Food safety evaluation for R-proteins introduced by biotechnology: A case study of VNT1 in late blight protected potatoes.

Resistance genes (R-genes) from wild potato species confer protection against disease and can be introduced into cultivated potato varieties using breeding or biotechnology. The R-gene, Rpi-vnt1, which encodes the VNT1 protein, protects against late blight, caused by Phytophthora infestans. Heterologous expression and purification of active VNT1 in quantities sufficient for regulatory biosafety studies was problematic, making it impractical to generate hazard characterization data. As a case study for R-proteins, a weight-of-evidence, tiered approach was used to evaluate the safety of VNT1. The hazard potential of VNT1 was identified from relevant safety information including history of safe use, bioinformatics, mode of action, expression levels, and dietary intake. From the assessment it was concluded that Tier II hazard characterization was not needed. R-proteins homologous to VNT1 and identified in edible crops, have a history of safe consumption. VNT1 does not share sequence identity with known allergens. Expression levels of R-proteins are generally low, and VNT1 was not detected in potato varieties expressing the Rpi-vnt1 gene. With minimal hazard and negligible exposure, the risks associated with consumption of R-proteins in late blight protected potatoes are exceedingly low. R-proteins introduced into potatoes to confer late blight protection are safe for consumption.

Development of an agricultural biotechnology crop product: testing from discovery to commercialization.

"Genetically modified" (GM) or "biotech" crops have been the most rapidly adopted agricultural technology in recent years. The development of a GM crop encompasses trait identification, gene isolation, plant cell transformation, plant regeneration, efficacy evaluation, commercial event identification, safety evaluation, and finally commercial authorization. This is a lengthy, complex, and resource-intensive process. Crops produced through biotechnology are the most highly studied food or food component consumed. Before commercialization, these products are shown to be as safe as conventional crops with respect to feed, food, and the environment. This paper describes this global process and the various analytical tests that must accompany the product during the course of development, throughout its market life, and beyond.

Assessment of possible allergenicity of hypothetical ORFs in common food crops using current bioinformatic guidelines and its implications for the safety assessment of GM crops.

Before a genetically modified (GM) crop can be commercialized it must pass through a rigorous regulatory process to verify that it is safe for human and animal consumption, and to the environment. One particular area of focus is the potential introduction of a known or cross-reactive allergen not previously present within the crop. The assessment of possible allergenicity uses the guidelines outlined by the Food and Agriculture Organization (FAO) and World Health Organization's (WHO) Codex Alimentarius Commission (Codex) to evaluate all newly expressed proteins. Some regulatory authorities have broadened the scope of the assessment to include all DNA reading frames between stop codons across the insert and spanning the insert/genomic DNA junctions. To investigate the utility of this bioinformatic assessment, all naturally occurring stop-to-stop frames in the non-transgenic genomes of maize, rice, and soybean, as well as the human genome, were compared against the AllergenOnline (www.allergenonline.org) database using the Codex criteria. We discovered thousands of frames that exceeded the Codex defined threshold for potential cross-reactivity suggesting that evaluating hypothetical ORFs (stop-to-stop frames) has questionable value for making decisions on the safety of GM crops.

Transformation of rice with long DNA-segments consisting of random genomic DNA or centromere-specific DNA.

Rice was transformed with either long DNA-segments of random genomic DNA from rice, or centromere-specific DNA sequences from either maize or rice. Despite the repetitive nature of the transgenic DNA sequences, the centromere-specific sequences were inserted largely intact and behave as simple Mendelian units. Between 4 and 5% of bombarded callus clusters were transformed when bombarded with just pCAMBIA 1305.2. Frequency of recovery dropped to 2-3% when BACs with random genomic inserts were co-bombarded with pCAMBIA, and fell to less than 1% when BACs with centromeric DNA inserts and pCAMBIA were co-bombarded. A similar effect was noted on regeneration frequency. Differences in transformation ability, regeneration and behavior of plants transgenic for BACs with random genomic DNA inserts, as compared to those with centromeric DNA inserts, suggests functional differences between these two types of DNA.

Phosphoserines on maize CENTROMERIC HISTONE H3 and histone H3 demarcate the centromere and pericentromere during chromosome segregation.

We have identified and characterized a 17- to 18-kD Ser50-phosphorylated form of maize (Zea mays) CENTROMERIC HISTONE H3 (phCENH3-Ser50). Immunostaining in both mitosis and meiosis indicates that CENH3-Ser50 phosphorylation begins in prophase/diplotene, increases to a maximum at prometaphase-metaphase, and drops during anaphase. Dephosphorylation is precipitous (approximately sixfold) at the metaphase-anaphase transition, suggesting a role in the spindle checkpoint. Although phCENH3-Ser50 lies within a region that lacks homology to any other known histone, its closest counterpart is the phospho-Ser28 residue of histone H3 (phH3-Ser28). CENH3-Ser50 and H3-Ser28 are phosphorylated with nearly identical kinetics, but the former is restricted to centromeres and the latter to pericentromeres. Opposing centromeres separate in prometaphase, whereas the phH3-Ser28-marked pericentromeres remain attached and coalesce into a well-defined tether that binds the centromeres together. We propose that a centromere-initiated wave of histone phosphorylation is an early step in defining the two major structural domains required for chromosome segregation: centromere (alignment, motility) and pericentromere (cohesion).

Centromere-encoded RNAs are integral components of the maize kinetochore.

RNA is involved in a variety of chromatin modification events, ranging from large-scale structural rearrangements to subtle local affects. Here, we extend the evidence for RNA-chromatin interactions to the centromere core. The data indicate that maize centromeric retrotransposons (CRMs) and satellite repeats (CentC) are not only transcribed, but that nearly half of the CRM and CentC RNA is tightly bound to centromeric histone H3 (CENH3), a key inner kinetochore protein. RNAs from another tandem repeat (180-bp knob sequence) or an abundant euchromatic retroelement (Opie) are undetectable within the same anti-CENH3 immune complexes. Both sense and antisense strands of CRM and CentC, but not small interfering RNAs homologous to either repeat, were found to coimmunoprecipitate with CENH3. The bulk of the immunoprecipitated RNA ranged in size from 40 to 200 nt. These data provide evidence for a pool of protected, single-stranded centromeric RNA within the centromere/kinetochore complex.