A General Surveillance plan to monitor for unanticipated adverse effects of genetically modified organisms (GMOs) on human health and the environment is required as part of the EU legislation for imported and cultivated GMOs.Imported GM products: Operators involved in the import, handling and processing of grain commodities entering the EU have monitoring procedures in place to survey products and are organised in associations across the EU Member States. They can therefore be considered as the best placed to monitor for potential unanticipated adverse effects linked to imports of GM products into the EU. Recognising this, the Plant Biotechnology Industry has established a collaboration to cover General Surveillance of grain commodity imports with the relevant associations, which is coordinated by EuropaBio, the European Association for Bioindustries.Cultivated GM plants: Monitoring for unanticipated adverse effects should take place in agronomic zones representative of commercial GM crop cultivation. Farmers are therefore considered to be the most valuable sources of information when it comes to General Surveillance of GM crop cultivation, due to their extensive experience with and direct involvement in cultivation. Acknowledging this, the Plant Biotechnology Industry has developed a harmonised general surveillance approach based on farmer questionnaires.
Applications for placing on the market of genetically modified organisms (GMOs) for import, food, feed and processing under Directive 2001/18/EC and Regulation (EC) No 1829/ 2003, have to include a monitoring plan conforming with Annex VII to Directive 2001/18/EC. One aspect of this monitoring plan is the need for general surveillance to identify the occurrence of adverse effects of the viable GMO or its use on human and animal health or the environment which were not anticipated in the environmental risk assessment (e.r.a.). Since international grain commodity trade consists of commingled products and the Plant Biotechnology Industry is not directly involved in commodity trade, authorisation holders under Directive 2001/18/EC and Regulation (EC) No 1829/2003 have been working together within the European Association of Bioindustries (EuropaBio) and with European trade associations representing relevant commodity trade operators to develop a harmonised general surveillance methodology for import and processing of viable GMOs. A harmonised industry general surveillance system was agreed upon by the Plant Biotechnology Industry members and the European trade associations and has been operational for several years. This harmonised industry general surveillance system has now been described in a harmonised industry monitoring plan. In line with Decision 2002/811/EC establishing guidance notes supplementing Annex VII to Directive 2001/18/EC and the Guidance Document of the Scientific Panel on Genetically Modified Organisms for the risk assessment of genetically modified plants and derived food and feed, this monitoring plan contains a detailed description of the agreed monitoring methodology together with other specifics, such as the baseline and controls for general surveillance, the time period over which general surveillance will be carried out, the use of existing networks and how the results of monitoring will be reported and reviewed.
Gene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.
The structure of "B18", an 18-residue fusogenic peptide from the sea urchin fertilization protein bindin, was investigated in several membrane-mimicking environments with circular dichroism and nuclear magnetic resonance spectroscopy. The fully conserved peptide sequence represents the minimal functional part of the 24 kDa protein, which can bind to membranes and induce fusion of lipid vesicles. The B18 peptide undergoes a coil-helix transition in the presence of TFE, showing a transient tendency to self-associate. Its NMR structure in 30% TFE exhibits two helical regions at either side, connected by a flexible loop. In DPC and SDS detergent micelles, this loop becomes distinctly bent, presumably due to the high degree of curvature of the micelles. The loop contains a histidine-rich motif for binding zinc, which is required for the fusogenic function of the peptide. Therefore, we monitored the structural response of B18 and of recombinant bindin toward this ion. Like TFE, and in a mutually cooperative manner, zinc induces a partially helical structure in both the peptide and the protein. Complex formation via the histidine residues rigidifies the flexible loop and is accompanied by self-association of the molecules. The data suggest that the zinc-bound functional state is a continuous amphipathic alpha-helix, bearing some resemblance to a leucine zipper. Two hydrophobic patches on one face could favorably penetrate into a membrane, while two arginines on the other face could interact with lipid phosphate groups. The three-dimensional model of the B18 sequence thus contributes to a better understanding of peptide-induced vesicle fusion in general, and of the lipid-protein interactions of sperm bindin in particular.
SummaryGene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.
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