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

<p style="text-align: justify;">Southern blot hybridization analysis was used to confirm the insertion of a single copy of the T-DNA region from plasmid pBI121fs and to confirm the integrity of the <i>prsv-cp</i> gene. Southern analysis also confirmed the lack of any plasmid backbone sequences within the X17-2 genome. The 5&#39; and 3&#39; genomic flanking sequences were cloned and sequenced, as was the T-DNA insert (less approx. 336 bp). The expected and observed nucleotide sequence of the T-DNA insert (from R5 generation) was 99.7% identical, with the differences occurring where the <i>prsv-cp</i> gene was ligated into the T-DNA. These differences appeared to have no consequence, however, the thymidine base originally incorporated into the leader sequence for the CP gene to create a frame-shift mutation was missing. Consequently, the <i>prsv-cp</i> gene is translatable and not non-translatable as intended, and western analyses indicated that the gene was expressed.</p>

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

<p style="text-align: justify;">The insert in line X17-2 has persisted as a single copy through six generations of progeny following the initial transformation and segregation analysis using kanamycin treatment of seedlings indicated that the inserted T-DNA segregated as a single locus in a Mendelian fashion.</p>

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

<p style="text-align: justify;">Expression of the PRSV CP in papaya line X17-2 was confirmed by enzyme linked immunosorbent assay (ELISA) using polyclonal anti-PRSV serum, and by western immunoblotting. The 35 kDa size of the expressed PRSV CP in line X17-2 was the same as predicted from the nucleotide sequence of PRSV RNA, although the natural CP is one of at least seven proteins that are post-translationally cleaved from the expressed poly-protein of PRSV in infected plants.</p>
