The 5' untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells.
We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS-7 cells revealed that the 5' untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5'utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5'utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3'utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site-directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in-frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5'utr was essential for the expression of GPx gene. However, the deletion of a part of the 3'utr and the site-directed mutation upstream of the Scy codon did not show drastic effects on the expression.
Kurata
H
H
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Kamoshita
K
K
Kawai
E
E
Sukenaga
Y
Y
Mizutani
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D005091:Exons; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D005979:Glutathione Peroxidase / Q000235:genetics*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D005979:Glutathione Peroxidase