Effect of the frame-shift mutation on expression of the M135R gene product. Panel (a). CAT-M135R fusion genes were constructed to demonstrate the effect of the +G insertion within the M135R gene on protein expression. Plasmids containing either the wt (pCATM135Rwt) or the frame-shift variant (pCATM135R+G) of the M135R sequence were constructed. The expected products from the plasmids containing just the CAT open reading frame (ORF) (i), the CAT ORF fused to the wt M135R ORF (ii) and CAT fused to the mutant M135R+G sequence (iii) are illustrated. Panel (b). The plasmids shown in panel (a) were transfected into BHK cells that had been infected with the recombinant vaccinia virus vTF7-3 which expresses the T7 RNA polymerase. Cell extracts were prepared 20 h later and analysed by SDS-PAGE and immunoblotting using rabbit anti-CAT antibodies. Detection was achieved using peroxidase-labelled anti-rabbit IgG and chemiluminescence reagents with a BioRad Chemidoc XRS. Lane 1, pGEMCATBX (without a termination codon at the end of CAT but translation terminates within the vector sequence); lane 2, pGEMCATBS (with a termination codon, see panel (a)); lanes 3 and 4, plasmids (pCATM135Rwt) containing the fused CAT and wt M135R ORFs (n.b., there is no termination codon at the end of the CAT sequence); lanes 5 and 6, plasmids (pCATM135Rwt+G) containing the fused CAT and mutant M135R+G sequences and lane 7, No DNA (negative control). The migration of molecular weight markers is indicated. The products corresponding to those expected from constructs (i), (ii) and (iii) shown in panel (a) are marked.
Belsham et al. Virology Journal 2010 7:7 doi:10.1186/1743-422X-7-7