['%', 'ae', 'Tobacco-Specific', 'Nitrosamines', 'Presentatiion', 'at', 'Third', 'Quarter', 'R&D:', 'Planning.', 'Meeting', '“ny,', '21', 'September', '1989', 'Only', 'that', 'aspect', 'of', 'the', 'TSNA', 'Program', 'devoted', 'to', 'biochemical', 'modifications', 'of', 'tobacco', 'was', 'discussed.', 'The', 'proximate', 'goal', 'of', 'that', 'strategy', 'is', 'to', 'prevent', 'biosynthesis', 'of', 'nicotine', 'in', 'the', 'tobacco', 'root', 'through', 'specific', 'genetic', 'transformation', 'of', 'the', 'nuclear', 'genome,', 'eliminating', 'thereby', 'the', 'production', 'of', 'one', 'or', 'more', 'enzymes', 'essential', 'to', 'the', 'alkaloid’s', 'formation.', 'We', 'have', 'chosen', 'the', 'enzyme', 'that', 'methylates', 'putrescine,', 'putrescine', 'N-', 'methyltransferase', '(PMT),', 'as', 'the', 'first', 'target', 'in', 'this', 'effort.', 'The', 'product', 'of', 'that', 'reaction,', 'N-methylputrescine,', 'is', 'subsequently', 'converted', 'to', 'N-', 'methylpyrrolinium', 'ion,', 'which', 'is', 'conjugated', 'with', 'nicotinic', 'acid', 'to', 'form', 'nicotine.', 'In', 'order', 'to', 'interfere', 'with', 'PMT', 'production', 'the', 'gene', 'that', 'encodes', 'its', 'synthesis', 'must', 'be', 'isolated', 'and', 'sequenced,', 'at', 'least', 'in', 'part.', 'Using', 'that', 'information,', '“antisense', 'RNA', 'technology"', 'can', 'be', 'applied', 'to', 'bring', 'about', 'hereditable', 'cessation', 'of', 'PMT', 'formation,', 'with', 'consequent', 'reduction', 'or', 'elimination', 'of', 'nicotine', 'accumulation', 'depending', 'upon', 'the', 'degree', 'of', 'reduction', 'of', 'PMT', 'activity.', 'The', 'gene', 'can', 'be', 'isolated', 'from', 'a', 'cDNA', 'library', 'from', 'tobacco', 'root', 'by', 'constructing', 'specific', 'oligonucleotide', 'probes', 'of', 'unique', 'base', 'sequence,', 'inferred', 'from', 'knowledge', 'of', 'the', 'primary', 'structure', '(amino', 'acid', 'sequence)', 'of', 'the', 'PMT', 'protein.', 'Hence,', 'PMT', 'must', 'first', 'be', 'isolated', 'to', 'homogeneity', 'and', 'sequenced.', 'We', 'anticipate', 'that', 'a', 'nanomole', 'of', 'the', 'protein', '(60', 'ug)', 'will', 'be', 'required', 'for', 'this', 'task.', 'Attempts', 'to', 'isolate', 'homogeneous', 'PMT', 'have', 'been', 'vitiated', 'by', 'the', 'cumulative', 'loss', 'of', 'the', 'enzyme', 'through', 'progressive', 'steps', 'of', 'purification,', 'yielding', 'only', 'about', '2%.', 'of', 'Starting', 'activity', 'through', 'an', 'empirically', 'developed', 'sequence', 'comprising', 'ammonium', 'sulfate', 'precipitation,', 'phenyl-Sepharose', 'chromatography,', 'S-adenosylhomocysteine', 'affinity', 'chromatography,', 'and', 'chromatography', 'using', 'aminoethyl-agarose.', 'Up', 'to', 'one', 'thousand-fold', 'enrichment', 'of', 'PMT', 'activity', 'was', 'achieved', 'by', 'this', 'scheme,', 'but', 'denaturing', 'electrophoresis', '(SDS-PAGE)', 'showed', 'that', 'many', 'contaminating', 'proteins', 'remained.', 'We', 'had', 'intended', 'to', 'identify', 'and', 'isolate', 'PMT', 'in', 'quantities', 'barely', 'sufficient', 'for', 'sequencing', 'by', 'means', 'of', 'two-dimensional', 'electrophoresis.', 'In', 'this', 'quarter,', 'however,', 'H.', 'Nakatani', 'innovated', 'a', 'method', 'for', 'PMT', 'purification', 'that', 'uses', 'putrescine', 'to', 'elute', 'the', 'protein', 'from', 'DEAE-Sepharose', 'in', 'a', 'highly', 'purified', 'state,', 'but', 'with', 'modest', 'yield,', 'Starting', 'with', 'the', 'complex', 'mixture', 'of', 'proteins', 'obtained', 'through', 'chromatography', 'with', 'phenyl-Sepharose.', 'We', 'hypothesize', 'that', 'the', 'mechanism', 'of', 'action', 'involves', 'formation', 'of', 'a', 'stable', 'enzyme-substrate', 'intermediate,', 'resulting', 'in', 'a', 'significant', 'increase', 'of', 'the', 'pI', 'value', 'of', 'the', 'complex', 'compared', 'to', 'that', 'of', 'the', 'free', 'enzyme.', 'The', 'procedure', 'is', 'undergoing', 'further', 'development', 'to', 'Stabilize', 'enzymatic', 'activity', 'so', 'as', 'to', 'enhance', 'yield,', 'but', 'we', 'are', 'much', 'encouraged', 'by', 'its', 'apparent', 'superior', 'power', 'of', 'resolution.', 'In', 'the', 'fourth', 'quarter,', 'up', 'to', '15', 'kg', 'of', 'root', 'material', 'will', 'be', 'processed', 'until', 'an', 'amount', 'of', 'homogeneous', 'PMT', 'protein', 'is', 'available', 'sufficient', 'to', 'carry', 'out', 'sequencing', 'at', 'an', 'external', 'facility', '(to', 'be', 'identified).', 'G9TEedce20e']