Herbicide target genes and methods

The invention relates to genes isolated from Arabidopsis that code for proteins essential for normal plant development. The invention also includes the methods of using these proteins to discover new herbicides, based on the essentiality of the genes for normal growth and development. The invention can also be used in a screening assay to identify inhibitors that are potential herbicides. The invention is also applied to the development of herbicide tolerant plants, plant tissues, plant seeds, and plant cells.

FIELD OF THE INVENTION
 The invention relates to genes isolated from Arabidopsis thaliana that
 encode proteins essential for plant growth and development. The invention
 also includes the methods of using these proteins as herbicide targets,
 based on the essentiality of these genes for normal growth and
 development. The invention is also useful as a screening assay to identify
 inhibitors that are potential herbicides. The invention may also be
 applied to the development of herbicide tolerant 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.
 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 through standard molecular
 biological techniques and utilized as herbicide targets in screens for
 novel inhibitors of the enzyme activity. The novel inhibitors discovered
 through such screens may then be used as herbicides to control undesirable
 vegetation.
 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).
 Notwithstanding the above-described advancements, there remains a
 persistent and ongoing problem with unwanted or detrimental vegetation
 growth (e.g. weeds). Furthermore, as the population continues to grow,
 there will be increasing food shortages. Therefore, there exists a long
 felt, yet unfulfilled need, to find new, effective, and economic
 herbicides.
 SUMMARY OF THE INVENTION
 It is an object of the invention to provide an effective and beneficial
 method to identify novel herbicides. A feature of the invention is the
 identification of a gene in A. thaliana, herein referred to as the 8388
 gene, which shows sequence similarity to DEAD box RNA helicase (Luking et
 al. (1998) Critical Reviews in Biochemistry and Molecular Biology, 33(4):
 259-296). A feature of the invention is the identification of a gene in A.
 thaliana, herein referred to as the 18048 gene, which shows sequence
 similarity to ADP-ribosylation factor (Art) genes (Regad et al. (1993)
 FEBS Lett. 25: 133-136; Bar-Peled et al. (1995) The Plant Cell, 7:
 667-676). A feature of the invention is the identification of a gene in A.
 thaliana, herein referred to as the 16713 gene, which shows sequence
 similarity to acetoacetyl coA thiolases (Vollack and Bach (1996) Plant
 Physiol. 111: 1097-1107; Hiser et al. (1994) J. Biol. Chem. 269:
 31383-31389; Fukao et al. (1990) J. Clin. Invest. 86: 2086-2092; Fukao et
 al. (1989) J. Biochem. 106: 197-204; Wilson et al. (1994) Nature 368:
 32-38). A feature of the invention is the identification of a gene in
 Arabidopsis, herein referred to as the 4144 gene, which encodes a protein
 with sequence similarity to chloroplast ATP synthase delta chain (Hermans
 et al. (1988) Plant Mol. Biol. 10: 323-330; Hoesche and Berzborn (1992)
 Biochimica et Biophysica Acta, 1171: 201-204; Hoesche and Berzbom (1993)
 Biochimica et Biophysica Acta, 1142: 293-305; Napier et al. (1992) Plant
 Mol. Biol. 20: 549-554). Another feature of the invention is the discovery
 that the 8388, 18048, 16713, and 4144 genes are essential for normal
 growth and development. An advantage of the present invention is that the
 newly discovered essential genes provide the basis for identity of a novel
 herbicidal mode of action which enables one skilled in the art to easily
 and rapidly discover novel inhibitors of gene function useful as
 herbicides.
 One object of the present invention is to provide essential genes in plants
 for assay development for inhibitory compounds with herbicidal activity.
 Genetic results show that when any one of the 8388, 18048, 16713, or 4144
 genes is mutated in Arabidopsis thaliana, the resulting phenotype is
 lethal in the homozygous state. This suggests a critical role for the gene
 products encoded by the 8388, 18048, 16713, and 4144 genes.
 Using T-DNA insertion mutagenesis, the inventors of the present invention
 have demonstrated that the activity of any one of the 8388, 18048, 16713,
 or 4144 gene products is essential for A. thaliana growth. This implies
 that chemicals, which inhibit the function of the 8388, 18048, 16713, or
 4144-encoded protein 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 protein encoded
 by the 8388, 18048, 16713, or 4144 gene sequence described below 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 novel nucleotide sequences derived from A.
 thaliana, designated the 8388, 18048, 16713, or 4144 genes. The nucleotide
 sequences of the coding regions for the cDNA clones are set forth in SEQ
 ID NO:1, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:21, respectively, and the
 corresponding amino acid sequences of the 8388, 18048, 16713, or
 4144-encoded protein are set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID
 NO:8, and SEQ ID NO:22, respectively. The present invention also includes
 nucleotide sequences substantially similar to those set forth in SEQ ID
 NO:1, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:21, respectively. The
 present invention also encompasses plant proteins whose amino acid
 sequence are substantially similar to the amino acid sequences set forth
 in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:22, respectively.
 The present invention also includes methods of using the 8388, 18048,
 16713, or 4144 gene products as herbicide targets, based on the
 essentiality of these genes for normal growth and development.
 Furthermore, the invention can be used in a screening assay to identify
 inhibitors of 8388, 18048, 16713, or 4144 gene function that are potential
 herbicides.
 In a preferred embodiment, the present invention relates to a method for
 identifying chemicals having the ability to inhibit 8388, 18048, 16713, or
 4144 activity in plants preferably comprising the steps of: a) obtaining
 transgenic plants, plant tissue, plant seeds or plant cells, preferably
 stably transformed, comprising a non-native nucleotide sequence encoding
 an enzyme having 8388, 18048, 16713, or 4144 activity and capable of
 overexpressing an enzymatically active 8388, 18048, 16713, or 4144 gene
 product (either full length or truncated but still active); b) applying a
 chemical to the transgenic plants, plant cells, tissues or parts and to
 the isogenic non-transformed plants, plant cells, tissues or parts; c)
 determining the growth or viability of the transgenic and non-transformed
 plants, plant cells, tissues after application of the chemical; d)
 comparing the growth or viability of the transgenic and non-transformed
 plants, plant cells, tissues after application of the chemical; and e)
 selecting chemicals that suppress the viability or growth of the
 non-transgenic plants, plant cells, tissues or parts, without
 significantly suppressing the growth of the viability or growth of the
 isogenic transgenic plants, plant cells, tissues or parts. In a preferred
 embodiment, the enzyme having 8388, 18048, 16713, or 4144 activity is
 encoded by a nucleotide sequence derived from a plant, preferably
 Arabidopsis thaliana, desirably identical or substantially similar to the
 nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7,
 and SEQ ID NO:21, respectively. In another embodiment, the enzyme having
 8388, 18048, 16713, or 4144 activity is encoded by a nucleotide sequence
 capable of encoding the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6,
 SEQ ID NO:8, and SEQ ID NO:22, respectively. In yet another embodiment,
 the enzyme having 8388, 18048, 16713, or 4144 activity has an amino acid
 sequence identical or substantially similar to the amino acid sequence set
 forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:22,
 respectively.
 The present invention further embodies plants, plant tissues, plant seeds,
 and plant cells that have modified 8388, 18048, 16713, or 4144 activity
 and that are therefore tolerant to inhibition by a herbicide at levels
 normally inhibitory to naturally occurring 8388, 18048, 16713, or
 4144-encoded activity. Herbicide tolerant plants encompassed by the
 invention include those that would otherwise be potential targets for
 8388, 18048, 16713, or 4144-inhibiting herbicides, particularly the
 agronomically important crops mentioned above. According to this
 embodiment, plants, plant tissue, plant seeds, or plant cells are
 transformed, preferably stably transformed, with a recombinant DNA
 molecule comprising a suitable promoter functional in plants operatively
 linked to a nucleotide sequence that encodes a modified 8388, 18048,
 16713, or 4144 gene that is tolerant to inhibition by a herbicide at a
 concentration that would normally inhibit the activity of wild-type,
 unmodified 8388, 18048, 16713, or 4144 gene product. Modified 8388, 18048,
 16713, or 4144 activity may also be conferred upon a plant by increasing
 expression of wild-type herbicide-sensitive 8388, 18048, 16713, or 4144
 protein by providing multiple copies of wild-type 8388, 18048, 16713, or
 4144 genes to the plant or by overexpression of wild-type 8388, 18048,
 16713, or 4144 genes under control of a stronger-than-wild-type promoter.
 The transgenic plants, plant tissue, plant seeds, or plant cells thus
 created are then selected using conventional techniques, whereby herbicide
 tolerant lines are isolated, characterized, and developed. Alternately,
 random or site-specific mutagenesis may be used to generate herbicide
 tolerant lines.
 Therefore, the present invention provides a plant, plant cell, plant seed,
 or plant tissue transformed with a DNA molecule comprising a nucleotide
 sequence isolated from a plant that encodes an enzyme having 8388, 18048,
 16713, or 4144 activity, wherein the DNA expresses the 8388, 18048, 16713,
 or 4144 activity and wherein the DNA molecule confers upon the plant,
 plant cell, plant seed, or plant tissue tolerance to a herbicide in
 amounts that normally inhibits naturally occurring 8388, 18048, 16713, or
 4144 activity. According to one example of this embodiment, the enzyme
 having 8388, 18048, 16713, or 4144 activity is encoded by a nucleotide
 sequence identical or substantially similar to the nucleotide sequence set
 forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:21,
 respectively, or has an amino acid sequence identical or substantially
 similar to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:6,
 SEQ ID NO:8, and SEQ ID NO:22, respectively.
 The invention also provides a method for suppressing the growth of a plant
 comprising the step of applying to the plant a chemical that inhibits the
 naturally occurring 8388, 18048, 16713, or 4144 activity in the plant. In
 a related aspect, the present invention is directed to a method for
 selectively suppressing the growth of undesired vegetation in a field
 containing a crop of planted crop seeds or plants, comprising the steps
 of: (a) optionally planting herbicide tolerant crops or crop seeds, which
 are plants or plant seeds that are tolerant to a herbicide that inhibits
 the naturally occurring 8388, 18048, 16713, or 4144 activity; and (b)
 applying to the herbicide tolerant crops or crop seeds and the undesired
 vegetation in the field a herbicide in amounts that inhibit naturally
 occurring 8388, 18048, 16713, or 4144 activity, wherein the herbicide
 suppresses the growth of the weeds without significantly suppressing the
 growth of the crops.
 The invention thus provides an isolated DNA molecule comprising a
 nucleotide sequence substantially similar to SEQ ID NO:1, SEQ ID NO:5, SEQ
 ID NO:7, or SEQ ID NO:21, respectively. In a preferred embodiment, the
 nucleotide sequence encodes an amino acid sequence substantially similar
 to SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:22, respectively.
 In another preferred embodiment, the nucleotide sequence is SEQ ID NO:1,
 SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:21, respectively. In yet another
 preferred embodiment, the nucleotide sequence encodes the amino acid
 sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:22,
 respectively. Preferably, the nucleotide sequence is a plant nucleotide
 sequence, which preferably encodes a polypeptide having 8388, 18048,
 16713, or 4144 activity, respectively.
 The invention further provides a polypeptide comprising an amino acid
 sequence encoded by a nucleotide sequence substantially similar to SEQ ID
 NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:21, respectively. Preferably,
 the amino acid sequence is encoded by SEQ ID NO:1, SEQ ID NO:5, SEQ ID
 NO:7, or SEQ ID NO:21, respectively. Preferably, the polypeptide comprises
 an amino acid sequence substantially similar to SEQ ID NO:2, SEQ ID NO:6,
 SEQ ID NO:8, or SEQ ID NO:22, respectively. Preferably the amino acid
 sequence is SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:22,
 respectively. The amino acid sequence preferably has 8388, 18048, 16713,
 or 4144 activity, respectively. In another preferred embodiment, the amino
 acid sequence comprises at least 20 consecutive amino acid residues of the
 amino acid sequence encoded by SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, or
 SEQ ID NO:21, respectively. Or, alternatively, the amino acid sequence
 comprises at least 20 consecutive amino acid residues of the amino acid
 sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:22,
 respectively. The invention further provides an expression cassette
 comprising a promoter operatively linked to a DNA molecule according to
 the present invention, a recombinant vector comprising an expression
 cassette according to the present invention, wherein said vector is
 preferably capable of being stably transformed into a host cell, a host
 cell comprising a DNA molecule according to the present invention, wherein
 said DNA molecule is preferably expressible in the cell. The host cell is
 preferably selected from the group consisting of an insect cell, a yeast
 cell, a prokaryotic cell and a plant cell. The invention further provides
 a plant or seed comprising a plant cell of the present invention, wherein
 the plant or seed is preferably tolerant to an inhibitor of 8388, 18048,
 16713, or 4144 activity, respectively.
 The invention further provides a process for making nucleotides sequences
 encoding gene products having altered 8388, 18048, 16713, or 4144
 activity, respectively, comprising: a) shuffling an unmodified nucleotide
 sequence of the present invention, b) expressing the resulting shuffled
 nucleotide sequences, and c) selecting for altered 8388, 18048, 16713, or
 4144 activity, respectively, as compared to the 8388, 18048, 16713, or
 4144 activity, respectively, of the gene product of said unmodified
 nucleotide sequence.
 In a preferred embodiment, the unmodified nucleotide sequence is identical
 or substantially similar to SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ
 ID NO:21, respectively, or a homolog thereof. The present invention
 further provides a DNA molecule comprising a shuffled nucleotide sequence
 obtainable by the process described above, a DNA molecule comprising a
 shuffled nucleotide sequence produced by the process described above.
 Preferably, a shuffled nucleotide sequence obtained by the process
 described above has enhanced tolerance to an inhibitor of 8388, 18048,
 16713, or 4144 activity, respectively. The invention further provides an
 expression cassette comprising a promoter operatively linked to a DNA
 molecule comprising a shuffled nucleotide sequence a recombinant vector
 comprising such an expression cassette, wherein said vector is preferably
 capable of being stably transformed into a host cell, a host cell
 comprising such an expression cassette, wherein said nucleotide sequence
 is preferably expressible in said cell. A preferred host cell is selected
 from the group consisting of an insect cell, a yeast cell, a prokaryotic
 cell and a plant cell. The invention further provides a plant or seed
 comprising such plant cell, wherein the plant is preferably tolerant to an
 inhibitor of 8388, 18048, 16713, or 4144 activity, respectively.
 The invention further provides a method for selecting compounds that
 interact with the protein encoded by SEQ ID NO:1, SEQ ID NO:5, SEQ ID
 NO:7, or SEQ ID NO:21, respectively, comprising: a) expressing a DNA
 molecule comprising SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7. or SEQ ID
 NO:21, respectively, or a sequence substantially similar to SEQ ID NO:1,
 SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:21, respectively, or a homolog
 thereof, to generate the corresponding protein, b) testing a compound
 suspected of having the ability to interact with the protein expressed in
 step (a), and c) selecting compounds that interact with the protein in
 step (b).
 The invention further provides a process of identifying an inhibitor of
 8388, 18048, 16713, or 4144 activity, respectively, comprising: a)
 introducing a DNA molecule comprising a nucleotide sequence of SEQ ID
 NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:21, respectively, and having
 8388, 18048, 16713, or 4144 activity, respectively, or nucleotide
 sequences substantially similar thereto, or a homolog thereof, into a
 plant cell, such that said sequence is functionally expressible at levels
 that are higher than wild-type expression levels, b) combining said plant
 cell with a compound to be tested for the ability to inhibit the 8388,
 18048, 16713, or 4144 activity, respectively, under conditions conducive
 to such inhibition, c) measuring plant cell growth under the conditions of
 step (b), d) comparing the growth of said plant cell with the growth of a
 plant cell having unaltered 8388, 18048, 16713, or 4144 activity,
 respectively, under identical conditions, and e) selecting said compound
 that inhibits plant cell growth in step (d).
