<p style="text-align: justify;">Phosphinothricin (PPT), the active ingredient of glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonium in susceptible plants within hours of application.</p>

<p style="text-align: justify;">The phosphinothricin tolerance gene engineered into AgrEvo&#39;s corn lines T14 and T25 codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. PAT has extremely high substrate specificity for L-PPT and dimethylphosphinothricin (DMPT), and experimental data clearly showed that neither L-PPT&#39;s analog L-glutamic acid, D-PPT, nor any other amino acid can be acetylated by the PAT enzyme.<br />
The synthetic PAT gene was derived from a PAT gene isolated from Streptomyces viridochromogenes, an aerobic soil bacteria. The PAT enzyme occurs naturally in the soil and acetyltransferases are ubiquitous in nature.</p>

<p style="text-align: justify;">The gene is linked to a constitutive promoter, and protein expression was detected in plant tissues, grain and silage. The PAT enzyme was not detected in protein extracts from the pollen.<br />
The PAT enzyme from T14 and T25 was compared to PAT expressed from the transformed canola line HCN-92 (see Decision Document DD95-01, &quot;Determination of Environmental Safety of AgrEvo Canada Inc&#39;s Glufosinate Ammonium-Tolerant Canola.&quot; The molecular weight and immunoreactivity of the enzyme from the plant derived sources was similar. When the plant derived PAT enzyme was compared to PAT enzyme from a bacterial expression system, the molecular weights were similar, indicating that the protein was unlikely to have been glycosylated or undergone other post translational modifications. Studies showed that the enzyme was inactivated within one minute when subjected to typical mammalian stomach conditions.</p>

<p style="text-align: justify;">Expression of the pat gene in T14 and T25 derived plants results in the production of the PAT protein in corn tissues at low levels (&lt; 0.003% of total crude protein). The expressed PAT protein mediates the rapid metabolism of glufosinate ammonium herbicide.</p>

<p style="text-align: justify;">Corn lines derived from T14 and T25 do not produce mRNA from the disrupted amp-r genes, nor do they produce beta-lactamase, the enzyme encoded by the bla gene. This demonstrates that the intoduced bla genes are indeed non-functional in T14 and T25 derived corn plants.</p>

<p style="text-align: justify;">The gene nucleotide sequence was provided. The nucleotide sequence showed no significant homology with any known toxins or allergens. A tissue cultured cell line of corn was used as a source of protoplasts. DNA containing the PAT gene was combined with the protoplasts. Cell colonies derived from this protoplast/DNA mixture were then regenerated and transformants were selected on a medium containing glufosinate ammonium.</p>

<p style="text-align: justify;">Two primary transformants, T14 and T25, were then backcrossed with both commercial public inbred lines and proprietary inbred lines of the yellow dent corn type. The data provided demonstrated the integration of one and three copies of the genetic sequence in transformation events T25 and T14, respectively.</p>

<p style="text-align: justify;">Liberty LinkTM corn lines are several generations removed from the two original transformation events.</p>
