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

<p style="text-align: justify;">Southern blot analyses of genomic DNA from GTSB77 confirmed the existence of a single copy of the T-DNA region containing complete functional copies of the CP4 EPSPS and GUS encoding genes, and a partial non-functional copy of the GOX encoding gene. This partial copy of the GOX encoding sequences was due to a 3&rsquo; truncation of the T-DNA insert, which also resulted in no integration of NPTII encoding sequences into the host genome.</p>

<p style="text-align: justify;">The truncated copy of the <i>goxv247</i> gene was fused with genomic sugar beet DNA sequences, forming a chimeric sequence consisting of 69% of the entire GOX coding sequence plus 130 bp of host sequence terminated with an identified translational stop codon. While mRNA transcripts from this chimeric sequence were identified in GTSB77, a protein fusion product was never detected, and plant tissue extracts contained no detectable GOX enzyme activity.</p>

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

<p style="text-align: justify;">Phenotypic segregation analysis of the glyphosate-tolerant trait and GUS activity were conducted over multiple generations and confirmed an inheritance pattern consistent with the stable transfer of a single dominant locus for these genes. These phenotypes were also shown to be stable over multiple generations in successive cropping.</p>

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

<p style="text-align: justify;">Sugar beet line GTSB77 expressed two novel proteins CP4 EPSPS and GUS. The CP4-EPSPS protein is 47.6 kDa in size and consists of a single polypeptide of 455 amino acids. The GUS protein was experimentally determined to have a molecular weight of 68.2kD.</p>

<p style="text-align: justify;">These proteins were detected in very low levels in root tissue of sugar beet GTSB77 (58 ppm and 0.5 ppm for CP4 EPSPS and GUS respectively). They were also present at higher levels in leaf and stem tissue (237 ppm and 3 ppm for CP4-EPSPS and GUS respectively).</p>

<p style="text-align: justify;">As previously mentioned, the chimeric sequence formed between the truncated <i>goxv247</i> gene and 130 bp of plant genome sequence gave rise to the possibility that a novel protein could be expressed. The expression of such a GOX-like protein was investigated by Western immunoblot analysis using antibodies raised to a 46 kDa protein (designated 34550) expressed in <i>E. coli</i> containing a cloned copy of the chimeric sequence. This protein was comprised of 89 amino acids from the CTP1 transit peptide, the N-terminal 299 amino acids from GOX, and 43 amino acids encoded by sugar beet genomic DNA. Western blot analysis, on plant tissue extracts from several locations, determined that protein 34550 was not present at a detection limit of 180 ppb in plant tissue.</p>

<p style="text-align: justify;">An ancillary study was undertaken to determine the presence of GOX activity by monitoring the production of glyoxylate, the breakdown product of glyphosate. No detectable GOX-like activity could be demonstrated for protein 34550 purified from <i>E. coli</i>. A similar study confirmed that sugar beet GTSB77 did not display GOX activity.</p>
