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Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters
Abstract:
The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.
Insights
Mutant p60v-src proteins from Rous sarcoma virus cause temperature-sensitive defects in cell transformation. Lesions in the tyrosine kinase domain are crucial for these defects, while the amino-terminal domain influences cell morphology.
Area of Science:
- Molecular biology
- Cellular biology
- Virology
Background:
- Rous sarcoma virus (RSV) encodes p60v-src, a protein tyrosine kinase that drives cell transformation.
- Temperature-sensitive (ts) mutants of RSV, like PA101 and PA104, have defects in p60v-src function.
- Understanding the structural basis of these defects is key to comprehending viral oncogenesis.
Purpose of the Study:
- To elucidate the structural determinants responsible for the transformation-defective phenotype of PA101 and PA104 p60v-src mutants.
- To investigate the roles of specific domains within p60v-src in regulating cell proliferation, morphology, and anchorage independence.
Main Methods:
- Molecular cloning and analysis of the v-src genes from PA101 and PA104 mutants.
- Construction of chimeric v-src genes by exchanging amino- and carboxy-terminal regions with wild-type v-src and chicken c-src.
- Expression of reconstructed viral DNA in infected cells at various temperatures to assess transformation parameters.
Main Results:
- Lesions within the tyrosine kinase domain of p60v-src mutants confer temperature sensitivity to cell proliferation, morphological alteration, and anchorage independence.
- The amino-terminal region of the PA101 mutant affects cell morphology in a temperature-independent manner.
- The kinase domain is essential for all three transformation parameters, while the amino-terminal domain specifically impacts cell morphology.
Conclusions:
- The tyrosine kinase domain of p60v-src is critical for mediating all tested transformation functions.
- The amino-terminal domain plays a distinct role in modulating cell morphology.
- Structure-function relationships of viral oncoproteins provide insights into cellular transformation mechanisms.