Figure 2.

rPHGPx nascent peptide generated by the coupled transcription-translation of rPHGPx (TGA) cDNA by using wheat germ extracts does not accumulate at the codon preceding the Sec codon when the Sec codon is recognized as nonsense. The structure of the SP6-rat(r)PHGPx cDNA and positions of important codons are shown. The diagonally stripped box specifies the bacteriophage SP6 promoter, open boxes represent PHGPx exons, and the darkened region of the last exon indicates the SECIS. ATG(27) specifies the translation initiation codon used primarily in somatic tissues (Pushpa-Rekha et al., 1995), TGA(72) specifies the sole Sec codon [which in derivative constructs was changed to either TGT(72) or TAA(72)], and TAA(197) specifies the normal termination codon. Test pSP-rPHGPx plasmids (9 μg) harboring either the usual Sec (TGA) codon, a Cys (TGT) codon, or a nonsense (TAA) codon and reference luciferase DNA (2 μg; Promega) were incubated in the presence of [³⁵S]methionine in 25 μl of the TNT Wheat Germ Lysate System (Promega). By so doing, cDNAs were transcribed by SP6 RNA polymerase, and product RNAs were then translated. A fraction (12 μl) was precipitated by reducing the pH to 5.0 by using acetic acid and subsequently electrophoresed in acrylamide. The level of PHGPx from each plasmid was normalized to the level of luciferase, and normalized values were then calculated relative to the amount of normalized protein from the TGA-containing construct (after accounting for the number of radioactive amino acids per molecule), which was defined as 1. The asterisk denotes an unidentified protein that was produced in extracts without exogenous DNA (−).