Cell Cycle (MY GOAT HIRO) Flashcards

Question

How to analyse cell cycle mutants (cdc)

Answer 1

Phenotypic analysis Classical genetics molecular genetic analysis

Answer 2

analysing the arrest stage of the mutants look at: -DNA contents -Visualise state of nucleus, chromosomes, spindles -biochemical analysis for proteins with cell-cycle functions

Answer 3

-making a diploid with a wt yeast to see if mutant is dominant or recessive usually recessive -complementation tests: cross with other cdc mutants if still temperature sensitive (no rescue) then both are mutants in the same gene

Answer 4

-Gene cloning: physical isolation of the WT version of mutated gene usually through complementation -to isolate a DNA fragment which rescues the mutation -Sequence determine DNA sequence >the predicted Amino acid sequence of gene product >may give clue about biochemical function

Answer 5

proteins/gene products involved in particular cell cycle events pathways which regulate particular cell cycle events overall control of cell cycle progression

Answer 6

in the middle of G1 cell decides whether it will divide or do something else -nutrition (if dividing w/out nutrition then cells would get smaller and smaller) -cell size -sexual signals (mating/meiosis)

Answer 7

withdraw some nutrients from an asynchronous culture: -Cells in early G1 all arrest in G1 -cells in late G1,S,G2,M can all continue with the division cycle theyre currently in but then arrest in the next G1 Middle of G1 is the important boundary START -chekcpoint for nutrient availability -if YES then keep going - Commited to division even if nutrients removed after until they next reach the checkpoint also check for size, sexual partner

Answer 8

budding yeast sometimes when a daughter cell buds off mother the mother cell is large enough to continue right away in the cell cycle BUT the daughter cell is too small so needs to wait until it reaches a larger size so that subsequent cycles produce sufficiently large cells and they dont keep shrinking over time

Answer 9

cells meet partner - "courtship" but the cell cycle continues until they reach the next start at start - with the mating factor present - causes them to arrest at Start in G1 can then begin mating/conjugation

Answer 10

the key kinase for start (in S cerevisiae i think) cdc28 mutants arrest at start in G1 normal cdc28 gene function required for passing start (ie commiting to division) encodes a Serine/Threonine protein kinase phosphorylates substrates leading to commiting to rest of cell cycle is the equivalent of cdc2 in S. pombe

Answer 11

Hit yeast w X-ray causes DNA damage causes cell to arrest right before mitosis in S-phase if a DNA break goes into mitosis chromosomes separate and some chunk of chromosome gets lost but if stop right before can allow for repair then continue with intact DNA

Answer 12

Mutants who fail to arrest - radiation sensitive (rad mutants) most rad mutants arrest in G2/M phase but then die as they cannot repair DNA rad9 mutants can repair DNA fine but lack the ability to arrest before M-phase so do not have time to repair before mitosis and so die because of that

Answer 13

DNA damage: G2/M transition DNA replication checkpoint: G2/M transition Spindle assembly checkpoint: Metaphase/Anaphase transition, protects against trying to divide with eg spindle defects and more keep the cell cycle under control to ensure it occurs right each time

Answer 14

-is yeast - easy to grow, manipulate, isolate mutants -grow by length extension cell length can be used as a marker for cell cycle -are more similar to higher eukaryotes in some aspects -mitosis more similar as spindle only forms in M-phase, and Cell division is symmetrical

Answer 15

slightly different so S. cerevisiae Cell cycle mutants become long are able to grow at restrictive temp BUT it blocks division so keep growing and become abnormally long used to isolate a lot of cdc mutants

Answer 16

wee mutants go into mitosis early in WT cell grows and enters mitosis at certain size but wee mutants do this at smaller size - too early

Answer 17

are regulatory cdc mutants can be any component missing BUT only regulatory parts speed it up when broken cdc mutants stop wee mutants (regulatory) speed it up

Answer 18

wee1 all mutants were recessive (loss of function) meaning wee1 is a NEGATIVE regulator (an inhibitor) of mitosis entry overexpression of wee1 delays or prevents entry into mitosis gives the long cdc mutant phenotype w no mitosis

