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

<p style="text-align: justify;">Information from Southern blots indicated that there are multiple copies of the T-DNA present in C5. Cloning and sequencing of genomic clones, together with PCR analysis, has revealed five inserted fragments all in close proximity and which behave as a single genetic locus. One of the fragments contains a complete insert, consisting of the PPV coat protein gene, the <i>nptII</i> gene and the <i>uidA</i> gene. The other four fragments contain only partial insertions with deletions, duplications and rearrangements observed. One fragment is comprised of an inverted repeat of the PPV coat protein gene, which is thought to be critical in generating the resistance trait.</p>

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

<p style="text-align: justify;">The structure of the inserted DNA has been evaluated in both vegetatively propagated plants and progeny of C5 with no changes noted, indicating that the locus is stable. In addition, expression of the resistance trait has been maintained during seven years of field trials with C5 clones grafted onto rootstocks, as well as <i>in vitro</i> and pot-grown seedlings of C5 crossed with cultivated varieties of plums. The plants remained free of virus despite very high inoculation pressure obtained by grafting PPV infected material onto the transgenic shoots and grafting transgenic shoots onto infected rootstocks.</p>

<p style="text-align: justify;">Using assays for the <i>uidA</i> gene product, together with PCR assays, the progeny of crosses made with C5 showed close to a 1:1 segregation pattern, indicating that the integrated DNA behaved as a single locus and was inherited in a Mendelian fashion.</p>

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

<p style="text-align: justify;">The <i>nptII</i> and <i>uidA</i> genes produce the NPT and GUS enzymes, respectively, in C5. Expression of these proteins was demonstrated not only by resistance to Kanamycin, provided by the NPT protein, but also by assays for the GUS protein using the colorimetric substrate, X-Gluc which is converted into a blue precipitate by the enzyme.</p>

<p style="text-align: justify;">There is no detectable protein produced by the PPV coat protein gene and assays for the messenger RNA from this gene were also negative, prompting the deduction that the resistance mechanism is through post-transcriptional gene silencing (PTGS). This was confirmed by nuclear run-on analysis which confirmed that mRNA from the PPV coat protein was being produced, but was not accumulating in the plant cells. Another characteristic of PTGS is the production of short interfering RNA molecules (siRNA) that are derived from double-stranded RNA degraded by a specific RNAseIII-like enzyme referred to as DICER. It is these siRNA molecules which are thought to provide the specificity of PTGS in silencing certain genes. Detection of siRNA specific to the PPV coat protein genes confirmed this mechanism of transgenic virus resistance in C5.</p>