 The invention further comprises a compound having herbicidal activity
 identifiable according to the process described immediately above.
 The invention further comprises:
 A process of identifying compounds having herbicidal activity comprising:
 a) combining a protein of the present invention and a compound to be tested
 for the ability to interact with said protein, under conditions conducive
 to interaction, b) selecting a compound identified in step (a) that is
 capable of interacting with said protein, c) applying identified compound
 in step (b) to a plant to test for herbicidal activity, and d) selecting
 compounds having herbicidal activity.
 The invention further comprises a compound having herbicidal activity
 identifiable according to the process described immediately above.
 The invention further comprises:
 A method for suppressing the growth of a plant comprising, applying to said
 plant a compound that inhibits the activity of a polypeptide of the
 present invention in an amount sufficient to suppress the growth of said
 plant.
 The invention further comprises:
 A method for recombinantly expressing a protein having 8388, 18048, 16713,
 or 4144 activity comprising introducing a nucleotide sequence encoding a
 protein having one of the above activities into a host cell and expressing
 the nucleotide sequence in the host cell. A preferred host cell is
 selected from the group consisting of an insect cell, a yeast cell, a
 prokaryotic cell and a plant cell. A preferred prokaryotic cell is a
 bacterial cell, e.g. E. coli.
 Other objects and advantages of the present invention will become apparent
 to those skilled in the art from a study of the following description of
 the invention and non-limiting examples.
 DEFINITIONS
 For clarity, certain terms used in the specification are defined and
 presented as follows:
 Cofactor: natural reactant, such as an organic molecule or a metal ion,
 required in an enzyme-catalyzed reaction. A co-factor is e.g. NAD(P),
 riboflavin (including FAD and FMN), folate, molybdopterin, thiamin,
 biotin, lipoic acid, pantothenic acid and coenzyme A,
 S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.
 Optionally, a co-factor can be regenerated and reused.
 DNA shuffling: DNA shuffling is a method to rapidly, easily and efficiently
 introduce mutations or rearrangements, preferably randomly, in a DNA
 molecule or to generate exchanges of DNA sequences between two or more DNA
 molecules, preferably randomly. The DNA molecule resulting from DNA
 shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA
 molecule derived from at least one template DNA molecule. The shuffled DNA
 encodes an enzyme modified with respect to the enzyme encoded by the
 template DNA, and preferably has an altered biological activity with
 respect to the enzyme encoded by the template DNA.
 Enzyme activity: means herein the ability of an enzyme to catalyze the
 conversion of a substrate into a product. A substrate for the enzyme
 comprises the natural substrate of the enzyme but also comprises analogues
 of the natural substrate which can also be converted by the enzyme into a
 product or into an analogue of a product. The activity of the enzyme is
 measured for example by determining the amount of product in the reaction
 after a certain period of time, or by determining the amount of substrate
 remaining in the reaction mixture after a certain period of time. The
 activity of the enzyme is also measured by determining the amount of an
 unused co-factor of the reaction remaining in the reaction mixture after a
 certain period of time or by determining the amount of used co-factor in
 the reaction mixture after a certain period of time. The activity of the
 enzyme is also measured by determining the amount of a donor of free
 energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl
 phosphate or phosphocreatine) remaining in the reaction mixture after a
 certain period of time or by determining the amount of a used donor of
 free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate or
 creatine) in the reaction mixture after a certain period of time.
 Herbicide: a chemical substance used to kill or suppress the growth of
 plants, plant cells, plant seeds, or plant tissues.
 Heterologous DNA Sequence: a DNA sequence not naturally associated with a
 host cell into which it is introduced, including non-naturally occurring
 multiple copies of a naturally occurring DNA sequence; and genetic
 constructs wherein an otherwise homologous DNA sequence is operatively
 linked to a non-native sequence.
 Homologous DNA Sequence: a DNA sequence naturally associated with a host
 cell into which it is introduced.
 Inhibitor: a chemical substance that causes abnormal growth, e.g., by
 inactivating the enzymatic activity of a protein such as a biosynthetic
 enzyme, receptor, signal transduction protein, structural gene product, or
 transport protein that is essential to the growth or survival of the
 plant. In the context of the instant invention, an inhibitor is a chemical
 substance that alters the enzymatic activity encoded by a nucleotide
 sequence of the present invention. More generally, an inhibitor causes
 abnormal growth of a host cell by interacting with the gene product
 encoded by the nucleotide sequence of the present invention.
 Isogenic: plants which are genetically identical, except that they may
 differ by the presence or absence of a heterologous DNA sequence.
 Isolated: in the context of the present invention, an isolated DNA molecule
 or an isolated enzyme is a DNA molecule or enzyme that, by the hand of
 man, exists apart from its native environment and is therefore not a
 product of nature. An isolated DNA molecule or enzyme may exist in a
 purified form or may exist in a non-native environment such as, for
 example, in a transgenic host cell.
 Mature protein: protein which is normally targeted to a cellular organelle,
 such as a chloroplast, and from which the transit peptide has been
 removed.
 Minimal Promoter: promoter elements, particularly a TATA element, that are
 inactive or that have greatly reduced promoter activity in the absence of
 upstream activation. In the presence of a suitable transcription factor,
 the minimal promoter functions to permit transcription.
 Modified Enzyme Activity: enzyme activity different from that which
 naturally occurs in a plant (i.e. enzyme activity that occurs naturally in
 the absence of direct or indirect manipulation of such activity by man),
 which is tolerant to inhibitors that inhibit the naturally occurring
 enzyme activity.
 Pre-protein: protein which is normally targeted to a cellular organelle,
 such as a chloroplast, and still comprising its transit peptide.
 Significant Increase: an increase in enzymatic activity that is larger than
 the margin of error inherent in the measurement technique, preferably an
 increase by about 2-fold or greater of the activity of the wild-type
 enzyme in the presence of the inhibitor, more preferably an increase by
 about 5-fold or greater, and most preferably an increase by about 10-fold
 or greater.
 Significantly less: means that the amount of a product of an enzymatic
 reaction is reduced by more than the margin of error inherent in the
 measurement technique, preferably a decrease by about 2-fold or greater of
 the activity of the wild-type enzyme in the absence of the inhibitor, more
 preferably an decrease by about 5-fold or greater, and most preferably an
 decrease by about 10-fold or greater.
 Substantially similar: with respect to the 8388 gene, in its broadest
 sense, the term "substantially similar", when used herein with respect to
 a nucleotide sequence, means a nucleotide sequence corresponding to a
 reference nucleotide sequence, wherein the corresponding sequence encodes
 a polypeptide having substantially the same structure and function as the
 polypeptide encoded by the reference nucleotide sequence, e.g. where only
 changes in amino acids not affecting the polypeptide function occur.
 Desirably the substantially similar nucleotide sequence encodes the
 polypeptide encoded by the reference nucleotide sequence. The term
 "substantially similar" is specifically intended to include nucleotide
 sequences wherein the sequence has been modified to optimize expression in
 particular cells. The percentage of identity between the substantially
 similar nucleotide sequence and the reference nucleotide sequence
 desirably is at least 65%, more desirably at least 75%, preferably at
 least 85%, more preferably at least 90%, still more preferably at least
 95%, yet still more preferably at least 99%. Sequence comparisons are
 carried out using a Smith-Waterman sequence alignment algorithm (see e.g.
 Waterman, M. S. Introduction to Computational Biology: Maps, sequences and
 genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or at
 http://www-hto.usc.edu/software/seqaln/index.html). The localS program,
 version 1.16, is used with following parameters: match: 1, mismatch
 penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide
 sequence "substantially similar" to reference nucleotide sequence
 hybridizes to the reference nucleotide sequence in 7% sodium dodecyl
 sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing
 in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium
 dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with
 washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more
 preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA
 at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
 As used herein the term "8388 gene" refers to a DNA molecule comprising
 SEQ ID NO:1 or comprising a nucleotide sequence substantially similar to
 SEQ ID NO:1. Homologs of the 8388 gene include nucleotide sequences that
 encode an amino acid sequence that is at least 25% identical to SEQ ID
 NO:2 as measured, using the parameters described below, wherein the amino
 acid sequence encoded by the homolog has the biological activity of the
 8388 protein.
 With respect to the 8388 protein, the term "substantially similar", when
 used herein with respect to a protein, means a protein corresponding to a
 reference protein, wherein the protein has substantially the same
 structure and function as the reference protein, e.g. where only changes
 in amino acids sequence not affecting the polypeptide function occur. When
 used for a protein or an amino acid sequence the percentage of identity
 between the substantially similar and the reference protein or amino acid
 sequence desirably is at least 65%, more desirably at least 75%,
 preferably at least 85%, more preferably at least 90%, still more
 preferably at least 95%, yet still more preferably at least 99%, using
 default BLAST analysis parameters BLAST 2.0.7. As used herein the term
 "8388 protein" refers to an amino acid sequence encoded by a DNA molecule
 comprising a nucleotide sequence substantially similar to SEQ ID NO:1.
 Homologs of the 8388 protein are amino acid sequences that are at (again
 here) least 25% identical to SEQ ID NO:2, as measured using the parameters
 described above, wherein the amino acid sequence encoded by the homolog
 has the biological activity ofthe 8388 protein.
 With respect to the 18048 gene, in its broadest sense, the term
 "substantially similar", when used herein with respect to a nucleotide
 sequence, means a nucleotide sequence corresponding to a reference
 nucleotide sequence, wherein the corresponding sequence encodes a
 polypeptide having substantially the same structure and function as the
 polypeptide encoded by the reference nucleotide sequence, e.g. where only
 changes in amino acids not affecting the polypeptide function occur.
 Desirably the substantially similar nucleotide sequence encodes the
 polypeptide encoded by the reference nucleotide sequence. The term
 "substantially similar" is specifically intended to include nucleotide
 sequences wherein the sequence has been modified to optimize expression in
 particular cells. The percentage of identity between the substantially
 similar nucleotide sequence and the reference nucleotide sequence
 desirably is at least 65%, more desirably at least 75%, preferably at
 least 85%, more preferably at least 90%, still more preferably at least
 95%, yet still more preferably at least 99%. Sequence comparisons are
 carried out using a Smith-Waterman sequence alignment algorithm (see e.g.
 Waterman, M. S. Introduction to Computational Biology: Maps, sequences and
 genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or at
 http://www-hto.usc.edu/software/seqaln/index.html). The local S program,
 version 1.16, is used with following parameters: match: 1, mismatch
 penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide
 sequence "substantially similar" to reference nucleotide sequence
 hybridizes to the reference nucleotide sequence in 7% sodium dodecyl
 sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing
 in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium
 dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with
 washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more
 preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA
 at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
 As used herein the term "18048 gene" refers to a DNA molecule comprising
 SEQ ID NO:5 or comprising a nucleotide sequence substantially similar to
 SEQ ID NO:5. Homologs of the 18048 gene include nucleotide sequences that
 encode an amino acid sequence that is at least 30% identical to SEQ ID
 NO:6 as measured, using the parameters described below, wherein the amino
 acid sequence encoded by the homolog has the biological activity of the
 18048 protein.
 With respect to the 18048 protein, the term "substantially similar", when
 used herein with respect to a protein, means a protein corresponding to a
 reference protein, wherein the protein has substantially the same
 structure and function as the reference protein, e.g. where only changes
 in amino acids sequence not affecting the polypeptide function occur. When
 used for a protein or an amino acid sequence the percentage of identity
 between the substantially similar and the reference protein or amino acid
 sequence desirably is at least 65%, more desirably at least 75%,
 preferably at least 85%, more preferably at least 90%, still more
 preferably at least 95%, yet still more preferably at least 99%, using
 default BLAST analysis parameters BLAST 2.0.7. As used herein the term
 "18048 protein" refers to an amino acid sequence encoded by a DNA molecule
 comprising a nucleotide sequence substantially similar to SEQ ID NO:5.
 Homologs of the 18048 protein are amino acid sequences that are at least
 30% identical to SEQ ID NO:6, as measured using the parameters described
 above, wherein the amino acid sequence encoded by the homolog has the
 biological activity of the 18048 protein.
 With respect to the 16713 gene, in its broadest sense, the term
 "substantially similar", when used herein with respect to a nucleotide
 sequence, means a nucleotide sequence corresponding to a reference
 nucleotide sequence, wherein the corresponding sequence encodes a
 polypeptide having substantially the same structure and function as the
 polypeptide encoded by the reference nucleotide sequence, e.g. where only
 changes in amino acids not affecting the polypeptide function occur.
 Desirably the substantially similar nucleotide sequence encodes the
 polypeptide encoded by the reference nucleotide sequence. The term
 "substantially similar" is specifically intended to include nucleotide
 sequences wherein the sequence has been modified to optimize expression in
 particular cells. The percentage of identity between the substantially
 similar nucleotide sequence and the reference nucleotide sequence
 desirably is at least 90%, more desirably at least 95%, yet still more
 preferably at least 99%. Sequence comparisons are carried out using a
 Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M. S.
 Introduction to Computational Biology: Maps, sequences and genomes.
 Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or at
 http://www-hto.usc.edu/software/seqaln/index.html). The localS program,
 version 1.16, is used with following parameters: match: 1, mismatch
 penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide
 sequence "substantially similar" to reference nucleotide sequence
 hybridizes to the reference nucleotide sequence in 7% sodium dodecyl
 sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing
 in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium
 dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with
 washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more
 preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA
 at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
 As used herein the term "16713 gene" refers to a DNA molecule comprising
 SEQ ID NO:7 or comprising a nucleotide sequence substantially similar to
 SEQ ID NO:7. Homologs of the 16713 gene include nucleotide sequences that
 encode an amino acid sequence that is at least 45% identical, preferably
 at least 55%, more preferably at least 65%, still more preferably at least
 75%, yet still more preferably at least 85% identical to SEQ ID NO:8 as
 measured, using the parameters described below, wherein the amino acid
 sequence encoded by the homolog has the biological activity of the 16713
 protein.
 With respect to the 16713 protein, the term "substantially similar", when
 used herein with respect to a protein, means a protein corresponding to a
 reference protein, wherein the protein has substantially the same
 structure and function as the reference protein, e.g. where only changes
 in amino acids sequence not affecting the polypeptide function occur. When
 used for a protein or an amino acid sequence the percentage of identity
 between the substantially similar and the reference protein or amino acid
 sequence desirably is at least 93%, still more preferably at least 95%,
 yet still more preferably at least 99%, using default BLAST analysis
 parameters BLAST 2.0.7. As used herein the term "16713 protein" refers to
 an amino acid sequence encoded by a DNA molecule comprising a nucleotide
 sequence substantially similar to SEQ ID NO:7. Homologs of the 16713
 protein are amino acid sequences that are at least 45% identical,
 preferably at least 55%, more preferably at least 65%, still more
 preferably at least 75%, yet still more preferably at least 85% identical
 to SEQ ID NO:8, as measured using the parameters described above, wherein
 the amino acid sequence encoded by the homolog has the biological activity
 of the 16713 protein.
 With respect to the 4144 gene, in its broadest sense, the term
 "substantially similar", when used herein with respect to a nucleotide
 sequence, means a nucleotide sequence corresponding to a reference
 nucleotide sequence, wherein the corresponding sequence encodes a
 polypeptide having substantially the same structure and function as the
 polypeptide encoded by the reference nucleotide sequence, e.g. where only
 changes in amino acids not affecting the polypeptide function occur.
