Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-06-05
2001-09-25
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S018000, C435S195000, C435S232000, C530S370000
Reexamination Certificate
active
06294345
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to enzymatic activity involved in ENR-A, CBL, UROD, PBGD, or CPPO in plants. In particular, the invention relates to plant genes that encode a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity. The invention has various utilities, including the recombinant production of polypeptides having ENR-A, CBL, UROD, PBGD, or CPPO activity in heterologous hosts, the screening of chemicals for herbicidal activity, and 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
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.
For example, present herbicides often impose special limitations on farming practices, and the time and method of application and stage of weed plant development often are critical for good weed control with such herbicides, thus creating farm management constraints. Furthermore, since only a few target enzymes are inhibited by currently used herbicides, various weed species are, or may become, resistant to these herbicides. For all of these reasons, the discovery and development of effective new herbicides, in particular those acting on novel target enzymes, is increasingly important.
Novel herbicides can now be discovered using high-throughput screens that implement recombinant DNA technology. Once identified, metabolic enzymes essential to plant growth and development can be recombinantly produced through standard molecular biological techniques and utilized as herbicide targets in screens for novel inhibitors of the enzyme's activity. The novel inhibitors discovered through such screens may then be used as herbicides to control undesirable vegetation. Such herbicides are also useful for selecting herbicide tolerant plants, and seed plants tolerant to the herbicide can be produced, for example by genetic engineering techniques. Thus, herbicides that exhibit greater potency, broader weed spectrum, and more rapid degradation in soil can be applied to crops that are resistant or tolerant to herbicides in order to kill weeds without attendant risk of damage to the crop.
Therefore, in order to meet the future food requirements of the world's growing population in a cost-effective and environmentally safe manner, there exists a long felt and unfulfilled need for novel target enzymes for herbicides, for new and better herbicides inhibiting such target enzymes and for plants tolerant to these new and better herbicides.
SUMMARY OF THE INVENTION
In view of these long felt yet unfulfilled needs, one object of the invention is to provide a method for identifying new or improved herbicides. Another object of the invention is to provide a method 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, and seed thereof, that are tolerant to such new or improved herbicides.
In furtherance of these and other objects, the present invention provides a DNA molecule comprising a nucleotide sequence, preferably isolated from a plant, that encodes a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity. The inventors are the first to demonstrate that the ENR-A, CBL, UROD, PBGD, or CPPO genes are essential for the growth of a plant, and is therefore good target enzymes for identifying new herbicides. According to one embodiment, the present invention provides a DNA molecule comprising a nucleotide sequence isolated from a plant that encodes the polypeptide set forth in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. For example, the DNA molecule of the invention may comprise a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, respectively. In another example, the DNA molecule of the invention comprises a nucleotide sequence that is substantially similar to any one of the coding sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, and that encodes a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively. Although a nucleotide sequence provided in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, is isolated from
Arabidopsis thaliana,
using the information provided by the present invention, other nucleotide sequences that encode a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity are obtained from other sources, e.g. from other plants, using standard methods known in the art.
The present invention also provides a nucleotide sequence construct comprising a promoter operatively linked to a DNA molecule of the invention. Further, the present invention provides methods to stably transform such a nucleotide sequence construct into a host cell, and host cells comprising such a nucleotide sequence construct, wherein the host cell is capable of expressing the DNA molecule encoding a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively. Any suitable cell may be used as a host cell, e.g. a bacterial cell, a yeast cell, or a plant cell.
In accordance with another embodiment, the present invention also relates to the recombinant production of a ENR-A, CBL, UROD, PBGD, or CPPO polypeptide and methods of use of ENR-A, CBL, UROD, PBGD, or CPPO in assays for identifying compounds that interact with ENR-A, CBL, UROD, PBGD, or CPPO polypeptide, respectively. In a preferred embodiment, the present invention provides a plant polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity useful for identifying inhibitors of ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively, in in vivo and in vitro assays. Preferably the isolated polypeptide of the present invention comprises an amino acid sequence substantially similar to any one of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, respectively. More preferably, this enzyme comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
The present invention further provides methods of using purified polypeptides having ENR-A, CBL, UROD, PBGD, or CPPO activity, preferably polypeptides derived from plant sources, in assays to screen for and identify compounds that interact with a ENR-A, CBL, UROD, PBGD, or CPPO polypeptide, respectively. Such compounds are preferably inhibitors of ENR-A, CBL, UROD, PBGD, or CPPO activity, and are potentially herbicides of future commercial interest. The inhibitors are used as herbicides to suppress the growth of undesirable vegetation 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.
Thus, an assay useful for identifying inhibitors of essential plant genes, such as plant ENR-A, CBL, UROD, PBGD, or CPPO genes, comprises the steps of:
a) reacting a plant ENR-A, CBL, UROD, PBGD, or CPPO enzyme, and a substrate thereof in the presence of a suspected inhibitor of the enzyme's function;
b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor to the rate of enzymatic activity under the same conditions in the absence of the suspected inhibitor; and
c) determining whether the suspected inhibitor inhibits the ENR-A, CBL, UROD, PBGD, or CPPO enzyme, respectively.
For example, the inhibitory effect on plant ENR-A, CBL, UROD, PBGD, or CPPO may be determined by a reduction or complete inhibition of ENR-A, CBL, UROD, PBGD, or CPPO activity in the assay. Such a determination may be ma
Bauer Michael William
Levin Joshua Zvi
Zheng Feng
Fronda Christian L.
Lebel Edouard G.
Meigs J. Timothy
Nashed Nashaat T.
Stults Larry W.
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