L10 - Genomic Instability Flashcards

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1
Q

Describe the disovery of p53

A

In 1975 - thought to be the basis of the decision to replicate the DNA

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2
Q

What virus was used to help discover p53

A

Papovirus - SV40

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3
Q

Describe the normal distribution of SV40 protein

A

Reside in the nucleus

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4
Q

Advantage of SV40 proteins

A

Immunogenic - they are easy to raise antibodies against

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5
Q

What is the relevant viral protein from SV40

A

Large T

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6
Q

Describe what happens when you transfect cells with viral SV40

What is the experimental method required to see this

A

Can see that proteins are constantly undergoing protein synthesis

Shown through the addition of a radio-AA

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7
Q

What was the next direction after the SV40 transfection

A

To ask wehther large T interacted wth any other host proteins

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8
Q

Results of immnoprecipitation experiemnts of cells TF with SV40 large T

A

In presence of antiserum or antiserum from control animal pre-immunisation
Only immunopreciptate in transformed SV40 cells where antibody present

Band around 54kD = p53

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9
Q

Purpose of a cellular transformation assay

A

Allows you to determine if cell is transformed in the presence of an oncogene

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10
Q

How would you tell if a cell had been transformed

A

If the cell shows anchorage independent growth … transformation … clone into an immune compromised mouse

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11
Q

Ras + P53 mutant

A

Normal number of colonies

CONTROL

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12
Q

Ras+P53

P53 FROM TISSUE CULTURE CELLS

A

Increased numbr of colonies … looks like p53 acting as an oncogene

BUT

Tissue culture cancer cell line - vast majority of p53 has been lost due to genomic instability

(In the case from the paper there was a Val135 mutation)

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13
Q

Ras + WT (true) P53

A

No colonies
Onogenic function of Ras has been supressed

P53 BEHAVES AS A TUMOUR SUPRESSOR GENE

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14
Q

What cancers display a p53 mut

A

ALL

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15
Q

What happens usually when TSGs are deleted in the embryo

What is done to get around this?

Is this the case for p53

A

Embryonic lethality
- Since many TSGs are regulators of cell numbers

Conditional KO approaches

Not the case for P53

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16
Q

Why are the Kaplan Meier plots for p53 mutations unusal?

A

If P53 is a TSG would expect to see Knudsens rules followed… but they aren’t

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17
Q

What is an epititope map

A

Pannel of antibodies that recognise p53 but each recognises it at a different site (epitope)

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18
Q

Describe the experiments that lead t the conclusion of p53 having a short half life

A

Label cells with radio-methionine
Harvest cells at 1 hour
Blot for p53 with Pab421
Can see after around 45 minutes alsmost all 421 expression is lost

p53 HAS A HIGHTURNOVER RATE

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19
Q

Descibe the effect of LargeT/p53 interaction has on Pab426 binding

A

With large T present p53 cant bind anymore

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20
Q

Describe the experiments looking at how much p53 was in the cells

Control vs large T TF

A

In normal 421 detects p53

But in SV40 transformed cells - 246 detectable p53 in non transfect but in SV40 246 cant detect p53

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21
Q

Is it the levels of p53 that changes in the SV40 transformed cells?

A

No…

Using a-p53 for a different epitiope … shows there is equal ammounts of p53

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22
Q

What sort of mutations occurs in p53

What is unusual about this?

A

Most are missense mutations

There is an unexpected bias in the mutations

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23
Q

Domains within p53 affect transcriptional activation of ….

A

Gal4

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24
Q

Describe the experiments looking to see if p53 engaages with DNA as a transcritpion factor

A

STRUGGLES TO DEMONSTRATE DNA BINDING

If have DNA binding domain and transcriptional activating domain = lots of gene expression

Take VP16 and fuse p53 to gal4 construct

If just use N-terminal p53 can show transcriptional activation

Suggests part had transcriptional activation

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25
Q

Evidence that p53 acts as a tetramer

A

Migrates as a 45kd and 145kd protein during glucose sucrose density gradient centrifugation

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26
Q

What does the tetrameric strucuture explain about p53 acting unusally as a TSG

A

In hets
Unlikely to get 4 subunits of total WT (only 1/16 chance)
Likely to have AT LEAST one mutant subunit (15/16)

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27
Q

Wt P53 is tthe

A

Guardian of the genome

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28
Q

mutant p53 at Pab246 site…

A

Loses its guardian role

29
Q

When is the guardian role of p53 lost?

A

Lost in the presence of large T or through missense mutations

30
Q

What happens to p53 levels if you irradiate the skin withUV

How was this seen

A

Increase in the levels of p53

Western blot experiments

31
Q

Describe an experiment to see what genes are promoted by p53

A

Null cell line is easy to come by since p53 is commonly mutated

Insert p53 into null line under control of glucocorticoid promoter
Apply dexamethasone …

Look for differences in the transcriptional output
ISOLATE mRNA

Subtractive hybridisation
Essentially to identify which genes arent present

32
Q

What was a gene that p53 promotes

A

WAF1

33
Q

WAF1 aka

A

p21

34
Q

What is p21

Effects of p21

A

CDK inhibitor

Inhibits CDK4/2/1

Controls the G1 –> S phase progression through p53

35
Q

Types of genes that p53 upregulates

A

Growth arres genes
DNA repair genes
Regulators of apoptosis
Anti-angiogenic proteins

