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Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants
Abstract:
The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.
Insights
Two mutations within the Rous sarcoma virus (RSV) src gene
Area of Science:
- Virology
- Molecular Biology
- Oncogenesis
Background:
- Rous sarcoma virus (RSV) is a retrovirus known to cause tumors.
- Temperature-sensitive (ts) mutants of RSV allow for the study of viral gene function at different temperatures.
- The src gene is crucial for RSV-induced cell transformation.
Purpose of the Study:
- To identify the specific mutations responsible for the temperature-sensitive phenotype in RSV src genes.
- To determine the contribution of individual mutations to the ts phenotype of cell transformation.
Main Methods:
- Analysis of src genes from four temperature-sensitive (ts) Rous sarcoma virus (RSV) mutants.
- Cloning of mutant src genes into a replication-competent RSV expression vector.
- In vitro recombination of mutant src genes with wild-type src sequences to assess individual mutation effects.
Main Results:
- Three Schmidt-Ruppin strain RSV mutants each had two mutations in the conserved kinase domain.
- A common mutation (Valine to Methionine at amino acid 461) was present in these three mutants.
- Single mutations alone did not confer temperature sensitivity, indicating a requirement for two mutations.
Conclusions:
- Two distinct mutations within the src kinase domain are necessary for the temperature-sensitive transformation phenotype in RSV.
- Specific amino acid changes in the src kinase domain are critical for regulating its function in cell transformation.