Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation.
Transformation of cells by Rous sarcoma virus results from the expression of the viral src gene product, pp60src. Site-directed mutagenesis techniques have been used to construct defined deletion mutations within the src gene of Prague A strain of Rous sarcoma virus. The deletion of DNA sequences at the Bg/II restriction site in the src gene yielded both transformation-defective mutants (tdCH4, 64, and 146) and a mutant temperature sensitive for morphological transformation (tsCH119). The genome of tsCH119 contains an in-phase deletion of approximately 160 base pairs, which mapped to the immediate 3' side of the Bg/II restriction site. Upon infection of chicken cells, tsCH119 encoded a structurally altered src protein, pp53src, containing a deletion of amino acid residues 202 to 255. Immune complexes containing pp53src isolated from tsCH119-infected cells grown at 41 degrees C exhibited only 50% less tyrosine-specific kinase activity than immune complexes isolated from cells grown at 35 degrees C. pp53src immunoprecipitated from tsCH119-infected cells grown at either 35 or 41 degrees C contained phosphoserine and phosphotyrosine. We suggest that tsCH119 represents a class of mutants containing mutations mapping within a functionally important domain of the src protein, distinct from the domain specifying the protein kinase activity.
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics* / Q000502:physiology; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D002642:Chick Embryo; D005814:Genes, Viral; D009154:Mutation; D015688:Oncogene Protein pp60(v-src); D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D014764:Viral Proteins; D011494:Protein Kinases; D015688:Oncogene Protein pp60(v-src)