is preparing the next cyclin CDK in the sequence
-the s-phase inhibitor is a target for ubiquitination by. SCF
-subsequent degradation activates the s-phase cyclin CDK allowing s-phase to begin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

G1/S and S-phase cyclin-CDK complexes

A

G1/S phase cyclin-CDK is responsible for preparing the cell for the upcoming M-phase.

  • targets of G1/S phase cyclin-CDK are TFs. that regulate the expression of genes coding for misosis including M-phase cyclins
  • other targets include proteins that mediate the process of centrosome duplication
  • S phase cyclin-CDK is necessary for activation and assembly of the replication complex at sites of origins of replication
  • phosphorylation of proteins associated with origins of replication ensure that the origins “fire” only once or are used only once per cell cycle
  • there must be only one replication complex per origin in each cell cycle
  • more than one would result in too many copies of segments of the DNA molecule
  • phosphorylation of the M-phase CDK inhibits activation. until the cell cycle is prepared to enter mitosis
  • one criteria for “readiness” is the completion of DNA replication
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

M-phase cyclin-CDK complex

A
  • the M-phase cyclin CDK complex has many diverse targets
  • each target is part of a dynamic change in cell behaviour that occurs during prophase
  • phosphorylation of chromosomal. proteins allows chromosome condensation
  • phosphorylation of nuclear lamins initiates nuclear envelope breakdown
  • phosphorylation of microtubule association proteins (MAPs) allows. the assembly of the mitotic spindles
  • phosphorylation of kinetochore proteins at chromosome centromeres allows chromosome spindle association; and phosphorylation of the APC complex prepares the cell for the serial progression through the phases of mitosis
  • ubiquitination and degradation of proteins occurs at 2 points in mitosis
    1) anaphase inhibitors are degraded to allow the metaphase to anaphase transition (MAT)
    2) mitotic cyclins. are degraded to allow the cell to exit mitosis (mitotic exit networks, MEN)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

identification of M-phase cyclin-CDK

A
  • the identification came from researchers studying the synchronized mitotic division seen in early embryos
  • studies were performed in both mammalian cells and xenopus. laevis oocytes
  • Masui and Marker identified a factor called the maturation promoting factor (MPF) that was able to include these cells to complete meiosis and initiate a series of mitotic divisions to form a blastocyst, before further cell division and differentiation allows the blastocyst to develop into a tadpole
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

MPF

A

cyclin and CDK heterodimer

-factor renamed to MPF and has shown to be the M-phase cyclin-CDK complex

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

sea urchin embryos

A

another useful model for studying mitosis

-undergo rapid synchronous mitotic divisons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

identification of cyclin B

A

in 1980, Hunt and Ruderman identified a collection of proteins that undergo cyclical synthesis and degradation during embryonic cell division cycles
-radiolabelled proteins were isolated at different time points after fertilization and then separated on. a polyacrylamide gel
x-axis is time points after fertilization. at which proteins were collected
y-axis is size of protein of the gel, largest proteins at the top as they migrate more slowly
-Hunt saw that some proteins remainder at a constant level throughout the series of cell divisions, whereas other proteins showed carrying levels of concentration-
-the [ ] was high and then. decreased only to be followed by an increase in [ ] again
-observation is that the 2 cycles are synchronized. Every increase in cyclin [ ] is followed by an increase in the number of. cells that are actively engaged in mitosis
-when the cyclin [ ] decreases, cells are not engaged in mitosis
-cycling protein turns out to be cyclin B, the protein regulating M-phase cyclin-CDK activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

cycles of cyclin B expression

A

cyclin B can be seen during interphase. and early mitosis, as the cell cycle proceeds, there is a rapid drop in cyclin B concentration that is associated with anaphase and exit from mitosis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

cyclin function

A

while MPF had kinase activity, cyclin has no enzymatic activity

  • in 1980s Murray and colleagues performed an invitro experiment to study the role of cyclin proteins
  • Murray’s lab isolated extracts from fertilized eggs that contained mRNAs and proteins necessary for cell division
  • the in-vitro experiments using cell extracts required 3 different assays or measurements of biological activities
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

3 assays

A

kinase activity
cyclin concentration
sperm chromosomes monitored for condensation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

kinase activity (MPF activity)

A

chromatin protein histone H1 phosphorylation
-used an assay for MPf activity
researchers simply assayed for phosphorylation of the target protein H1

