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

<p style="text-align: justify;">The <i>Pvu</i>II fragment derived from plasmid pAC321 was integrated at a single locus and as one single copy in the soybean genome. The complete CV127 insert sequence is 4758 bp in length.</p>

<p style="text-align: justify;">No DNA sequences other than those derived from the <i>Pvu</i>II transformation fragment were integrated into the CV127 genome. Southern blot analyses clearly indicated that no elements derived from the backbone of the plasmid pAC321 either linked or unlinked to the insert were detected in the genome of CV127. The insert in CV127 comprises a single functional copy of the <i>csr1-2</i> gene as revealed by Southern blot analyses of and by cloning and sequencing of the insert, as well as genomic flanking DNA.</p>

<p style="text-align: justify;">DNA sequence analysis revealed that the <i>csr1-2</i> gene cassette contains three point mutations relative to the <i>Pvu</i>II linear DNA fragment of pAC321. One of the point mutations is a G to A mutation at position 272 in the <i>csr1-2</i> gene, which results in an amino acid change from arginine to lysine. This is a conservative amino acid substitution and has no impact on the herbicide tolerance or enzymatic properties of the AtAHAS protein. The other two mutations are genetically silent.</p>

<p style="text-align: justify;">Sequence analysis revealed that parts of the <i>Pvu</i>II transformation fragment are not contained within the transgene insert in CV127. Deletions of unannotated Arabidopsis genomic DNA occurred both at the 5&rsquo; end and 3&rsquo; end during insertion into the soybean genome.</p>

<p style="text-align: justify;"><b>Stability and Inheritance:</b></p>

<p style="text-align: justify;">The integration of one single insert into the nuclear soybean genome was confirmed by segregation data of the <i>csr1-2</i> gene which showed that tolerance to the herbicide is conferred by a single gene and that this trait is inherited according to classical Mendelian genetics.</p>

<p style="text-align: justify;"><b>Protein Expression Levels:</b></p>

<p style="text-align: justify;">Expression levels of the AHAS protein in different tissues of CV127 soybean plants were determined by enzyme-linked immunosorbent assay using AHAS-specific antibodies. Generally, expression levels of the AtAHASL protein in CV127 soybean are extremely low, especially at later stages of plant growth and development. AHAS enzyme activity is highest in young and growing plant tissues (leaves and whole plants at the V2 growth stage) where the need for branched chain and other amino acids is greatest due to the higher level of de novo protein synthesis and declines as tissues mature. Expression of AHAS decreased with age of the plant and AHAS was only barely detectable or undetectable in roots, older leaves, and grain.</p>
