[Food safety of GMOs]. / Les risques alimentaires des OGM.

In this presentation, we review the complexity of the different biological events which occur during life cell cycles. Indeed transgenesis is not an unknown event for cells. In the second part of this article, the complex and complete evaluation process destined to assure the food safety of GMOs, before they are released on the market, is describd. Some ansers to questions frequently asked about the GMOs are given. It is concludedthat GMOs are probably more safe than their conventional non-GM counterpart.

The thioredoxin h system: potential applications.

The thioredoxin h system has the specific capability to reduce intramolecular disulfide bonds of proteins, thereby modifying their tertiary structure. It is involved in many processes: in the activation or deactivation of enzymes and enzyme inhibitors and in the germination process. This system can be used to improve the breadmaking quality of wheat by strengthening the dough. It can also decrease the epitope accessibility, then modifying the response of the IgE immune system. Transgenic barley and wheat have been created to confirm the functionality of the NADP-dependent thioredoxin h system.

Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat.

In hexaploid bread wheat ( Triticum aestivum L. em. Thell), ten members of the IWMMN ( International Wheat Microsatellites Mapping Network) collaborated in extending the microsatellite (SSR = simple sequence repeat) genetic map. Among a much larger number of microsatellite primer pairs developed as a part of the WMC ( Wheat Microsatellite Consortium), 58 out of 176 primer pairs tested were found to be polymorphic between the parents of the ITMI ( International Triticeae Mapping Initiative) mapping population W7984 x Opata 85 (ITMI pop). This population was used earlier for the construction of RFLP ( Restriction Fragment Length Polymorphism) maps in bread wheat (ITMI map). Using the ITMI pop and a framework map (having 266 anchor markers) prepared for this purpose, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (no marker on chromosome 6D). These 66 mapped microsatellite (SSR) loci add to the existing 384 microsatellite loci earlier mapped in bread wheat.

Optimization of the wheat puroindoline-a production in Pichia pastoris.

AIMS: A recombinant puroindoline-a (rPIN-a) was produced using the methylotrophic yeast Pichia pastoris. METHODS AND RESULTS: In fed-batch culture, the production of rPIN-a decreased after 24 h of methanol induction. Most of the rPIN-a was not soluble in the culture medium remaining bound to the cell walls. Soluble and membrane-bound rPIN-a were quantified by ELISA after Triton X-114 phase partitioning. In order to improve the production of rPIN-a, the influence of pH, specific growth rate and the addition of TX-114 was tested on two independent continuous cultures. The production of rPIN-a was improved when continuous culture was carried out at 29 degrees C under acid conditions (pH 5) with a low dilution rate (D=0.025 h(-1)). The addition of 0.01% TX-114 to the medium inverted the ratio between the secreted and the membrane-bound rPIN-a. CONCLUSION: When a continuous culture was carried out under optimized conditions, the rPIN-a production yield was increased 10-fold to 14 mg l(-1) and 80% of the rPIN-a was soluble. SIGNIFICANCE AND IMPACT OF THE STUDY: This study would be helpful to optimize the expression of other membrane-bound proteins in P. pastoris.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic rice seeds.

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to -1068, -388, -210 or -124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the uidA reporter gene encoding beta-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the uidA reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the puroindoline-b promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5' promoter deletions identified the region between -388 and -210 as essential for endosperm expression, and the region between -210 and -124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes.

Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat.

The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.

High-level secretion of a wheat lipid transfer protein in Pichia pastoris.

Plant nonspecific lipid transfer proteins are small basic proteins with eight cysteine residues, all engaged in disulfide bonds. The sequence encoding the wheat 9-kDa LTP was cloned into the secretion vector pYAM7SP8 giving rise to pYTdltp4.90. Production in shake-flasks and a fermentor led to the synthesis of two major species of LTP: a larger than expected species of 14 kDa and a species of 10 kDa, close to the expected size of wheat LTP. When production was carried out in a fermentor with regulation of pH, oxygen level, and feed rate of carbon source, the 10-kDa species was the main protein at the end-point of culture. The recombinant wheat LTP (rLTP), secreted at a level of 720 mg/liter into the culture medium, is soluble. The rLTP was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography, with a recovery yield of 36%. However, the molecular mass of rLTP, determined by mass spectrometry, is 9996 Da, while its naturally occurring counterpart has a molecular mass of 9607 Da. This discrepancy in size corresponds to a protein carrying three extra amino acids (DKR) at its N-terminal end, and this was confirmed by sequencing. In vitro lipid transfer activity showed that rLTP behaves in a similar way to the naturally occurring protein. These data indicate that Pichia pastoris is an efficient system for production of large quantities of soluble and biologically active rLTP for structure/function analysis.

Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli.

Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h, respectively, were isolated from mid-maturation seed cDNA libraries. The T aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5% similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three-step procedure including heat treatment, anion-exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13,391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N-terminal end sequence AASAAT. Using the 5,5'-dithiobis(2-nitrobenzoic acid)-reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP:thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed alpha-amylase inhibitors (chloroform/methanol-soluble proteins). The presence of a putative transmembrane domain at the N-terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.

