L10 - Genomic Instability Flashcards
Describe the disovery of p53
In 1975 - thought to be the basis of the decision to replicate the DNA
What virus was used to help discover p53
Papovirus - SV40
Describe the normal distribution of SV40 protein
Reside in the nucleus
Advantage of SV40 proteins
Immunogenic - they are easy to raise antibodies against
What is the relevant viral protein from SV40
Large T
Describe what happens when you transfect cells with viral SV40
What is the experimental method required to see this
Can see that proteins are constantly undergoing protein synthesis
Shown through the addition of a radio-AA
What was the next direction after the SV40 transfection
To ask wehther large T interacted wth any other host proteins
Results of immnoprecipitation experiemnts of cells TF with SV40 large T
In presence of antiserum or antiserum from control animal pre-immunisation
Only immunopreciptate in transformed SV40 cells where antibody present
Band around 54kD = p53
Purpose of a cellular transformation assay
Allows you to determine if cell is transformed in the presence of an oncogene
How would you tell if a cell had been transformed
If the cell shows anchorage independent growth … transformation … clone into an immune compromised mouse
Ras + P53 mutant
Normal number of colonies
CONTROL
Ras+P53
P53 FROM TISSUE CULTURE CELLS
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)
Ras + WT (true) P53
No colonies
Onogenic function of Ras has been supressed
P53 BEHAVES AS A TUMOUR SUPRESSOR GENE
What cancers display a p53 mut
ALL
What happens usually when TSGs are deleted in the embryo
What is done to get around this?
Is this the case for p53
Embryonic lethality
- Since many TSGs are regulators of cell numbers
Conditional KO approaches
Not the case for P53
Why are the Kaplan Meier plots for p53 mutations unusal?
If P53 is a TSG would expect to see Knudsens rules followed… but they aren’t
What is an epititope map
Pannel of antibodies that recognise p53 but each recognises it at a different site (epitope)
Describe the experiments that lead t the conclusion of p53 having a short half life
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
Descibe the effect of LargeT/p53 interaction has on Pab426 binding
With large T present p53 cant bind anymore
Describe the experiments looking at how much p53 was in the cells
Control vs large T TF
In normal 421 detects p53
But in SV40 transformed cells - 246 detectable p53 in non transfect but in SV40 246 cant detect p53
Is it the levels of p53 that changes in the SV40 transformed cells?
No…
Using a-p53 for a different epitiope … shows there is equal ammounts of p53
What sort of mutations occurs in p53
What is unusual about this?
Most are missense mutations
There is an unexpected bias in the mutations
Domains within p53 affect transcriptional activation of ….
Gal4
Describe the experiments looking to see if p53 engaages with DNA as a transcritpion factor
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
Evidence that p53 acts as a tetramer
Migrates as a 45kd and 145kd protein during glucose sucrose density gradient centrifugation
What does the tetrameric strucuture explain about p53 acting unusally as a TSG
In hets
Unlikely to get 4 subunits of total WT (only 1/16 chance)
Likely to have AT LEAST one mutant subunit (15/16)
Wt P53 is tthe
Guardian of the genome