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

<p style="text-align: justify;">Southern blot analysis of the genomic DNA of TC-6275 demonstrated the integration of a single copy of the T-DNA of PHP12537. The analysis confirmed the intact insertion of the <i>bar</i> gene and its promoter and terminator sequences, as well as the <i>cry</i>1F gene and its terminator sequences; however, the analysis revealed that the promoter region of the <i>cry</i>1F gene transcription unit was truncated at the 5&rsquo; end. Cry1F was constitutively expressed despite the incomplete insertion of the promoter (<i>v.</i> <b>Expressed Material</b>). The sequences outside of the T-DNA border, including the spectinomycin resistance (<i>spc</i>) and tetracycline (<i>tet</i>) resistance genes, were not integrated into the genome of TC-6275.</p>

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

<p style="text-align: justify;">Southern blot analysis was also used to verify the stability of the T-DNA insert. The analysis demonstrated the stability of the insert containing the <i>cry</i>1F and <i>bar</i> genes, across three generations of TC-6275. Segregation analysis also demonstrated a stable insertion consistent with a Mendelian inheritance pattern for a dominant trait.</p>

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

<p style="text-align: justify;">The levels of expression of the Cry1F protein were quantified in various tissues of TC-6275 maize using enzyme-linked immunosorbent assay (ELISA) technique. Samples were taken from 6 field trials conducted at 3 locations in Chile from 2001 to 2002. Cry1F was found to be expressed throughout the plant and was quantified in samples collected at five growth stages (V9: vegetative, 9 leaves developed; R1: silking; R4: dough stage; physiological maturity; senescence). Mean levels of Cry1F, expressed on a dry weight basis, were: 16.7 ng/mg in leaves at V9; 6.22 and 7.17 ng/mg in the whole plant at V9 and R1, respectively; 11.0 ng/mg in stalks at R1; 6.26 ng/mg in forage at R4; and 2.47 ng/mg in the whole plant at senescence. Mean levels of Cry1F in pollen were 3.67 ng/mg, and 1.14 ng/mg in grain at physiological maturity.</p>

<p style="text-align: justify;">Western blots of SDS-PAGE (sodium dodecylsulfate polyacrylamide) gel electrophoresis separated Cry1F protein expressed in TC-6275 revealed a 65kD moiety corresponding to the trypsin-resistant core of the Cry1F delta-endotoxin. The amino acid sequences 1 to 605 of the maize-expressed Cry1F were found to be identical to those of the native delta-endotoxin, except for a single amino acid substitution. These sequences represent the portion of the full length (1174 amino acids) Cry1F protein that remains in the insect gut after digestion by proteases.</p>

<p style="text-align: justify;">The Cry1F protein used in toxicity studies, such as those conducted on non-target organisms, was produced microbially using <i>Pseudomonas fluorescens</i> into which the modified <i>cry</i>1F gene had been inserted. Cry1F expressed in <i>P. fluorescens</i> was demonstrated to be biochemically equivalent to the Cry1F expressed in TC-6275.</p>

<p style="text-align: justify;">The PAT protein was detected and characterized in the same samples of TC-6275 that were tested for the expression of Cry1F. The mean levels of PAT in leaves were: 323 ng/mg at V9, 674 ng/mg at R1, 682 ng/mg at R4, and 0 ng/mg at senescence; in roots: 112 ng/mg at V9, 253 ng/mg at R1, 223 ng/mg at R4, and 41 ng/mg at senescence; in stalks, 282 ng/mg at R1; and in whole plants: 5 ng/mg at V9, 72 ng/mg at R1 and 18 ng/mg at senescence. Levels in forage harvested at R4 were 7 ng/mg. Grain, harvested at physiological maturity, contained 23 ng/mg. The levels of PAT in pollen were below the level of quantification.</p>
