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Translation controls the expression level of a chimaeric reporter gene

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

Expression of a rhizobium phaseoli sym plasmid in R. trifolii and agrobacterium tumefaciens: incompatibility with a R. trifolii sym plasmid

Microbial BiotechnologyPlant science

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An Easy and Efficient Procedure for the Isolation of Pure DNA Restriction Fragments from Agarose Gels

A new procedure is developed to isolate DNA from agarose gels. Using a kind of blotting technique, DNA is isolated from the gel. It is shown that the isolated DNA can be used for fragmentation by restriction endonucleases, synthesis of complementary RNA by DNA-dependent RNA polymerase from Escherichia coli and nick translation. The procedure gives a high recovery and is easy to perform.