Lecture 2

Question 1

First tumour virus discovered

Answer 1

Rous Sarcoma Virus

Question 2

How was RSV discovered ??

Answer 2

• Chicken with sarcoma in breast muscel
• Remove sarcoma and break up
• Grind with sand
• collect filtrate passed through fine pore filter
• inject filtrate into young chicken
• Observe sarcoma in injected chicken.

Question 3

Howard Temin & Harry Rubin showed:

Answer 3

• the virus RSV persisted
• carried out a productive lifecycle in cultured cells (live viral progeny)
• cells displayed traits similar to cancerous cells.

Question 4

Effects of Tumour Viruses on Cells

Answer 4

Altered morphology

Anchorage independent growth:

Question 5

Altered morphology (

Answer 5

(RSV in Chick embryo fibroblasts):
• Increased thickness of cell layer
• Loss of contact inhibition
• Rounded morphology

Question 6

Anchorage independent growth:

Answer 6

• In semi solid medium such as methylcellulose / agarose.
• Cells form foci. (lumps of cells)
Severe Compromised Immuno-deficient mouse (SCID)
• Lack Thymus
• Nude (no hair)
• Will accept non-self cells
• Strong predictor of tumourigenic capacity of the cells.

Question 7

Xenograft model show

Answer 7

Xenograft model show primary cells injected into mice themselves don’t cause tumours unless already infected with RSV
Evidence viruses cause cancer – most not

Question 8

RSV

Answer 8

RSV is a retrovirus – sequencing of genome

• Virus - 3 genes gag/pol/env
• 4 genes Src – retrovirus that cause cancer

Question 9

How does a RNA virus persist in the cell through successive cell growth cycles?

Answer 9

• Temin first suggested the idea of reserve transcription of the viral genome to a DNA intermediate in the 1960s.
• Support for the hypothesis grew on the discovery by Temin and David Baltimore of the enzyme Reverse Transcriptase in 1970.

Question 10

How does RSV work ??

Answer 10

Dna gets transcribed by cellular machinery
Integration into DNA
DNA inserted randomly and disrutpting regulatory genetic processes
- see diagram

Question 11

Discovery of viruses led to development of what ?/

Answer 11

Onocogenes

Question 12

Viral theory of cancer

Answer 12

Guinea pig retrovirus – data produced showing retroviral seq imbedded in DNA of cell or tissue and can excise from it and propagate – maybe conceptually nuclei acid insult activate a process DNA that is Latent in genome excised and activated inducing viral production

Question 13

• Human Immunodeficiency Virus (HIV)

Answer 13

associated with Non-Hodgkin’s Lymphoma and HIV.

Question 14

• Epstein Barr Virus (EBV)

Answer 14

associated with lymphoma and nasopharyngeal cancers.

Question 15

mutagenesis

Answer 15

concept

Something happens to a gene and as a result cells are transformed

Question 16

Mutagenesis experiment showing transformation

Answer 16

Cells in petri dish embedded in agar undertaking anchorage independent growth
Form colonies visually
– contact inhibition stop as soon as they touch other cells
Cells undergoing transformation aren’t affected by contact inhibition – overgrow each other

Question 17

Bob Weinberg

Answer 17

Transfection technique

Question 18

Transfection technique - over view

Answer 18

Take transformed fibroblast and expose to methylcholanthrene (mutagenic drug)
Under normal conditions- take up DNA

Question 19

Transfection technique

Answer 19

• chemically transformed mouse fribroblast
• add DNA
• inject into normal mouse fibroblast
• formation of a focus of morphologically transformed cells
• inject into mouse host
• Tumour - evidence something has happened to DNA

Question 20

Work out percentage of genome taken up transformed cell

Answer 20

about 30 genes introduced into each cell

Question 21

Michael Wigler

Answer 21

First cellular oncogene identified is c-Ras

Question 22

Michael wigler Experiment

Answer 22

Chemically attached gene to DNA from transformed cell – now with attached essential gene
Might have an oncogene – trying to find it in sea of all the others
Take mixture and put back into mouse fibroblast- foci form from oncogene
Tumour- shows its gone in
Then extract DNA back out – why attached gene essential – can distinguish transfected DNA
Introduce into bacteria without essential gene – only bacteria that grow are those that have tagged DNA that contains oncogene sequence
Select for the only one tagged- keep repeating extract DNA and pass back through Bacteria
Fraction of oncogene

Question 23

What was the first cellular oncogene to be identified ??

Answer 23

RAS

Question 24

Southern blotting

Answer 24

• use elecrtophoresis
• agarose gel
• Labelled RNA/DNa
• Sealed bag around gel and labeled probe in buffer
• Labelled Probe hybridises complementary DNA / RNA Band visualised bu autoradiography.
• nitrocellulose gel - binds to molecules
• remove nitrocellulose paper with tightly bound nucleic acid.
• If complimentary to any sequence in the nitrocellulose- anneals at right temp get specific binding then wash away
• Radioactive spot correspond to particular RNA – isolated

Question 25

Transformed NIH3T3 Genomic DNA interrogated

with H-Ras viral oncogene probe

Answer 25

-implied cancer had a gene similar to human or mice but just mutating it makes it so similar to a viral oncogene probe which showed that humans are related to viruses

Question 26

Transfored NIH3T3

Answer 26

NIH 3T3 cells were transformed with DNA from a human bladder carcinoma. DNA was extracted and hybridised with probes produced using H-Ras gene from murine sarcoma virus

Question 27

H- Ras genes

Answer 27

Extra gene - along with gag pol env

Question 28

How does oncogene ras differ from normal ras ?

Answer 28

sequence - extra gene
Single base change convert cellular ras into a oncogene – glycine to valine
Sufficient to form tumorigenesis

Question 29

Anchorage independent growth definition

Answer 29

A cell that has lost the need for anchorage dependence, which is essential for cell growth, division, and spreading. Supplement. Cells that have become anchorage-independent are said to have transformed or have become neoplastic in nature.