Food safety evaluation of crops produced through genetic engineering--how to reduce unintended effects?

Scientists started applying genetic engineering techniques to improve crops two decades ago; about 70 varieties obtained via genetic engineering have been approved to date. Although genetic engineering offers the most precise and controllable genetic modification of crops in entire history of plant improvement, the site of insertion of a desirable gene cannot be predicted during the application of this technology. As a consequence, unintended effects might occur due to activation or silencing of genes, giving rise to allergic reactions or toxicity. Therefore, extensive chemical, biochemical and nutritional analyses are performed on each new genetically engineered variety. Since the unintended effects may be predictable on the basis of what is known about the insertion place of the transgenic DNA, an important aim of plant biotechnology is to define techniques for the insertion of transgene into the predetermined chromosomal position (gene targeting). Although gene targeting cannot be applied routinely in crop plants, given the recent advances, that goal may be reached in the near future.

Virus-like particle formation and translational start site choice of the plant retrotransposon Tto1.

Ty1/copia group retrotransposon Tto1 from tobacco was put under control of an inducible promoter for expression in Arabidopsis thaliana. The system was used to analyze intermediates of the transposition process. The Tto1 RNA 5' region has a complex structure and contains several AUG codons. We therefore sought to experimentally define the translation initiation site. Constructs starting at various positions within the structural gag region were expressed in planta and functionally characterized. We found that gag proteins starting at the first ATG of the gag-pol ORF (ATG1), but also those starting at the third ATG of the gag-pol ORF (ATG3), can form virus-like particles (VLPs). However, gag protein expressed by the inducible Tto1 element had a size similar to gag starting at ATG1, and mutation of ATG1 in the inducible element abolished reverse transcription. This suggested that translation initiation at ATG1 is essential for the Tto1 life cycle. To support this conjecture, gag protein starting at ATG1, or gag protein shortened amino-terminally by nine amino acids (starting at the second ATG of the gag region, ATG2), was co-expressed with Tto1 carrying mutations at ATG1 and ATG2. Trans-complementation of the defective Tto element by gag starting at ATG1, but not by gag starting at ATG2, defines ATG1 as the functional translation initiation site.

Two classes of 5S rDNA unit arrays of the silver fir, Abies alba Mill.: structure, localization and evolution.

The structure and organization of the 5S ribosomal DNA units of the silver fir, Abies alba Mill., as well as their position in the chromosome complement were investigated. PCR amplification of the gene and nontranscribed spacer region, sequence analysis and Southern hybridization, using a homologous probe, detected DNA sequences of approximately 550 bp and 700 bp. Sequence analysis of the spacers revealed that the difference in length between the sequences occurred in the middle spacer region as a result of the amplification of a 75-bp sequence of the short unit class, which is organized in four 54- to 68-bp tandem repeats in the long spacer unit. The 5S rDNA transcribed region is 120 bp long and shows high sequence similarity with other gymnosperm species. The comparative analysis of 5' and 3' flanking sequences of 5S rRNA genes of silver fir and other gymnosperms indicates that A. alba spacer units have the same rate of evolution and are more closely related to Larix and Pseudotsuga than to Pinus and Picea. Southern hybridization and fluorescence in situ hybridization of metaphase chromosomes of A. alba suggest that the short and long spacer units are organized as separate tandem arrays at two chromosomal loci on chromosomes V and XI.

Unorthodox mRNA start site to extend the highly structured leader of retrotransposon Tto1 mRNA increases transposition rate.

Retroelement RNAs serve as templates for both translation and reverse transcription into extrachromosomal DNA. DNA copies may be inserted into the host genome to multiply element sequences. This transpositional activity of retroelements is usually restricted to specific conditions, particularly to conditions that impose stress on the host organism. In this work, we examined how the mRNA initiation point, and features of primary and secondary structure, of tobacco retrotransposon Tto1 RNA influence its transpositional activity. We found that the most abundant Tto1 RNA is not a substrate for reverse transcription. It is poorly translated, and its 5'-end does not contain a region of redundancy with the most prominent 3'-end. In contrast, expression of an mRNA with the 5'-end extended by 28 nucleotides allows translation and gives rise to transposition events in the heterologous host, Arabidopsis thaliana. In addition, the presence of extended hairpins and of two short open reading frames in the 5'-leader sequence of Tto1 mRNA suggests that translation does not involve ribosome scanning from the mRNA 5'-end to the translation initiation site.