1. Nuclear Male Sterility<br>The male sterility gene codes for the barnase ribonuclease (RNAse). Male sterility results from 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 the other plant tissues tested: leaves, immature kernels, roots, dry and germinating seeds. <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>The gene and its associated regulatory sequences are the same as those of the canola (B. napus) lines MS1 and MS8 that were authorized for unconfined release by AAFC on April 28, 1995 and October 21, 1996 (please see DD95-04 and DD96-17).<br>2. Glufosinate Ammonium Tolerance<br>Phosphinothricin (PPT), the active ingredient of the herbicide 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 MS3 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 (DMPT), but cannot acetylate L-PPT's analog L-glutamic acid, D-PPT, nor any protein amino acid. <br>Expression levels of PAT were estimated through the quantification of RNA transcripts: 0.05 pg/µg of RNA was found in leaves and immature kernels, while none was detected in roots, dry and germinating seeds. A spectrophotometric assay showed no evidence for the presence of PAT in MS3 kernels. <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. <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>3. Development Method<br>The Zea mays inbred line H99 was transformed by electroporation of immature embryos in the presence of a preparation of linearized plasmids. One plasmid carried the genes of interest (male sterility and herbicide tolerance), while the other plasmid, called helper plasmid, carried genes useful at the development stage (e.g. a bacterial marker gene, not expressed in plants).<br>4. Stable Integration into the Plant's Genome<br>The data provided showed that there was incorporation of three copies of the plasmid carrying the genes of interest, and a partial copy of the helper plasmid, all inserted at a single insertion site. None of the coding sequences of the helper plasmid are expressed in MS3. <br>Segregation was predictable over at least six generations observed and showed that transformation resulted in integration at one single dominant locus. <br>Comparisons between the original transgenic plant and plants from four and six generations away showed no difference in the gene integration pattern.