1911 Peyton Rous published a paper on the treatment of syphilis;Peyton Rous published a paper on the treatment of syphilis discovered an RNA-containing virus.discovered an RNA-containing virus 1960 Similar viruses were found to be associated with mammary tumors and leukemias in rodents and cats. Question 1Question 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 RNA-dependent DNA polymerase was discovered independently by David Baltimore, Temin and Satoshi Mizutani.RNA-dependent DNA polymerase was discovered independently by David Baltimore, Temin and Satoshi Mizutani Question 2Question 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. cDNA sarc 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 cDNA sarc 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.

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? Questions

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 tthe product of src --- pp60 src Research on the function of src ——the discovery of its product

 pp60 src  Localization (by electronmicroscopic immunocytochemistry) pp60 src is localized at the plasma membrane of the cell and is particularly concentrated at the sites of gap junctions. Research on the function of src ——the discovery of its product

Function of pp60 src

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

Research on HUMAN BEINGS

Which genes in a cell, when activated by mutation or some other mechanism, are responsible for causing the cell to become malignant? Question

 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. A transfection experiment

Researches on human oncogene

precise changes in the human RAS that lead to its activation as an oncogene

RAS can be activated to induce transformation by two totally different pathways:  increase its expression  alter the amino acid sequence of its encoded polypeptide Mechanism of RAS

A glycine residue plays a critical role in the structure and function of this protein 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 cancer- associated mutant Ras proteins to accumulate.

Summary