 Desirably the substantially similar nucleotide sequence encodes the
 polypeptide encoded by the reference nucleotide sequence. The term
 "substantially similar" is specifically intended to include nucleotide
 sequences wherein the sequence has been modified to optimize expression in
 particular cells. The percentage of identity between the substantially
 similar nucleotide sequence and the reference nucleotide sequence
 desirably is at least 65%, more desirably at least 75%, preferably at
 least 85%, more preferably at least 90%, still more preferably at least
 95%, yet still more preferably at least 99%. Sequence comparisons are
 carried out using a Smith-Waterman sequence alignment algorithm (see e.g.
 Waterman, M. S. Introduction to Computational Biology: Maps, sequences and
 genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0, or at
 http://www-hto.usc.edu/software/seqaln/index.html). The localS program,
 version 1.16, is used with following parameters: match: 1, mismatch
 penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide
 sequence "substantially similar" to reference nucleotide sequence
 hybridizes to the reference nucleotide sequence in 7% sodium dodecyl
 sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing
 in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium
 dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with
 washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in
 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree.
 C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more
 preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA
 at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
 As used herein the term "4144 gene" refers to a DNA molecule comprising
 SEQ ID NO:21 or comprising a nucleotide sequence substantially similar to
 SEQ ID NO:21. Homlologs of the 4144 gene include nucleotide sequences that
 encode an amino acid sequence that is at least 30% identical to SEQ ID
 NO:22 as measured using the parameters described below, wherein the amino
 acid sequence encoded by the homolog has the biological activity of the
 4144 protein.
 With respect to the 4144 protein, the term "substantially similar", when
 used herein with respect to a protein, means a protein corresponding to a
 reference protein, wherein the protein has substantially the same
 structure and function as the reference protein, e.g. where only changes
 in amino acids sequence not affecting the polypeptide function occur. When
 used for a protein or an amino acid sequence the percentage of identity
 between the substantially similar and the reference protein or amino acid
 sequence desirably is at least 65%, more desirably at least 75%,
 preferably at least 85%, more preferably at least 90%, still more
 preferably at least 95%, yet still more preferably at least 99%, using
 default BLAST analysis parameters. As used herein the term "4144 protein"
 refers to an amino acid sequence encoded by a DNA molecule comprising a
 nucleotide sequence substantially similar to SEQ ID NO:21. Homologs of the
 4144 protein are amino acid sequences that are at least 30% identical to
 SEQ ID NO:22, as measured using the parameters described above, wherein
 the amino acid sequence encoded by the homolog has the biological activity
 of the 4144 protein.
 One skilled in the art is also familiar with other analysis tools, such as
 GAP analysis, to determine the percentage of identity between the
 "substantially similar" and the reference nucleotide sequence, or protein
 or amino acid sequence. In the present invention, "substantially similar"
 is therefore also determined using default GAP analysis parameters with
 the University of Wisconsin GCG, SEQWEB application of GAP, based on the
 algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol.
 Biol. 48: 443-453).
 Thus, in the context of the "8388 gene" and using GAP analysis as described
 above, "substantially similar" refers to nucleotide sequences that encode
 a protein having at least 37% identity, more preferably at least 50%
 identity, still more preferably at least 65% identity, still more
 preferably at least 75% identity, still more preferably at least 85%
 identity, still more preferably at least 95% identity, yet still more
 preferably at least 99% identity to SEQ ID NO:2. Further, using GAP
 analysis as described above, "homologs of the 8388 gene" include
 nucleotide sequences that encode an amino acid sequence that has at least
 29% identity to SEQ ID NO:2, more preferably at least 35% identity, still
 more preferably at least 45% identity, still more preferably at least 55%
 identity, yet still more preferably at least 65% identity, still more
 preferably at least 75% identity, yet still more preferably at least 85%
 identity to SEQ ID NO:2, wherein the amino acid sequence encoded by the
 homolog has the biological activity of the 8388 protein.
 When using GAP analysis as described above with respect to a protein or an
 amino acid sequence and in the context of the "8388 gene", the percentage
 of identity between the "substantially similar" protein or amino acid
 sequence and the reference protein or amino acid sequence (in this case
 SEQ ID NO:2) is at least 37%, more preferably at least 50%, still more
 preferably at least 65%, still more preferably at least 75%, still more
 preferably at least 85%, still more preferably at least 95%, yet still
 more preferably at least 99%. "Homologs of the 8388 protein" include amino
 acid sequences that are at least 29% identical to SEQ ID NO:2, more
 preferably at least 35% identical, still more preferably at least 45%
 identical, still more preferably at least 55% identical, yet still more
 preferably at least 65% identical, still more preferably at least 75%
 identical, yet still more preferably at least 85% identical to SEQ ID
 NO:2, wherein homologs of the 8388 protein have the biological activity of
 the 8388 protein.
 Thus, in the context of the "18048 gene" and using GAP analysis as
 described above, "substantially similar" refers to nucleotide sequences
 that encode a protein having at least 64% identity, more preferably at
 least 70% identity, still more preferably at least 75% identity, still
 more preferably at least 85% identity, still more preferably at least 95%
 identity, yet still more preferably at least 99% identity to SEQ ID NO:6.
 Further, using GAP analysis as described above, "homologs of the 18048
 gene" include nucleotide sequences that encode an amino acid sequence that
 has at least 45% identity to SEQ ID NO:6, more preferably at least 50%
 identity, still more preferably at least 55% identity, still more
 preferably at least 60% identity, yet still more preferably at least 65%
 identity, still more preferably at least 75% identity, yet still more
 preferably at least 85% identity to SEQ ID NO:6, wherein the amino acid
 sequence encoded by the homolog has the biological activity of the 18048
 protein.
 When using GAP analysis as described above with respect to a protein or an
 amino acid sequence and in the context of the "18048 gene", the percentage
 of identity between the "substantially similar" protein or amino acid
 sequence and the reference protein or amino acid sequence (in this case
 SEQ ID NO:6) is at least 64%, more preferably at least 70%, still more
 preferably at least 75%, still more preferably at least 85%, still more
 preferably at least 95%, yet still more preferably at least 99%. "Homologs
 of the 18048 protein" include amino acid sequences that are at least 45%
 identical to SEQ ID NO:6, more preferably at least 50% identical, still
 more preferably at least 55% identical, still more preferably at least 60%
 identical, yet still more preferably at least 65% identical, still more
 preferably at least 75% identical, yet still more preferably at least 85%
 identical to SEQ ID NO:6, wherein homologs of the 18048 protein have the
 biological activity of the 18048 protein.
 Thus, in the context of the "16713 gene" and using GAP analysis as
 described above, "substantially similar" refers to nucleotide sequences
 that encode a protein having at least 93% identity, more preferably at
 least 95% identity, still more preferably at least 99% identity to SEQ ID
 NO:8. Further, using GAP analysis as described above, "homologs of the
 16713 gene" include nucleotide sequences that encode an amino acid
 sequence that has at least 45% identity to SEQ ID NO:8, more preferably at
 least 50% identity, still more preferably at least 55% identity, still
 more preferably at least 60% identity, yet still more preferably at least
 70% identity, still more preferably at least 85% identity, yet still more
 preferably at least 90% identity to SEQ ID NO:8, wherein the amino acid
 sequence encoded by the homolog has the biological activity of the 16713
 protein.
 When using GAP analysis as described above with respect to a protein or an
 amino acid sequence and in the context of the "16713 gene", the percentage
 of identity between the "substantially similar" protein or amino acid
 sequence and the reference protein or amino acid sequence (in this case
 SEQ ID NO:8) is at least 93%, more preferably at least 95%, still more
 preferably at least 99%. "Homologs of the 16713 protein" include amino
 acid sequences that are at least 45% identical to SEQ ID NO:8, more
 preferably at least 50% identical, still more preferably at least 55%
 identical, still more preferably at least 60% identical, yet still more
 preferably at least 70% identical, still more preferably at least 85%
 identical, yet still more preferably at least 95% identical to SEQ ID
 NO:8, wherein honiologs of the 16713 protein have the biological activity
 of the 16713 protein.
 Thus, in the context of the "4144 gene" and using GAP analysis as described
 above, "substantially similar" refers to nucleotide sequences that encode
 a protein having at least 89% identity, more preferably at least 90%
 identity, still more preferably at least 95% identity, yet still more
 preferably at least 99% identity to SEQ ID NO:22. Further, using GAP
 analysis as described above, "homologs of the 4144 gene" include
 nucleotide sequences that encode an amino acid sequence that has at least
 45% identity to SEQ ID NO:22, more preferably at least 50% identity, still
 more preferably at least 55% identity, still more preferably at least 60%
 identity, yet still more preferably at least 65% identity, still more
 preferably at least 75% identity, yet still more preferably at least 85%
 identity to SEQ ID NO:22, wherein the amino acid sequence encoded by the
 homolog has the biological activity of the 4144 protein.
 When using GAP analysis as described above with respect to a protein or an
 amino acid sequence and in the context of the "4144 gene", the percentage
 of identity between the "substantially similar" protein or amino acid
 sequence and the reference protein or amino acid sequence (in this case
 SEQ ID NO:22) is at least 89%, more preferably at least 90%, still more
 preferably at least 95%, yet still more preferably at least 99%. "Homologs
 of the 4144 protein" include amino acid sequences that are at least 45%
 identical to SEQ ID NO:22, more preferably at least 50% identical, still
 more preferably at least 55% identical, still more preferably at least 60%
 identical, yet still more preferably at least 65% identical, still more
 preferably at least 75% identical, yet still more preferably at least 85%
 identical to SEQ ID NO:8, wherein homologs of the 4144 protein have the
 biological activity of the 4144 protein.
 Substrate: a substrate is the molecule that an enzyme naturally recognizes
 and converts to a product in the biochemical pathway in which the enzyme
 naturally carries out its function, or is a modified version of the
 molecule, which is also recognized by the enzyme and is converted by the
 enzyme to a product in an enzymatic reaction similar to the
 naturally-occurring reaction.
 Tolerance: the ability to continue essentially normal growth or function
 when exposed to an inhibitor or herbicide in an amount sufficient to
 suppress the normal growth or function of native, unmodified plants.
 Transformation: a process for introducing heterologous DNA into a cell,
 tissue, or plant. Transformed cells, tissues, or plants are understood to
 encompass not only the end product of a transformation process, but also
 transgenic progeny thereof.
 Transgenic: stably transformed with a recombinant DNA molecule that
 preferably comprises a suitable promoter operatively linked to a DNA
 sequence of interest.
 BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
 SEQ ID NO:1 Genomic DNA, single exon, coding sequence for the Arabidopsis
 thaliana 8388 gene
 SEQ ID NO:2 amino acid sequence encoded by the Arabidopsis thaliana 8388
 DNA sequence shown in SEQ ID NO:1
 SEQ ID NO:3 complete cDNA sequence, including 5' UTR, coding region, and 3'
 UTR sequences, for the Arabidopsis thaliana 8388 gene
 SEQ ID NO:4 amino acid sequence encoded by the Arabidopsis thaliana 8388
 cDNA sequence shown in SEQ ID NO:3
 SEQ ID NO:5 cDNA coding sequence for the Arabidopsis thaliana 18048 gene
 SEQ ID NO:6 amino acid sequence encoded by the Arabidopsis thaliana 18048
 DNA sequence shown in SEQ ID NO:5
 SEQ ID NO:7 cDNA coding sequence for thc Arabidopsis thaliana 16713 gene
 SEQ ID NO:8 amino acid sequence encoded by the Arabidopsis thaliana 16713
 DNA sequence shown in SEQ ID NO:7
 SEQ ID NO:9 oligonucleotide CA50
 SEQ ID NO:10 oligonucleotide CA51
 SEQ ID NO:11 oligonucleotide CA52
 SEQ ID NO:12 oligonucleotide CA53
 SEQ ID NO:13 oligonucleotide CA54
 SEQ ID NO:14 oligonucleotide CA55
 SEQ ID NO:15 oligonucleotide CA66
 SEQ ID NO:16 oligonucleotide CA67
 SEQ ID NO:17 oligonucleotide CA68
 SEQ ID NO:18 oligonucleotide JM33
 SEQ ID NO:19 oligonucleotide JM34
 SEQ ID NO:20 oligonucleotide JM35
 SEQ ID NO:21 cDNA coding sequence for the Arabidopsis 4144 gene
 SEQ ID NO:22 amino acid sequence encoded by the Arabidopsis 4144 DNA
 sequence shown in SEQ ID NO:21
 SEQ ID NO:23 genomic sequence of the Arabidopsis 4144 gene
 SEQ ID NO:24 5' UTR from the cDNA sequence for the Arabidopsis 4144 gene
 SEQ ID NO:25 3' UTR from the cDNA sequence for the Arabidopsis 4144 gene
 SEQ ID NO:26 oligonucleotide slp346
 DETAILED DESCRIPTION OF THE INVENTION
 I.a. Essentiality of the 8388, 18048, and 16713 Genes in Arabidopsis
 thaliana Demonstrated by T-DNA Insertion Mutagenesis
 As shown in the examples below, the identification of a novel gene
 structure, as well as the essentiality of the 8388, 18048, and 16713 genes
 for normal plant growth and development, have been demonstrated for the
 first time in Arabidopsis using T-DNA insertion mutagenesis. Having
 established the essentiality of 8388, 18048, and 16713 function in plants
 and having identified the genes encoding these cssential activities, the
 inventors thereby provide an important and sought after tool for new
 herbicide development.
 Essential genes are identified through the isolation of lethal mutants
 blocked in early development. Examples of lethal mutants include those
 blocked in the formation of the male or female gametes or embryo.
 Gametophytic mutants are found by examining T1 insertion lines for the
 presence of 50% aborted pollen grains or ovules. Embryo defective mutants
 produce 25% defective seeds following self-pollination of T1 plants (see
 Errampalli et al. 1991, Plant Cell 3:149-157; Castle et al. 1993, Mol Gen
 Genet 241:504-514).
 When a line is identified as segregating for an embryo lethal mutation, it
 is determined if the resistance marker in the T-DNA co-segregates with the
 lethality (Errampalli et al. (1991) The Plant Cell, 3:149-157).
 Cosegregation analysis is done by placing the seeds on media containing
 the selective agent and scoring the seedlings for resistance or
 sensitivity to the agent. Examples of selective agents used are hygromycin
 or phosphinothricin. About 35 (8388), 35 (18048), and 38 (16713) resistant
 seedlings are transplanted to soil and their progeny are examined for the
 segregation of the embryo-lethal phenotype. In the case in which the T-DNA
 insertion disrupts an essential gene, there is cosegregation of the
 resistance phenotype and the embryo-lethal phenotype in every plant.
 Therefore, in such a case, all resistant plants segregate for the lethal
 phenotype in the next generation; this result indicates that each of the
 resistant plants is heterozygous for the mutation and hemizygous for the
 T-DNA insert causing the mutation. For those lines showing cosegregation
 of the T-DNA resistance marker and the lethal phenotype, PCR-based
 approaches, such as TAIL PCR (Liu and Whittier (1995), Genomics, 25:
 674-681) vectorette PCR (Riley et al. (1990) Nucleic Acids Research, 18:
 2887-2890), or a strategy such as the Genome Walker system (CLONTECH
 Laboratories, Inc, Palo Alto, Calif.), may be used to directly amplify
 plant DNA/T-DNA border fragments. Each of these techniques takes advantage
 of the fact that the DNA sequence of the insertion element is known, and
 can routinely be used to recover small (less than 5 kb) fragments adjacent
 to the known sequence. Alternatively, plasmid rescue may be used to
 isolate the plant DNA/T-DNA border fragments. Southern blot analysis may
 be performed as an initial step in the characterization of the molecular
 nature of each insertion. Southern blots are done with genomic DNA
 isolated from heterozygotes and using probes capable of hybridizing with
 the T-DNA vector DNA.
