Effects of 3' untranslated and intergenic regions on gene expression in Trypanosoma cruzi.
The effects of 3' untranslated regions (UTR) and intergenic regions (INT), from various Trypanosoma cruzi stage-specific and constitutive genes, on the expression of the reporter firefly luciferase gene (luc), were studied using stable episomal transformation. The 3' UTR influenced luciferase expression by changing the steady-state level and/or the translation efficiency of luc mRNA. Glycoprotein 72 gene (gp72), glycoprotein 85 gene (gp85) or amastin gene (ama) 3' UTR decreased the luc mRNA level 6- to 14-fold, compared to the glyceraldehyde 1-phosphate dehydrogenase gene (gapdh) 3' UTR, in epimastigotes. Luciferase activity decreased in parallel with the luc mRNA level in transformants utilizing the gp85 or ama 3' UTR, whereas luc mRNA containing the gp72 3' UTR showed approximately 5-fold higher translation efficiency than luc mRNA containing a minimal 3' UTR. In amastigotes, the inhibitory effect of the ama 3' UTR observed in other life cycle stages was abolished and luciferase expression was stimulated 16-fold. The overall stage-specific difference mediated by the ama 3' UTR, between epimastigotes and amastigotes, was approximately 100-fold. INT, which was expected to influence polyadenylation efficiency, of gapdh, gp72, or heat shock protein 60 gene inserted after gapdh 3' UTR increased luc mRNA 2- to 8-fold, whereas gp85 INT slightly decreased luc mRNA. By separating effects attributable to the 3' UTR and INT, this study shows the effects of 3' UTR on RNA levels and translational efficiency in T. cruzi.
Nozaki
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Laboratory of Molecular Parasitology, Rockefeller University, New York, NY 10021, USA.
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D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D017931:DNA Primers; D015870:Gene Expression; D017125:Genes, Protozoan; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D007438:Introns; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D015800:Protozoan Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases