Figure 3.

RNA blot hybridization indicates that pmCMV-PHGPx harboring either the TGA Sec codon or one of several nonsense codons generates mRNA that is reduced in abundance in NIH3T3 and H35 cells. Structures of the mCMV-PHGPx gene and positions mutated in the various derivative alleles are shown. The diagonally stripped box specifies the mCMV promoter, open boxes represent PHGPx exons, intervening lines represent introns, and the right-most bold line represents PHGPx 3′-flanking DNA. Codons for translation initiation, Sec, and translation termination are specified as determined for rPHGPx sequences (see legend to Figure 2). NIH3T3 or H35 cells that had been propagated in MEM plus 10% FBS were either not transfected (−) or transiently transfected with the designated pmCMV-PHGPx test plasmid and the pmCMV-TPI reference plasmid, total RNA was isolated, and PHGPx and TPI transcripts were quantitated by blot hybridization. The level of each PHGPx mRNA was normalized to the level of TPI mRNA and subsequently calculated as a percentage of the normalized level of PHGPx UGU (72) mRNA, which was defined as 100. Values did nor vary by >7–8% in three independently performed experiments.