Answer 19

wee2 mutant is a dominant allele of cdc2 a HYPERACTIVE version hyperactivity of the gene causes early entry into mitosis so is a POSITIVE regulator (inducer) of mitosis loss of function mutations of cdc2 gene delayed mitosis giving long cells

Answer 20

recessive cdc25 mutants give arrest in G2 - long cells in S. pombe overexpression gave the wee phenotype - early mitosis so cdc25 is a positive regulator/inducer of mitosis

Answer 21

wee1 (mitosis inhibitor) inhibits cdc2 activity cdc25 activates cdc2 activity cdc2 drives transition into mitosis

Answer 22

fused cell has 2 nuclei from 2 diff cells fuse mitotic cell with interphase cell either: 1. M cell has mitotic inducer present fuse with I cell that does not once fused the mitotic inducer can diffuse and induce mitosis in interphase nucleus 2. mitotic inhibitor exists in interphase cell diffuses in fused cell mitotic chromosomes inhibited giving two interphase nuclei 3. non-diffusible chromosome/nuclear factor: cant diffuse (maybe tightly associated w chromosomes) -no diffusion in fused cell mitotic and interphase chromosomes stay as they are 1 is correct - the DIFFUSIBLE MITOTIC INDUCER

Answer 23

immature oocyte matures and begins meiosis then arrests at metaphase of 2nd division continues after fertilisation -immature oocyte resembles G2 phase in cell -mataphase of meiosis II resembles M phase in cell can collect many of these cells as they are arrested at both of these points (Meiosis only begins from immature oocyte when exposed to hormones) M phase cytoplasm can induce maturation of the immature oocytes into "m-phase"

Answer 24

present in the cytoplasm of the meiosis II arrested oocyte is able to auto-activate so active MPF induces MPF-precursors in immature oocyte to become active MPF

Answer 25

fertilise mature oocyte can take cytoplasm from different stages of dividing embryonic cells inject into immature oocyte and observe ability to induce maturation/MPF activation MPF activation is high during mitosis is when the inducing capabilities of the cytoplasm is highest MPF activity is found late in G2 and in M-phase is conserved in inducing mitosis AND meiosis

Answer 26

is a mitotic inducer in different contexts: Maturation promoting factor Mitosis promoting factor M-phase promoting factor

Answer 27

cleavage divisions during early ebryonic divisions very little protein synthesis BUT if it protein synthesis is inibited - no divisions take place so this little bit of synth is important for cell division

Answer 28

introduce radioactive AAs to the early embryo cells to label any proteins currently being synthesised run proteins from cell on gel use autoragiograph to identify bands corresponding to the proteins synthesised during these divisions discovered Cyclins bands become stronger towards mitosis peak then come back down in interphase -Cyclin A -Cyclin B

Answer 29

continuous synthesis during interphase levels of the protein peak at beginning of M-phase then degraded in M-phase - needed to exit mitosis

Answer 30

Fission yeast: cdc2 (and wee1/cdc25) mammal: diffusible mitotic inducer xenopus: MPF sea urchins: cyclins

Answer 31

the same protein product controls Start and Mitosis cdc2 (S. pombe) and cdc28 (s. cerevisiae) encode similar protein kinases conserved are functionally homologous -expressing one species' gene in a mutant of the other rescues the phenotype

Answer 32

S. pombe cdc2 mutant introduce a mixture of vectors with human cDNA introduces the DNA into the yeast cdc2 mutants then plate them many recovered a random gene from the cDNA and were still temperature sensitive however some were rescued by the human cdc2 homologue - were able to grow at restrictive temperature -pick out colony -sequence the cDNA that integrated encodes a protein kinase similar to cdc2 called Hs cdc2 (homo sapiens) ALL eukaryotes have a cdc2 homologue

Answer 33

only knew that active MPF from unfertilised M-phase like egg could induce maturation in other cells purify protein with same activity as MPF: -homogenise oocytes -fractionate the homogenate -separetes protein according to size (big proteins fall faster) -can inject different fractions into immature oocytes and look for maturation activity -have narrowed down fraction containing MPF -take this fraction and separate on another column -can cycle this process -gets purer and purer -takes ages tho :/

Answer 34

consists of a heterodimer fo cdc2 and cyclin B xenopus homologues this complex regulates G2/M transition in ALL eukaryotes

