Download - The Discovery of Oncogenes
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The Discovery of
Oncogenes
LinZhu 1040800040
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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)
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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)
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Virus budding
• RNA-containing viral particles budding from the cell.
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Experiment 1:remove the tumors
grind (研磨 ) the cells
centrifuged
remove the supernatant
fluid through filters
Injected the filtrate into
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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.
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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
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David Baltimore
• In 1970
• Discover RNA-dependant DNA polymerase which later known as reverse transcriptase.
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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?
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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
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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)
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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
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Conclusion 2
• DNA sensitive to deoxyribonuclease
• RNA sensitive to alkaline hydrolysis
• Conclusion:
The reaction product was DNA.
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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.
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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.
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Over turned Central Dogma
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Harold Varmus & J.Michael Bishop
• Discover the reverse transcriptase
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
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New questions
• These findings raised new questions
• Welcome QU.