The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex

Plasmid pMSl50, encoding the v-sis gene under control of the Rous sarcoma virus promoter, was used as a parental clone to make the fusion proteins. The DNA sequence encoding amino acids 238-271 of v-sis is easily removed from pMS150 as a BstEII-ClaI fragment, allowing for insertion of novel sequences to create various fusion constructs. Optimized oligonucleotide synthesis and purification were as previously described (Xu et al., 1993) . The complementary oligonucleotides for each fusion protein were designed so that, when annealed, 5' BstEII and 3' ClaI overhangs were produced. Oligonucleotides were then ligated with vector DNA (pMS150) previously digested with BstEII and ClaI and purified by agarose gel electrophoresis. Recombinant clones were recovered and the sequences of the oligonucleotides were confirmed by dideoxy nucleotide sequencing.