Cellular oncogenes

Question 1

What is RSV?

Answer 1

Rous sarcoma virus

virus identified in 1910 in chickens causing sarcomas

Question 2

What are the effects of cancerous viruses on cells?

Answer 2

Altered morphology

anchorage independent growth

Question 3

What does altered morphology mean?

Answer 3

The thickness of the cells increases, as cells grow over each other
this is unusual as when cells touch each other they usually stop growing- contact inhibition- but this is lost in the infected cells
the cells have a more rounded morphology

Question 4

What is anchorage independent growth?

Answer 4

cells growing with a lack of a solid substrate to attach to, they can grow in a semi solid medium
the cells form foci

Question 5

How did they infect mice with RSV?

Answer 5

They infected fibroblasts with the virus and injected them into immuno-deficient mice
the mice developed tumours as a result

Question 6

What genes do retroviruses in general have, how does this compare to the RSV genome?

Answer 6

General genes- gag, pol, env

RSV has an extra gene- src

Question 7

When did the theory of reverse transcription come about? What are the stages of reverse transcription

Answer 7

The 60’s- the theory grew when reverse transcriptase was discovered

1. entry of the viral particle into the cell and shedding of its envelope
2. reverse transcriptase creates DNA-DNA double helixes out of the viral RNA
3. integration of the viral DNA into the host genome
4. transcription of viral DNA
5. assembly of new viral particles, all with reverse transcriptase found in the protein coats

Question 8

Which predictions about viral cancer were proven wrong?

Answer 8

There was no evidence of clusters of infectious outbreaks

was not possible to isolate viral particles from human tumour cells (is for RSV)

Question 9

Examples of viruses and the cancers they cause

Answer 9

Hepatitis C virus= colon and prostate cancer
Human immunodeficiency virus= non-hodgkins lymphoma
Epstein barr virus= nasopharyngeal cancer

Question 10

Which theory overtook the viral cancer theory?

Answer 10

Mutagenesis theory

Question 11

What are the principles of transformation of cells in tissue culture?

Answer 11

cells in agar undertaking anchorage independent growth, forming colonies and aggregrates, lacking contact inhibition

Question 12

Which experiment lead to the discovery of oncogenes?

Answer 12

Transfection experiment
damaging DNA, reintroducing the DNA into healthy mouse blastocysts, seeing the creation of transformed cells, injecting these cells into a mouse (xenograft), and seeing the formation of tumours

Question 13

How did they identify the first oncogene, and what was it?

Answer 13

(MUTATED) C-Ras
experi= extracting DNA from transformed cells, attaching essential bacterial gene, inserting into mouse fibroblasts, extracting DNA from transformed cells- repeat the process= purification
final step- introduce DNA into bacteria, bacterial with the essential bacterial gene will survive. Extract non-bacterial DNA from the surviving bacteria, and sequence

Question 14

What are the principles of southern blotting?

Answer 14

DNA electrophoresis on agar gel
sheet of nitrocellulose on top
sheets of paper on top
buffer placed under gel
capillary action- moves DNA from the gel onto the membrane
hybridise the DNA with a labelled probe
= identify a specific DNA sequence using the probe

Question 15

Southern blotting of murine cells transformed with human bladder carcinomas- and hybridised with the H-ras oncogene from the murine sarcoma virus- caused what?

Answer 15

Implied that the gene in cancer causing viruses- is similiar to the mutated genes in humans and mice

Question 16

Whats the difference between c-Ras and h/k-Ras?

Answer 16

A single base change, glycine is changed to valine
c-Ras- no tumours- glycine
h/k-Ras- tumours- valine