The Discovery of

Oncogenes

LinZhu 1040800040

Three stages of the discovery

• In 1911, found the virus.(Peyton Rous)

• In 1970, Over turned Central Dogma.

(David Baltimore & Temin & Satoshi Mizutani)

• In 1970s, isolate the gene.(Harold Varmus,J.Michael Bishop,Dominique Stehelin)

Peyton Rous (1879-1970)

• In 1911

• prove that some spontaneous chicken tumours, to all appearances classical neoplasms, are actually started off and driven by viruses (Rous sarcoma virus)

Virus budding

• RNA-containing viral particles budding from the cell.

Experiment 1:remove the tumors

grind (研磨 ) the cells

centrifuged

remove the supernatant

fluid through filters

Injected the filtrate into

Result &Conclusion 1:

• Result :significant percentage of the injected animals developed the tumor.

• Conclusion :Tumor could be transmitted from one animal to another by a “filterable”virus.

Other experiments

• Tumor passed through

mother egg offspring

• Transmitted from cell to cell by means of mitosis without having any obvious effect on the behavior of the cells

David Baltimore

• In 1970

• Discover RNA-dependant DNA polymerase which later known as reverse transcriptase.

Problems 2

Whether the viral genome is passed from parents to progeny

• as free RNA molecules

or

• is somehow integrated into the DNA of the host cell?

Experiment 2

R-MLV 小鼠白血病病毒 & RSV肉瘤病毒Incubate

• DNA polymerase, Mg2+,NaCl

• dithiothreitol（二硫苏糖醇 )

• all four deoxyribonucleosid triphosphates(四种脱氧核苷三磷酸盐 )

• TTP was radioactively labeled

Characterization of the Polymerase Product

• Pancreatic ribonuclease: effected

( 脱氧核糖核酸酶 )• Micrococcal nuclease: effected

（微球菌核酸酶）• Pancreatic ribonuclease: uneffected

（核糖核酸梅）• Alkalin hydrolysis: uneffected

（碱水解）

Characterization of the Polymerase Product

EXPT.

Treatment Acid-insoluble Radioactivity

Percentage Undigested Product

1 Untreated 1,425 (100)

Pancreatic ribonuclease

235 9

Micrococcal nuclease 69 5

Pancreatic ribonuclease

1,361 96

2 Untreated 1,644 (100)

Alkalin hydrolysis 1,684 100

Conclusion 2

• DNA sensitive to deoxyribonuclease

• RNA sensitive to alkaline hydrolysis

• Conclusion:

The reaction product was DNA.

Result Conclusion 2:

• Result:The DNA-polymerizing enzyme was found to co-sediment with the mature virus particles.

• Conclusion:It was part of the viron itself and not an enzyme donated by the host cell.

Conclusion 2

• Viral RNA as template for synthesis of a DNA copy ,

• DNA copy as a template for the synthesis of viral mRNAs required for infection and transformation.

Over turned Central Dogma

Harold Varmus & J.Michael Bishop

• Discover the reverse transcriptase

The Discovery of Oncogenes

• During the 1970s, attention turned to the identification of the genes carried by

tumor viruses that were responsible for transformation and the mechanism of action of the gene products.

Problems• WHY? mutant strains of viruses could be isolated

that retained the ability to grow in host cells, but were unable to transform the cell into one exhibiting malignant properties.

• HOW? Thus, the capacity to transform a cell resided

in a restricted portion of the viral genome.

Experiment 3

• Isolate mutant strains of the avian sarcoma virus(ASV)鸟类肉瘤病毒 .

• Unable to induce sarcomas in chickens or to transform fibroblasts in culture.

• The responsible gene is called “ src ”

• Isolate the gene

Isolate cDNAsarc

• RNA from the genomes of complete virions was used as a template for the formation of a radioactively labeled, single-stranded, complementary DNA (cDNA) using reverse transcriptase.

• The labeled cDNA was the hybridized to RNA obtained from one of the deletion mutants.

Isolate cDNAsarc

• DNA failed to hybridize to the RNA

= the genome that had been deleted from the transformation-defective mutant

• thus were presumed to contain the gene required by the virus to cause transformation.

• separated by column chromatography.

Conclution

• the gene is a part of the cells’ normal genome.

• not ture viral genes

• cellular genes that were picked up by RNA tumor viruses during a previous infection.

src sequences

• Src sequence is present in all of the avian species

• suggests that the sequence has been conserved during avian evolution and, thus, is presumed to govern a basic activity of normal cells.

src sequences

• In a subsequent study, it was found that cDNAsarc binds to DNA from all vertebrate classes,including mammals, but not to the DNA from sea urchins, fruit flies, or bacteria.

• It plays some critical function in the cells of all vertebrates.

New questions

• These findings raised new questions

• Welcome QU.