<p style="text-align: justify;"><b>The Introduced DNA</b><br />
The incorporated DNA was characterized using a combination of Southern blot analyses, polymerase chain reaction (PCR) amplification of specific sequences, and nucleotide sequencing of the entire inserted fragment, including flanking sequences from the host genome. In addition, bioinformatics analyses were conducted on the inserted sequence, including host genome junction regions, to demonstrate the lack of any unforeseen, or chimeric, open reading frames (ORFs) that could potentially result in the expression of unanticipated novel proteins.<br />
<br />
These analyses demonstrated the introduction of single copy of the transforming DNA at a single insertion site within the host genome. Some anomalies were, however, observed:</p>

<ol>
	<li>In addition to a complete copy of the introduced DNA, the insert also included a 217 bp fragment (50 bp of polylinker sequence + 167 bp of the enhancer region of the rice actin promoter) inversely linked to the 3? terminus of the introduced DNA.</li>
	<li>Nucleotide sequencing indicated that the sequence of the CP4 EPSPS gene within the second (3? proximal) cassette differed by two nucleotides from the inserted sequence. This gave rise to a single amino acid substitution at position 214 of the expressed protein (leucine --&gt; proline; variant protein referred to as: CP4 EPSPS L214P).</li>
	<li>Analyses of specific PCR products from the 3? terminus of the inserted DNA revealed that an additional segment comprising 305 bp of chloroplast DNA had been co-integrated. Bioinformatics analysis indicated that this sequence corresponded to a portion of the maize DNA-directed RNA polymerase alpha-subunit and ribosomal S11 protein. The source of this DNA was believed to be the chloroplast of the transformed cell.</li>
</ol>

<p style="text-align: justify;">&nbsp;</p>

<p style="text-align: justify;"><b>Genetic Stability of the Introduced Trait</b><br />
Southern blot analyses of genomic DNA isolated from plants over six generations of crossing and three generations of self-pollination were used to confirm that the introduced DNA was stably inherited and segregated as a single locus according to Mendelian genetics. Multigenerational stable expression of the glyphosate tolerant trait was demonstrated using bioassay (tolerance to glyphosate application) and enzyme linked immunosorbent assay (ELISA) to measure CP4 EPSPS protein concentration.<br />
<br />
<b>Expressed Material</b><br />
Expression of full-length (approx. 47 kDa) CP4 EPSPS protein was confirmed by Western immunoblot analysis and protein concentrations were estimated using ELISA. Plant samples from line NK603 and the non-transformed parental control line were collected from six non-replicated and two replicated field sites during the 1998 growing season and assayed using a double antibody sandwich (DAS)-ELISA employing monoclonal anti-CP4 EPSPS antibody as the capture antibody and a horseradish peroxidase-conjugated anti-CP4 EPSPS polyclonal antibody as the detection reagent. Mean CP4 EPSPS protein levels (across all sites) were 25.6 ?/g (fwt; range 18.0 ? 31.2) and 10.9 ?/g (fwt; range 6.9 ? 15.6) for forage and grain tissue, respectively.</p>
