<!-- Generated by XStandard version 3.0.0.0 on 2012-10-23T14:40:19 --><p>A modified wild type maize gene which imparts field level tolerance to glyphosate, the active ingredient in Roundup herbicide, was introduced into the GA21 corn line. <br>A plant-derived coding sequence expressing an optimized chloroplast transit peptide was fused to the modified maize gene. This peptide facilitates the import of the newly translated modified maize EPSPS enzyme into the chloroplast, the site of both the shikimate pathway and glyphosate mode-of-action. <br>The inserted gene codes for a version of the native 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. The wild type version of this maize enzyme is sensitive to glyphosate. The herbicide disrupts this essential pathway, leading to growth suppression or death of the plant. A modified version of the plant enzyme was created with site directed mutagenesis. The altered enzyme is glyphosate tolerant and allows production of aromatic amino acids in the presence of the herbicide. The expression of the novel enzyme in the plant was quantified. <br>The expressed novel enzyme is a single polypeptide with a 99.3% sequence identity to the wild type. <br>The modified maize EPSPS (mEPSPS) was detected by western blot. Western blot data indicated that the EPSPS expression in the transformed plants was an order of magnitude higher than the non-transformed plants. A gene coding for the mEPSPS was expressed in bacteria and the resulting enzyme compared to the plant derived mEPSPS by Western blot. The enzymes expressed from the two sources were shown to be identical. The microbial derived enzyme was used to evaluate the enzyme kinetics, to perform toxicology studies and as a standard in the determination of protein expression from the modified plant. <br>The expression of the mEPSPS gene was evaluated in forage and in grain from plants at 5 field locations. Pooled grain samples from 9-16 ears were analyzed. Expression of EPSPS protein averaged 3.2 µg/ fresh weight and ranged from 1.4-4.9 µg/g fwt . Expression of the wild-type EPSPS in grain was undetectable at all sites. Expression of EPSPS protein in forage (entire plant minus the roots) averaged 118.7 µg/g fwt. and ranged from 46.6-210.4 µg/g fwt.. The wild type EPSPS was detected at 4 out of 5 sites but not at levels high enough to quantify. <br>The mEPSPS protein was not stable against digestion. The enzyme was completely digested within 15 seconds in simulated gastric fluid (pepsin) and within 1 minute in simulated intestinal fluid (pancreatin). Results were shown on western blots. <br>No toxicity from the protein was apparent in animal studies. A database of protein toxin sequences was assembled from public databases and compared to the amino acid sequence of the mEPSPS. Using the &quot;FASTA&quot; computer program, no biologically relevant sequence similarities were detected between mEPSPS and known toxins. A database of 219 allergen and gliadin sequences was constructed from public domain databases. No significant sequence similarities between mEPSPS and known allergens and gliadins were discovered. Data were presented that showed that the protein was not glycosylated, a property common to many allergens. EPSPS is an enzyme that is ubiquitous in nature, and therefore would not be expected to be toxic or allergenic.</p>