Answer 35

cyclin dependent kinases: requires cyclin subunit to be present to be catalytically active cdc2 = cdk1 diff cyclins complex wit diff Cdks to form the binary kinase

Answer 36

cdc2 levels are constant throughout the cell cycle instead its kinase activity is regulated by cyclin B levels going up and down cdc2 kinase activity only happens when cyclin B levels go up then decreases when cyclin B levels drop though even when cyclin B is bound - Cdc2 activity still isnt very high there is another level of control

Answer 37

in all eukaryotes cdc25 is an activator wee1 is an inhibitor of cdc2 cdc25 gene encodes a protein phosphatase wee1 gene encodes a protein kinase -phosphorylated cdc2 is inactive - wee1 product phosphorylates and inactivates it -cdc25 phosphatase removes this Pi and allows it to activate

Answer 38

wee1 activity higher in G2 so cdc2/cyclin B complex is inactive cdc25 activity increases when M-phase begins removes phosphate allows cdc2/cyclin B to activate cdc2/cyclin B activity inhibits wee1 so positive feedback once cdc25 activates it cdc2/cyclin B complex can now phosphorylate substrates for beginning mitosis

Answer 39

Cdk is the same at these stages but complexes w a different cyclin Cdk1:G1-cyclin - progress past Start Cdk1:G1/S-cyclin - takes part during G1 Cdk1:S-cyclin - progress into S-phase Cdk1:Cyclin B(M-cyclin) regulates G2->M

Answer 40

Cdk1:G1-cyclin - phosphorylates targets which activate transcription of G1/S-cyclin and S-cyclin G1/S-cyclin is active before S-cyclin -because S-cyclin is usually inhibited -BUT the G1/S-cyclin/Cdk1 complex destroys the S-cyclin inhibitor

Answer 41

Cki bind Cdk/cyclin complexes and inactivate them the Cki inhibiting Cdk1/S-cyclin needs ti be destroyed before S-phase can commence -Cdk1:G1/S-cyclin phosphorylates this Cki -allows Cdk1/S-cyclin to be active and progress to S-phase

Answer 42

in G1->S-phase progression: -CKIs are degraded in M-phase: -Cyclin B is degraded to exit mitosis

Answer 43

marks protein for degradation by the proteasome ubiquitin chains added onto protein once enough ubiquitin is attached, protein is now marked for degradation Ubiquitin is added by ubiquitin ligase (E3) selectively attach ubiquitin, determining specificity and timing of degradation once chain is formed then localisation to proteasome for degradation is automoatic

Answer 44

active only in M-phase and G1 ubiquitinaltes Cyclin A, B, other proteins is an E3 which directs degradation during M-phase

Answer 45

is an E3 to direct degradation during G1 active only in G1 ubiquitinates the phosphorylated CKIs from the Cdk1/S-cyclin complex allowing it to be degraded

Answer 46

yeast has just one Cdk however mammals have multiple Cdks as well as mutliple cyclins some cyclins also have multiple forms >increases complexity of the system also difficulty of genetic analysis: -harder to KO in diploid mammal cell than in haploid yeast -RNAi and CRISPR developments help w this >but was harder in beginning

Answer 47

limiting factor is cyclin levels Cdk levels normally constant so amount of cyclin determines the amount fo complex and hence the activity of the corresponding Cdk SO overexpression of cyclins can be a way of determining their activity from the altering of the phenotype

Answer 48

can cause artifacts can cause them to begin complexing with Cdks that it normally does not

Answer 49

Cyclin E: Shorter G1 Cyclin D: also shorter G1 Cyclins D and E are essential to G1->S progression

Answer 50

when there was no good method to KO a gene: Inject Ab that binds and interferes with cyclin function (eg blocking Cdk binding) -can use this to determine when different cyclins are important in cell cycle Inject Cyclin D Ab: No DNA replication, cells stay in G1 - dont enter S-phase evidence for cyclin D being essential for progress into S-phase

Answer 51

G1-Cdk complex: Cdk4, Cdk6 + cyclin D G1/S-Cdk complex: Cdk2 + Cyclin E S-Cdk complex: Cdk2 + Cyclin A M-Cdk complex: Cdk1(=cdc2) + Cyclin B

