Frameshifting for expression of C.elegans antizyme was investigated in heterologous systems. Two constructs containing the entire antizyme cDNA, one with the wildtype sequence and one with a single nucleotide deletion that fuses ORF1 to ORF2 in-frame (in-frame control), were transcribed in vitro and the RNA was translated in rabbit reticulocyte lysate. The products were examined by SDS±PAGE (Figure 7) . The main product from both constructs has an apparent M r of 21 kDa, slightly greater than the predicted M r of 17.7 kDa [aberrant, slower than expected, mobility is observed with antizyme proteins from other species (Ivanov et al., 1998a) ]. From the ratio of wild-type to in-frame product, we estimate that the ef®ciency of frameshifting of C.elegans antizyme in reticulocyte lysate is~0.8%, which is somewhat lower than SPA frameshifting in the same system. Addition of spermidine to the translation reactions almost doubles the ef®ciency of frameshifting to~1.5% (the exact numbers are not easy to determine because of dif®culty in de®ning background values). The frameshifting properties of C.elegans antizyme mRNA were also tested in vivo in S.pombe cells. A sequence including all but the ®rst codon of ORF1 plus 180 nucleotides downstream was inserted between GST and lacZ of the PIU-LAC plasmid. Comparison of the b-galactosidase activity of cells (Dspa::LEU2 strain) transformed with the wild-type construct and the in-frame control constructs indicated 3.5% +1 frameshifting. From the frameshifting observed in the heterologous systems, as well as the sequence considerations discussed above, we conclude that expression of this C.elegans gene requires ribosomal frameshifting.