Download Conclusion 2

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 RNAdependant 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:
(脱氧核糖核酸酶)
• Micrococcal nuclease:
（微球菌核酸酶）
• Pancreatic ribonuclease:
（核糖核酸梅）
• Alkalin hydrolysis:
（碱水解）
effected
effected
uneffected
uneffected
Characterization of the
Polymerase Product
EX
PT.
Treatment
Acid-insoluble
Radioactivity
1
Untreated
Pancreatic
ribonuclease
Micrococcal nuclease
Pancreatic
ribonuclease
1,425
235
Percentage
Undigested
Product
(100)
9
69
1,361
5
96
Untreated
Alkalin hydrolysis
1,644
1,684
(100)
100
2
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.