• The discovery of Reverse transcriptase
• Experiment about the discover of src
• Src gene &something about human oncogene
History
In 1911 Peyton Rous
a paper less than one page in length
chicken sarcoma
Propagated from one hen to another
By a “filterable virus”
ExperimentRemove the tumor( 瘤组织 )
→ground→centrifuged
→remove the supernatant
→through filter( 滤器 )
→inject into recipient hen
Found that a significant percentage of the injected animals developed the tumor.
1960sThe virus discovered by Rous is an RNA-cont
aining virus
It could be seen within the tumor cells and also budding from the cell surface
Figure 1
It transmitted through the fertilized egg from mother to offspring
These provided evidence that the viral genome can be inherited through the gametes
Cell to cell
Without having any obvious effect on the behavior of the cell
QuestionsOne of the major questions concerning the ver
tical transmission of RNA tumor was whether the viral genome is passed from parents to progeny( 子孙 )as
• Free RNA molecules
• Integrated( 整合 ) into the DNA of the host cell
But
Require the synthesis of DNA
• Howard Temin suggested
tumor viruses occurs by means of a DNA intermediate-a provirus-a template
• But need a enzyme
In 1970 the enzyme was discovered
After that • A experiment suggesting that it was part of t
he virion itself and not an enzyme donated by the host cell.
• And the viral RNA was providing the template for synthesis of a DNA copy.
During the 1970
• Genes carried by tumor viruses
Responsible for transformation & the mechanism of action of the gene products.
• Mutant strain of viruses
retained the ability to grow in host cells,
but unable to transform the cell into malignant properties.
Thus
The capacity to transform a cell resided in a restricted portion of the viral genome.
So The researchers began by isolating mutant
strains of the avian sarcoma virus(ASV)
First
• Deletions of 10 to 20 percent of the genome
that render the virus Unable to induce sarcomas or to transform fibroblast in culture
The gene which is missing in these mutant referred to as src
Second • The reseachers use the RNA from the gen
omes of complete virions was used as template for the formation of a radioactively labeled ,single-stranded complementary (cDNA).Then use the labeled cDNA fragment hybridized to RNA obtained from one of the deletion mutant
• Those DNA fragment that failed to hybridize to the RNA represent of the genome that had been deleted from the transformation-defective mutant
• Thus Presumed to contain the gene required by the virus to cause transformation.
• Once isolated labeled cDNA hybridizes to DNA extracted from cells of avian species contain DNA sequence that is closely related to src
Find
• That a gene carried by a tumor virus that cause cell transformation is actully present in the DNA of normol genome.
• So the result indicated that the viral genome are not true viral genes
• But rather are cellular genes that were picked up by RNA tumor viruses during a previous infection.
• 反转录 RNA 病毒感染宿主细胞将病毒 RNA 反转录成双链 DNA ,然后在细胞原癌基因旁整合。病毒成熟前,病毒 DNA 转录成 RNA 同时原癌基因也转录下来,后经突变成为病毒 RNA 一部分。
Conclusion• The src sequence is present in all of the avi
an species and the sequence has been conserved during avian evolution.
• So the src gene is not only present in the RNA of the ASV genome but also in the genome of the chicken cells.
Question
• What is the Function of src gene product• How does src alter the behavior of normal c
ell that already possesses a copy of the cellular gene
Then • Precipitation the RSV-antibodies • Synthesis of the protein using the isolated viral ge
ne as a template
Immunoprecipitation find src codes for an enzyme that possesses protein k
inase activity.Antibodies localized on the inner surface of the plas
ma membrane.Src gene product in this part of the cell.
These were the first studies to elucidate(解释 ) the function of an oncogene.
The viral version of the gene may induce transformation because it functions at a higher level of activity than the cellular version.
Cellular genes could be converted into oncogenes in two different ways:
1.Incorporated into the genome of a virus
2.By carcinogenic chemicals
Through the experiment found
In1981
• Attention turned to human cancer• Once the transforming gene from human bladder c
ancer cells had been isolated and cloned.• In the same human genes-RAS-can cause a wide ra
nge of different tumors.• RAS gene that leads to its activation as an oncogen
e.• The change of amino acid lead the change of the p
rotein.
• RAS gene is a proto-oncogene that, like SRC, can be activated by linkage to a viral promoter.
• RAS induce by two way:
1.increasing its expression
2.altering the amino acid sequence of its encode polypeptide.
The research led to the realization
• The oncogene is a celluar sequence that is acquired by the virus
• An oncogene can cause cancer without the involvement of a viral genome.