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

<p style="text-align: justify;">Molecular characterization of MON89034 by Southern blot analyses demonstrated that the DNA inserted into the genome is present at a single locus and contains one functional copy of the <i>cry1A.105</i> and the <i>cry2Ab2</i> expression cassettes. Furthermore, no backbone plasmid DNA or <i>nptII</i> sequences were detected in these analyses. The organization of the elements within the insert in MON89034 was confirmed by DNA sequencing analyses. PCR primers were designed to amplify seven overlapping regions of DNA that span the entire length of the insert (9317 bp), and the amplified DNA fragments were subjected to DNA sequencing analyses. The results confirmed that the sequence of the DNA insert in MON89034 matched the designed, corresponding sequences in PV-ZMIR245 with one exception.</p>

<p style="text-align: justify;">This exception is that the <i>e35S</i> promoter that regulates expression of the <i>cry1A.105</i> gene has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border sequence in MON89034. This molecular rearrangement can be explained by a recombination event which occurred, either prior to or during the process of T-DNA transfer to the plant cell, between the DNA sequences near the 35S promoters in T-DNA I and T-DNA II. Due to this recombination event, the reconstituted <i>e35S</i> promoter in MON89034 no longer has the duplicated enhancer elements compared to the original <i>e35S</i> promoter in PV-ZMIR245. Despite the deletion of the enhance elements, the Cry1A.105 protein expression levels in MON89034 are still sufficiently high under the regulation of the modified <i>e35S</i> promoter to deliver the required efficacy against target insect pests.</p>

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

<p style="text-align: justify;">Analysis of the stability of the integrated DNA demonstrated that the unique Southern blot fingerprint of MON89034 was maintained through seven generations of conventional breeding, thereby confirming the stability of the insert. Additionally, the Southern blo analysis of multiple generations in the MON89034 breeding history indicated that there were no detectable T-DNA II elements other than those which are common to T-DNA I, i.e., 35S promoter, <i>nos</i> 3&acute; end sequence and the Left Border sequence. Furthermore, these generations were shown not to contain any detectable backbone sequence from plasmid PVZMIR245. The stability was further confirmed by the fact that the inheritance of the lepidopteran protection trait in MON89034 follows Mendelian segregation principle.</p>

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

<p style="text-align: justify;">The expression levels of the Cry1A.105 and Cry2Ab2 proteins in different tissues of MON89034 are relatively low. Therefore, it was necessary to produce the proteins in a high-expressing, recombinant microorganism in order to obtain sufficient quantities of the protein for safety studies. Recombinant Cry1A.105 and Cry2Ab2 proteins were produced in <i>Escherichia coli</i>, with the sequences engineered to match the Cry1A.105 and Cry2Ab2 proteins produced in MON89034. The equivalence of the physicochemical characteristics and functional activity between the MON89034-produced and <i>E. coli</i>-produced proteins was confirmed by a panel of analytical techniques, including sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), glycosylation analysis, and assays of biological activity.</p>

<p style="text-align: justify;">The levels of the Cry1A.105 and Cry2Ab2 proteins in various tissues of MON89034 were determined using enzyme-linked immunosorbent assays (ELISA). To produce the tissues for analysis, MON89034 and conventional maize were planted at five field locations during the 2005 growing season. In tissues harvested throughout the growing season, mean Cry1A.105 protein levels across all sites varied from 72-520 ?g/g dwt in leaf, 11-79 ?g/g dwt in root, 5.9 ?g/g dwt in grain and 42-380 ?g/g dwt in whole plant. In tissues harvested throughout the growing season, mean Cry2Ab2 protein levels across all sites varied from 130-180 ?g/g dwt in leaf, 21-58 ?g/g dwt in root, 1.3 ?g/g dwt in grain and 38-130 ?g/g dwt in whole plant. In general, levels of both proteins declined in vegetative tissues over the growing season.</p>