 Using the results of the Southern analysis, appropriate restriction enzymes
 are chosen to perform plasmid rescue in order to molecularly clone
 Arabidopsis thaliana genomic DNA flanking one or both sides of the T-DNA
 insertion. Plasmids obtained in this manner are analyzed by restriction
 enzyme digestion to sort the plasmids into classes based on their
 digestion pattern. For each class of plasmid clone, the DNA sequence is
 determined.
 The resulting sequences, obtained by any of the above outlined approaches,
 are analyzed for the presence of non-T-DNA vector sequences. When such
 sequences are found, they are used to search DNA and protein databases
 using the BLAST and BLAST2 programs (Altschul et al. (1990) J Mol. Biol.
 215: 403-410; Altschul et al (1997) Nucleic Acid Res. 25:3389-3402).
 Additional genomic and cDNA sequences for each gene are identified by
 standard molecular biology procedures.
 One method of confirming that the disrupted gene is the cause of the mutant
 phenotype is to transform a wild-type form of the gene into the mutant
 plant. Another method is identification of a second mutant allele showing
 a lethal phenotype. Alternatively, the mutant is phenocopied by
 specifically reducing expression of the disrupted gene in transgenic
 plants expressing an antisense version of the gene behind a synthetic
 promoter (Guyer et al. (1998) Genetics, 149: 633-639). Thus, for example,
 two other revertant alleles disrupting the 8388 gene are obtained by T-DNA
 insertion (mutants no. 14652 and 29863). Also, another mutant allele of
 the 18048 gene is obtained by EMS mutagenesis (mutant no. ttn5-2 with a
 mutation at base 195 of the coding sequence changing a Trp codon (TGG) to
 a stop codon (TGA)).
 I.b. Essentiality of the 4144 Gene in Arabidopsis Demonstrated by T-DNA
 Insertion Mutagenesis
 As shown in the examples below, the identification of a novel gene
 structure, as well as the essentiality of the 4144 gene for normal plant
 growth and development, have been demonstrated for the first time in
 Arabidopsis using T-DNA insertion mutagenesis. Having established the
 essentiality of 4144 function in plants and having identified the gene
 encoding this essential activity, the inventors thereby provide an
 important and sought after tool for new herbicide development.
 Arabidopsis insertional mutant lines segregating for seedling lethal
 mutations are identified as a first step in the identification of
 essential proteins. Starting with T2 seeds collected from single T1 plants
 containing T-DNA insertions in their genomes, those lines segregating
 homozygous seedling lethal seedlings are identified. These lines are found
 by placing seeds onto minimal plant growth media, which contains the
 fungicides benomyl and maxim, and screening for inviable seedlings after 7
 and 14 days in the light at room temperature. Inviable phenotypes include
 altered pigmentation or altered morphology. These phenotypes are observed
 either on plates directly or in soil following transplantation of
 seedlings.
 When a line is identified as segregating a seedling lethal, it is
 determined if the resistance marker in the T-DNA co-segregates with the
 lethality (Errampalli et al. (1991) The Plant Cell, 3:149-157).
 Co-segregation analysis is done by placing the seeds on media containing
 the selective agent and scoring the seedlings for resistance or
 sensitivity to the agent. Examples of selective agents used are hygromycin
 or phosphinothricin. About 35 resistant seedlings are transplanted to soil
 and their progeny are examined for the segregation of the seedling lethal.
 In the case in which the T-DNA insertion disrupts an essential gene, there
 is co-segregation of the resistance phenotype and the seedling lethal
 phenotype in every plant. Therefore, in such a case, all resistant plants
 segregate seedling lethals in the next generation; this result indicates
 that each of the resistant plants is heterozygous for the DNA causing both
 phenotypes.
 For those lines showing co-segregation of the T-DNA resistance marker and
 the seedling lethal phenotype, Southern analysis is performed as an
 initial step in the characterization of the molecular nature of each
 insertion. Southerns are done with genomic DNA isolated from heterozygotes
 and using probes capable of hybridizing with the T-DNA vector DNA. Using
 the results of the Southern analysis, appropriate restriction enzymes are
 chosen to perform plasmid rescue in order to molecularly clone Arabidopsis
 genomic DNA flanking one or both sides of the T-DNA insertion. Plasmids
 obtained in this manner are analyzed by restriction enzyme digestion to
 sort the plasmids into classes based on their digestion pattern. For each
 class of plasmid clone, the DNA sequence is determined. The resulting
 sequences are analyzed for the presence of non-T-DNA vector sequences.
 When such sequences are found, they are used to search DNA and protein
 databases using the BLAST and BLAST2 programs (Altschul et al. (1990) J
 Mol. Biol. 215: 403-410; Altschul et al (1997) Nucleic Acid Res.
 25:3389-3402). Additional genomic and cDNA sequences for each gene are
 identified by standard molecular biology procedures.
 II. Sequence of the Arabidopsis 8388, 18048, 16713 and 4144 Genes
 The Arabidopsis 8388 gene is identified by isolating DNA flanking the T-DNA
 border from the tagged embryo-lethal line #8388. Arabidopsis DNA flanking
 the T-DNA border is identical to regions of two sequenced EST clones from
 Arabidopsis (accession numbers H77096 and R30603). The inventors are the
 first to demonstrate that the 8388 gene product is essential for normal
 growth and development in plants, as well as defining the function of the
 8388 gene product through protein homology. The present invention
 discloses the cDNA nucleotide sequence of the Arabidopsis 8388 gene as
 well as the amino acid sequence of the Arabidopsis 8388 protein. The
 nucleotide sequence corresponding to the genomic DNA, single exon , coding
 region is set forth in SEQ ID NO:1, and the amino acid sequence encoding
 the protein is set forth in SEQ ID NO:2. The nucleotide sequence
 corresponding to the complete cDNA, which includes 5' UTR and coding and
 3' UTR sequences, is set forth in SEQ ID NO:3. The present invention also
 encompasses an isolated amino acid sequence derived from a plant, wherein
 said amino acid sequence is identical or substantially similar to the
 amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID
 NO:1, wherein said amino acid sequence has 8388 activity. Using BLASTX
 (2.0.7) programs with the default settings, the sequence of the 8388 gene
 shows similarity to DEAD box RNA helicase. Notable species similarities
 include: human EIF-4A-I [Genbank peptide accession #417180]; mouse EIF-4A
 [Genbank peptide accession #72888]; mouse EIF-4A-I [Genbank peptide
 accession #90965]; and rabbit EIF-4A-I [Genbank peptide accession
 #266336].
 The Arabidopsis 18048 gene is identified by isolating DNA flanking the
 T-DNA border from the tagged embryo-lethal line #18048. Arabidopsis DNA
 flanking the T-DNA border is identical to a sequenced BAC clone (T30D6,
 accession number AC006439). The inventors are the first to demonstrate
 that the 18048 gene product is essential for normal growth and development
 in plants, as well as defining the function of the 18048 gene product
 through protein homology. The present invention discloses the cDNA
 nucleotide sequence of the Arabidopsis 18048 gene as well as the amino
 acid sequence of the Arabidopsis 18048 protein. The nucleotide sequence
 corresponding to the cDNA coding region is set forth in SEQ ID NO:5, and
 the amino acid sequence encoding the protein is set forth in SEQ ID NO:6.
 The present invention also encompasses an isolated amino acid sequence
 derived from a plant, wherein said amino acid sequence is identical or
 substantially similar to the amino acid sequence encoded by the nucleotide
 sequence set forth in SEQ ID NO:5, wherein said amino acid sequence has
 18048 activity. Using BLASTX (2.0.8) programs with the default settings,
 the sequence of the 18048 gene shows similarity to ADP-ribosylation factor
 genes. Notable species similarities include: human [accession #NP.sub.--
 001658], rat [accession #O08697], Drosophila [accession #Q06849],
 Caenorhabditis elegans [accession #CAA90353], Schizosaccharomyces pombe
 [accession #Q09767], maize [accession #P49076], and soybean [accession
 number AAD17207].
 The Arabidopsis 16713 gene is identified by isolating DNA flanking the
 T-DNA border from the tagged embryo-lethal line #16713. Arabidopsis DNA
 flanking the T-DNA border is identical to a portion of sequence to the P1
 clone MIF21 (Accession #AB023039). Annotation suggests that a gene is
 present in the region disrupted by the T-DNA. BLAST-N searches using
 default settings, using the annotated gene region, reveals public EST
 clones with sequence identity to the predicted gene, indicating that this
 region contains an expressed gene. The EST clones are: 144H12T7, 184O20T7,
 126L22T7, VBVWD08, 204J9T7, 129A14, and 174A7T7. The inventors are the
 first to demonstrate that the 16713 gene product is essential for normal
 growth and development in plants, as well as defining the function of the
 16713 gene product through protein homology. The present invention
 discloses the cDNA nucleotide sequence of the Arabidopsis 16713 gene as
 well as the amino acid sequence of the Arabidopsis 16713 protein. The
 nucleotide sequence corresponding to the cDNA coding region is set forth
 in SEQ ID NO:7, and the amino acid sequence encoding the protein is set
 forth in SEQ ID NO:8. The present invention also encompasses an isolated
 amino acid sequence derived from a plant, wherein said amino acid sequence
 is identical or substantially similar to the amino acid sequence encoded
 by the nucleotide sequence set forth in SEQ ID NO:1, wherein said amino
 acid sequence has 16713 activity. Using BLASTX (1.4.11) programs with the
 default settings, the sequence of the 16713 gene shows similarity to
 acetoacetyl coA thiolase genes. Notable species similarities include:
 radish (accession #CAA55006), maize (accession #AAD44539), yeast
 (accession #P41338), human (accession #BAA14278), rat (accession
 #BAA03016), Caenorhabditis elegans (accession #AAA82403), and E. coli
 (accession number Q46939).
 The Arabidopsis 4144 gene is identified by isolating DNA flanking the T-DNA
 border from the tagged seedling-lethal line #4144. A region of the
 Arabidopsis DNA flanking the T-DNA border shows 100% identity to
 preliminary Arabidopsis genomic sequence (designated: Preliminary CSHL076
 T25P22-99.03.10-68148.seq; found at
 http://genome-www2.stanford.edu/cgi-bin/AtDB/
 getseq?database=cshlprel&item=CSHL076). The inventors are the first to
 demonstrate that the 4144 gene product is essential for normal growth and
 development in plants, as well as defining the function of the 4144 gene
 through protein homology. The present invention discloses the cDNA coding
 nucleotide sequence of the Arabidopsis 4144 gene as well as the amino acid
 sequence of the Arabidopsis 4144 protein. The nucleotide sequence
 corresponding to the genomic DNA is set forth in SEQ ID NO:23.
 III. Recombinant Production of 8388, 18048, 16713, and 4144 Activities and
 Uses Thereof
 For recombinant production of 8388, 18048, 16713, or 4144 activity in a
 host organism, a nucleotide sequence encoding a protein having one of the
 above activities is inserted into an expression cassette designed for the
 chosen host and introduced into the host where it is recombinantly
 produced. For example, SEQ ID NO:1, or nucleotide sequences substantially
 similar to SEQ ID NO:1, or homologs of the 8388 coding sequence can be
 used for the recombinant production of a protein having 8388 activity. For
 example, SEQ ID NO:5, or nucleotide sequences substantially similar to SEQ
 ID NO:5, or homologs of the 18048 coding sequence can be used for the
 recombinant production of a protein having 18048 activity. For example,
 SEQ ID NO:7, or nucleotide sequences substantially similar to SEQ ID NO:7,
 or homologs of the 16713 coding sequence can be used for the recombinant
 production of a protein having 16713 activity. For example, SEQ ID NO:21,
 or nucleotide sequences substantially similar to SEQ ID NO:21, or homologs
 of the 4144 coding sequence can be used for the recombinant production of
 a protein having 4144 activity. The choice of specific regulatory
 sequences such as promoter, signal sequence, 5' and 3' untranslated
 sequences, and enhancer appropriate for the chosen host is within the
 level of skill of the routineer in the art. The resultant molecule,
 containing the individual elements operably linked in proper reading
 frame, may be inserted into a vector capable of being transformed into the
 host cell. Suitable expression vectors and methods for recombinant
 production of proteins are well known for host organisms such as E. coli,
 yeast, and insect cells (see, e.g., Luckow and Summers, Bio/Technol. 6: 47
 (1988), and baculovirus expression vectors, e.g., those derived from the
 genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A
 preferred baculovirus/insect system is pAcHLT (Pharmingen, San Diego,
 Calif.) used to transfect Spodoptera frugiperda Sf9 cells (ATCC) in the
 presence of linear Autographa californica baculovirus DNA (Pharmigen, San
 Diego, Calif.). The resulting virus is used to infect HighFive Tricoplusia
 ni cells (Invitrogen, La Jolla, Calif.).
 In a preferred embodiment, the nucleotide sequence encoding a protein
 having 8388, 18048, 16713, or 4144 activity is derived from an eukaryote,
 such as a mammal, a fly or a yeast, but is preferably derived from a
 plant. In a further preferred embodiment, the nucleotide sequence is
 identical or substantially similar to the nucleotide sequence set forth in
 SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:21, respectively, or
 encodes a protein having 8388, 18048, 16713, or 4144 activity,
 respectively, whose amino acid sequence is identical or substantially
 similar to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:6,
 SEQ ID NO:8, or SEQ ID NO:22, respectively. The nucleotide sequence set
 forth in SEQ ID NO:1 encodes the Arabidopsis 8388 protein, whose amino
 acid sequence is set forth in SEQ ID NO:2. The nucleotide sequence set
 forth in SEQ ID NO:5 encodes the Arabidopsis 18048 protein, whose amino
 acid sequence is set forth in SEQ ID NO:6. The nucleotide sequence set
 forth in SEQ ID NO:7 encodes the Arabidopsis 16713 protein, whose amino
 acid sequence is set forth in SEQ ID NO:8. The nucleotide sequence set
 forth in SEQ ID NO:21 encodes the Arabidopsis 4144 protein, whose amino
 acid sequence is set forth in SEQ ID NO:22. In another preferred
 embodiment, the nucleotide sequences are derived from a prokaryote,
 preferably a bacteria, e.g. E. coli. Recombinantly produced protein having
 8388, 18048, 16713, or 4144 activity is isolated and purified using a
 variety of standard techniques. The actual techniques that may be used
 will vary depending upon the host organism used, whether the protein is
 designed for secretion, and other such factors familiar to the skilled
 artisan (see, e.g. chapter 16 of Ausubel, F. et al., "Current Protocols in
 Molecular Biology", pub. by John Wiley & Sons, Inc. (1994).
 Assays Utilizing the 8388, 18048, 16713, or 4144 Protein
 Recombinantly produced 8388, 18048, 16713, or 4144 proteins having 8388,
 18048, 16713, or 4144 activities, respectively, are useful for a variety
 of purposes. For example, they can be used in in vitro assays to screen
 known herbicidal chemicals whose target has not been identified to
 determine if they inhibit 8388, 18048, 16713, or 4144. Such in vitro
 assays may also be used as more general screens to identify chemicals that
 inhibit such enzymatic activity and that are therefore novel herbicide
 candidates. Alternatively, recombinantly produced 8388, 18048, 16713, or
 4144 proteins having 8388, 18048, 16713, or 4144 activity may be used to
 elucidate the complex structure of these molecules and to further
 characterize their association with known inhibitors in order to
 rationally design new inhibitory herbicides as well as herbicide tolerant
 forms of the enzymes.
 In vitro Inhibitor Assay
 An in vitro assay usefiul for identifying inhibitors of enzymes encoded by
 essential plant genes, such as, e.g. 3-ketoacyl-CoA thiolase, comprises
 the steps of: a) reacting an enzyme, e.g. an enzyme having 3-ketoacyl-CoA
 thiolase activity and the substrate thereof in the presence of a suspected
 inhibitor of the enzyme's function; b) comparing the rate of enzymatic
 activities in the presence of the suspected inhibitor to the rate of
 enzymatic activities under the same conditions in the absence of the
 suspected inhibitor; and c) determining whether the suspected inhibitor
 inhibits the enzyme activity, e.g. the 3-ketoacyl-CoA thiolase activity.
