The signal sequence of the p62 protein of Semliki Forest virus is involved in initiation but not in completing chain translocation

Constructions of pGEM2alphaGX and pGEM2dhfrX. For the construction of the final fusion protein-coding plasmids used in this study we first had to make plasmids pGEM2alphaGX, which are derived from pGEM2alphaG and pGEM2dhfrX, which are derived from pGEM2dhfr (Zerial et al., 1986) . Plasmid pGEM2alphaG contains a 548 bp-long Nco I-Pst I fragment encompassing the entire chimpanzee alpha-globin coding region between the Hinc II and Pst I sites of the polylinker of the plasmid pGEM1 (Pmmega Biotech). The Nco I site contains the translation initiation codon from alpha-globin (Zerial et al., 1986 ). An Xho I site, allowing subsequent in-frame ligations of SFV sequences, had to be introduced in pGEM2alphaG. Therefore, this plasmid was cut (upstream of the Nco I site) with Sac I, the 3' sticky ends removed with "1"4 DNA Folymerase, an Xho I octamer linker introduced and, after cutting with Xho I, the plasmid was religated at low DNA concentration (1 #g/ml). Plasmid pGEM2alphaGX then contains the 2,057 bp-long Xbo I-Pvu I fragment needed for the construction of the fusion protein-coding plasmid pC62alphaG.