<!-- Generated by XStandard version 3.0.0.0 on 2012-10-23T14:40:19 -->
<p style="text-align: justify;"><b>The Introduced DNA</b><br />
Southern blot analysis of genomic DNA from GA21 demonstrated the integration at a single site on an 18.5 kb DNA restriction fragment containing three complete copies in tandem of the plasmid fragment used in the transformation, plus three incomplete copies. The copy at the 5&#39; end of the insert was shown to include the a partial rice actin promoter and a full length mEPSPS encoding gene with the NOS 3&#39; untranslated region. Following the three complete copies is a full length rice actin promoter element followed by a partial mEPSPS coding sequence. A rice actin promoter element is located at the 3&#39; end of the insert. Transcription of the intact mEPSPS encoding gene(s) was determined by Northern blot analysis of mRNA isolated from GA21.</p>

<p style="text-align: justify;">Because the 3&#39;-terminal region of the inserted DNA was truncated after a rice actin promoter, there was the potential of creating a chimeric open reading frame (ORF) that included plant DNA sequences. Based on DNA sequence data, two putative overlapping open reading frames, ORF-1 (97 amino acids) and ORF-2 (19 amino acids) were identified. However, Northern blot analysis using poly (A+) RNA prepared from leaf tissue of maize line GA21, probed with maize genomic DNA sequence flanking the insert, demonstrated that there was no detectable RNA transcript of this region.</p>

<p style="text-align: justify;"><b>Genetic Stability of the Introduced Trait</b><br />
The stability of the inserted DNA was assessed from Southern blots of genomic DNA obtained from 5 generations of progeny from successive backcrossing of the transgenic line. The introduced trait segregated as a single locus according to Mendelian rules of inheritance.</p>

<p style="text-align: justify;"><b>Expressed Material</b></p>

<p style="text-align: justify;">The deduced amino acid sequence of the mEPSPS (445 amino acids) was 99.3% identical to that of the wild-type enzyme from maize. Western blot analysis of GA21 tissue extracts confirmed the expression of a 47.4 kDa protein, which was the size predicted for full-length mEPSPS protein and also corresponded to the size of this protein when expressed in bacterial cells transformed with a plasmid containing the mEPSPS encoding gene.</p>

<p style="text-align: justify;">The expression of the mEPSPS gene was quantitated by enzyme linked immunosorbent assay (ELISA) of samples of forage and grain from plants at 5 field locations. Pooled grain samples from 9-16 ears were analyzed. The mEPSPS protein was expressed in all tissues of the modified plant (roots, stem, leaves and pollen). Levels of mEPSPS protein in grain averaged 3.2 1.7 ?/ fresh weight (fwt). Expression of the wild-type EPSPS in grain was undetectable at all sites. Expression of mEPSPS protein in forage (entire plant minus the roots) averaged 118.7 ?/g fwt, ranging from 46.6-210.4 ?/g fwt. In forage, the wild type EPSPS was detected at 4 out of 5 sites but not at levels high enough to quantify.</p>

<p style="text-align: justify;">Studies to evaluate the activity of the mEPSPS and potential toxicity were performed on the bacterial expressed form of the enzyme. <i>Escherichia coli</i> were transformed with a plasmid containing the same mEPSPS encoding gene that was introduced into GA21 maize. Bridging data on immunological cross-reactivity and physiochemical properties were provided to demonstrate equivalence between the bacterial and plant expressed forms of the mEPSPS enzyme. Enzyme kinetics and substrate specificity analysis demonstrated that the bacterial expressed mEPSPS had the same substrate specificity as the wild-type maize enzyme.</p>

<p>&nbsp;</p>