 The inhibitory effect, e.g. on 3-ketoacyl-CoA thiolase, is determined by a
 reduction or complete inhibition of product formation in the assay. In a
 preferred embodiment, such a determination is made by comparing, in the
 presence and absence of the candidate inhibitor, the amount of product
 formed in the in vitro assay using fluorescence or absorbance detection. A
 preferred substrate for 3-ketoacyl-CoA thiolase is Acetoacetyl-CoA
 (AcAc-CoA). Additional substrates include palmitoyl coenzyme A, myristoyl
 coenzyme A, or lauroyl coenzyme A.
 In vitro Inhibitor Assays: Discovery of Small Molecule Ligand that
 Interacts with the Gene Product of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7,
 or SEQ ID NO:21
 Once a protein has been identified as a potential herbicide target, the
 next step is to develop an assay that allows screening large number of
 chemicals to determine which ones interact with the protein. Although it
 is straightforward to develop assays for proteins of known function,
 developing assays with proteins of unknown functions is more difficult.
 This difficulty can be overcome by using technologies that can detect
 interactions between a protein and a compound without knowing the
 biological function of the protein. A short description of three methods
 is presented, including fluorescence correlation spectroscopy,
 surface-enhanced laser desorption/ionization, and biacore technologies.
 Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972
 but it is only in recent years that the technology to perform FCS became
 available (Madge et al. (1972) Phys. Rev. Lett., 29: 705-708; Maiti et al.
 (1997) Proc. Natl. Acad. Sci. USA, 94: 11753-11757). FCS measures the
 average diffusion rate of a fluorescent molecule within a small sample
 volume. The sample size can be as low as 10.sup.3 fluorescent molecules
 and the sample volume as low as the cytoplasm of a single bacterium. The
 diffusion rate is a function of the mass of the molecule and decreases as
 the mass increases. FCS can therefore be applied to protein-ligand
 interaction analysis by measuring the change in mass and therefore in
 diffusion rate of a molecule upon binding. In a typical experiment, the
 target to be analyzed is expressed as a recombinant protein with a
 sequence tag, such as a poly-histidine sequence, inserted at the N or
 C-terminus. The expression takes place in E. coli, yeast or insect cells.
 The protein is purified by chromatography. For example, the poly-histidine
 tag can be used to bind the expressed protein to a metal chelate column
 such as Ni2+ chelated on iminodiacetic acid agarose. The protein is then
 labeled with a fluorescent tag such as carboxytetramethylrhodamine or
 BODIPY.RTM. (Molecular Probes, Eugene, Oreg.). The protein is then exposed
 in solution to the potential ligand, and its diffusion rate is determined
 by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood,
 N.Y.). Ligand binding is determined by changes in the diffusion rate of
 the protein.
 Surface-Enhanced Laser Desorption/Ionization (SELDI) was invented by
 Hutchens and Yip during the late 1980's (Hutchens and Yip (1993) Rapid
 Commun. Mass Spectrom. 7: 576-580). When coupled to a time-of-flight mass
 spectrometer (TOF), SELDI provides a mean to rapidly analyze molecules
 retained on a chip. It can be applied to ligand-protein interaction
 analysis by covalently binding the target protein on the chip and analyze
 by MS the small molecules that bind to this protein (Worrall et al. (1998)
 Anal. Biochem. 70: 750-756). In a typical experiment, the target to be
 analyzed is expressed as described for FCS. The purified protein is then
 used in the assay without further preparation. It is bound to the SELDI
 chip either by utilizing the poly-histidine tag or by other interaction
 such as ion exchange or hydrophobic interaction. The chip thus prepared is
 then exposed to the potential ligand via, for example, a delivery system
 capable to pipet the ligands in a sequential manner (autosampler). The
 chip is then submitted to washes of increasing stringency, for example a
 series of washes with buffer solutions containing an increasing ionic
 strength. After each wash, the bound material is analyzed by submitting
 the chip to SELDI-TOF. Ligands that specifically bind the target will be
 identified by the stringency of the wash needed to elute them.
 Biacore relies on changes in the refractive index at the surface layer upon
 binding of a ligand to a protein immobilized on the layer. In this system,
 a collection of small ligands is injected sequentially in a 2-5 microlitre
 cell with the immobilized protein. Binding is detected by surface plasmon
 resonance (SPR) by recording laser light refracting from the surface. In
 general, the refractive index change for a given change of mass
 concentration at the surface layer, is practically the same for all
 proteins and peptides, allowing a single method to be applicable for any
 protein (Liedberg et al. (1983) Sensors Actuators 4: 299-304; Malmquist
 (1993) Nature, 361: 186-187). In a typical experiment, the target to be
 analyzed is expressed as described for FCS. The purified protein is then
 used in the assay without further preparation. It is bound to the Biacore
 chip either by utilizing the poly-histidine tag or by other interaction
 such as ion exchange or hydrophobic interaction. The chip thus prepared is
 then exposed to the potential ligand via the delivery system incorporated
 in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands
 in a sequential manner (autosampler). The SPR signal on the chip is
 recorded and changes in the refractive index indicate an interaction
 between the immobilized target and the ligand. Analysis of the signal
 kinetics on rate and off rate allows the discrimination between
 non-specific and specific interaction.
 IV. In vivo Inhibitor Assay
 In one embodiment, a suspected herbicide, for example identified by in
 vitro screening, is applied to plants at various concentrations. The
 suspected herbicide is preferably sprayed on the plants. After application
 of the suspected herbicide, its effect on the plants, for example death or
 suppression of growth is recorded.
 In another embodiment, an in vivo screening assay for inhibitors of the
 8388, 18048, 16713, or 4144 activity uses transgenic plants, plant tissue,
 plant seeds or plant cells capable of overexpressing a nucleotide sequence
 having 8388, 18048, 16713, or 4144 activity, wherein the 8388, 18048,
 16713, or 4144 gene product is enzymatically active in the transgenic
 plants, plant tissue, plant seeds or plant cells. The nucleotide sequence
 is preferably derived from an eukaryote, such as a yeast, but is
 preferably derived from a plant. In a further preferred embodiment, the
 nucleotide sequence is identical or substantially similar to the
 nucleotide sequence set forth in SEQ ID NO:1, or encodes an enzyme having
 8388 activity, whose amino acid sequence is identical or substantially
 similar to the amino acid sequence set forth in SEQ ID NO:2. In a further
 preferred embodiment, the nucleotide sequence is identical or
 substantially similar to the nucleotide sequence set forth in SEQ ID NO:5,
 or encodes an enzyme having 18048 activity, whose amino acid sequence is
 identical or substantially similar to the amino acid sequence set forth in
 SEQ ID NO:6. In a further preferred embodiment, the nucleotide sequence is
 identical or substantially similar to the nucleotide sequence set forth in
 SEQ ID NO:7, or encodes an enzyme having 16713 activity, whose amino acid
 sequence is identical or substantially similar to the amino acid sequence
 set forth in SEQ ID NO:8. In a further preferred embodiment, the
 nucleotide sequence is identical or substantially similar to the
 nucleotide sequence set forth in SEQ ID NO:21, or encodes an enzyme having
 4144 activity, whose amino acid sequence is identical or substantially
 similar to the amino acid sequence set forth in SEQ ID NO:22. In another
 preferred embodiment, the nucleotide sequence is derived from a
 prokaryote, preferably a bacteria, e.g. E. coli.
 A chemical is then applied to the transgenic plants, plant tissue, plant
 seeds or plant cells and to the isogenic non-transgenic plants, plant
 tissue, plant seeds or plant cells, and the growth or viability of the
 transgenic and non-transformed plants, plant tissue, plant seeds or plant
 cells arc determined after application of the chemical and compared.
 Compounds capable of inhibiting the growth of the non-transgenic plants,
 but not affecting the growth of the transgenic plants are selected as
 specific inhibitors of 8388, 18048, 16713, or 4144 activity.
 V. Herbicide Tolerant Plants
 The present invention is further directed to plants, plant tissue, plant
 seeds, and plant cells tolerant to herbicides that inhibit the naturally
 occurring 8388, 18048, 16713, or 4144 activity in these plants, wherein
 the tolerance is conferred by an altered 8388, 18048, 16713, or 4144
 activity. Altered 8388, 18048, 16713, or 4144 activity may be conferred
 upon a plant according to the invention by increasing expression of
 wild-type herbicide-sensitive 8388, 18048, 16713, or 4144 gene, for
 example by providing additional wild-type 8388, 18048, 16713, or 4144
 genes and/or by overexpressing the endogenous 8388, 18048, 16713, or 4144
 gene, for example by driving expression with a strong promoter. Altered
 8388, 18048, 16713, or 4144 activity also may be accomplished by
 expressing nucleotide sequences that are substantially similar to SEQ ID
 NO:1, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:21, respectively, or homologs
 in a plant. Still further altered 8388, 18048, 16713, or 4144 activity is
 conferred on a plant by expressing modified herbicide-tolerant 8388,
 18048, 16713, or 4144 genes in the plant. Combinations of these techniques
 may also be used. Representative plants include any plants to which these
 herbicides are applied for their normally intended purpose. Preferred are
 agronomically important crops such as cotton, soybean, oilseed rape, sugar
 beet, maize, rice, wheat, barley, oats, rye, sorghum, millet, turf,
 forage, turf grasses, and the like.
 A. Increased Expression of Wild-Type 8388, 18048, 16713, or 4144
 Achieving altered 8388, 18048, 16713, or 4144 activity through increased
 expression results in a level of 8388, 18048, 16713, or 4144 activity in
 the plant cell at least sufficient to overcome growth inhibition caused by
 the herbicide when applied in amounts sufficient to inhibit normal growth
 of control plants. The level of expressed enzyme generally is at least two
 times, preferably at least five times, and more preferably at least ten
 times the natively expressed amount. Increased expression may be due to
 multiple copies of a wild-type 8388, 18048, 16713, or 4144 gene; multiple
 occurrences of the coding sequence within the gene (i.e. gene
 amplification) or a mutation in the non-coding, regulatory sequence of the
 endogenous gene in the plant cell. Plants having such altered gene
 activity can be obtained by direct selection in plants by methods known in
 the art (see, e.g. U.S. Pat. No. 5,162,602, and U.S. Pat. No. 4,761,373,
 and references cited therein). These plants also may be obtained by
 genetic engineering techniques known in the art. Increased expression of a
 herbicide-sensitive 8388, 18048, 16713, or 4144 gene can also be
 accomplished by transforming a plant cell with a recombinant or chimeric
 DNA molecule comprising a promoter capable of driving expression of an
 associated structural gene in a plant cell operatively linked to a
 homologous or heterologous structural gene encoding the 8388, 18048,
 16713, or 4144 protein or a homolog thereof. Preferably, the
 transformation is stable, thereby providing a heritable transgenic trait.
 B. Expression of Modified Herbicide-Tolerant 8388, 18048, 16713, or 4144
 Proteins
 According to this embodiment, plants, plant tissue, plant seeds, or plant
 cells are stably transformed with a recombinant DNA molecule comprising a
 suitable promoter functional in plants operatively linked to a coding
 sequence encoding a herbicide tolerant form of the 8388, 18048, 16713, or
 4144 protein. A herbicide tolerant form of the enzyme has at least one
 amino acid substitution, addition or deletion that confers tolerance to a
 herbicide that inhibits the unmodified, naturally occurring form of the
 enzyme. The transgenic plants, plant tissue, plant seeds, or plant cells
 thus created are then selected by conventional selection techniques,
 whereby herbicide tolerant lines are isolated, characterized, and
 developed. Below are described methods for obtaining genes that encode
 herbicide tolerant forms of 8388, 18048, 16713, or 4144 protein.
 One general strategy involves direct or indirect mutagenesis procedures on
 microbes. For instance, a genetically manipulatable microbe such as E.
 coli or S. cerevisiae may be subjected to random mutagenesis in vivo with
 mutagens such as UV light or ethyl or methyl methane sulfonate.
 Mutagenesis procedures are described, for example, in Miller, Experiments
 in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,
 N.Y. (1972); Davis et al., Advanced Bacterial Genetics, Cold Spring Harbor
 Laboratory, Cold Spring Harbor, N.Y. (1980); Sherman et al., Methods in
 Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
 (1983); and U.S. Pat. No. 4,975,374. The microbe selected for mutagenesis
 contains a normal, inhibitor-sensitive 8388, 18048, 16713, or 4144 gene
 and is dependent upon the activity conferTed by this gene. The mutagenized
 cells are grown in the presence of the inhibitor at concentrations that
 inhibit the unmodified gene. Colonies of the mutagenized microbe that grow
 better than the unmutagenized microbe in the presence of the inhibitor
 (i.e. exhibit resistance to the inhibitor) are selected for further
 analysis. 8388, 18048, 16713, or 4144 genes conferring tolerance to the
 inhibitor are isolated from these colonies, either by cloning or by PCR
 amplification, and their sequences are elucidated. Sequences encoding
 altered gene products are then cloned back into the microbe to confirm
 their ability to confer inhibitor tolerance.
 A method of obtaining mutant herbicide-tolerant alleles of a plant 8388,
 18048, 16713, or 4144 gene involves direct selection in plants. For
 example, the effect of a mutagenized 8388, 18048, 16713, or 4144 gene on
 the growth inhibition of plants such as Arabidopsis, soybean, or maize is
 determined by plating seeds sterilized by art-recognized methods on plates
 on a simple minimal salts medium containing increasing concentrations of
 the inhibitor. Such concentrations are in the range of 0.001, 0.003, 0.01,
 0.03, 0.1, 0.3, 1, 3, 10, 30, 110, 300, 1000 and 3000 parts per million
 (ppm). The lowest dose at which significant growth inhibition can be
 reproducibly detected is used for subsequent experiments. Determination of
 the lowest dose is routine in the art.
 Mutagenesis of plant material is utilized to increase the frequency at
 which resistant alleles occur in the selected population. Mutagenized seed
 material is derived from a variety of sources, including chemical or
 physical mutagenesis or seeds, or chemical or physical mutagenesis or
 pollen (Neuffer, In Maize for Biological Research Sheridan, ed. Univ.
 Press, Grand Forks, N.Dak., pp. 61-64 (1982)), which is then used to
 fertilize plants and the resulting M.sub.1 mutant seeds collected.
 Typically for Arabidopsis, M.sub.2 seeds (Lehle Seeds, Tucson, Ariz.),
 which are progeny seeds of plants grown from seeds mutagenized with
 chemicals, such as ethyl methane sulfonate, or with physical agents, such
 as gamma rays or fast neutrons, are plated at densities of up to 10,000
 seeds/plate (10 cm diameter) on minimal salts medium containing an
 appropriate concentration of inhibitor to select for tolerance. Seedlings
 that continue to grow and remain green 7-21 days after plating are
 transplanted to soil and grown to maturity and seed set. Progeny of these
 seeds are tested for tolerance to the herbicide. If the tolerance trait is
 dominant, plants whose seed segregate 3:1/resistant:sensitive are presumed
 to have been heterozygous for the resistance at the M.sub.2 generation.
 Plants that give rise to all resistant seed are presumed to have been
 homozygous for the resistance at the M.sub.2 generation. Such mutagenesis
 on intact seeds and screening of their M2 progeny seed can also be carried
 out on other species, for instance soybean (see, e.g. U.S. Pat. No.
 5,084,082). Alternatively, mutant seeds to be screened for herbicide
 tolerance are obtained as a result of fertilization with pollen
 mutagenized by chemical or physical means.
