L2 oncogenes Flashcards
what is an oncogene?
A cancer inducing gene which can transform cells.
Usually caused from a transition of a proto-oncogene to an oncogene by a single mutation
when and what was the first tumour virus to be discovered?
1910 - Rous Sarcoma virus (RSV)
How was it concluded that in chicken breast sarcomas there must be a transferrable agent? (RSV)
- Chicken breat sarcoma tissue/,muscle was broken up and homogenised with sand
- filtrate was collected
- filtrate injected into young chickens - got sarcoma
(shows must be a transmissible agent in the filtrate (RSV-virus)
what did Temin and Rubin show about RSV?
They showed that the RSV persisted in in cell culture when infected - cells showed similar traits to cancerous cells
what effects do tumour viruses have on cells?
1) altered morphology - increased thickness of cell layer, loss of contact inhibition, rounded morphology
2) anchorage independent growth - cells form foci
What is meant by anchorage independent growth?
- cells do not require the need to grow/divide on a flattened surface - can grow on top of each other and form foci (large clumps of growing cells)
- anchor independence correlates with tumorigenecity
- TGF-B can induce anchorage independent growth
what happens when foci are transplanted into SCID mice?
- predicts tumorigenecity of cells
- tumour develops
what do SCID mice stand for?
severe compromised immunodeficent mice
lack thymus, no hair, accept non-celf cells
what time of virus is RSV and what are its featured?
Retrovirus - has an RNA genome
- has envelope and proteins/caspid
- reverse transcriptase
- has an extra gene Src (has 4 genes not 3)
How is RSV different to ALV?
RSV has an extra gene Src
both have gag, pol and envelope
How does the RNA virus of RSV persist in cell through successive growth cycles?
Reverse transcription of viral RNA genome into host DNA
- Virus enters into cells and sheds envelope
- Virus RNA and reverse transcriptase is released int the cell
- RT causes RNA to become DNA - then DNA double stranded helix forms
- Integration of DNA copy into the host chromosome - PRO-VIRUS
- translation of RNA causes production of many viral particles (envelope, capsid proteins, RT)- assembly of new viral particles - the virus proliferates and infects new cells.
how can viruses aquire extra host sequences?
when viruses come out of genome - if not spliced properly can pull out parts of host DNA - acquire oncogene?
- viruses can acquire an oncogene e.g. Src
why is the theory that proposed mutagens activate latent pro-viruses already in host DNA INCORRECT?
- proved incorrect as
- do not get clusters of infectious outbreaks of cancer
- cannot isolate virus particles from human tumour cells (if this was correct there should be viral particles in all tumours - there wasn’t - this cannot be the root cause of cancer)
who discoered reverse transcriptase and linked this to RSV?
Temin and David Baltimore
Give an example of other virsuses which can cause human cancer
Epstein Barr virus - nasopharyngeal cancer
Hepatitis C virus - hepato celluar cancer
HIV - non-hodkins lymphoma
What is a method that can be used to detect oncogones?
Transfection studies
- expose C3H10T1/1 cells to 3-methylcholanthrene
- in prescence of calcium phosphate cells take up foreign DNA
- extract DNA from chemically transformed fibroblasts and inject into normal mouse fibroblasts-(NIH3T3 cells)
- focus of morphological transformed cells forms
- inject morpholigcally transformed cells into mouse
- tumour forms (cells likely to have taken up oncogene (formed from mutations induced by carcinogens-single mutant allele formed)
how much of genome is taken up during transfection and what could be concluded from this?
0.1% of genome.
10 -6 is probability of 2 unlinked genes having an effect (unlikely more than one gene is mutated)
- more likely a single gene is responsible for transformation (not 2) - oncogenes?
- single mutant allele formed by carcinogens
why are transfection experiments important
They show that oncogenes can arise in the genomes of cells through mechanisms which have no connection with viral infection - e.g. carcinogens used not viruses
what was the first cellular proto-oncogene found?
C-ras
How can cellular oncogenes be identified?
Through repeated rounds of DNA extraction and transfection
- Extract DNA from foci
- Add essential bacterial gene
- split population of DNA into separate tubes
- inject DNA from different tubes into cells/fibroblasts and see if from foci
- extract DNA from cells which form foci
- repeat rounds of transfection
- ideally will end up with pure poplaiton of oncogene and bacterial gene
- remove bacterial gene and identify / sequence oncogene.
what is a proto-oncogene?
normal gene, which when altered by a mutation becomes an oncogene
what happens in southern blotting?
southern blotting identifies specific sequences of DNA
- fragments separate and are transferred onto a nitrocellulose membrane via the use of a buffer . assay via hybridisation of radioactive/ labelled probes to identify similar sequences
what did wigler do to find sequence similar to h-RAS?
- took viral oncogene probe developed from H-ras and probed transformed mouse fibroblasts (NIH3T3 transfromed cells with DNA from human bladder carcinoma) to find sequence highly similar
- oncogene in transformed NIH3T3 cells has high stringency to viral oncogene h-ras
what does K-ras anneal to?
K-ras viral oncogenic probe annelas to oncogene from human colon carcinoma cell line
how does oncogenic Ras differ from nromal ras?
- oncogenic Ras has a G-T base change - point mutation at position 12
- glycine - valine aa change
- causes proto-oncogene to become an oncogene
How is burkitts lyphome caused?
By TRANSLOCATION AND AMPLIFICATION
- chromosomes become swapped when cut
- myc gene moves from chromosome 8 to chromosome 14, downstream of IgH promoter
- IgH promoter is strong promoter - on a lot of the time - means there is high expression levels of myc gene
- misregulated expression of myc
- causes proliferaiton and cancer phenotype
- western blot shows high L-myc over time in tumour cells.
Why does the mutation at position 12 on ras have proliferative effects?
Position 12 (glycine) is in area where there is GTPase catalytic activity.
- mutation at position 12 changes conformation and negatively affects GAPS
- this means Ras/GTPase cannot change into an inactive form so stays bound to GTP- active ras drive porilferation
what occurs in a 1/3 of glioblastomas?
The EGF receptor is decapitated/truncated
- missing the ECD
- this means the ICD remains activated even if the ligand/EGF is not present
what can you do to determine how similar a prob and sequence are?
increase the temperature and salt concentration - weaker binding/ less similar matches come apart at lower temperatures.
How similar are H-Ras probe and DNA/oncogene of transformed cells?
are highly identical
- viral oncogene identical to a cellular oncogene - same thing?
- evolutionally virus taken oncogene from organisms???