36
Q

Specific alarm signals provoke p53 mediated

A

Inhibition of proliferation

Triggering of apoptosis

37
Q

Cytostatic and pro-apoptotic functions of p53 represent a threat to incipient cancer cells for which the following are necessary conditions

A

Hypoxia
Genomic damage
Signalling excess

38
Q

Inactive forms of p53 enable

A

Oncogene activation wo apoptosis
Higher tolerance of anoxia
Sloppy oversight of chromosomee inegrity

39
Q

What is the double minutee phenotype

A

Double minute chromosomes seen in fibroblasts NIH-3T3

Cells able to spontaenously transform and produce tumours
Identified and cloned genes OE in these cells

40
Q

What genes are OE in the double minute phenotype

A

Mdm1-3

41
Q

OE Mdm1-3 sufficient to cause

A

Transformation

42
Q

Coprecipitation of ___ and ____

A

Mdm2 and p53

43
Q

Mdm2 plays an important role in …

A

Inducing the destruction of p53

44
Q

Increasing mdm2 lead to …… in a …

A

P53 not being present

CONCENTRATION DEPENDENT MANNER

45
Q

What was the control when looking at the p53 / mdm2 relationship

A

Using mutant p53 which is lacking the N-terminal

46
Q

Mdm2 is a

A

Ubiquitin ligase

47
Q

What does Mdm2 do to p53

A

polyubiquitinate marking for proteasomal degradation

48
Q

if you increase Mdm2 … p53 ______

What is Mdm2 acting as`

A

p53 will decrease

Mdm2 acting here as an oncogene

49
Q

Give some examples of dependency points … explain each one

Are dependency events absolute?

A

Bacteriophage assembly
Later component assembly dependent on the correct assembly of earlier ones

Drosophila polytene chr
Huge - exist since multiple reps without division , SHOWS depdency events are not aboslute

Polyploidy
Multipel chr per cell
SHOWS depdency events are not aboslute

NO

50
Q

Outline the paradigm for S/M checkpoint control in Yeast

A

Cells lacking in Rad9 healthy in the absence of extrinsic interference

51
Q

What is CDC9

What is the effect of losing Cdc9?

A

DNA ligase - responsible for sealing the nick between okazaki fragments

WO Cdc9 cant get a sigar phosphate backbone all of the way through the molecule

52
Q

Describe the mutation in the Yeast paradigm

What were the mutants crossed with

A

Temperature dependent Cdc9 mutation

Too high - null
Permissive - Cdc9 will work

Done this was since conventional Cdc9 KO would be lethal

Then cross Cdc9 mutants with Rad9 mutants

53
Q

Descirbe the Cdc9 ts mut/ Rad9
What would you expect to seen … what is actually seen

At the permissive temperature

At the restrictive temperature

A

Normal

Would expect them to be stuck in S phase
Since checkpoint contols should prevent the advance of the cell cycle when replication is incomplete
BUT ACTUALLY SEE CELLS ADVANICING INTO M PHASE

54
Q

Describe the viability determination of these cells

A

Wt
Cdc9 ts - stuck in S phase but still viable
Rad9 - viable
Cdc9ts Rad9 - because no Rad9 cells going into G2 and M without properly replicated DNA

55
Q

Rad9 is the ….

A

Founder of the S/M checkpoint in yeast

56
Q

Describe the experiments to demonstrate the S/M checkpoint in mammalian cells

A

Hamster cells with ts mut
At restricitve temp ith DNA rep inhibitor of colchicine (blocks cells in M)

Restrive - fragmentation of chromosomes caused by improper segregation
Permissive - condensed chromosomes

57
Q

Loss of checkpoint control may also be induced with

A

Caffenine

58
Q

All metozoan systems have a ______- componenet at the S/M checkpoint

A

Caffeine sensitive

59
Q

What does caffeine interfere with

A

Transducers and effectors

60
Q

Describe the replication fork sensors

A

At the replication fork …

Stalling due to lesion leads to the assembly of a complex sensor system
Important to ensure integrity during replication
Occurs ALOT

61
Q

ATR is ________ sensnitve and is related to ____

A

Caffeine sensitive

Rad3

62
Q

ATR is a

A

Protein kinase - very complex strucutre and is an atypical protein kinase

TRANSDUCTION DEVICE WHICH IS CAFFEINE SENSITIVE

63
Q

Describe the mechanism involving ATr that occurs following DNA damage

A

Replication stress leads to ATR being turned ON
Assembly of the sensor
Turns on ATR
Turns on Chk1
+wee1 , -cdc25
Cdk1 is OFF and entry into mitosis is blocked

64
Q

What does the inherited loss of checkpoint proteins demonstrate

A

The role of checkpoints in supressing cancers (spontaneous and inherited)

65
Q

Checkpoint signalling is essential for

A

P53 function

66
Q

What is cisplatin

A

Chemotoxic agent

Is a chemotherapy drugs used to solid tumours - induces apoptosis

67
Q

Describe what happens when cisplatin is applied

A

p53 is phosphorylated at serine-15 - N terminal P53 (criticial for Mdm2 interaction) - this prevents the binding of Mdm2 and p53 is not degraded

68
Q

Describe what happens when a dominant negative form of ATR is used

A

No phos and activation of p53

Mdm2 able to ubiquitanted and degrade p53