29
Q

cyclin concentration

A

measured cyclin B protein [ ] in the extracts on a gel

-antibody to cyclin B

30
Q

sperm chromosomes

A
  • were monitored for condensation
  • looked for behaviours typical of a cell undergoing mitosis
  • in this case, in the absence of a cell, they looked at the behaviour of sperm nuclei added to the in vitro cell extracts
31
Q

first in vitro experiment

-untreated extract

A

cell extracts from mitotically dividing embryonic cells contain mRNAs, proteins and all of the cellular components necessary for protein synthesis

  • sperm cell nuclei were added to these extracts
  • in the cell free extracts, nuclei underwent cyclical behaviours typical of. embryonic cell division
  • the sperm nuclei underwent events typical of early mitosis such as chromosome condensation and nuclear envelop breakdown and late mitosis such as condensation and nuclear envelope reformation
  • overlaid ontop of this is the [ ] of cyclin B and the assay for kinase activity
  • can see that with an increase in cyclin B there is an increase in MPF activity
  • at the peak of MPF activity, chromosomes are condensing, a behaviour typical of Henry into mitosis
  • as the cyclin B [ ] goes. down, there. is a decrease in MPF activity with this decrease comes behaviours typical of exit from mitosis including chromosome decondensation
  • the synchronized cycling of these in vitro behaviours can continue in the absence of a cell for as many as 10 cycles
32
Q

second in vitro experiment

-RNase treated extract

A
  • exoeriment was repeated, but now the extracts are treated with low [ ] of RNase
  • this treatment completely removes all of the mRNAs from the extracts, but leaves the tRNAs and rRNAs necessary for protein. synthesis in tact
  • now there is no increase in cyclin B protein [ ] and. also no increase in MPF activity and no mitotic events
33
Q

third in vitro experiment

-RNase treated extract + wild type cyclin B mRNA

A
  • to test whether cyclin B is necessary for MPF activity, experiment was repeated with the RNase treatment, but also the subsequent addition of just 1 kind of mRNA for cyclin B
  • the synchronized behaviours of the original extract are completely responred
  • the only protein synthesized is cyclin B and we can see the cycling protein [ ]
  • can also see cycling CDK activity and the mitotic behaviours
  • cyclin B osscilations are sufficient to response cycling CDK activity
  • while this suggests that mitotic cycles require cyclin B, it does not show that cyclin B needs to oscillate
34
Q

fourth in vitro experiment

-RNase treated extract + non-degradable cycling B mRNA

A

-a signle kind of. mRNA is added to the RNAse treated extract
-in this case, the mRNA codes for a variant of cyclin B that cannot be degraded
-instead, cycling B levels remain high
correspondingly, CDK activity remains high. and the sperm nuclei enters something similar to mitotic arrest
-the chromosomes remain in a condensed state typical of early mitosis

35
Q

cyclin degradation and cyclin CDK inactivation

A
  • experiments revealed the relationship between cyclin B synthesis and degradation and the regulation of CDK activity in the control of mitotic events
  • images show a florescent DNA stain and a fluorescently labelled tubulin
36
Q

degradation of cyclin B

A
  • RNase treated extracts with added cyclin B mRNA
  • from 0-40 mins can see the condensed chromosomes undergoing anaphase (15 mins) and telophase (chromosomes condensation at 40 mins)
  • at the same time, the mitotic spindle can be seen. assembling and dissasembling
37
Q

failure to degrade cycling B

A
  • RNase treated extracts with added mRNA for the non degradable cyclin B
  • while the chromosomes undergo anaphase, they fail to decondense and the mitotic spindle does not dissassemble
  • see that cyclin B must be degraded to allow a cell to exit mitosis
  • synthesis of cyclin B is needed to activate CDK and the removal of cyclin B is needed to turn of CDK
38
Q

cyclin B degradation by APC-cdh1

A
  • the APC-cdc20 and APC-cdh1 complexes are E3 ligases that target cyclin B for ubiquitination and degradation via the proteasome
  • the destruction of cyclin B inactivates CDK activity and. allows the cell to proceed through and exit mitosis in preparation for the next cycle of cell divison
  • although degradation of cyclin B begins at anaphase, mediated through APC-cdc20 ubiquitination, the degrataion of cyclin B that allows exit from the cell cycle occurs via APC-cdh1 mediated ubiquitination
39
Q

recognition of cyclin B by APC-cdc20

A
  • in a study by Kirshner, a short peptide sequence found near the N-terminus of cyclin B, was identified as the recognition sequence for APC-cdc20
  • sequence is called the destruction box (D-box)
40
Q

