<p style="text-align: justify;"><b>The Introduced DNA</b></p>

<p style="text-align: justify;">Southern blot analysis of genomic DNA from GTS 40-3-2 demonstrated that there were two sites of integration, one site containing a functional copy of CP4 EPSPS gene and a second site containing a non-functional segment of the CP4 EPSPS gene. The <i>gus</i> gene was not integrated into the host genome and there were no antibiotic resistance marker genes introduced into GTS 40-3-2.</p>

<p style="text-align: justify;">Southern blots, chromosome walking and DNA sequencing were used to analyze the insertion sites. The largest insert, containing the function CP4 EPSPS gene, contained a deletion in the enhancer region of the E35S promoter and the remainder of the E35S promoter was functional. A single copy of the CP4 EPSPS gene (1365 bp) was integrated and analysis of the 3&#39; terminus revealed a complete <i>nos</i> 3&#39; transcriptional termination element, rather than a portion of <i>nos</i> as previously reported. Also, an additional 250 bp sequence corresponding to a portion of the CP4 EPSPS gene was recently detected adjacent to the 3&#39; end of the <i>nos</i> 3&#39; element, which was not detected in earlier studies (Additional information submitted by Monsanto to regulatory authorities in the spring of 2000).</p>

<p style="text-align: justify;">Analysis of the second insert site showed the presence of a 72 bp sequence corresponding to a portion of the CP4 EPSPS gene. Sequence analysis, western and northern blotting confirmed that neither the additional 250 bp sequence identified in the first insertion site nor the 72 bp fragment inserted at a second site were functional. Pedigree analyses confirmed that these segments of CP4 EPSPS were present in GTS 40-3-2 soybeans evaluated during all safety assessment studies and are present in commercial varieties.</p>

<p style="text-align: justify;"><b>Genetic Stability of the Introduced Trait</b></p>

<p style="text-align: justify;">DNA analyses over six generations demonstrated that the CP4 EPSPS gene was stably inserted. Observations over multiple generations showed the CP4 EPSPS gene was no longer segregating, demonstrating that soybean line GTS 40-3-was homozygous for the herbicide tolerance trait. These multi-generation studies on GTS 40-3-2 also demonstrated that the 72 bp CP4 EPSPS segment co-segregated with the primary insert, indicating that the two sites of integration are close linked and behave as a single locus.</p>

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

<p style="text-align: justify;">Expression of the full-length CP4 EPSPS gene, which encodes a 456 amino acid polypeptide (46 kDa), was confirmed by Western immunoblot analysis and the levels of expression were quantitated using enzyme linked immunosorbent assay (ELISA). The concentrations of CP4 EPSPS averaged 239 &micro;g/g fresh weight tissue in seeds and 495 &micro;g/g f.w.t. in leaves.</p>

<p style="text-align: justify;">Soybean line GTS 40-3-2 was found to express only the full length mRNA and complete CP4 EPSPS protein. The additional DNA segments, the 72 bp CP4 EPSPS segment that comprises the second insert or the 250 bp of the CP4 EPSPS element adjacent to the 3&#39; end of NOS on the functional insert, were both non-functional and did not result in the expression of any mRNA or protein. These results were expected as neither segment contained a promoter or terminator sequence, and only the full-length mRNA and full-length CP4 EPSPS protein were detected.</p>