Answer 52

most adult cells not dividing referred to as quiescent metabolically active but not dividing in terms of DNA content - are in G1 like state some quiescent cells can be stimulated to divide can switch on and off when needed

Answer 53

cells taken from a person and put in culture: -will all stop dividing after certain amount of divisions -Senescence -have to take cells many time sometimes can get an immortalised cell line that can divide forever -not 100% normal as have mutation that immortalises them -but the cell cycle in some immortal cell lines are still normal so can be used for cell cycle research

Answer 54

require them to divide without them they enter a quiescent state Serum usually added to culture media contains multiple active components required for cell proliferation -growth factors or mitogens

Answer 55

remove growth factors if cell is in early G1 then they will stay in G1 dont go into S-phase or divide if cell is in later G1 - cell still goes into S then divides (same for S, G2, M cells) so within G1 there is a boundary -before which cells need Growth factors to divide -but after passing it they are commited to continuing through that cycle this point is the Restriction point (R) in mammal cells V similar to concept of Start in Yeast

Answer 56

say that cells in the quiescent state at R are in G0 not G1 as it can take hours to return to cell cycle after growth factors reintroduced better to think of it as cell going into diff state outside of cell cycle G0 molecularly different to G1

Answer 57

Cell cycle control: -cell is going through cycle -and then there is eg DNA damage -cell stops there before it is fixed >Mainly controlled through INTERNAL signals Proliferation control: -cell goes between being in cell cycle, or out of it (G0) -mainly determined by external signals (growth factors...) these two controls are linked at R point

Answer 58

-Growth factor binds Receptor Tyrosine Kinase -activates Ras G-protein -activates 3 kinases in sequence: >Raf (MAP3K) >MAP2K >MAPK -MAPK stimulation: >stimulates general metabolism to grow cell >AND transcription of cell cycle genes - triggers passage of R point

Answer 59

the MAPK activates transcription of Cyclin D (unstable so only present with mitogen signalling), a G1 cyclin complexes to form active Cdk4/6-Cyclin D as soon as transcription stops: -Cyclin D levels drop quick -need high transcription to keep high Cyclin D levels Cdk4/6-cyclin D complex actovates G1/S-Cdk and S-Cdk by activating trasncription of corresponding cyclins

Answer 60

Cdk4/6-Cyclin D (G1-Cdk) adds two phosphates to Rb protein this inactivates it causing it to change conformation and free up the TF E2F free active E2f can go to nucleus and activate transcription of Cyclin E and A Allows for activation of G1/S-Cdk and S-Cdk i think the same Cki stuff happens with active G1/S-Cdk and S-Cdk as in yeast

Answer 61

in G0: -Cdk and cyclin levels down -Cki levels up -assures cells are fully arrested Transition from G0-G1: -Cdks and cylcins resynthesised -Ckis destroyed by SCF (a ubiquitin ligase/E3) >this takes time - hence the lag for cells to exit G0 after serum added

Answer 62

after serum re-added 2 waves of trancription -Early response genes transcribed fast, within hours -Delayed response genes transcribed later Early response genes encode the TFs for delayed response genes so if add a protein synthesis inhibitor: -Early gene transcription is independent of protein synth -however the transcribed mRNAs cannot be translated -the TF products cannot go on to activate delayed response genes -so no delayed response transcription

Answer 63

MAPK phosporylates targets those targets activate early response genes early response genes encode TFs

Answer 64

actiavted by the TFs from early response encode cyclin D E2F SCF subunits components necessary for progression through G1 into S

Answer 65

clone of cells accumulating multiple mutations to gain anti-social behaviour eg: -uncontrolled proliferation -invade other tissues -survive and proliferate in foreign sites -genetically unstable cell cycel misregulation important part of cancer

Answer 66

Proto-oncogenes -present in normal cells -can gain hyperactive mutations -oncogene activated (induces cancer) -these genes induce proliferation Tumour suppressor genes: -loss of function mutation happens in gene that normally inhibits proliferation -induces cancer -usually recessive mutation need both loss of function alleles many anti cancer drugs target cell cycle proteins

Answer 67

Growth factors RTKs Ras G-protein early response gene TFs Cyclin D

Cell Cycle (MY GOAT HIRO) Flashcards

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