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. 1998 May;18(5):2932–2939. doi: 10.1128/mcb.18.5.2932

FIG. 4.

FIG. 4

(A) Structures of the mCMV-GPx1 gene and derivative alleles. The shaded box represents the 550-bp XbaI-EcoRI fragment that harbors the mCMV promoter. The open boxes represent exons, the intervening line represents the single 216-bp intron, and the right-most line represents 3′ flanking DNA. ATG(0), TGA(46), and TAA(201) represent, respectively, the initiation codon, Sec codon, and termination codon. Mutations that convert the Sec codon to either a Cys codon (TGC) or a premature termination codon (TAA) are indicated below the gene structure. (B) The level of mCMV-GPx1 mRNA in total RNA of NIH 3T3 cells grown in Se-deficient medium (−) is 50% of the level in NIH 3T3 cells grown in Se-supplemented (+) medium. Cells were either untransfected or transiently transfected with pmCMV-GPx1 (25 μg) and the reference plasmid pmCMV-G1 (25 μg). Total RNA was isolated, and RT-PCR was used to quantitate mCMV-GPx1 and mCMV-G1 mRNAs. The left-most five lanes contain twofold serial dilutions of RNA from Se-supplemented cells in order to demonstrate a linear relationship between the amounts of input RNA and RT-PCR products. The level of mCMV-GPx1 mRNA was normalized to the level of mCMV-G1 mRNA. The normalized value for mCMV-GPx1 mRNA in Se-deficient cells was then calculated as a percentage of the normalized value for mCMV-GPx1 mRNA in Se-supplemented cells, which was considered to be 100%. The values from two independently performed experiments did not differ by more than 3%. (C) The ratio of cytoplasmic mCMV-GPx1 mRNA to nuclear mCMV-GPx1 mRNA is decreased in Se-deficient NIH 3T3 cells by a decay pathway that is dependent on recognition of the Sec codon as a termination codon. Transfections and analyses of RNA were as described in the legend to Fig. 4B, except that nuclear and cytoplasmic RNAs were purified and analyzed as described in the legend to Fig. 2. The left-most four lanes contain twofold dilutions of cytoplasmic RNA from Se-supplemented NIH 3T3 cells transiently transfected with pmCMV-GPx1-TGC(46). GPx1, GPx1-TAA(46), and GPx1-TGC(46) signify pmCMV-GPx1 plasmids harboring at position 46 the wild-type sequence (TGA), a nonsense codon (TAA), and a Cys codon (TGC), respectively. The two right-most lanes, which reflect PCR analyses of intronless pmCMV-GPx1 DNA and pmCMV-GPx1 DNA, provide molecular weight standards for pre-mRNA and mRNA, respectively.