<b>1. Nuclear Male Sterility</b><br>The male sterility gene codes for 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 only detected in the tapetum cell layer of anthers, only in early stages of their development. It was not detected in other plant tissues. <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 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, other than with ribonucleases from other bacilli. No resemblance with potential toxins or allergens was observed. <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><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 production of the enzyme only occurs at a specific stage during anther development in the tapetum cell layer of the anther. <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><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 MS1 and RF1 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. <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><b>4. Kanamycin Resistance</b><br>Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell. <br>The kanamycin resistance gene codes for the enzyme neomycin phosphotransferase (NPTII) that prevents kanamycin from binding to ribosomes, thereby rendering the cells resistant. The gene was isolated from Escherichia coli, a facultative anaerobic member of the Enterobacteriaceae usually found in the lower part of the intestinal tract of warm blooded animals. The full nucleotide sequence of the gene was provided. <br>The gene is linked to a weak constitutive promoter. The enzyme was not detected in unprocessed honey or pollen samples. <br>The expressed enzyme was compared to the bacterial protein: molecular weights were similar, indicating that the protein had not been glycosylated nor had it undergone post-transcriptional modifications. <br>The nucleotide sequence showed no significant homology with any known toxins or allergens. 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, other than with other peptides that have a similar function (aminoglycoside 3'-phosphotransferases, kanamycin kinases, streptomycin 3'-kinases). No resemblance with potential toxins or allergens was observed.<br>5. Development Method<br>Brassica napus cultivar Drakkar" was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector. The vector contained the 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 MS1 and RF1. <br><b>6. Stable Integration into the Plant's 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>Deletion studies at the insertion site showed no evidence that a native plant gene was interrupted. The insertion site was found to be located in the B. rapa portion of the amphidiploid B. rapa/B. oleracea genome of B. napus. <br>Segregation was predictable over all generations observed and showed that transformation resulted in integration at one single dominant locus. <br>MS1 and RF1 are several generations removed from the original transformants. Comparisons between the original transgenic plant and these lines show no difference in the presence and expression of the genes nor in the insertion site.