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

<p style="text-align: justify;">Southern blot analysis of the genomic DNA from COT102 revealed the incorporation of single intact copies of the <i>vip3A(a)</i> and aph4 genes, along with intact copies of their respective regulatory sequences. Results of these analyses also demonstrated that none of the vector backbone sequences, including the streptomycin adenyltransferase (<i>aadA</i>) gene, were incorporated into the genomic DNA.</p>

<p style="text-align: justify;">The synthetic <i>vip3A(a)</i> gene in COT102 encodes a 789-amino acid polypeptide virtually identical to the protein encoded by the gene from <i>B. thruringiensis</i> strain AB88. The <i>vip3A(a)</i> gene was synthesized to optimize its expression in plants and encodes for glutamine at position 284 instead of lysine. The biochemical properties of the modified VIP3A are identical to that of the bacterial protein. Studies conducted with trypsin and lepidopteran gut proteases demonstrated that the 60 kDa toxic core is cleaved from the full-length 89 kDa VIP3A protein, similar to the native bacterial protein.</p>

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

<p style="text-align: justify;">The stability of the inserted insect resistance trait in COT102 was assessed over five generations using Southern blot analysis and segregation analysis. Statistical analysis of the segregation data demonstrated the stability of the trait over the five generations, and a Mendelian inheritance typical of a single dominant trait.</p>

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

<p style="text-align: justify;">The levels of VIP3A and APH4 proteins in COT102 were quantified using enzyme-linked immunosorbant assay (ELISA) methods. Cotton tissue was sampled at six growth stages, from field trials at 3 locations in 2002. Mean whole plant levels of VIP3A declined with advancing growth stage: 12.11&micro;g/g at the 4-leaf stage, 5 - 5.28 &micro;g/g at first bloom and first open boll respectively, and 1.75 &micro;u/g at pre-harvest. VIP3A concentrations were the highest in leaves, averaging 15.31 &micro;g/g at 4-leaf stage, 17.55 &micro;g/g at squaring, 9.73 &micro;g/g at first bloom, 5.29 &micro;g/g at peak bloom, 8.82 &micro;g/g at first open boll, and 4.73 &micro;g/g at pre-harvest. A maximum of 136 &micro;g/g was detected in leaves at squaring. Levels of VIP3A were the lowest in roots (1.39 &micro;g/g at the 4-leaf stage, 1.5 &micro;g/g at peak bloom, and 1.18 &micro;g/g at pre-harvest), as well as in bolls (1.0 &micro;g/g at peak bloom, and below the level of quantification [The APH4 protein was not consistently detected leaves, squares, roots, bolls, seeds and nectar, and when detected, was below the lower limit of quantification for this protein (150 ng/g dry weight). However, the protein was quantified in pollen at 2.3 &micro;g/g.</p>