D-box

A

-RxxLxxxxN/Q
where x is any aa, R is arginine, L is leucine and N/Q is asparginine or glutamine
-required for recognition and ubiquitination of cycling B
-necessary for degradation
-mutations that replace any one of the conserved aa residues will produce a stable, non degradable form of cyclin B
-the D-box is sufficient for. degradation; the addition of the D-box to a stable protein such as GFP will make that protein undergo cyclical degradation akin to that seen for cyclin B

41
Q

other APC targets

A

experiment was repeated with a the addition of a short peptide, the D-box peptide

  • DNA is monitored through the addition of a fluorescent DNA stain
  • for each [ ] the nucleus is examined at different time points
  • when no peptide is added, see typical chromosome behaviours. at 15 mins, chromosomes are compact and in anaphase. at 35 mins, the chromosomes are decondensing and in telophase
  • when 20 mg/ml of D-box. is added, the cell behaviours are delayed. at 15 mins, chromosomes still in metaphase not anaphase. at 35 mins, the chromosomes are completing anaphase but still condensed
  • the dealt is increased when 40 ug/ml of D-box is added. at 15 mins, chromosomes are still in metaphase, at 35 mins, chromosomes are only just entering anaphase
  • whjen 60 ug/ml and 80 ug/ml are added, the cells are arrested at 15 and 35 mins
  • suggests that the APC has a second target in addition to cyclin B. This target seems to be an anaphase inhibitor
  • when the anaphase inhibitor is not degraded bc the APC is too busy binding to the D-box peptide, anaphase does not occur
42
Q

anaphase inhibitor (securin)

A
  • name comes from its function
  • it indirectly ensures that the 2 sister chromatids are secured together prior to anaphase
  • the cohesion complex is a collection of cohesion proteins (SMC1, SMC3 and SCC1) that attach the sister chromatids together at replication
  • this association persists as the cell enters mitosis and the chromosomes condense
  • in metaphase, the cohesion complex can be seen holding the 2 replicated DNA molecules
43
Q

separase

A

able to initiate separation of sister chromatids by cleaving one of the cohesion proteins. SCC1

  • this protease is kept inactive through its association with securin proteins
  • it is in this way that securin secures sister chromatid association
44
Q

activation of APC-cdc20

A

enables E3 ligase to target securin for ubiquitination and degradation

  • once securin is removed, the separate protein is activated
  • active separate can now target SCC1 for cleavage at a single peptide recognition sequence
  • this one cut breaks apart the cohesion complex and the sister chromatids attached to opposite spindles poles can be pulled apart
45
Q

role of APC-cdc20 in degradation of securin

A

one. of the APC subunits is a. target of cyclin B-CDK kinase activity
- at entry into mitosis, the cell is preparing for the metaphase to anaphase transition
- the addition of the cdc20 specificity factor is necessary.y for the ability of. APC to target securin for ubiquitination

46
Q

APC-cdh1 ubiquitinylates cyclin B

A

APC-cdh1 degrades cyclin B at the end of mitosis

  • cdc20 is exchanged for cdh1 after. anaphase in order to change the target specificity of APC
  • one of the APC subunits is a target for cyclin B-CDK kinase activity
  • in this case, cyclin B CKD is preparing for its own inactivation by activating APC at the beginning of mitosis
  • after telophase APC ubiquitinylates cyclin B; cyclin B degradation inactivates the M-phase cyclin
  • the addition of the cdh1 specificity factor is necessary for the ability of APC to target cyclin B for ubiquitinylation
  • in the absence of active cyclin B-CDK, phosphatases, dephosphorylate APC inactivating it in G1
47
Q

SFC ubiquitinylates the S-phase inhibitor, Sic 1

A

SFC. is an acronym for 3 proteins identified as part of the complex in different species (Skp, Cullin, F-box containing complex)
-in mid G1, the S-phase CDK complex is inactive, allowing the cell time to grow in preparation for mitosis
-the S-phase CDK complex. must be kept inactive until the cell is ready
-this is accomplished through the association of an inhibitor with the cyclin CDK
-there are many such inhibitors that bind to and inactivate a cyclin CDK complex, while still keeping the complex intact
-in this case, the S-phase inhibitor is called Sic 1, which is a target of the G1 cyclin CDK
-The phosphorylation of Sic 1 allows it to be recognized by. the SCF E3 ligase, ubiquitinylated and degraded
-removal of Sic 1 activates the S-phase cyclin CDK and the cell. proceeds through into s-phase
the cell is committed to entry into S-phase once the sic 1 protein is degraded