 Confirmation that the genetic basis of the herbicide tolerance is a 8388,
 18048, 16713, or 4144 gene is ascertained as exemplified below. First,
 alleles of the 8388, 18048, 16713, or 4144 gene from plants exhibiting
 resistance to the inhibitor are isolated using PCR with primers based
 either upon the Arabidopsis cDNA coding sequences shown in SEQ ID NO:1,
 SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:21, respectively, or, more
 preferably, based upon the unaltered 8388, 18048, 16713, or 4144 gene
 sequence from the plant used to generate tolerant alleles. After
 sequencing the alleles to determine the presence of mutations in the
 coding sequence, the alleles are tested for their ability to confer
 tolerance to the inhibitor on plants into which the putative
 tolerance-conferring alleles have been transformed. These plants can be
 either Arabidopsis plants or any other plant whose growth is susceptible
 to the 8388, 18048, 16713, or 4144 inhibitors. Second, the inserted 8388,
 18048, 16713, or 4144 genes are mapped relative to known restriction
 fragment length polymorphisms (RFLPs) (See, for example, Chang et al.
 Proc. Natl. Acad, Sci, USA 85: 6856-6860 (1988); Nam et al., Plant Cell 1:
 699-705 (1989), cleaved amplified polymorphic sequences (CAPS) (Konieczny
 and Ausubel (1993) The Plant Journal, 4(2): 403-410), or SSLPs (Bell and
 Ecker (1994) Genomics, 19: 137-144). The 8388, 18048, 16713, or 4144
 inhibitor tolerance trait is independently mapped using the same markers.
 When tolerance is due to a mutation in that 8388, 18048, 16713, or 4144
 gene, the tolerance trait maps to a position indistinguishable from the
 position of the 8388, 18048, 16713, or 4144 gene.
 Another method of obtaining herbicide-tolerant alleles of a 8388, 18048,
 16713, or 4144 gene is by selection in plant cell cultures. Explants of
 plant tissue, e.g. embryos, leaf disks, etc. or actively growing calluis
 or suspension cultures of a plant of interest are grown on medium in the
 presence of increasing concentrations of the inhibitory herbicide or an
 analogous inhibitor suitable for use in a laboratory environment. Varying
 degrees of growth are recorded in different cultures. In certain cultures,
 fast-growing variant colonies arise that continue to grow even in the
 presence of normally inhibitory concentrations of inhibitor. The frequency
 with which such faster-growing variants occur can be increased by
 treatment with a chemical or physical mutagen before exposing the tissues
 or cells to the inhibitor. Putative tolerance-conferring alleles of the
 8388, 18048, 16713, or 4144 gene are isolated and tested as described in
 the foregoing paragraphs. Those alleles identified as conferring herbicide
 tolerance may then be engineered for optimal expression and transformed
 into the plant. Alternatively, plants can be regenerated from the tissue
 or cell cultures containing these alleles.
 Still another method involves mutagenesis of wild-type, herbicide sensitive
 plant 8388, 18048, 16713, or 4144 genes in bacteria or yeast, followed by
 culturing the microbe on medium that contains inhibitory concentrations
 (i.e. sufficient to cause abnormal growth, inhibit growth or cause cell
 death) of the inhibitor, and then selecting those colonies that grow
 normally in the presence of the inhibitor. More specifically, a plant
 cDNA, such as the Arabidopsis cDNA encoding the 8388, 18048, 16713, or
 4144 protein, is cloned into a microbe that otherwise lacks the 8388,
 18048, 16713, or 4144 activity. The transformed microbe is then subjected
 to in vivo mutagenesis or to in vitro mutagenesis by any of several
 chemical or enzymatic methods known in the art, e.g. sodium bisulfite
 (Shortle et al., Methods Enzymol. 100:457-468 (1983); methoxylamine
 (Kadonaga et al., Nucleic Acids Res. 13:1733-1745 (1985);
 oligonucleotide-directed saturation mutagenesis (Hutchinson et al., Proc.
 Natl. Acacl. Sci. USA, 83:710-714 (1986); or various polymerase
 misincorporation strategies (see, e.g. Shortle et al., Proc. Natl. Acad.
 Sci. USA, 79:1588-1592 (1982); Shiraishi et al., Gene 64:313-319 (1988);
 and Leung et al., Technique 1:11-15 (1989). Colonies that grow normally in
 the presence of normally inhibitory concentrations of inhibitor are picked
 and purified by repeated restreaking. Their plasmids are purified and
 tested for the ability to confer tolerance to the inhibitor by
 retransforming them into the microbe lacking 8388, 18048, 16713, or 4144
 activity. The DNA sequences of cDNA inserts fromi plasmids that pass this
 test are then determined.
 Herbicide resistant 8388, 18048, 16713, or 4144 proteins are also obtained
 using methods involving in vitro recombination, also called DNA shuffling.
 By DNA shuffling, mutations, preferably random mutations, are introduced
 into nucleotide sequences encoding 8388, 18048, 16713, or 4144 activity.
 DNA shuffling also leads to the recombination and rearrangement of
 sequences within a 8388, 18048, 16713, or 4144 gene or to recombination
 and exchange of sequences between two or more different of 8388, 18048,
 16713, or 4144 genes. These methods allow for the production of millions
 of mutated 8388, 18048, 16713, or 4144 coding sequences. The mutated
 genes, or shuffled genes, are screened for desirable properties, e.g.
 improved tolerance to herbicides and for mutations that provide broad
 spectrum tolerance to the different classes of inhibitor chemistry. Such
 screens are well within the skills of a routineer in the art.
 In a preferred embodiment, a mutagenized 8388, 18048, 16713, or 4144 gene
 is formed from at least one template 8388, 18048, 16713, or 4144 gene,
 wherein the template 8388, 18048, 16713, or 4144 gene has been cleaved
 into double-stranded random fragments of a desired size, and comprising
 the steps of adding to the resultant population of double-stranded random
 fragments one or more single or double-stranded oligonucleotides, wherein
 said oligonucleotides comprise an area of identity and an area of
 heterology to the double-stranded random fragments; denaturing the
 resultant mixture of double-stranded random fragments and oligonucleotides
 into single-stranded fragments; incubating the resultant population of
 single-stranded fragments with a polymerase under conditions which result
 in the annealing of said single-stranded fragments at said areas of
 identity to form pairs of annealed fragments, said areas of identity being
 sufficient for one member of a pair to prime replication of the other,
 thereby forming a mutagenized double-stranded polynucleotide; and
 repeating the second and third steps for at least two further cycles,
 wherein the resultant mixture in the second step of a further cycle
 includes the mutagenized double-stranded polynucleotide from the third
 step of the previous cycle, and the further cycle forms a further
 mutagenized double-stranded polynucleotide, wherein the mutagenized
 polynucleotide is a mutated 8388, 18048, 16713, or 4144 gene having
 enhanced tolerance to a herbicide which inhibits naturally occurring 8388,
 18048, 16713, or 4144 activity. In a preferred embodiment, the
 concentration of a single species of double-stranded random fragment in
 the population of double-stranded random fragments is less than 1% by
 weight of the total DNA. In a further preferred embodiment, the template
 double-stranded polynucleotide comprises at least about 100 species of
 polynucleotides. In another preferred embodiment, the size of the
 double-stranded random fragments is from about 5 bp to 5 kb. In a further
 preferred embodiment, the fourth step of the method comprises repeating
 the second and the third steps for at least 10 cycles. Such method is
 described e.g. in Stemmer et al. (1994) Nature 370: 389-391, in U.S. Pat.
 No. 5,605,793, U.S. Pat. No. 5,811,238 and in Crameri et al. (1998) Nature
 391: 288-291, as well as in WO 97/20078, and these references are
 incorporated herein by reference.
 In another preferred embodiment, any combination of two or more different
 8388, 18048, 16713, or 4144 genes are mutagenized in vitro by a staggered
 extension process (StEP), as described e.g. in Zhao et al. (1998) Nature
 Biotechnology 16: 258-261. The two or more 8388, 18048, 16713, or 4144
 genes are used as template for PCR amplification with the extension cycles
 of the PCR reaction preferably carried out at a lower temperature than the
 optimal polymerization temperature of the polymerase. For example, when a
 thermostable polymerase with an optimal temperature of approximately
 72.degree. C. is used, the temperature for the extension reaction is
 desirably below 72.degree. C., more desirably below 65.degree. C.,
 preferably below 60.degree. C., more preferably the temperature for the
 extension reaction is 55.degree. C. Additionally, the duration of the
 extension reaction of the PCR cycles is desirably shorter than usually
 carried out in the art, more desirably it is less than 30 seconds,
 preferably it is less than 15 seconds, more preferably the duration of the
 extension reaction is 5 seconds. Only a short DNA fragment is polymerized
 in each extension reaction, allowing template switch of the extension
 products between the starting DNA molecules after each cycle of
 denaturation and annealing, thereby generating diversity among the
 extension products. The optimal number of cycles in the PCR reaction
 depends on the length of the 8388, 18048, 16713, or 4144 genes to be
 mutagenized but desirably over 40 cycles, more desirably over 60 cycles,
 preferably over 80 cycles are used. Optimal extension conditions and the
 optimal number of PCR cycles for every combination of 8388, 18048, 16713,
 or 4144 genes are determined as described in using procedures well-known
 in the art. The other parameters for the PCR reaction are essentially the
 same as commonly used in the art. The primers for the amplification
 reaction are preferably designed to anneal to DNA sequences located
 outside of the 8388, 18048, 16713, or 4144 genes, e.g. to DNA sequences of
 a vector comprising the 8388, 18048, 16713, or 4144 genes, whereby the
 different 8388, 18048, 16713, or 4144 genes used in the PCR reaction are
 preferably comprised in separate vectors. The primers desirably anneal to
 sequences located less than 500 bp away from 8388, 18048, 16713, or 4144
 sequences, preferably less than 200 bp away from the 8388, 18048, 16713,
 or 4144 sequences, more preferably less than 120 bp away from the 8388,
 18048, 16713, or 4144 sequences. Preferably, the 8388, 18048, 16713, or
 4144 sequences are surrounded by restriction sites, which are included in
 the DNA sequence amplified during the PCR reaction, thereby facilitating
 the cloning of the amplified products into a suitable vector.
 In another preferred embodiment, fragments of 8388, 18048, 16713, or 4144
 genes having cohesive ends are produced as described in WO 98/05765. The
 cohesive ends are produced by ligating a first oligonucleotide
 corresponding to a part of a 8388, 18048, 16713, or 4144 gene to a second
 oligonucleotide not present in the gene or corresponding to a part of the
 gene not adjoining to the part of the gene corresponding to the first
 oligonucleotide, wherein the second oligonucleotide contains at least one
 ribonucleotide. A double-stranded DNA is produced using the first
 oligonucleotide as template and the second oligonucleotide as primer. The
 ribonucleotide is cleaved and removed. The nucleotide(s) located 5' to the
 ribonucleotide is also removed, resulting in double-stranded fragments
 having cohesive ends. Such fragments are randomly reassembled by ligation
 to obtain novel combinations of gene sequences.
 In yet another embodiment, herbicide-resistant 8388, 18048, 16713, or 4144
 proteins are produced using the incremental truncation for the creation of
 hybrid enzymes (ITCHY), as described in Ostermejer et al. (1999) Nature
 Biotechnology 17:1205-1209), and this reference is incorporated herein by
 reference.
 Any 8388, 18048, 16713, or 4144 gene or any combination of 8388, 18048,
 16713, or 4144 genes is used for in vitro recombination in the context of
 the present invention, for example, a 8388, 18048, 16713, or 4144 gene
 derived from a plant, such as, e.g. Arabidopsis thaliana, e.g. a 8388,
 18048, 16713, or 4144 gene set forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID
 NO:7, or SEQ ID NO:21, respectively. A 8388-like gene from E. coli, yeast,
 human, or mouse (Luking et al. (1998) Critical Reviews in Biochemistry and
 Molecular Biology, 33 (4): 259-296), a 18048-like gene from human or
 Drosophila (Clark et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90 (19):
 8952-8956 or other like genes), a 16713-like gene (Vollack and Bach (1996)
 Plant Physiol. 111: 1097-1107 or other like genes), all of which are
 incorporated herein by reference. Whole 8388, 18048, 16713, or 4144 genes
 or portions thereof are used in the context of the present invention. The
 library of mutated 8388, 18048, 16713, or 4144 genes obtained by the
 methods described above are cloned into appropriate expression vectors and
 the resulting vectors are transformed into an appropriate host, for
 example an algae like Chlamydomonas, a yeast or a bacteria. An appropriate
 host is preferably a host that otherwise lacks 8388, 18048, 16713, or 4144
 activity, for example E. coli. Host cells transformed with the vectors
 comprising the library of mutated 8388, 18048, 16713, or 4144 genes are
 cultured on medium that contains inhibitory concentrations of the
 inhibitor and those colonies that grow in the presence of the inhibitor
 are selected. Colonies that grow in the presence of normally inhibitory
 concentrations of inhibitor are picked and purified by repeated
 restreaking. Their plasmids are purified and the DNA sequences of cDNA
 inserts from plasmids that pass this test are then determined.
 An assay for identifying a modified 8388, 18048, 16713, or 4144 gene that
 is tolerant to an inhibitor may be performed in the same manner as the
 assay to identify inhibitors of the 8388, 18048, 16713, or 4144 activity
 (Inhibitor Assay, above) with the following modifications: First, a mutant
 8388, 18048, 16713, or 4144 protein is substituted in one of the reaction
 mixtures for the wild-type 8388, 18048, 16713, or 4144 protein of the
 inhibitor assay. Second, an inhibitor of wild-type enzyme is present in
 both reaction mixtures. Third, mutated activity (activity in the presence
 of inhibitor and mutated enzyme) and unmutated activity (activity in the
 presence of inhibitor and wild-type enzyme) are compared to determine
 whether a significant increase in enzymatic activity is observed in the
 mutated activity when compared to the unmutated activity. Mutated activity
 is any measure of activity of the mutated enzyme while in the presence of
 a suitable substrate and the inhibitor. Unmutated activity is any measure
 of activity of the wild-type enzyme while in the presence of a suitable
 substrate and the inhibitor.
 In addition to being used to create herbicide-tolerant plants, genes
 encoding herbicide tolerant 8388, 18048, 16713, or 4144 protein can also
 be used as selectable markers in plant cell transformation methods. For
 example, plants, plant tissue, plant seeds, or plant cells transformed
 with a heterologous DNA sequence can also be transformed with a sequence
 encoding an altered 8388, 18048, 16713, or 4144 activity capable of being
 expressed by the plant. The transformed cells are transferred to medium
 containing an inhibitor of the enzyme in an amount sufficient to inhibit
 the growth or survivability of plant cells not expressing the modified
 coding sequence, wherein only the transformed cells will grow. The method
 is applicable to any plant cell capable of being transformed with a
 modified 8388, 18048, 16713, or 4144 gene, and can be used with any
 heterologous DNA sequence of interest. Expression of the heterologous DNA
 sequence and the modified gene can be driven by the same promoter
 functional in plant cells, or by separate promoters.
 VI. Plant Transformation Technology
 A wild-type or herbicide-tolerant form of the 8388, 18048, 16713, or 4144
 gene, or homologs thereof, can be incorporated in plant or bacterial cells
 using conventional recombinant DNA technology. Generally, this involves
 inserting a DNA molecule encoding the 8388, 18048, 16713, or 4144 gene
 into an expression system to which the DNA molecule is heterologous (i.e.,
 not normally present) using standard cloning procedures known in the art.
