Transcript src

The Discovery of Oncogene
曲颖（1040800020）
What is the function of the src gene product?
How does src alter the behavior of normal cell
that already possesses a copy of the cellular
gene?
Identification of the product of the src gene
Prepare the
antibodies from
RSV-infected
animals
Precipitate(沉淀) the
protein from extracts
of transformed cells
by antibodies
Synthesize the protein using the isolated viral
gene as a template in a cell-free proteinsynthesizing system
A protein of 60,000 daltons—pp60src
Function of the product of the src gene
When pp60src was incubated with [32P]ATP,
radioactive phosphate groups were transferred to
the heavy chains of the associated antibody
molecules used in the immunoprecipitation(免疫共
沉淀).
src gene codes for an enzyme that possesses
protein kinase activity.
Orientation of the product of the src gene
Cells infected with ASV were fixed, sectioned, and
incubated with ferritin(铁蛋白)-labeled antibodies
against pp60src, the antibodies were found to be
localized on the inner surface of the plasma
membrane,
suggesting a concentration of the src gene
product in this part of the cell.
What is the function of the scr gene product?
src gene product:
 A protein of 60,000 daltons—pp60src
 An enzyme that possesses protein
kinase activity.
 Concentrate on the inner surface of
the plasma membrane
 The number of phosphorylated tyrosine
residues in proteins of RSV-transformed cells
was approximately eight times higher than that
of control cells.
Viral vesion of the gene may induce
transformation because it functions at a higher
level of activity than the cellular version.
How does src alter the behavior of normal cell
that already possesses a copy of the cellular
gene?
＊ An increased activity of an oncogene product
could be a key to converting a normal cell into a
malignant cell .
＊ Malignant cell could also be induced by an
oncogene that contained an altered nucleotide
sequence.
DNA transfection
Obtain 15 different malignant cell lines that were derived from
mouse cells that had been treated with a carcinogenic(致癌的)
chemical. (Made malignant without exposing them to viruses .)
DNA from each of these cell lines was extracted
Transfect a type of nonmalignant mouse fibroblast called
an NIH3T3 cell.
The fibroblasts were grown in vitro.
The cultrues were screened for the formation of clumps(foci) that
contained cells that had been transformed by the added DNA.
Demonstration of
Oncogenes by Gene Transfer
DNA, isolated from tumor cells
(growing in culture), is mixed with
calcium phosphate and added to
normal mouse cells under
conditions where the DNA can
enter the cells (this process is
called transfection). If an oncogene
is present in the DNA, a small
number of the normal cells will
become oncogenically transformed
(i.e. “tumor-like”). If these
transformed cells are isolated and
injected into a mouse, they grow to
form a tumor.
Five of the fifteen cell lines tested yielded
DNA that could transform the recipient
NIH3T3 cells (DNA from normal cells
lacked this capability) .
Carcinogenic chemicals produced
alterations in the nucleotide sequences of
genes that gave the altered genes the
ability to transform other cells.
Attention changed to human cancer
DNA isolated from human tumor cells can
transform mouse NIH3T3 cells following
transfection.
No evidence of viral DNA was detected in
these cells.
Some human cancer cells contain an activated
oncogene that can be transmitted to other
cells ,causing their transformation.
Three different laboratories reported
the same isolation and cloning of an
unidentified gene from human bladder
carcinoma (膀胱癌) cells that can
transform mouse NIH3T3 fibroblasts
（成纤维细胞）——named ras that is
carried by the Harvey sarcoma virus,
which is a rat RNA tumor virus.
Nucleotide sequence analysis indicated that the
DNA from the malignant bladder cells is
activated as a result of a single base
substitution within the coding region of the gene.
A guanine(G)-containing nucleotide at a specific
site in the DNA of the proto-oncogene had been
converted to a thymidine(T) in the activated
oncogene.
This base substitution results in the replacement
of a valine(缬氨酸) for a glycine（甘氨酸） as the
twelfth amino acid residue of the polypeptide.
The change in the viral gene substitutes an
arginine for the normal glycine. It was apparent
that this particular glycine residue plays a critical
role in the structures and function of this protein.
Thus RAS can be activated to induce
transformation by two totally different
pathways: either by increasing its
expression or by altering the amino acid
sequence of its encoded polypeptide.
Both the RAS gene and SRC gene
are proto-oncogenes.
The Biological Role of Protooncogene
Products
The discovery of protooncogenes in normal
cells raises questions regarding their role in
normal cellular processes.
Are protooncogenes silently sitting within
normal cells “waiting” to cause cancer, or do
these genes play a role in normal
physiological processes?
Research shows that the protooncogene and its
product plays key role in some physiological
process. The
processes in which
protooncogenes are involved is quite varied;
however, most protooncogenes
have been
found to be
involved in the mechanisms
that govern the growth
and
differentiation of cells.
The
biochemical function of protooncogene products is
even
more varied.
The expression of oncogenes (literally
“cancer genes”) within cells is a crucial
event in the early stages of tumor
formation. Oncogenes can arise in cells
via two mechanisms: infection of cells by
tumor viruses and conversion (mutation)
of cellular protooncogenes to oncogenes.
These discoveries have important
implications for the prevention, detection
and treatment of cancer.
That three decades of intensive research in oncology and related basic
sciences has not led to a cure for cancer may seem disappointing to the
general public; it has, however, led scientists to a much better
understanding of the ‘problem’ of cancer. The discovery of the genetic
basis for tumorigenesis, along with the advent of ‘biotechnology’ holds
great promise that the next thirty years will bring both more effective
anti-tumor therapies and greatly improved diagnosis of tumors.
We believe that one day we will defeat cancer
eventually !
1-9-2007