The p2luc reporter was a kind gift from Professor John Atkins at the University of Utah (36) , and the pRL-SV40 vector was purchased from Promega. Oligonucleotides containing the slippery sequence (TTTAAAC), spacer (GGGTT) and the stimulator sequences were chemically synthesized. They were amplified by forward and reverse primers containing BsrGI and BsaAI sites, respectively, and ligated into the BsrGI/BsaAI sites (1392/1426) of pRL-SV40 vectors treated with restriction enzymes. Oligonucleotides containing DU177, hTR-5 0 hp, hTR5 0 hp-L1c and hTR174T5 0 hp sequences were also inserted into p2luc reporter in a similar way, except for the use of SalI and BamHI restriction sites instead. A À1frame stop codon was inserted into the DU177-containing p2luc reporter, thus generating a premature À1-frame protein product, to facilitate the À1 PRF assays of DU177-related variants in vitro (37) . All of the mutants were constructed using the quick-change mutagenesis kit from Stratgene, and the identities of all cloned and mutated genes were confirmed by DNA sequencing analysis.