To test overexpression of SPA, two versions of the gene were cloned into pREP3 expression vector behind a strong, thiamine-repressible promoter (nmt1). One had the wild- type SPA sequence while in the second, ORF1 and ORF2 are fused in-frame. SPA wild type and an SPA deletion strain were transformed with each of the overexpression constructs. Derepression of the nmt1 promoter is a gradual process since it requires dilution of the intracellular pool of thiamine (the repressor) through cell division. After 2.5 days of exponential growth under derepressed conditions, yeast strains transformed with either SPA overexpression construct show signi®cant increases in doubling time ( Figure 4A ). The growth inhibition is greater with the construct expressing the in-frame version of SPA and after prolonged incubation (5±7 days); these cells cease growth and accumulate in G 1 as determined bȳ ow cytometry (data not shown). The fact that the inframe overexpression construct, which differs by a single nucleotide from the wild-type construct, confers a more severe phenotype is consistent with the hypothesis that translational frameshifting is required for expression of SPA. The growth phenotype associated with SPA overexpression is only partially relieved by adding 100 mM putrescine to the media (1 mM had no further effect) (data not shown). To see whether the slower growth is correlated with aberrant polyamine levels the polyamine contents of the deletion strain carrying in-frame SPA overexpression vector were measured under derepressed and repressed conditions, in both cases after 2 days of exponential growth ( Figure 4B ). As expected, overexpression of SPA results in signi®cant reduction in the intracellular levels of all four polyamines. After longer (4±5 days) incubation under derepressed conditions, no putrescine and cadaverine can be detected (data not shown).