 The vector contains the necessary elements for the transcription and
 translation of the inserted protein-coding sequences in a host cell
 containing the vector. A large number of vector systems known in the art
 can be used, such as plasmids, bacteriophage viruses and other modified
 viruses. The components of the expression system may also be modified to
 increase expression. For example, truncated sequences, nucleotide
 substitutions, nucleotide optimization or other modifications may be
 employed. Expression systems known in the art can be used to transform
 virtually any crop plant cell under suitable conditions. A heterologous
 DNA sequence comprising a wild-type or herbicide-tolerant form of the
 8388, 18048, 16713, or 4144 gene is preferably stably transformed and
 integrated into the genome of the host cells. In another preferred
 embodiment, the heterologous DNA sequence comprising a wild-type or
 herbicide-tolerant form of the 8388, 18048, 16713, or 4144 gene located on
 a self-replicating vector. Examples of self-replicating vectors are
 viruses, in particular gemini viruses. Transformed cells can be
 regenerated into whole plants such that the chosen form of the 8388,
 18048, 16713, or 4144 gene confers herbicide tolerance in the transgenic
 plants.
 A. Requirements for Construction of Plant Expression Cassettes
 Gene sequences intended for expression in transgenic plants are first
 assembled in expression cassettes behind a suitable promoter expressible
 in plants. The expression cassettes may also comprise any further
 sequences required or selected for the expression of the heterologous DNA
 sequence. Such sequences include, but are not restricted to, transcription
 terminators, extraneous sequences to enhance expression such as introns,
 vital sequences, and sequences intended for the targeting of the gene
 product to specific organelles and cell compartments. These expression
 cassettes can then be easily transferred to the plant transformation
 vectors described infra. The following is a description of various
 components of typical expression cassettes.
 1. Promoters
 The selection of the promoter used in expression cassettes will determine
 the spatial and temporal expression pattern of the heterologous DNA
 sequence in the plant transformed with this DNA sequence. Selected
 promoters will express heterologous DNA sequences in specific cell types
 (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in
 specific tissues or organs (roots, leaves or flowers, for example) and the
 selection will reflect the desired location of accumulation of the gene
 product. Alternatively, the selected promoter may drive expression of the
 gene under various inducing conditions. Promoters vary in their strength,
 i.e., ability to promote transcription. Depending upon the host cell
 system utilized, any one of a number of suitable promoters known in the
 art can be used. For example, for constitutive expression, the CaMV 35S
 promoter, the rice actin promoter, or the ubiquitin promoter may be used.
 For regulatable expression, the chemically inducible PR-1 promoter from
 tobacco or Arabidopsis may be used (see, e.g., U.S. Pat. No. 5,689,044).
 2. Transcriptional Terminators
 A variety of transcriptional terminators are available for use in
 expression cassettes. These are responsible for the termination of
 transcription beyond the heterologous DNA sequence and its correct
 polyadenylation. Appropriate transcriptional terminators are those that
 are known to function in plants and include the CaMV 35S terminator, the
 tml terminator, the nopaline synthase terminator and the pea rbcS E9
 terminator. These can be used in both monocotyledonous and dicotyledonous
 plants.
 3. Sequences for the Enhancement or Regulation of Expression
 Numerous sequences have been found to enhance gene expression from within
 the transcriptional unit and these sequences can be used in conjunction
 with the genes of this invention to increase their expression in
 transgenic plants. For example, various intron sequences such as introns
 of the maize AdhI gene have been shown to enhance expression, particularly
 in monocotyledonous cells. In addition, a number of non-translated leader
 sequences derived from viruses are also known to enhance expression, and
 these are particularly effective in dicotyledonous cells.
 4. Coding Sequence Optimization
 The coding sequence of the selected gene may be genetically engineered by
 altering the coding sequence for optimal expression in the crop species of
 interest. Methods for modifying coding sequences to achieve optimal
 expression in a particular crop species are well known (see, e.g. Perlak
 et al., Proc. Natl. Acaci. Sci. USA 88: 3324 (1991); and Koziel et al,
 Bio/technol. 11: 194 (1993)).
 5. Targeting of the Gene Product Within the Cell
 Various mechanisms for targeting gene products are known to exist in plants
 and the sequences controlling the functioning of these mechanisms have
 been characterized in some detail. For example, the targeting of gene
 products to the chloroplast is controlled by a signal sequence found at
 the amino terminal end of various proteins which is cleaved during
 chloroplast import to yield the mature protein (e.g. Comai et al. J. Biol.
 Chem. 263: 15104-15109 (1988)). Other gene products are localized to other
 organelles such as the mitochondrion and the peroxisome (e.g. Unger et al.
 Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products
 can also be manipulated to effect the targeting of heterologous products
 encoded by DNA sequences to these organelles. In addition, sequences have
 been characterized which cause the targeting of products encoded by DNA
 sequences to other cell compartments. Amino terminal sequences are
 responsible for targeting to the ER, the apoplast, and extracellular
 secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783
 (1990)). Additionally, amino terminal sequences in conjunction with
 carboxy terminal sequences are responsible for vacuolar targeting of gene
 products (Shinshi et aL Plant Molec. Biol. 14: 357-368 (1990)). By the
 fusion of the appropriate targeting sequences described above to
 heterologous DNA sequences of interest it is possible to direct this
 product to any organelle or cell compartment.
 B. Construction of Plant Transformation Vectors
 Numerous transformation vectors available for plant transformation are
 known to those of ordinary skill in the plant transformation arts, and the
 genes pertinent to this invention can be used in conjunction with any such
 vectors. The selection of vector will depend upon the preferred
 transformation technique and the target species for transformation. For
 certain target species, different antibiotic or herbicide selection
 markers may be preferred. Selection markers used routinely in
 transformation include the nptII gene, which confers resistance to
 kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268
 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which
 confers resistance to the herbicide phosphinothricin (White et al., Nucl.
 Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631
 (1990)), the hph gene, which confers resistance to the antibiotic
 hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), the manA
 gene, which allows for positive selection in the presence of mannose
 (Miles and Guest (1984) Gene, 32:41-48; U.S. Pat. No. 5,767,378), and the
 dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO
 J. 2(7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance
 to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642).
 1. Vectors Suitable for Agrobacterium Transformation
 Many vectors are available for transformation using Agrobacterium
 tumefaciens. These typically carry at least one T-DNA border sequence and
 include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Typical
 vectors suitable for Agrobacterium transformation include the binary
 vectors pCIB200 and pCIB2001, as well as the binary vector pCIB10 and
 hygromycin selection derivatives thereof. (See, for example, U.S. Pat. No.
 5,639,949).
 2. Vectors Suitable for non-Agrobacterium Transformation
 Transformation without the use of Agrobacterium tumefaciens circumvents the
 requirement for T-DNA sequences in the chosen transformation vector and
 consequently vectors lacking these sequences can be utilized in addition
 to vectors such as the ones described above which contain T-DNA sequences.
 Transformation techniques that do not rely on Agrobacterium include
 transformation via particle bombardment, protoplast uptake (e.g. PEG and
 electroporation) and microinjection. The choice of vector depends largely
 on the preferred selection for the species being transformed. Typical
 vectors suitable for non-Agrobacterium transformation include pCIB3064,
 pSOG19, and pSOG35. (See, for example, U.S. Pat. No. 5,639,949).
 C. Transformation Techniques
 Once the coding sequence of interest has been cloned into an expression
 system, it is transformed into a plant cell. Methods for transformation
 and regeneration of plants are well known in the art. For example, Ti
 plasmid vectors have been utilized for the delivery of foreign DNA, as
 well as direct DNA uptake, liposomes, electroporation, micro-injection,
 and microprojectiles. In addition, bacteria from the genus Agrobacterium
 can be utilized to transform plant cells.
 Transformation techniques for dicotyledons are well known in the art and
 include Agrobacterium-based techniques and techniques that do not require
 Agrobacterium. Non-Agrobacterium techniques involve the uptake of
 exogenous genetic material directly by protoplasts or cells. This can be
 accomplished by PEG- or electroporation-mediated uptake, particle
 bombardment-mediated delivery, or microinjection. In each case the
 transformed cells are regenerated to whole plants using standard
 techniques known in the art.
 Transformation of most monocotyledon species has now also become routine.
 Preferred techniques include direct gene transfer into protoplasts using
 PEG or electroporation techniques, particle bombardment into callus
 tissue, as well as Agrobacterium-mediated transformation.
 D. Plastid Transformation
 In another preferred embodiment, a nucleotide sequence encoding a
 polypeptide having 8388, 18048, 16713, or 4144 activity is directly
 transformed into the plastid genome. Plastid expression, in which genes
 are inserted by homologous recombination into the several thousand copies
 of the circular plastid genome present in each plant cell, takes advantage
 of the enormous copy number advantage over nuclear-expressed genes to
 permit expression levels that can readily exceed 10% of the total soluble
 plant protein. In a preferred embodiment, the nucleotide sequence is
 inserted into a plastid targeting vector and transformed into the plastid
 genome of a desired plant host. Plants homoplasmic for plastid genomes
 containing the nucleotide sequence are obtained, and are preferentially
 capable of high expression of the nucleotide sequence.
 Plastid transformation technology is for example extensively described in
 U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,877,462 in PCT
 application no. WO 95/16783 and WO 97/32977, and in McBride et al. (1994)
 Proc. Natl. Acad. Sci. USA 91, 7301-7305, all incorporated herein by
 reference in their entirety. The basic technique for plastid
 transformation involves introducing regions of cloned plastid DNA flanking
 a selectable marker together with the nucleotide sequence into a suitable
 target tissue, e.g., using biolistics or protoplast transformation (e.g.,
 calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking
 regions, termed targeting sequences, facilitate homologous recombination
 with the plastid genome and thus allow the replacement or modification of
 specific regions of the plastome. Initially, point mutations in the
 chloroplast 16S rRNA and rps12 genes conferring resistance to
 spectinomycin and/or streptomycin are utilized as selectable markers for
 transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc.
 Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992)
 Plant Cell 4, 39-45). The presence of cloning sites between these markers
 allowed creation of a plastid targeting vector for introduction of foreign
 genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606).
 Substantial increases in transformation frequency are obtained by
 replacement of the recessive rRNA or r-protein antibiotic resistance genes
 with a dominant selectable marker, the bacterial aadA gene encoding the
 spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase
 (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917).
 Other selectable markers useful for plastid transformation are known in
 the art and encompassed within the scope of the invention.
 VII. Breeding
 The wild-type or altered form of a 8388, 18048, 16713, or 4144 gene of the
 present invention can be utilized to confer herbicide tolerance to a wide
 variety of plant cells, including those of gymnosperms, monocots, and
 dicots. Although the gene can be inserted into any plant cell falling
 within these broad classes, it is particularly useful in crop plant cells,
 such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato,
 sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli,
 turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper,
 celery, canot, squash, pumpkin, zucchini, cucumber, apple, pear, quince,
 melon, plum, cherry, peach, nectarine, apricot, strawberry, grape,
 raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean,
 tobacco, tomato, sorghum and sugarcane.
 The high-level expression of a wild-type 8388, 18048, 16713, or 4144 gene
 and/or the expression of herbicide-tolerant forms of a 8388, 18048, 16713,
 or 4144 gene conferring herbicide tolerance in plants, in combination with
 other characteristics important for production and quality, can be
 incorporated into plant lines through breeding approaches and techniques
 known in the art.
 Where a herbicide tolerant 8388, 18048, 16713, or 4144 gene allele is
 obtained by direct selection in a crop plant or plant cell culture from
 which a crop plant can be regenerated, it is moved into commercial
 varieties using traditional breeding techniques to develop a herbicide
 tolerant crop without the need for genetically engineering the allele and
 transforming it into the plant.

The invention will be further described by reference to the following
 detailed examples. These examples are provided for purposes of
 illustration only, and are not intended to be limiting unless otherwise
 specified.
 EXAMPLES
 Standard recombinant DNA and molecular cloning techniques used here are
 well known in the art and are described by Sambrook, el al, Molecular
 Cloning, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
 N.Y. (1989) and by T. J. Silhavy, M. L. Bermnan, and L. W. Enquist,
 Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring
 Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in
 Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience
 (1987), Reiter, et al., Methods in Arabidopsis Research, World Scientific
 Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer
 Academic Publishers (1998). These references describe the standard
 techniques used for all steps in tagging and cloning genes from T-DNA
 mutagenized populations of Arabiclopsis: plant infection and
 transformation; screening for the identification of seedling mutants;
 cosegregation analysis; and plasmid rescue.
 Example 1
 Plant Infection and Transformation in Tagged Embryo-Lethal Lines 8388,
 18048, and 16713
 Arabidopsis plants (strain Columbia) are inverted, and their leaves are
 vacuum-infiltrated with Agrobacterium (1.times. dilution of Agrobacterium
 grown to OD600 of 0.8 in 10 mM MgCl.sub.2). T1 seed is collected from
 these plants, and germinated on an agar-solidified medium containing (50
 ug/ml Basta) or sprayed in soil (400 .mu.g/ml Basta). Typically, 0.1% to
 1.0% of the plants contain T-DNA inserts in a population of T1
 transformants. Furthermore, the plants that survive on Basta selection are
 hemizygous for the T-DNA insertion and thus the Basta selectable marker.
 Mutants blocked in growth or development are identified by examining T2
 progeny using an embryo screen and recovering those plants that contained
 25% aborted seeds. Using segregation analysis of T2 individuals,
 approximately one-third of the mutants are tagged.
 Example 2
 Embryo Screen for the Identification of Mutants Blocked in Early
 Development from Tagged Embryo-Lethal Lines 8388, 18048, and 16713
 Essential genes are identified through the isolation of lethal mutants
 blocked in early development. Examples of lethal mutants include those
 blocked in the formation of the male or female gametes, embryo, or
 resulting seedling. Gametophytic mutants are found by examining T1
 insertion lines for the presence of 50% aborted pollen grains or ovules.
 Embryo defective lethal mutants produce 25% defective seeds following
 self-pollination of T1 plants (see Errampalli et al. 1991, Plant Cell
 3:149-157; Castle et al. 1993, Mol Gen Genet 241:504-514). Seedling lethal
 mutants segregate for 25% seedli ngs that exhibit a lethal phenotype.
 The T1 line #8388 shows 250% defective seeds that contain embryos that are
 normal in size and shape, but completely lack normal pigmentation, i.e.
 they are albino. Similarly, defective seeds are normal in size and shape,
 and are white, rather than green, in mature siliques.
 The T1 line #18048 shows 25% defective seeds that contain embryos that
 abort very early in development soon after fertilization.
 The T1 line #16713 shows 25% defective seeds that contain embryos that
 abort very early in development soon after fertilization.
 Example 3
 Cosegregation Analysis for Tagged Embryo-Lethal Lines 8388,18048, and 16713
 The linkage of the mutation to the T-DNA insert is established after
 identifying a transformed line segregating for a lethal phenotype of
 interest. A line segregating with a single functional insert will
 segregate for resistance in the ratio of 2:1 (resistance:sensitive) to the
 selectable marker Basta. In this case, one-quarter of the T2 progeny will
 fail to germinate due to embryo lethality, resulting in a reduction of the
 normal 3:1 ratio to 2:1. Each of the Basta resistant progeny are therefore
 heterozygous for the mutation if the T-DNA insert is causing the mutant
 phenotype. To confirm cosegregation of the T-DNA and the mutant phenotype,
 Basta resistant progeny are transplanted to soil and screened again for
 the presence of 25% aborted seeds.
 For 8388, each of the 23 progeny examined contains approximately 25%
 aborted seeds with the expected phenotype. These results confirm that
 there is no evidence for recombination between the T-DNA and the mutation.
 Single plant southern blot analysis suggests that the T-DNA insertion in
 line #8388 con sists of a simple insertion.
 For 18048, each of the 23 progeny examined contains approximately 25%
 aborted seeds with the expected phenotype. These results confirm that
 there is no evidence for recombination between the T-DNA and the mutation.
