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

<p style="text-align: justify;">Southern blot analyses of genomic DNA from each of the male-sterile lines confirmed that all of the lines contained one complete copy of <i>dam</i> and <i>pat</i> at a single insertion site. In addition, each line contained partial and/or rearranged copies of either the <i>pat</i> or <i>dam</i> genes. For example, line 676 contained a second <i>pat</i> gene at a separate insertion site; line 678 contained two additional copies of <i>dam</i> and one additional partial copy of <i>pat</i>; and line 680 contained three additional partial copies of <i>dam</i>. Similar analyses were also used to verify that, as predicted, the antibiotic resistance marker gene (<i>bla</i>) was not incorporated into any of the male-sterile lines.</p>

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

<p style="text-align: justify;">Genetic analysis of maize lines 676, 678, 680 demonstrated that the <i>pat</i> and <i>dam</i> genes segregated as a single genetic locus according to Mendelian rules of inheritance.</p>

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

<p style="text-align: justify;">The production of <i>dam</i>-specific mRNA in each of these lines was tested by Northern blot analysis, and detectable amounts were found only in anther tissue (tetrad release stage) obtained from line 678. The inability to detect <i>dam</i> mRNA in similar tissue from the other two lines was attributed to the fact that the expressed DAM enzyme results in cell ablation and destruction.</p>

<p style="text-align: justify;">The levels of PAT protein expression were determined in grain and leaf tissue from each line and found to range between 204-278 &micro;g/g total protein and 601-717 &micro;g/g total protein in leaf tissue from lines 676 and 678, respectively. Concentrations of PAT were below the limit of detection (20 &micro;g/g total protein) in both grain and leaf tissue from line 680. Grain from lines 676 and 678 contained low levels of PAT protein, ranging from undetectable to 19.2 &micro;g/g total protein and 14.8 &micro;g/g total protein, respectively.</p>
