The expression pattern of the test and control constructs was analysed to understand the basis for the observed upregulation in yeast. Cassettes containing the reporters and the studied intercistronic sequences were expressed and purified as N-terminal histidine fusions. This allowed us to capture all the N-terminally intact products. The affinity-purified proteins were separated in SDS-PAGE gels, and visualized either by Coomassie staining (data not shown), or by western blotting with the CfMV-specific anti-VPg antibodies. The expected b-galactosidase-CfMV fusions terminating at the end of the 0-frame in the test constructs were detected. Also, the longer transframe b-galactosidase-CfMV-firefly luciferase fusion proteins were present in both the test and the inframe constructs ( Figure 3) . Comparison of the Coomassie-stained gels with the western blots revealed that the antisera recognized the products terminating at the CfMV-encoding regions better than the transframe products. Furthermore, the small size of the CfMV-specific region in the pYES2/NT-Am decreased the binding of the antibodies to these inframe control fusions. Thus, this data were not suitable for quantitative analysis of À1 PRF. Interestingly, an additional protein, which reacted with CfMV-specific antisera, was co-purified from the cells expressing pYES2/NT-Bm and pYES2/NT-Cm inframe controls ( Figure 3 ). The size of these fusions suggested that translation had terminated approximately at the site for À1 PRF signals. If such putative termination products were also present in cells expressing the test constructs, the correctly terminated 0-frame products in the western blots masked these products.