L2 - cellular oncogenes Flashcards
What is RSV?
A virus first identified in chickens, which when injected into an animal causes the formation of tumours. It is a retrovirus that uses reverse transcriptase to integrate itself into the host cell genome, creating more copies of itself.
What affect does RSV have on cells?
- Altered morphology
- Anchorage independent growth
- In nude mice - lots of tumours
What are the predictions of a viral theory of cancer?
The infectious theory should see;
- cancer occurring in clusters/outbreaks
- Isolation of virus particles from tumour cells
What was the first oncogene identified?
C-RAS (look at notes for how it was identified)
What did Howard Temin and Harry Rubin show?
- Virus RSV persisted
- productive lifecycle in cultured cells
- cells displayed traits similar to cancer cells
How does RSV persist through cell growth cycles?
Reverse transcription of RNA into DNA which is then incorporated into the genome. This is done via reverse transcriptase
What does the viral theory of cancer suggest?
An infectious theory of cancer
HYPOTHESIS
Chemical/physical aspects (mutagens) activate latent pro-virus in cells
How was the viral theory of cancer tests?
Cells were treated with 5-bromo-2’-deoxyuridine. This mutagen will cause an increase in the expression of endogenous retroviral seq that can then be detected by Northern blotting and probes
What is the mutagenic theory of cancer?
Mutagenesis alters the cellular DNA which causes the transformation of cells
How can we exploit transfection?
- chemically transform fibroblasts by exposing them to DNA mutating drugs
- Extract DNA
- Inject the DNA into normal fibro and look for foci
- inject these cells into nude mouse - look for tumour
What did the experiments with transfection show us?
0.1% of the genome was taken up per cell (30 genes) therefore it was unlikely that more than one gene was mutated in these cells
How was c-Ras identified?
- Extract DNA from foci
- Tag DNA with essential bacterial gene
- Inset DNA into virgin fibro and look for foci
- Extract DNA of foci and grow bact with extracted DNA
Only tagged DNA will permit survival - Extract DNA of surviving bact and inject into virgin fibro
Repeat over and over - increase purity of oncogene in fraction until there is a single piece of DNA left - seq
First gene to be seq was c-Ras
What did southern blotting of c-Ras DNA show?
After southern blotting, probes were applied to the NC gel. A probe for the H-Ras viral oncogene bound to c-Ras. This showed that the c-Ras in transformed cells had a similar genetic sequence to the H-Ras viral oncogene
What sort of mutations activate the oncogenic form of Ras?
Point mutations at aa 12,13 and 61
What mutation activates the EGFR?
Truncation mutation affecting the extracellular domain. The EC domain normally inhibits the intracellular signalling and is itself inhibited by binding of EGF. When the EC domain is missing the intracellular signalling pathway is constitutively active