Characterization of a barley gene coding for an alpha-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment.

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.

Linkage between RFLP markers and genes affecting kernel hardness in wheat.

A molecular-marker linkage map of wheat (Triticum aestivum L. em. Thell) provides a powerful tool for identifying genomic regions influencing breadmaking quality. A variance analysis for kernel hardness was conducted using 114 recombinant inbred lines (F7) from a cross between a synthetic and a cultivated wheat. The major gene involved in kernel hardness, ha (hard), known to be on chromosome arm 5DS, was found to be closely linked with the locus Xmta9 corresponding to the gene of puroindoline-a. This locus explained around 63% of the phenotypic variability but there was no evidence that puroindoline-a is the product of Ha (soft). Four additional regions located on chromosomes 2A, 2D, 5B, and 6D were shown to have single-factor effects on hardness, while three others situated on chromosomes 5A, 6D and 7A had interaction effects. Positive alleles were contributed by both parents. A three-marker model explains about 75% of the variation for this trait.

Purification and activity of a wheat 9-kDa lipid transfer protein expressed in Escherichia coli as a fusion with the maltose binding protein.

The cDNA encoding a wheat (Triticum durum) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage. Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion. However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.

Characterization of the Triticum durum Desf. chloroform-methanol-soluble protein family.

The CM (chloroform-methanol-soluble) proteins are low-molecular-weight cysteine-rich proteins that are found in wheat and barley endosperms. A cDNA clone encoding a Triticum durum (T. durum) CM3 protein has been isolated from a mid-maturation seed cDNA library. The T. durum CM3 protein is synthesized as a precursor including a signal peptide (SP) of 25 residues. Northern blot analysis shows that in developing seed the highest level of CM3 protein mRNA is detected at mid-maturation. The hybridization patterns obtained by Southern blot analysis indicated that T. durum CM proteins are encoded by a small multigene family. The similarity between the wheat and barley CM proteins encoded by homologous chromosomes is much higher than that between each of the three members of the T. durum family. All CM proteins contain ten cysteine residues organized in a conserved cysteine motif.

Expression of a cDNA encoding the wheat CM16 protein in Escherichia coli.

The wheat kernel CM16 protein, a subunit of the heterotetrameric insect alpha-amylase inhibitor that has been involved in the technological quality of wheat-products, was produced in Escherichia coli. Cloning of the cDNA part encoding the mature protein in a pET expression plasmid, under the control of a promoter for the bacteriophage T7 RNA polymerase, allows the synthesis of large amounts of the CM16 protein in the bacteria. Upon induction with isopropyl thiogalactopyranoside the recombinant protein accumulates in insoluble inclusion bodies. Solubilization with 6 M urea containing 0.5 mM dithiothreitol, followed by slow elimination of the denaturing agents by step dialysis, results in a significant recovery of the recombinant protein in a soluble, monomeric form. Characterization of the protein was done by automated Edman degradation and total amino acid determination. The recombinant protein in comparison with the one isolated from wheat exhibits a Met extension at the N-terminus that was introduced in the construction for translation initiation. The CM16 protein produced in this manner has the advantage over wheat purified protein of not being contaminated with other proteins from the same family and constitutes adequate material for further analysis of the technological properties of the protein in wheat-derived products.

Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression.

From a mid-maturation seed cDNA library we have isolated cDNA clones encoding two Triticum aestivum puroindolines. Puroindoline-a and puroindoline-b, which are 55% similar, are basic, cystine-rich and tryptophan-rich proteins. Puroindolines are synthesized as preproproteins which include N- and C-terminal propeptides which could be involved in their vacuolar localization. The mature proteins have a molecular mass of 13 kDa and a calculated isoelectric point greater than 10. A notable feature of the primary structure of puroindolines is the presence of a tryptophan-rich domain which also contains basic residues. A similar tryptophan-rich domain was found within an oat seed protein and a mammalian antimicrobial peptide. The ten cysteine residues of puroindolines are organized in a cysteine skeleton which shows similarity to the cysteine skeleton of other wheat seed cystine-rich proteins. Northern blot analysis showed that puroindoline genes are specifically expressed in T. aestivum developing seeds. No puroindoline transcripts as well as no related genes were detected in Triticum durum. The identity of puroindolines to wheat starch-granule associated proteins is discussed as well as the potential role of puroindolines in the plant defence mechanism.

Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X-114 phase partitioning.

A new basic protein has been isolated from wheat endosperm by Triton X-114 phase partitioning. It contains five disulfide bridges and is composed of equal amounts of a polypeptide chain of 115 amino acid residues and of the same chain with a C-terminus dipeptide extension. The most striking sequence feature is the presence of a unique tryptophan-rich domain so that this protein isolated from wheat seeds has been named puroindoline. The similar phase partitioning behavior in Triton X-114 of this basic cystine-rich protein and of purothionins suggests that puroindoline may also be a membranotoxin that might play a role in the defense mechanism of plants against microbial pathogens.