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Translation controls the expression level of a chimaeric reporter gene

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Author: Hensgens, L.A.M. · Fornerod, M.W.J. · Rueb, S. · Winkler, A.A. · Veen, S. van der · Schilperoort, R.A.
Type:article
Date:1992
Institution: Centrum voor Plantenfysiologisch Onderzoek
Source:Plant Molecular Biology, 5, 20, 921-938
Identifier: 231978
doi: doi:10.1007/BF00027163
Keywords: β-D-glucuronidase · Gene expression · Initiation of translation · Mannopine synthase promoter · Beta glucuronidase · Chimeric protein · DNA · Mannopine synthase · Oligodeoxyribonucleotide · Agrobacterium tumefaciens · Biosynthesis · Enzymology · Gene expression regulation · Genetic transcription · Genetics · Isolation and purification · Metabolism · Molecular genetics · Nucleotide sequence · Plant · Promoter region · Estriction mapping · RNA translation · Tobacco · Transgenic plant · Western blotting · Agrobacterium tumefaciens · Base Sequence · Blotting, Southern · Blotting, Western · Chimeric Proteins · DNA · Gene Expression Regulation, Enzymologic · Glucuronidase · Hydro-Lyases · Molecular Sequence Data · Oligodeoxyribonucleotides · Plants, Genetically Modified · Plants, Toxic · Promoter Regions (Genetics) · Reading Frames · Restriction Mapping · RNA, Messenger · Support, Non-U.S. Gov't · Tobacco · Transcription, Genetic · Translation, Genetic

Abstract

Transcriptional and translational fusions between the reading frame of the β-D-glucuronidase gene (gusA) and the 2′ as well as the 1′ promoter of mannopine synthase (mas), a TR 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. © 1992 Kluwer Academic Publishers. Chemicals/CAS: beta glucuronidase, 9001-45-0; DNA, 9007-49-2; hydrolyase, 9044-86-4; Chimeric Proteins; DNA, 9007-49-2; Glucuronidase, EC 3.2.1.31; Hydro-Lyases, EC 4.2.1.; mannopine synthase, EC 4.2.1.-; Oligodeoxyribonucleotides; RNA, Messenger