48
Q

genetic screen

A

unbiased search for genes that are involved in a particular mechanism

  • if you are interested in identifying genes required for the precise regulation of the cell cycle, you start by assuming that every gene in the genome is a potential candidate
  • you create mutations randomly in every gene
  • thousands of. different mutations are created, but on average there is one mutation. per cell
  • look at phenotypic effects of the mutation
  • a mutation that affects the cell cycle could inhibit cell division or cause cell division to occur to rapidly
49
Q

genetic mutations

A

created at random, any gene may be mutated

-each mutation can potentially have a different phenotype

50
Q

temperature sensitive mutant

A
  • a temperature sensitive (TS) mutation is a special class of mutation
  • mutated gene codes for a TS protein
  • this is a protein that folds at the permissive temperature of 24 degrees but misfolds at the restrictive temperature of 37 degrees
  • this allows the researcher to change the temperature and turn on and off protein function
51
Q

fission yeast (S. pombe)

A

model organism

  • see elongated rod shaped cells that are actively dividing in cultire
  • cells undergo nuclear division followed by cytokinesis
52
Q

genetic screen for TS cell cycle mutants

A
  • mutation in cell cycle regulators (cell division cycle mutants or CDC mutants) can have 1 of 2 effects on the cells
    1) one phenotype is the elongated cell
  • cells are delayed in G2 and keep growing instead of entering mitosis, as a result, cells grow longer than they should
    2) another phenotype is the wee phenotype
  • these cells transition into mitosis prematurely and as a result, the cells are smaller than normal
53
Q

mutants in cdc2

A
  • loss of function mutation causes an elongated phenotype

- a gain of function mutation causes wee phenotype (dominant)

54
Q

identifying mitotic CDK in s.pombe (cdc2)

A
  • in the absence. of the cdc2 protein, the cell fails to divide
  • when cdc2 protein function is increased, the cell divides too early. and too frequently
  • suggests it is a ket regulator for entry into mitosis and acts to promote cell division
  • cdc2 is a. 32 kDa protein with kinase activity
  • forms a heterodimer with another cyclin cdc13
  • looks like cdc2 might be cyclin0dependent kinase of the MPF
55
Q

cdc13

A

also identified as a cell cycle regulator in a genetic screen

  • required for mitosis
  • loss of function mutations in cdc13 cause the elongated phenotype and dominant mutations. produce the wee phenotype
  • the cdc13 protein is found to increase and decrease in concentrations during the cell cycle
56
Q

cdc2-cdc13

A

a heterodimer of 2 proteins that are the s.pombe MPF

  • in fission yeast, there is only 1 CDK, cdc2 and 1 cyclin cdc13
  • this complex functions as not only the m-phase cyclin-CDK but also the S and G phase cyclin CDK as well
57
Q

cdc25 mutants

A
  • many other temperature-sensitive mutants were identified in genes coding for cell cycle regulators
  • a loss of function cdc25 mutation produces an elongated cell
  • a dominant cdc25 mutation causes the wee cell phenotype
  • since a lack of cdc25 inhibits entry into M-phase it is likely that cdc25 is normally an activator of MPF and promotes entry into M-phase
  • this is supported by the observation that the gain of function mutation caused premature entry. into M-phase
  • cdc25 is an activator of the cell cycle that activates the MPF complex
58
Q

wee1 mutants

A
  • gain of function mutation causes the elongated phenotype
  • loss of functions mutation causes the wee phenotype
  • since lack of wee1 causes premature entry into M-phase, it is likely that wee1 is normally an inhibitor of MPF and delays entry into M-phase
  • this is supported by the observation that the gain of function mutations prevent entry into M-phase
  • wee1 is an inhibitor of the cell cycle that inactivates the MPF complex
59
Q

regulation of MPF activity

A
  • wee1 and cdc25 are 2 regulatory of MPF activity
  • wee1 is an inhibitor, it is a tyrosine kinase that phosphorylates tyrosine 15 on cdc2 and inactivated the MPF complex
  • cdc25 is an activator, it is a phosphatase that reverses this phosphorylation, dephosphorylation tyrosine 15 and activating MPF
60
Q

activation of MPF

A

MPF is regulated by phosphorylation and dephosphorylation

1) first step in regulation of MPF is the synthesis of mitotic cyclin
- in the absence of cyclin, the CDK (cdc2) is. inactive
- assicoation between the cyclin and the CDK is required for activation
- 2 aa residues on cdc2 are important in the next step in phosphoregulation of MPF
a) wee kinase that phosphorylates tyrosine 15 (this is inhibitory phosphorylation that keeps MPF inactive)
b) CAK kinase, phosphorylates threonine 161. while this is an activating phosphorylation, MPF is still inactive due to the phosphorylated tyrosine 15
c) cdc25 phosphatase activator which dephosphorylation tyrosine 15, thus activating cdc2 kinase

