We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9 kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.

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An assay for nitrate that utilizes the dissimilatory nitrate reductase from Escherichia coli, strain B, and formate to reduce quantitatively nitrate to nitrite is presented. The nitrite formed is measured by the standard diazotization method. Procedures for culture of E. coli, extraction of the enzyme, and assay are given. Since the assay is specific and sensitive (0.1 µg of NOr-N in 10-ml volume) it can be used to determine nitrate in partially clarified plant extracts or similar preparations. The advantages of enzymatic rather than chemical reduction of nitrate prior to determination of nitrite by diazotization in estimating nitrate content of plant and soil extracts are specificity and reproducibility (Lowe and Hamilton, 1967). The assay described by Lowe and Hamilton is most satisfactory, with the exception that problems arise in obtaining adequate amounts of usable soybean nodules that can serve as a source of the dissimilatory nitrate reductase. Because of the space and time it takes (6 to 8 weeks) to produce soybeans with nodules, and the inability to obtain reproducibly nodules with the dissimilatory enzyme, or to culture in vitro Rhizobium japonicum, it seemed desirable to attempt to use bacteria as a source of the enzyme. Previous work of Taniguchi and Itazaki (1959) and Fewson and Nicholas (1961a,b) suggested E. coli, Pseudomonas aeruginosa, or Micrococcus denitrificans as suitable sources of dissimilatory nitrate reductase. Because stocks of E. coli strain B were readily available and preliminary work showed that this bacteria was a good source of the enzyme, it was selected for study. The work of Ruiz-Herrera and DeMoss (1969) indicates that other strains of E. coli would also be sources of the enzyme.The objective of the work was to show that E. coli strain B could be readily cultured to provide a convenient source for the dissimilatory nitrate reductase used in quantitative determination of nitrate, as previously described by Lowe and Hamilton (1967). MATERIALS AND METHODSMany of the procedures used were adapted from the manuscript of Taniguchi and Itazaki (1960).Stock Cultures. Escherichia coli strain B is stored on nutrient agar slants. The nutrient agar contains (in g/1.): bactopeptone, 5.0; beef extract, 3.0; and bactoagar, 15.0 (obtainable from Difco Labs, Detroit, Mich.). The final pH of this mixture is approximately 7.0. After solubilization of the components by warming and gentle agitation, the material is transferred to test tubes and sterilized for 20 min at 120°C and cooled. The organism is streaked on the solidified slants and allowed to grow for 3 to 4 hr at 37°C prior to storage at 2°C. Growth Medium. The growth medium contains (in g/1.): bactopeptone, 10.0; beef extract, 1.0; casamino acids (vitamin free), 3.0; yeast extract, 3.0 (all obtainable from Difco Labs); K2HP04, 2.0; KNOa, 1.0; and glucose, 20.0.