Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-06-22
2001-08-07
Nelson, Amy (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C536S023600, C435S252300, C435S320100, C435S419000
Reexamination Certificate
active
06271445
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for screening herbicidal compounds which inhibit the enzymatic activity of 5′-phosphoribosyl-5-aminoimidazole (AIR) synthetase, an enzyme involved in de novo purine biosynthesis. The invention also relates to the use of thereby identified herbicidal chemicals to control the growth of undesired vegetation. The invention may also be applied to the development of herbicide tolerance in plants, plant tissues, plant seeds, and plant cells.
BACKGROUND OF THE INVENTION
I. AIR Synthetase
The AIR synthetase is an enzymatic step in the de novo purine biosynthesis pathway, which leads to the synthesis of the purine nucleotides IMP, AMP and GMP. De novo purine biosynthesis plays a central role in the nitrogen assimilation pathway and is conserved among bacteria, yeast, Drosophila and mammals (Schnorr et al. (1994) The Plant Journal, 6: 113-121). The AIR synthetase enzymatic activity corresponds to the fifth step in the pathway and catalyzes the conversion of 5′-phosphoribosyl-N-formylglycinamidine (FGAM) to 5′-phosphoribosyl-5-aminoimidazole (AIR). In
E. coli
, this step is carried out by a protein encoded by the purM gene. Recently, an Arabidopsis c-DNA encoding an enzyme having AIR synthetase activity has been cloned and its sequence has been determined (Senecoff and Meagher (1993) Plant Physiol. 102: 387-399; Schnorr et al. (1994) The Plant Journal, 6: 113-121).
II. Herbicide Discovery
The use of herbicides to control undesirable vegetation such as weeds in crop fields has become almost a universal practice. The herbicide market exceeds 15 billion dollars annually. Despite this extensive use, weed control remains a significant and costly problem for farmers.
Effective use of herbicides requires sound management. For instance, the time and method of application and stage of weed plant development are critical to getting good weed control with herbicides. Since various weed species are resistant to herbicides, the production of effective new herbicides becomes increasingly important. Novel herbicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Metabolic enzymes found to be essential to plant growth and development can be recombinantly produced though standard molecular biological techniques and utilized as herbicide targets in screens for novel inhibitors of the enzymes' activity. The novel inhibitors discovered through such screens may then be used as herbicides to control undesirable vegetation.
III. Herbicide Tolerant Plants
Herbicides that exhibit greater potency, broader weed spectrum, and more rapid degradation in soil can also, unfortunately, have greater crop phytotoxicity. One solution applied to this problem has been to develop crops that are resistant or tolerant to herbicides. Crop hybrids or varieties tolerant to the herbicides allow for the use of the herbicides to kill weeds without attendant risk of damage to the crop. Development of tolerance can allow application of a herbicide to a crop where its use was previously precluded or limited (e.g. to pre-emergence use) due to sensitivity of the crop to the herbicide. For example, U.S. Pat. No. 4,761,373 to Anderson et al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides. The resistance is conferred by an altered acetohydroxyacid synthase (AHAS) enzyme. U.S. Pat. No. 4,975,374 to Goodman et al. relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS, e.g. phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,013,659 to Bedbrook et al. is directed to plants expressing a mutant acetolactate synthase that renders the plants resistant to inhibition by sulfonylurea herbicides. U.S. Pat. No. 5,162,602 to Somers et al. discloses plants tolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The tolerance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).
SUMMARY OF THE INVENTION
One object of the present invention is to provide methods for identifying new or improved herbicides. Another object of the invention is to provide methods for using such new or improved herbicides to suppress the growth of plants such as weeds. Still another object of the invention is to provide improved crop plants that are tolerant to such new or improved herbicides.
Using an antisense validation system which allows for the inactivation of expression of an endogenous gene, the inventors of the present invention have demonstrated that the 5′-phosphoribosyl-5-aminoimidazole (AIR) synthetase activity is essential in plants. This implies that chemicals which inhibit AIR synthetase in plants are likely to have detrimental effects on plants and are potentially good herbicide candidates. The present invention therefore provides methods of using a purified AIR synthetase to identify inhibitors thereof, which can then be used as herbicides to suppress the growth of undesirable vegetation, e.g. in fields where crops are grown, particularly agronomically important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, and soybeans.
The present invention discloses for the first time the correct nucleotide sequence of the Arabidopsis AIR synthetase gene. The nucleotide sequence encoding the pre-protein is set forth in SEQ ID NO:1 and the nucleotide sequence encoding the putative mature protein is set forth in SEQ ID NO:3. The correct amino acid sequence of the Arabidopsis AIR synthetase pre-protein is set forth in SEQ ID NO:2 and of the correct amino acid sequence of the putative mature Arabidopsis AIR synthetase is set forth in SEQ ID NO:4. The present invention also encompasses plant enzymes having AIR synthetase activity and whose amino acid sequence are substantially similar to the amino acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:4.
In a preferred embodiment, the present invention describes a method for identifying a chemical to be tested for the ability to inhibit plant growth or viability, comprising the steps of: (a) combining an enzyme having AIR synthetase activity in a first reaction mixture with a substrate of AIR synthetase under conditions in which the enzyme is capable of catalyzing the synthesis of AIR; (b) combining the chemical to be tested and the enzyme in a second reaction mixture with a substrate of AIR synthetase under the same conditions and for the same period of time as in the first reaction mixture; (c) determining the activity of the enzyme in the first and second reaction mixtures; and (d) selecting the chemical to be tested for the ability to inhibit plant growth or viability when the activity of the enzyme in the second reaction mixture is less, desirably significantly less, than the activity of the enzyme in the first reaction mixture. In a preferred embodiment, the substrate of AIR synthetase is 5′-phosphoribosyl-N-formylglycinamidine (FGAM) and in a further preferred embodiment, the substrate of AIR synthetase is &bgr;-FGAM. In another preferred embodiment, the enzyme having AIR synthetase activity is derived from a plant and more preferably, is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3. In another embodiment, the AIR synthetase enzyme is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In yet another embodiment, the AIR synthetase enzyme has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4. In another preferred embodiment, the chemical is capable of inhibiting the growth or viability of a plant by inhibiting the activity of AIR synthetase in the plant. In yet another preferred embodiment, the activity of the enzyme is determine
Guyer David Charles
Potter Sharon Lee
Subramanian Venkiteswaran
Walters Eric
Ward Eric Russell
Kruse David H
Lebel Edouard G.
Meigs J. Timothy
Nelson Amy
Stults Larry W.
LandOfFree
Nucleic acid molecules encoding... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid molecules encoding..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid molecules encoding... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2546100