Transcript src

1911 Peyton Rous published a paper on the treatment of syphilis;
discovered an RNA-containing virus.
1960 Similar viruses were found to be associated with mammary
tumors and leukemias in rodents and cats.
Question 1: Vertical transmission of RNA tumor viruses was 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.
1970 RNA-dependent DNA polymerase was discovered
independently by David Baltimore , Temin and Satoshi Mizutani.
Question 2: Identification of the genes carried by tumor viruses
that were responsible for transformation and the mechanism of
action of the gene products.
Peyton Rous: Working with a chicken sarcoma that could be
propagated from one hen to another by inoculating a host of
the same strain with pieces of tumor tissue.
A series of experiments that strongly
suggested that the tumor could be transmitted
from one animal to another by a “filterable
virus”.
Filterable virus: A term that had been coined a decade or so
earlier to describe pathogenic agents that were small enough
to pass through filters that were impermeable to bacteria.
BACK
RNA-containing viral particles could be seen within the tumor
cells and also budding from the cell surface.
The gene(s) causing tumors in these inbred strains are
transmitted vertically, that is, through the fertilized egg from
mother to offspring, so that the adults of each generation
invariably develop the tumor.
Conclusion: The viral genome can be inherited through the
gametes and subsequently transmitted from cell to cell by
means of mitosis without having any obvious effect on the
behavior of the cells.
BACK
Evidence to question 1 indicated:
Infection and transformation by these viruses required the
synthesis of DNA.
Howard Temin (University of Wisconsin)
The replication of RNA tumor viruses occurs by means of a DNA
intermediate—a provirus—which then serves as a template for
the synthesis of viral RNA.
RNA-dependent DNA polymerase
BACK
The DNA-polymerizing enzyme was found to co-sediment with
the mature virus particles, suggesting that it was part of the
virion itself and not an enzyme donated by the host cell.
Conclusion: The viral RNA was providing the template for
synthesis of a DNA copy, which presumably served as a template
for the synthesis of viral mRNAs required for infection and
transformation.
The discovery of reverse transcriptase overturned the
longstanding concept originally proposed by Francis Crick and
known as the Central Dogma.
BACK
Harold Varmus, J. Michael Bishop, Dominique Stehelin, and their co-workers
at the University of California, San Francisco
They isolated mutant strains of the avian sarcoma virus(ASV) carrying deletions
of 10 to 20 percent of the genome that render the virus unable to induce
sarcomas .
The first gene studied responsible for transformation is src—sarcoma by using
experimental strategies, such as hybridization and column chromatography.
cDNAsarc corresponded to approximately 16 percent of the viral genome(1600
nucleotides out of a total genomic length of 10,000 nucleotides).
This cDNA fragment hybridizes to DNA extracted from cells of a variety of avian
species indicating that the cellular genomes of these birds contain a DNA
sequence that is closely related to src.
Indication: The transforming genes of the viral genome (the
oncogenes) are not true viral genes, but rather are cellular
genes that were picked up by RNA tumor viruses during a
previous infection.
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.
Conclusion: The src gene is not only present in the RNA of the
ASV genome and the genome of the chicken cells it can infect,
but a homologous gene is also present in the DNA of distantly
related vertebrates, suggesting that it plays some critical
function in the cells of all vertebrates.
Questions
1. What is the function of the src gene
product?
2. How does the presence of the v- src
alter the behavior of a normal cell
that possesses a copy of c- src?
Research on the function of src
——the discovery of its product
Method
1. Within cell: precipitation by antibodies prepared
from RSV- infected animals
2. Cell-free: synthesis of the protein using the
isolated viral gene as a template
 the product of src --- pp60src
Research on the function of src
——the discovery of its product
 pp60src
 Localization
(by
electronmicroscopic
immunocytochemistry)
pp60src is localized at
the plasma membrane
of the cell and is
particularly
concentrated at the
sites of gap junctions.
Function of pp60src
 The src gene codes for an protein kinase.
 pp60src transferred phosphate groups to
tyrosine residues on the substrate protein
rather than to serine or threonine residues.
 Not only did the product of v-src code for a
tyrosine protein kinase, so too did c-src.
 The viral src gene product had a higher activity
than cellular version.
Question 2.
How does the presence of the v- src
alter the behavior of a normal cell
that possesses a copy of c- scr?
Transformation mechanism
an excess of
normal protein
alter the biochemical
properties of the gene
product
Research on HUMAN BEINGS
Question
Which genes in a cell, when activated
by mutation or some other
mechanism, are responsible for
causing the cell to become malignant?
A transfection experiment
 13 different human tumors tested
 two provided DNA that was capable of
transforming mouse fibroblasts
 no evidence of viral DNA detected in these cells
Conclusion
Human cancer cells contain an activated oncogene
that can be trasmittd to other cells, causing their
transformation.
Researches on human oncogene
 Step 1--- Isolation and analysis of the oncogene in
human body
Step 2--- Determine the relationship between
human oncogene and viral oncogene
 Conclusion
The oncogene from human bladder carcinomas
that transforms NIH3T3 cells is
(named ras) that is carried by the
Harvey sarcoma virus
precise changes in the human
RAS that lead to its activation
as an oncogene
Mechanism of RAS
RAS can be activated to induce
transformation by two totally different
pathways:
 increase its expression
 alter the amino acid sequence of its
encoded polypeptide
Precise changes
A glycine
residue plays a
critical role in
the structure
and function of
this protein
Precise changes
IN HUMAN
G（proto-oncogene）
→T（activated oncogene）
12th amino acid residue ：
Gly → Val
IN VIRUS
Gly → Arg
Precise changes
P-Loop：GTP binding domain
The Gly to Val mutation
at residue 12 renders the
GTPase domain of Ras
insensitive to inactivation by
GAP , causing cancerassociated mutant Ras
proteins to accumulate.
Summary