Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
2001-01-04
2003-11-25
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C530S350000, C530S370000, C536S023100, C536S023200, C536S023600, C536S024100, C800S278000
Reexamination Certificate
active
06653531
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in chorismate biosynthesis in plants and seeds.
BACKGROUND OF THE INVENTION
Chorismate biosynthesis involves the last few steps in the common pathway for the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan. The last common step of biosynthesis of aromatic amino acids produced via the shikimic acid pathway is catalyzed by chorismate synthase which produces chorismate from 5-enolpyruvylshikimate 3-phosphate. The enzyme requires reduced FMN as a cofactor. There are two forms of the enzyme described in tomato. Tomato chorismate synthase 1 and 2 have 88% identity at the amino acid level and are expressed differentially in flowers, roots and stems (Gorlach, J. and Schmid, J. (1993)
Plant Mol Biol
23:707-716).
Manipulating either the amount or activity of this enzyme would afford manipulation of the ratio of aromatic to non-aromatic amino acids in plants, including corn, rice, soybean and wheat. This enzyme should also be useful for high throughput screening of compounds suitable for use as herbicides.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding chorismate synthase. Specifically, this invention concerns an isolated nucleic acid fragment encoding a chorismate synthase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a chorismate synthase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding chorismate synthase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a chorismate synthase.
In another embodiment, the instant invention relates to a chimeric gene encoding a chorismate synthase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a chorismate synthase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a chorismate synthase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants. An additional embodiment of the instant invention concerns a method of altering the level of expression of a chorismate synthase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a chorismate synthase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of chorismate synthase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a chorismate synthase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a chorismate synthase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a chorismate synthase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of chorismate synthase in the transformed host cell; (c) optionally purifying the chorismate synthase expressed by the transformed host cell; (d) treating the chorismate synthase with a compound to be tested; and (e) comparing the activity of the chorismate synthase that has been treated with a test compound to the activity of an untreated chorismate synthase, thereby selecting compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
Chorismate Synthase
SEQ ID NO:
Plant
Clone Designation
(Nucleotide)
(Amino Acid)
Corn
chpc24.pk0002.h1:fis
1
2
Soybean
sl1.pk0143.g5:fis
3
4
Wheat
wre1n.pk0094.e6
5
6
Corn
csi1n.pk0050.d11:fis
7
8
Rice
rls48.pk0033.g1
9
10
Rice
rls72.pk0029.g8
11
12
Soybean
ses9c.pk001.j6
13
14
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in
Nucleic Acids Research
13:3021-3030 (1985) and in the
Biochemical Journal
219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
DETAILED DESCRIPTION OF THE INVENTION
In the context of this disclosure, a number of terms shall be utilized. As used herein, a “nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.
For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalen
Cahoon Rebecca E.
Falco Saverio Carl
Bui Phuong T.
E. I. du Pont de Nemours and Company
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