61
Q

achieving final active MPF

A

could have been achieved using just the CAK kinase

  • a cell with a double mutation lacking wee1 and cdc25 activity can still divide
  • cell division is a little slow and cells in culture become unsynchronized but ultimately division occurs
  • phosphorylation of Y15 seems to increase the affinity of the CAK protein for the target substrate
  • the multiple cell cycle regulators seem to increase the efficiency of cell cycle regulation
62
Q

budding yeast (s. cerevisae)

A

difference between budding yeast and fission yeast is the formation a daughter bud off of the mother cell in G1 before the cell enters S-phase
-despite some differences in the appearance of the cell cycle, the same cell cycle regulators are used

63
Q

TS-loss of function mutations arrest in G1 of budding yeast

A

mutations that disrupt the cell cycle of budding yeast cause the cell to arrest in G1

  • as a result, the cell forms a daughter bud, but fails to proceed into s-phase
  • cdc28 is the budding yeast homolog of the fission yeast cdc2
  • as in fission yeast, there is just a single CDK that regulates cell cycle transitions in the budding yeast
64
Q

functional complementation

A

another technique used to identify genes coding for cell cycle regulators

  • principle of the technique is to screen genes to identify 1 that can. rescue the phenotype caused by a mutation and restore wildtype behaviour
  • usually the resulting gene that is added is the wildtype version of the mutated gene that caused the phenotype
  • also useful for studying functional homologs between species
65
Q

functional complementation step 1

A
  • need a TS mutant that causes a cell cycle defect
  • at permissive temp (25 degrees) cells divide and the culture grows
  • at the restrictive temp (35degrees) the cells arrest at G1
66
Q

functional complementation step 2

A
  • the mutation that caused the phenotype is unknown
  • in order to identify the effected gene, genes are added back at random to restore the ability of the cells to divide
  • a cDNA library is a collection of genes created for an organism and stored in bacterial cells
  • cDNA = complementary DNA
  • the cDNA genes are copies of the mRNAs expressed in. an organism, simple versions of the genes that represent spliced versions without introns
  • the library is created by isolating mRNAs and using an enzyme (reverse transcriptase) to make DNA copies of the messages
  • in this way, a cDNA library does not represent the whole genome, but the collection of mRNAs expressed at a particular time
  • in this case, a cDNA library for mitotically dividing cells should be used as it will contain copies of genes coding for cell cycle regulators
  • the cells are grown at the restrictive temp so that they are unable to divide
  • as each cDNA. is added, the phenotype is monitored. The addition of gene X or Y did not change the phenotype, the cells were still in G1 arrest, but the addition of gene Z restored normal cell division even at the restrictive temp
  • gene Z is said to have rescued the mutant phenotype
  • the most likely explanation. is that gene z is a wild type cDNA copy of the mutated gene that caused cell cycle arrest
67
Q

functional complementation step 3

A
  • go back. to cDNA library contained in bacterial cells and identify gene Z
  • the bacteria is carrying gene Z in a small extra, circular DNA called a plasmid
  • isolating the plasmid from the bacterial cell allows the researcher to sequence the cDNA
  • the gene. was named cdc28
  • cdc28 gene in budding yeast. and cdc2 gene in fission yeast code for the same protein cdk1
  • budding yeast cdc28 can be rescued by supplying the wildtype fission yeast cdc2 to the cells
  • highlights conservation of the mechanisms of the cell cycle control
68
Q

cdc28

A

codes for the single CDK in budding yeasty

  • it interacts with a cyclin to form an active heterodimer that targets for phosphorylation proteins that are necessary for DNA synthesis
  • often, instead of the term MPF, we would refer to this as an S-phase promoting factor or SPF
  • while there is a single CDK in s.cerevisiae, there are multiple cyclins, cln 1, 2 and 3 are considered to be G1/s-phase cyclins and associate with CDK 1 to form the SPF
  • Clb 1 and 2 are M-phase cyclins that are more similar to cyclin B
  • a clb cyclin associates with CDK 1 to form MPF
69
Q

conservation of cell cycle regulation

A

principles are conserved across all eukaryotic systems

  • in vertebrates, there are multiple CDKs and multiple cyclins, however, we see homologous cyclin-CDK complexes at each step in the cell cycle
  • in addition, homologous of the kinases and phosphatases that regulate MPF activity have been identified across diverse eukaryotic species

Cell Biology (6 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions