<b>1. Glyphosate Tolerance</b> <br>Glyphosate, the active ingredient of Roundup®, acts as a competitive inhibitor of 5-enol-pyruvylshikimate-3-phosphate synthase (EPSP synthase), an essential enzyme of the shikimate biochemical pathway, involved in the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan. This inhibition results in growth suppression and plant death. <br>The inserted glyphosate tolerance gene codes for a bacterial version of this essential enzyme, ubiquitous in plants, fungi and microorganisms. This enzyme is highly insensitive to inhibition by glyphosate and therefore fulfills the aromatic amino acid metabolic needs of the plant. <br>A plant-derived DNA sequence coding for a chloroplast transit peptide was co-introduced with the glyphosate tolerance gene. This peptide facilitates the import of the newly translated EPSP synthase into chloroplasts, where both the shikimate pathway and glyphosate site of action are located. The transit peptide is degraded rapidly and completely by proteases after the import has occurred. <br>The gene, associated with its transit peptide coding sequence, is linked to a strong constitutive promoter. Protein expression was quantified, and averaged 0.239 µg/mg (f.w.) in seeds and 0.495 µg/mg (f.w.) in leaves. <br>The full nucleotide sequence of the inserted gene was provided. The resulting enzyme was shown to be functionally and structurally similar to bacterial enzymes of the same type frequently found in food and feed products. Molecular weights were similar, indicating that the protein had not been glycosylated, nor had it undergone post-transcriptional modifications. <br>Studies showed that the introduced EPSP synthase was not heat stable, and that activity was lost after incubation at 65°C for 15 minutes. It was not stable in the presence of proteases in mammalian digestive tract, and was inactivated during processing required to manufacture soybeans into feed ingredients. <br>EPSP synthase is ubiquitous in nature and is not expected to be toxic or allergenic to non-target organisms. When subjected to comparative analyses with three genetic sequence databases of toxic or allergenic polypeptides, the enzyme amino acid sequence showed no significant homology with any known toxin or allergen. <br><b>2. Development Method </b><br>The soybean commercial variety A5403 (Asgrow Seed Co.) was transformed using particle bombardment, with a modified E. coli plasmid vector containing the genes coding for glyphosate tolerance and for production of ß-glucuronidase, a screenable marker. The original transformant selected showed two insertion sites, one with the screenable marker, and the other with the glyphosate tolerance gene. These two sites subsequently segregated independently in the following sexual generation, and line GTS 40-3-2, upon analysis, was found to contain just one insertion site in which is integrated the glyphosate tolerance gene only. <br><b>3. Stable Integration into the Plant's Genome </b><br>The data provided showed that there was no incorporation of any coding region from outside the fusion gene in the original plasmid vector, consisting of the glyphosate tolerance gene, combined with the transit peptide coding sequence, in addition to their regulatory sequences. Only one copy was integrated at the single insertion site. <br>Following generations demonstrated no further segregation of the fusion gene described above, showing that line GTS 40-3-2 was homozygous for that fusion gene. DNA analyses over six generations showed that the insertion was stable. <br><b>4. Updated Information (Office of Food Biotechnology, Health Canada)</b> <br>On May 19, Monsanto Canada submitted additional information to Health Canada on the detection of additional DNA in their Roundup Ready soybean (glyphosate tolerant soybean GTS 40-3-2). The company had detected the additional DNA as a result of their routine quality control program. Roundup Ready Soybean was previously approved by Health Canada in 1996 for sale as food in the Canadian marketplace. Health Canada regulators conducted a thorough and rigorous review of the supplementary data and found no health or safety concerns for humans. The new data demonstrates that the additional DNA was present in the original seeds approved in 1996 and in all other progeny derived from the original line. The newly detected DNA does not express mRNA or proteins and only the expected full length mRNA and CP4 EPSPS protein is found in Roundup Ready soybean. This soybean has undergone a thorough process of testing, analysis and assessment to ensure that it is wholesome, nutritious and above all, safe to eat. <br><b>5. Updated Information From Monsanto</b> <br>In 1999, Monsanto published detailed methods for the detection of Roundup Ready soybeans in processed samples using immuno-detection (ELISA and western blotting) of CP4 EPSPS, the protein conferring the Roundup Ready trait. While this work was being completed, and in response to emerging needs for detection methods on a global basis, Monsanto also developed DNA-based line identification methods, first using high sensitivity Southern blotting, and now using DNA sequence tags based on insert/genomic DNA junctions. <br>Monsanto previously reported to regulatory agencies (in 1994) and published in the scientific literature (in 1995) the molecular map of the functional insert of the CP4 EPSPS cassette as required by regulatory agencies at that time. Using more sensitive Southern blotting methods, a second 72 bp DNA insert (from CP4 EPSPS) was found. This non-functional second insert co-segregates with the functional insert. As a result of DNA sequencing of the insert/genomic DNA, an additional 250 bp segment of CP4 EPSPS DNA was also identified immediately 3' to the transcriptional termination signal of the primary CP4 EPSPS gene cassette. Sequence analysis, western and northern blotting confirmed that these two segments are non-functional. Pedigree analyses confirmed that these segments of CP4 EPSPS were present in RR soybeans evaluated during all safety assessment studies and are present in commercial varieties. DNA sequence determination of these inserts more precisely describes the molecular details of the inserted DNA in RR soybeans. This information does not impact the completed food and environmental safety assessments.