Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo

Abstract

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.