The SPA gene was ampli®ed using the following primers: 5¢-CAAAACAAGTTTTCATTATTGGTTTTTTTTAAATCAATCCCC (sense) and 5¢-CGTAAATCCAATCTAAATTTAATCTTCAACTAA-ATCATGAAAAGCCTC (antisense). The S.pombe cDNA library used as a template in the ampli®cation was kindly provided by R.Rowley (University of Utah). The C.elegans antizyme gene was ampli®ed using the following primers: 5¢-CCCAGGAATTCCTCGAGTATTTTGA-GTATAATTTTAC (sense) and 5¢-CGGCCGCTCGAGTTAGACCTT-GTAGCTCATGATG (antisense). This same ampli®ed DNA was used to make the constructs for in vitro transcription and translation of C.elegans antizyme by cloning it into pTZ18U plasmid using the SacI and HindIII sites incorporated in the two primers. The in-frame construct was made using a two-step PCR. The cDNA sequences of O.volvulus and P.paci®cus antizyme genes were obtained by performing 5¢ and 3¢ RACE PCR with cDNA libraries, which were kindly provided by Ralf Sommer (P.paci®cus) and Susan Haynes (O.volvulus). The SPA overexpression constructs were made by amplifying the gene with the primers 5¢-GCATCCGAATTCCCAAATCCAAGCATCATACGCC (sense) and 5¢-GCATCCGGATCCGCCAGTGTTCTTACTTTGAGA-TGC (antisense), and then inserting BamHI-digested product between the MscI and BamHI sites of pREP3 plasmid. The in-frame construct was made by two-step PCR and subsequently all in-frame SPA constructs described below were made by one-step PCR using this plasmid's DNA as a template. To make the constructs for frameshift assays in S.pombe, DNA fragments with a given nucleotide length (as described in the main text), were ampli®ed from both the SPA and C.elegans antizyme constructs described above. These fragments were then cloned between the KpnI and BstEII sites of PIU-LAC plasmid (Ivanov et al., 1998c) . The PCR primers included an`AC' spacer between the 5¢ cloning site (BstEII) and the antizyme sequences in order to correct the reading frame. The in vivo frameshifting assays in S.pombe (strains ura4-D18 leu1-32 ade6-M216 h ± and Dspa::LEU2 ura4-D18 leu1-32 ade6-M216 h ± ) were done as described (Ivanov et al., 1998c) . The plasmid for GST±SPA expression was made by PCR amplifying SPA (all but the ®rst codon of ORF1 through the downstream ORF2) from an in-frame template and cloning the product into the EcoRI and XhoI restriction sites of pGEX-5X-3 plasmid. The antizyme frameshift site in the BMV-coatantizyme fusion construct (C3NE) (Matsufuji et al., 1995) was mutated with a two-step PCR. To generate the two knockout strains, Dspa::URA4 and Dspa::LEU2, both ORFs of SPA were replaced exactly with the ORF of either URA4 or LEU2. To accomplish this, two pairs of primers ampli®ed URA4 and LEU2 such that 50±60 nucleotides, which normallȳ ank the two ORFs of SPA,¯ank the ORFs of the two genes. The ampli®ed DNA products were gel puri®ed and 2 mg of each were used to electroporate into ura4-D18 leu1-32 ade6-M216 h ± cells. URA + and LEU + transformants were selected by growth on URA ± and LEU ± media, respectively. PCR screen and partial sequencing, with primers¯anking the regions used for the homologous recombination, con®rmed the SPA disruptions. All DNA clones were sequenced with automated sequencing machines (ABI 100).