Translation controls the expression level of chimaeric reporter gene

• Published in: Plant molecular biology Vol. 20; no. 5; pp. 921 - 938
• Main Authors: Hensgens, L.A.M. (Leiden Univ. (Netherlands). Inst. of Molecular Plant Sciences), Fornerod, M.W.J, Rueb, S, Winkler, A.A, Veen, S. van der, Schilperoort, R.A
• Format: Journal Article
• Language: English
• Published: Netherlands 01-12-1992
• Subjects: AGROBACTERIUM TUMEFACIENS; Agrobacterium tumefaciens - enzymology; Agrobacterium tumefaciens - genetics; ARN MENSAJERO; ARN MESSAGER; Base Sequence; beta-glucuronidase; Blotting, Southern; Blotting, Western; DNA - genetics; DNA - isolation & purification; enzyme activity; EXPRESION GENICA; EXPRESSION DES GENES; FEUILLE; GENE; GENE EXPRESSION; Gene Expression Regulation, Enzymologic; GENES; genetic regulation; genetic transformation; genetically modified organisms; Glucuronidase - biosynthesis; Glucuronidase - genetics; gusa gene; HOJAS; Hydro-Lyases - biosynthesis; Hydro-Lyases - genetics; LEAVES; ligases; mannopine synthase; mas gene; MESSENGER RNA; Molecular Sequence Data; NICOTIANA; Nicotiana - enzymology; Nicotiana - genetics; Nicotiana tabacum; Oligodeoxyribonucleotides; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants, Genetically Modified; Plants, Toxic; promoter regions; Promoter Regions, Genetic; Protein Biosynthesis; RACINE; RAICES; Reading Frames; recombinant DNA; Recombinant Fusion Proteins - biosynthesis; reporter genes; Restriction Mapping; RNA, Messenger - metabolism; ROOTS; STEMS; TALLO; TIGE; Transcription, Genetic; TRANSGENIC PLANTS; translation
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/bf00027163

Transcriptional and translational fusions between the reading frame of the beta-D-glucuronidase gene (gusA) and the 2' as well as the 1' promoter of mannopine synthase (mas), a T(R) locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level. In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves. In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.