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

<p style="text-align: justify;">Southern blot analysis was used to determine the insert and copy number of the <i>amy797E</i> and <i>pmi</i> genes and to confirm the absence of DNA sequence from outside the T-DNA borders of the transformation vector. Genomic DNA used for Southern blot analysis was isolated from pooled leaf tissue from ten plants representing the backcross four (BC4) generation of Event 3272 and 10 plants representing negative segregants. All plants used for genomic DNA isolation were confirmed for the presence and absence of <i>amy797E</i> and <i>pmi</i> genes using PCR. The Southern analyses demonstrated that event 3272 contains a single copy of the <i>amy797E</i> and <i>pmi</i> genes inserted as a single piece of T-DNA. The Southern analyses also confirmed the absence of DNA from the plasmid backbone.</p>

<p style="text-align: justify;">To further characterise the integrity of the inserted T-DNA, the nucleotide sequence of the entire T-DNA insert was determined and compared to the DNA sequence of the transforming plasmid (pNOV7013). The final consensus sequence was determined by combining the sequence data from six individual clones to generate one consensus sequence for Event 3272. In total, 6100 bp of T-DNA was inserted into the corn genome. The consensus sequence data for Event 3272 T-DNA demonstrated the overall integrity of the insert and confirmed that the functional elements within the insert had been maintained. Sequence analysis revealed that some truncation occurred at the right and left borders of the T-DNA insert, most likely during the transformation process. The right border portion of the T-DNA insert was truncated by 23 bp and the left border portion by 7 bp. These deletions do not affect the normal function of the <i>amy797E</i> and <i>pmi</i> gene expression cassettes.</p>

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

<p style="text-align: justify;">Southern analyses were done to demonstrate that the insert in Event 3272 is stable over a number of generations. Genomic DNA for Southern analysis was isolated from pooled leaf tissue of ten plants each from three generations &ndash; BC1, BC2 and BC3 &ndash; and probed with an <i>amy797E</i>-specific probe. Negative segregants from the BC3 generation were used as controls. The hybridization pattern obtained with the <i>amy797E</i>-specific probe was found to be identical over the three generations examined.</p>

<p style="text-align: justify;">The inheritance pattern of the T-DNA insert in Event 3272 was confirmed on individual plants from the BC1, BC2, BC3 and BC4 generations, analysed for the presence of the <i>amy797E</i> gene by TaqMan&reg; PCR. The expected Mendelian inheritance ratio of positive and negative plants for a hemizygous trait in these populations is 1:1. The goodness of-fit of the observed genotypic ratio to the expected genotypic ratio was tested using Chi Square analysis, with Yates correction factor, and the hypothesis that the genetic trait is behaving in a Mendelian fashion was accepted for all generations.</p>

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

<p style="text-align: justify;">The alpha-amylase activity of the AMY797E protein was compared to that of generic commercial <i>Bacillus</i> alpha-amylase using studies such as dose-equivalents, HPLC analysis of the size distribution of starch hydrolysis products, HPLC analysis of residual sugars and organic compounds, and ethanol production yield. These studies demonstrated that the AMY797E alpha-amylase is functionally equivalent in starch hydrolysis to other commercial alpha-amylases.</p>

<p style="text-align: justify;">AMY797E protein was purified from grain of Event 3272 using a high temperature extraction process. Following separation of the extracted protein by SDS-PAGE, coomassie blue staining revealed a major band of an approximate molecular mass of 50.2 kDa (consistent with the predicted molecular mass of the mature AMY797E) and a minor low molecular mass band of The PMI protein produced in Event 3272-derived plants is encoded by the native <i>pmi</i> gene from <i>E. coli</i> (strain K-12). The protein consists of 391 amino acids and has a molecular weight of ca. 45 kDa. Plant cells expressing the <i>pmi</i> gene are capable of survival and growth in the presence of mannose as the only or primary carbon source. Under the same conditions, plant cells lacking PMI accumulate mannose-6-phosphate fail to grow. The PMI protein was extracted from leaf tissue from Event 3272 and its size, immunoreactivity, and specific enzymatic activity were compared to <i>E. coli</i> PMI. The PMI protein expressed in <i>E. coli</i> is identical in amino acid sequence to that encoded by the vector used to transform corn producing line 3272, with the exception of 16 amino acids added to the N-terminus as a result of the <i>E. coli</i> expression vector.</p>

<p style="text-align: justify;">Samples of leaf extracts from Event 3272 and <i>E. coli</i> produced PMI were subjected to SDS-PAGE, followed by immunoblotting. A single immunoreactive band corresponding to a molecular mass of 42.8 kDa was detected in four separate leaf extracts. The <i>E. coli</i>-expressed PMI protein showed one major band with a slightly lower mobility compared to the plant expressed PMI, consistent with a slightly higher predicted molecular mass of 44.4 kDa (resulting from the additional 16 amino acids at the N-terminus). The mean enzymatic activity of PMI in leaf extracts ranged from 81.6 &ndash; 112.6 U/mg PMI, corresponding to an overall mean enzymatic activity of 96.9 &plusmn; 14.2 U/mg PMI. <i>E. coli</i> produced PMI had a slightly lower mean enzymatic activity of 52.9 &plusmn; 0.0 U/mg PMI. The two enzyme activities are comparable.</p>

<p style="text-align: justify;">Mean AMY797E concentrations measured in grain from the maturity stage across four backcross generations were ca. 1044 - 1264 ?g/g fresh weight (fw; 1147 - 1389 ?g/g dry weight; dw). AMY797E levels were essentially undetectable in leaves and pollen although it was detected at low levels in the roots of some whorl stage plants (&lt; 0.1 ?g/g fw).</p>

<p style="text-align: justify;">Mean PMI concentrations in leaves from the anthesis stage across four backcross generations were ca. 6.6 - 9.3 ?g/g fw (25.6 - 35.7 ?g/g dw). PMI was detected at low levels in most of the plant tissues analyzed, except for some senescence-stage samples. Mean PMI levels measured in kernels at all developmental stages ranged from ca.</p>
