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Identification of an amino terminal domain required for the transforming activity of the Rous sarcoma virus src
1Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Abstract:
Transformation of chicken cells by Rous sarcoma virus (RSV) requires the functional expression of the viral src protein, a tyrosine protein kinase, pp60src. Variants of RSV containing deletions within the amino terminal one-third of the src protein have been identified that exhibit either temperature-sensitive or transformation-defective phenotype when used to infect chicken embryo cells. To define the regions within the amino terminal portion of pp60src that influence morphological transformation, a series of overlapping deletion mutations in the src gene of Prague A RSV (Pr A RSV) were constructed and their biological and biochemical properties were analyzed. Deletions within the src gene which remove amino acid residues 38 to 142 had minimal effects on the ability of the mutant viruses to induce cellular transformation. However, deletions, which impinged upon the region of the src gene encoding residues 142 to 169, inhibited cellular transformation. A variant containing a deletion of amino acid residues 169 to 225, was temperature sensitive for transformation. Structurally altered src proteins recovered from cells infected with transformation-defective variants exhibited a somewhat reduced tyrosine protein kinase activities when assayed in the immune complex kinase assay. Analysis of the in vivo phosphorylation of a pp60src substrate, the 36-kDa protein, revealed virtually wild-type levels of phosphorylation in cells infected with the transformation-defective mutants. These studies suggest that the region of the Pr A RSV src protein delineated by amino acid residues 142 to 169 is essential for initiation and maintenance of morphological transformation of chicken cells in culture.
Insights
The Rous sarcoma virus (RSV) src protein is crucial for cell transformation. Specific regions, particularly amino acids 142-169, are essential for Prague A RSV (Pr A RSV) to induce and maintain cell transformation.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Rous sarcoma virus (RSV) transformation of chicken cells depends on the viral src protein (pp60src), a tyrosine protein kinase.
- RSV variants with deletions in the N-terminal region of src exhibit temperature-sensitive or transformation-defective phenotypes.
Purpose of the Study:
- To pinpoint the specific regions within the N-terminal portion of pp60src that are critical for morphological transformation.
- To analyze the biological and biochemical properties of deletion mutants in the Prague A RSV (Pr A RSV) src gene.
Main Methods:
- Construction of a series of overlapping deletion mutations in the src gene of Prague A RSV.
- Analysis of biological properties, including cellular transformation.
- Biochemical analysis of src protein kinase activity and in vivo substrate phosphorylation.
Main Results:
- Deletions affecting amino acid residues 142-169 significantly inhibited cellular transformation.
- A deletion mutant (residues 169-225) displayed temperature-sensitive transformation.
- Mutant src proteins showed reduced tyrosine kinase activity, but in vivo phosphorylation of a key substrate (36-kDa protein) remained largely unaffected.
Conclusions:
- The region spanning amino acid residues 142-169 of the Pr A RSV src protein is indispensable for initiating and sustaining morphological transformation in chicken cells.
- While kinase activity is important, specific structural elements within this region are critical for the transformation process.