<b>1. Nuclear Male Sterility</b><br>The male sterility gene encodes the barnase ribonuclease (RNAse). Male sterility is caused by the production of this enzyme at a specific stage during anther development in the tapetum cell layer of the anther. The RNAse affects RNA production, disrupting normal cell functioning and arresting early anther development. <br>The gene is linked to an anther-specific promoter, and the enzyme was detected only in early stages of development of the tapetum cell layer of anthers. It was not detected in other plant tissues. <br>The barnase gene was isolated from Bacillus amyloliquefaciens, a common soil bacterium frequently used as a source for industrial enzymes. The enzyme is therefore naturally occurring in the soil. More generally, ribonucleases are very commonly found in various organisms including bacteria and plants. <br>The full nucleotide sequence of the gene was provided. Barnase is a small single-domain protein, containing no disulfide bonds, metalion cofactors or other non-peptide components. It unfolds completely into an inactive form when heated. When subjected to comparative analyses using a database of polypeptide sequences, the enzyme amino acid sequence did not show significant homology with other proteins present in the database other than bacilli ribonucleases. No resemblance with potential toxins or allergens was observed. <br>The gene and its associated regulatory sequences are the same as those of the line MS1 that was authorized for unconfined release by AAFC on April 28, 1995 (please see DD95-04).<br><b>2. Fertility Restoration</b><br>The fertility restoration gene codes for the barstar enzyme. This enzyme is a ribonuclease inhibitor and specifically inhibits the barnase RNAse. Barnase and its inhibitor barstar form a one-to-one complex, in which the RNAse is inactivated. The barnase-barstar complex is very stable in the absence of a denaturant and the inhibition is very specific. <br>The barstar gene was isolated from Bacillus amyloliquefaciens, a common soil bacterium frequently used as a source for industrial enzymes. The enzyme is therefore naturally occurring in the soil. More generally, ribonuclease inhibitors are very commonly found in various organisms including bacteria and plants. <br>The gene is linked to an anther-specific promoter, and the enzyme is only produced at a specific stage during anther development in the tapetum cell layer of the anther. It was not detected in other plant tissues. <br>The full nucleotide sequence of the gene was provided. Barstar is a small single-domain protein that unfolds completely into an inactive form when heated. When subjected to comparative analyses using the FASTDB algorithm of Intelligenetics with three databases of polypeptide sequences, the enzyme amino acid sequence did not show significant homology with other proteins present in the databases. No resemblance with potential toxins or allergens was observed. <br>The gene and its associated regulatory sequences are identical to those of the line RF1 that was authorized for unconfined release by AAFC on April 28, 1995 (please see DD95-04).<br><b>3. Glufosinate Ammonium Tolerance</b><br>Phosphinothricin (PPT), the active ingredient of glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonia in susceptible plants within hours of application. <br>The phosphinothricin tolerance gene engineered into MS8 and RF3 codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. PAT has extremely high substrate specificity for L-PPT and dimethylphosphinothricin (DMTT), but cannot acetylate L-PPTs analog L-glutamic acid, D-PPT, nor any protein amino acid. Expression levels of PAT varied from 0.04 mg/g (f.w.) of protein in seeds, to 1.80 mg/g (f.w.) of protein in leaves. <br>The gene was isolated from Streptomyces hygroscopicus, an aerobic soil actinomycete. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature. <br>A plant derived coding sequence expressing a chloroplast transit peptide was co-introduced with the gene. This peptide facilitates the import of the newly translated enzyme into chloroplasts. The PAT enzyme was detected in leaves, but not in flower buds or seeds. <br>The nucleotide sequence of the gene was provided. When subjected to comparative analyses using the FASTDB algorithm of Intelligenetics with three databases of polypeptide sequences, the enzyme amino acid sequence did not show significant homology with other proteins present in the databases, except with other phosphinothricin acetyltransferases originating from different organisms. No resemblance with potential toxins or allergens was observed. <br>The gene and its associated regulatory sequences were identical to those of the lines MS1 and RF1 that were authorized for unconfined release by AAFC on April 28, 1995 (please see DD95-04).<br><b>4. Development Method</b><br>Brassica napus cultivar "Drakkar" was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector. The vector contained the transfer DNA (T-DNA) region of an Agrobacterium plasmid from which disease-causing genes were removed and replaced with the genes of interest. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was shown to be the case in MS8 and RF3. <br><b>5. Stable Integration into the Plants' Genomes</b><br>The data provided clearly showed that there was no incorporation of any coding region from outside the T-DNA borders and that only one copy was integrated at a single insertion site. <br>The insertion site was very well characterized and determined to be located in the B. oleracea portion of the amphidiploid B. rapa/B. oleracea genome of B. napus for RF3, and in the B. rapa genome of B. napus for MS8. <br>Segregation was predictable over all generations observed and showed that transformation resulted in integration at one single dominant locus. <br>Comparisons between the original transformants and derived lines several generations away from these transformants show no difference in the presence and expression of the genes nor in the insertion site.