DERIVATION AND ANALYSIS OF MUTANTS IN THE TRANSFORMING GENE OF HARVEY MURINE SARCOMA VIRUS

The Harvey murine sarcoma virus (HaMSV) has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of Herpes simplex type 1 virus in a downstream linkage with the P21 ras gene of HaMSV. Virus (HaMSVtk) rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The HAT selectability of the thymidine kinase gene has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Clones 9 and 20 are characterized by genomic message containing both Harvey ras and thymidine kinase information. Clone 45 produces a more attenuated non-ras, thymidine kinase message. All three produce varying amounts of a subgenomic non-ras thymidine kinase message. Only clone 9 encodes a p22 immunologically related to p21. The clone 9 mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Observations made in these studies have led to the conclusion that the p21 of HaMSV consists of at least two domains. One domain specifies the guanine nucleotide-binding activity of p21 and the second is involved in membrane association in transformed cells. The processing, fatty acid binding, and membrane association of this newly developed mutant protein are explored in this context.