Bacillus thuringiensis var. kurstaki HD-1(B.t.k.) is a common gram-positive soil- borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the ð-endotoxin Cry1A(b) which is active against certain lepidopteran insects, such as ECB. This protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects. B.t.k. based foliar insecticides have been registered for over 30 years in Canada and have a long history of safe use. <br>A synthetic cry1A(b) gene was developed for maximum expression in corn, and introduced into line MON802. The gene codes for a protein similar to the Bacillus thuringiensis var. kurstaki HD-1 insecticidal crystal protein. The protein is insecticidal to lepidopteran larvae after cleavage to a bio-active, trypsin resistant core. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually results in cell lysis. Specific lepidopteran pests of corn sensitive to the protein are ECB and corn earworm. <br>The cry1A(b) gene is linked to a strong constitutive promoter. Average protein expression in micro-grams protein per gram fresh weight tissue evaluated by ELISA in leaf (six locations), grain (six locations) and whole plants (two locations) are as follows:<br>- Leaf: 9.55 microgram Bt per gram fresh weight<br>- Whole plant: 1.35 micrograms Bt per gram fresh weight<br>- Grain: 3.2 micrograms per gram fresh weight <br>The Cry1A(b) protein was shown to degrade readily in the environment. The plant expressed protein had DT50 and DT90 values (time to degrade to 50% and 90 % of the original bioactivity) of 1.6 and 15 days respectively. <br>Protein allergens are normally resistant to digestion unlike the Cry1A(b) protein which was shown to degrade readily in simulated gastric fluid. Unlike many known allergens the insecticidal protein is not glycosylated. A search for amino acid sequence similarity between the Cry1A(b) protein and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies. A search of a similarly constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the Cry1A(b) protein, with the exception of homologies to other B.t. insecticidal proteins. <br>Corn products are an important alternative to wheat flour for individuals afflicted with coeliac disease, an immune mediated food intolerance for which wheat gliadins have been implicated as the causal agent. In light of the importance of corn products to these individuals, a sequence similarity search was conducted and no amino acid sequence homologies between the Cry1A(b) protein and gliadins were detected. <br>The full nucleotide sequence and corresponding amino acid sequence were provided. <br>The native HD-1 protein has a molecular weight of 131 kD. The inserted, plant expressed cryIA(b) gene codes for a full length protein of 1156 amino acids which is processed by proteases into the 600 amino acid tryptic core fragment which is insecticidally active. After activation by trypsin to the insecticidal form, the resulting proteolytic fragments were compared to the bacterial proteins and shown to be of similar molecular weight, immunological reactivity and trypsin resistance. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein. Two genes have been introduced into the corn line MON802, which in combination provide field level tolerance to glyphosate, the active ingredient in Roundup® herbicide. <br>A plant-derived coding sequence expressing a chloroplast transit peptide was co- introduced with each of the Roundup ReadyTM genes. This peptide facilitates the import of the newly translated Roundup ReadyTM enzymes into the chloroplasts, the site of both the shikimate pathway and glyphosate mode-of-action. <br>Both genes associated with their transit peptide coding sequence are linked to the same constitutive promoter. <br>The first gene codes for a bacterial version of a plant enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), involved in the shikimate biochemical pathway for the production of the aromatic amino acids. The plant version of this enzyme, ubiquitous in nature, is sensitive to glyphosate - the herbicide disrupts this essential pathway, leading to growth suppression or death of the plant. However, the bacterial derived version of this enzyme is highly insensitive to glyphosate and fulfils the aromatic amino acid needs of the plant. The product of this gene was quantified and averaged:<br>- Leaf tissue - 26.99 micrograms protein/ gram (fresh weight)<br>- Whole plant - 1.85 micrograms protein/ gram (fresh weight)<br>- Grain - 2.27 micrograms protein/ gram (fresh weight) <br>The second gene codes for glyphosate oxidase, also a bacterial enzyme from a species ubiquitous in nature. Glyphosate oxidase degrades glyphosate, thereby deactivating the herbicidal effect. The average levels of glyphosate oxidase were determined:<br>- Leaf Tissue - 10.18 micrograms protein / gram fresh weight<br>- Whole plant - 1.68 micrograms protein / gram fresh weight<br>- Grain - not detectable <br>The Roundup ReadyTM proteins showed no significant homology with any known toxins or allergens. EPSPS is an enzyme that is ubiquitous in nature, and therefore would not be expected to be toxic or allergenic. For the second novel enzyme, glyphosate oxidase, Monsanto describes experiments with a variety of substrates that show that it has a narrow substrate specificity, and appears not to affect any plant specific pathways. Both proteins are inactivated by heat, and by proteases in simulated mammalian gastric and intestinal fluids. MON802 was co-transformed with two vectors, one carrying a synthetic cry1A(b) gene and the second bearing the two herbicide resistance genes. The two plasmid vectors were introduced by microprojectile bombardment into cultured plant cells. Glyphosate tolerant transformed cells were selected, then cultured in tissue culture medium for plant regeneration. Southern analysis indicated that there are two sites of integration of the introduced DNA. Backcross data is consistant with that of a single locus behaving in a Mendelian fashion, this suggests that the two insert sites are closely linked. <br>The traits have remained stable for six generations as shown by assays for Cry1A(b), and herbicide tolerance.