 Single plant Southern blot analysis suggests that the insertion in line
 #18048 consists of a at least three tandem T-DNA elements. Cosegregation
 analysis shows that Basta resistance and the mutant phenotype in line
 18048 exhibit complete linkage in 94 selfed progeny from a selfed
 heterozygote.
 For 16713, each of the 38 progeny examined contains approximately 25%
 aborted seeds with the expected phenotype. These results confirm that
 there is no evidence for recombination between the T-DNA and the mutation.
 Cosegregation analysis shows that Basta resistance and the mutant
 phenotype in line 16713 exhivit complete linkage in 38 selfed progeny from
 a selfed heterozygote.
 Example 4a
 Plasmid Rescue from Tagged Embryo-Lethal Line 8388
 Arabidopsis genomic DNA is isolated as described Reiter et al in Methods in
 Arabidopsis Research, World Scientific Press (1992). Genomic DNA is
 digested with a restriction endonuclease and ligatcd overnight. After
 ligation, the DNA is transformed into competent E. coli strain XL-1 Blue,
 DH10B, DH5 alpha, or the like, and colonies are selected on semi-solid
 medium containing ampicillin. Resistant colonies are picked into liquid
 medium with ampicillin and grown overnight. Plasmid DNA is isolated and
 digested with the rescue enzyme and analyzed on agarose gels containing
 ethidium bromide for visualization. Plasmids that represent different size
 classes are sequenced using primers that flank the plant DNA portion of
 the rescue element and the sequence is analyzed to determine what portion
 is plant DNA and what gene has been disrupted.
 One method of confirming that the disrupted gene is the cause of the mutant
 phenotype is to transform a wild-type form of the gene into the mutant
 plant. Alternatively, the mutant is phenocopied by specifically reducing
 expression of the disrupted gene in transgenic plants expressing an
 antisense version of the gene behind a synthetic promoter (Guyer et al.
 (1998) Genetics, 149: 633-639).
 Example 4b
 Plasmid Rescue from Tagged Embryo-Lethal Line 18048
 Arabidopsis genomic DNA is isolated as described in Reiter et al in Methods
 in Arabidopsis Research, World Scientific Press (1992). Genomic DNA is
 digested with a restriction endonuclease and ligated overnight. After
 ligation, the DNA is transformed into competent E. coli strain XL-1 Blue,
 DH10B, DH5 alpha, or the like, and colonies are selected on semi-solid
 medium containing ampicillin. Resistant colonies are picked into liquid
 medium with ampicillin and grown overnight. Plasmid DNA is isolated and
 digested with the rescue enzyme and analyzed on agarose gels containing
 ethidium bromide for visualization. Plasmids that represent different size
 classes are sequenced using primers that flank the plant DNA portion of
 the rescue element and the sequence is analyzed to determine what portion
 is plant DNA and what gene has been disrupted.
 One method of confirming that the disrupted gene is the cause of the mutant
 phenotype is to transform a wild-type form of the gene into the mutant
 plant. Alternatively, the mutant is phenocopied by specifically reducing
 expression of the disrupted gene in transgenic plants expressing an
 antisense version of the gene behind a synthetic promoter (Guyer et al.
 (1998) Genetics, 149: 633-639).
 DNA flanking the borders of line #18048 is isolated using modifications to
 the Genome Walker kit (CLONTECH Laboratories, Palo Alto, Calif.). In
 general, DNA from the heterozygous mutant is digested with several
 different blunt cutting restriction endonucleases in parallel. The
 protocol is modified by using four enzymes that do not have a recognition
 site in the T-DNA insertion element. Adapters are ligated onto the ends of
 restriction fragments. These separate digests and ligations constitute
 different libraries of adapter-ligated restriction fragments. The
 libraries are used as template DNA in a PCR-based approach to specifically
 amplify the borders flanking the T-DNA insert. To achieve specificity,
 nested PCR primers from either the right border or left border of the
 T-DNA are used in combination with adapter PCR primers in a series of PCR
 reaction reactions to amplify plant DNA flanking the T-DNA insertion. The
 PCR products are sequenced, or cloned and sequenced.
 Example 4c
 Border Rescue from Tagged Embryo-Lethal Line 16713
 Arabidopsis genomic DNA is isolated as described in Reiter et al in Methods
 in Arabidoysis Research, World Scientific Press (1992), DNA flanking the
 borders of line #16713 is isolated using TAIL PCR. A series of 12 TAIL PCR
 reactions are performed on DNA from line #16713; 6 arbitrary degenerate
 primers (CA50 primer: 5' NGT CGA SWG ANA WGA A 3': SEQ ID NO:9 (128-fold,
 AD2 from Liu et al. (1995) The Plant Journal, 8: 457-463); CA51 primer: 5'
 TGW GNA GSA NCA SAG A 3': SEQ ID NO:10 (128-fold derivative of AD1 from
 Liu and Whittier (1995) Genomics, 25: 674-681); CA52 primer: 5' AGW GNA
 GWA NCA WAG G 3': SEQ ID NO:11(128-fold, AD2 from Liu and Whittier (1995)
 Genomics, 25:674-681); CA53 primer: 5' STT GNT AST NCT NTG C 3': SEQ ID
 NO:12 (256-fold, AD5 from Tsugeki et al. (1996) The Plant Journal, 10:
 479-489); CA54 primer: 5' NTC GAS TWT SGW GTT 3': SEQ ID NO:13 (64-fold,
 AD1 from Liu et al. (1995) The Plant Journal, 8: 457-463); and CA55
 primer: 5' WGT GNA GWA NCA NAG A 3': SEQ ID NO:14 (256-fold, AD3 from Liu
 et al. (1995) The Plant Journal, 8: 457-463) are used in combination with
 two sets of nested, and T-DNA specific primers for the right border (CA66
 primer: 5' ATT AGG CAC CCC AGG CTT TAC ACT TTA TG 3': SEQ ID NO:15
 (pCSA104 right border primary primer); CA67 primer: 5' GTA TGT TGT GTG GAA
 TTG TGA GCG GAT AAC 3': SEQ ID NO:16 (pCSA104 right border secondary
 primer); and CA68 primer: 5' TAA CAA TTT CAC ACA GGA AAC AGC TAT GAC 3':
 SEQ ID NO:17 (pCSA104 right border tertiary primer) as well as for the
 left border (JM33 primer: 5' TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT ACA C
 3': SEQ ID NO:18 (pCSA104 left border tertiary primer; JM34 primer: 5' GCT
 TCC TAT TAT ATC TTC CCA AAT TAC CAA TAC A 3': SEQ ID NO:19 (pCSA104 left
 border secondary primer); and JM35 primer: 5' GCC TTT TCA GAA ATG GAT AAA
 TAG CCT TGC TTC C 3': SEQ ID NO:20 (pCSA104 left border primary primer) of
 the T-DNA region ofpCSA104.
 A total of seven products are obtained from the left border and eight
 products from the right border. PCR primers specific to the genomic region
 are then designed and used to confirm the border products obtained by TAIL
 PCR.
 Example 5a
 Sequence Analysis of Tagged Embryo-Lethal Line #8388 From the Insertional
 Mutant Collection
 Analysis of Arabidopsis thaliana genomic DNA sequence flanking the right
 border region of the T-DNA insert in line 8388 reveals a single exon open
 reading frame of 1,656 bp (SEQ ID NO:1). Arabidopsis thaliana genomic DNA
 flanking the T-DNA border is identical to the ESTs 166E6T7 (Genbank
 Accession #R30603) and 203E14T7 (Genbank Accession #H77096) and to
 portions of the genomic survey sequences T19C17TR (Genbank Accession
 #B28763) F13K23-Sp6 (Genbank Accession #B10372). Sequence of the open
 reading frame used as a BLASTX 2.0.7 query yielded the hits listed in the
 chart below.

GenPept Accession # % Identity % Similarity
 AAD20136.sup.4 36.554 46.214
 S00986.sup.1 31.852 46.173
 1170507.sup.2 29.923 44.501
 BAA19295.sup.3 35.250 45.750
 .sup.1 eIF-4A I from mouse (note: human, rabbit, and mouse eIF4A I are
 identical at the amino acid level, and therefore give identical scores)
 .sup.2 eIF-4A-3 from Nicotiana plumbaginifolia.
 .sup.3 ATP dependent RNA helicase DEAD homolog from Bacillus subtilis.
 .sup.4 autoaggregation-mediating protein from Lactobacillus reuteri..
 Example 5b
 Sequence Analysis of Tagged Embryo-Lethal Line #18048 From the Insertional
 Mutant Collection
 In the case of line #18048, there are multiple, tandemly arrayed T-DNA
 elements with left border sequences facing outward into plant DNA on both
 sides of the insert. Using the GenomeWalker strategy and left
 border-specific primers, a set of four independent PCR fragments are
 obtained and sequenced. Each of these four fragments shares sequence
 identity to the same region of a sequenced BAC clone (T30D6, accession
 number AC006439). Note that the BAC clone sequence is completed and is
 annotated by the public Arabidopsis Genome Sequencing project. Our
 sequences, both genomic and cDNA, match the predicted sequence exactly.
 Comparison of the recovered fragments with the T30D6 BAC clone sequence
 reveals that a 13 base deletion occurred upon insertion of the T-DNA in
 this mutant.
 Analysis of the DNA sequence from the recovered borders reveals a high
 degree of homology to members of the ADP ribosylation factor (Arf) family
 of genes. Further inspection of recovered border fragments reveals that
 the T-DNA has inserted in the middle of the coding region for a gene that
 encodes a protein with greater than 60% identity to Arf-like (Arl)
 proteins from Drosophila, human, and rat. Sequence of the protein (SEQ ID
 NO:6) used as a BLASTP 2.0.8 query yields the hits listed in the chart
 below.

Genbank Accession # % Identity % Similarity
 NP_001658.sup.1 64.130 72.283
 O08697.sup.2 63.043 72.283
 Q06849.sup.3 61.413 70.652
 CAA90353.sup.4 55.676 68.108
 Q09767.sup.5 48.370 66.304
 P49076.sup.6 48.876 60.112
 AAD17207.sup.7 47.458 58.757
 .sup.1 pARL2 protein from human
 .sup.2 ARL2_RAT protein from rat
 .sup.3 ARL2_DROME protein from Drosophila
 .sup.4 RFM_CAEEL protein from C. elegans
 .sup.5 ARL_SCHPO protein from S. pombe
 .sup.6 ARF_MAIZE protein from maize
 .sup.7 GMARF protein from soybean
 Example 5c
 Sequence Analysis of Tagged Embryo-Lethal Line #16713 From the Insertional
 Mutant Collection
 The sequence of the TAIL PCR border products matches the sequence from the
 P1 clone MIF21. All 15 TAIL PCR border products represent the same genomic
 region of the P1 clone MIF21 (Accession #AB023239). Further analysis of
 these products reveals a 44 base pair deletion that occurred upon T-DNA
 insertion in line #16713, corresponding to base number 46123 through
 46167, of the P1 clone MIF21.
 Analysis of the DNA sequence from the recovered borders reveals a high
 degree of homology to members of the acetoacetyl coA thiolase genes.
 Further inspection of recovered border fragments reveals that the T-DNA
 has inserted in the middle of the coding region for a gene that encodes a
 protein with greater than 50% identity to acetoacetyl-CoA thiolase
 proteins from radish, corn, yeast, human, and rat. Using GAP (Seq Web
 version 10.0, GCG), pairwise comparisons of the protein sequence (SEQ ID
 NO:8) and input sequences shown below give a measure of similarity between
 SEQ ID NO:8 and the indicated sequence; and are summarized below.

Genbank Accession # % Identity % Similarity
 CAA55006.sup.1 93.0 94.0
 AAD44539.sup.2 74.0 82.4
 P41338.sup.3 54.9 64.3
 BAA14278.sup.4 51.5 60.9
 BAA03016.sup.5 51.6 61.2
 AAA82403.sup.6 49.0 57.1
 Q46939.sup.7 45.6 55.9
 .sup.1 cytosolic acetoacetyl-coenzyme A thiolase from radish
 .sup.2 acetoacetyl CoA thiolase from maize
 .sup.3 acetoacetyl CoA thiolase from S. cerevisiae
 .sup.4 mitochondrial acetoacetyl-coenzyme A thiolase from human
 .sup.5 mitochondrial acetoacetyl-CoA thiolase from rat
 .sup.6 acetyl-CoA thiolase from C. elegans
 .sup.7 acetoacetyl-CoA thiolase from E. coli
 Example 5d
 Sequence Analysis of Tagged Seedling--Lethal Line #4144 From the T-DNA
 Mutagenized Population of Arabidopsis
 The plasmid rescue technique is used to molecularly clone Arabidlopsis
 flanking DNA from one or both sides of the T-DNA insertion(s). Plasmnids
 obtained in this manner are analyzed by restriction enzyme digestion to
 sort the plasmids into classes based on their digestion pattern. For each
 class of plasmid clone, the DNA sequence is determined. The resulting
 sequences are analyzed for the presence of non-T-DNA vector sequence. The
 plasmids recovered from the plasmid rescue protocol are sequenced using
 the slp346 primer (5' GCGGACATCTACATTTTTGA 3'; SEQ ID NO:26). Primer
 slp346 provides information on the flanking sequence immediately adjacent
 to the left T-DNA border. The plasmid rescue is validated via PCR of
 template genomic DNA from a heterozygote for the 4144 insertion mutation.
 The experiment uses a primer anchored in the predicted flanking sequence
 and the sip346 primer. Finding a PCR product of the appropriate size,
 based on the sequence of the plasmid rescue clone confirms a valid rescue.
 The sequence obtained from the above clone is used in BLASTx and BLASTn
 searches against nucleotide databases. (Altschul et al. (1990) J Mol.
 Biol. 215:403-410; Altschul et al. (1997) Nucleic Acids Res.
 25:3389-3402). The BLASTx results show that the translated plant flanking
 sequence shows similarity to the chloroplast ATP synthase delta chain from
 a number of organisms including spinach (SWISS PROT P11402), pea (SWISS
 PROT Q02758), millet (SWISS PROT Q07300), corn (PIR S43729), and tobacco
 (SWISS PROT P32980). The BLASTn results show the rescued flanking sequence
 to be identical to preliminary genomic sequence CSHL076
 T25P22-99.03.10-68148.seq. (found at
 http://genome-www2.stanford.edu/cgi-bin/AtDB/
 getseq?database=cshlprel&item=CSHL076). The region of genomic DNA where
 the T-DNA insertion occurred includes bases #26,159 through #27,088 of the
 annotated CSHL076 T25P22-99.03.10-68148. sequence, resulting in a seventy
 nine-base deletion. The BLASTn results also show the rescued flanking
 sequence is similar to Arabiclopsis sequences from EST cDNA clones 71D2T7
 (GenBank T45339), GBGe205 (GenBank Z26062 and Z28994), 174J16T7 (GenBank
 AA712658), 116O10T7 (GenBank T42797), and 121M24T7 (GenBank AA721953).
 From our own sequencing of EST 71D2, we identify the ORF of the cDNA
 sequence as that in SEQ ID NO:21. These data indicate that there are no
 introns in this gene.
 The sequence obtained from the above clone is used in GAP searches against
 protein databases, and the following results are obtained. B. rapa
 (GenBank #BAA11390): 89.5%, spinach (SWISS PROT #P11402): 54.1%, pea
 (SWISS PROT #Q02758): 57.9%, tobacco (SWISS PROT #P32980): 63.9%, millet
 (SWISS PROT #Q07300): 49.4%, and maize (PIR #S43729): 58.3%. The sequence
 obtained from the above clone is used in GAP searches against nucleotide
 databases, and the following result is obtained: B. rapa (DDBJ #D78493):
 82.1%.
 Example 6a
 Isolation and Identification of 8388 cDNA Coding Region
 The cDNA clone 166E6 is obtained from the Michigan State University EST
 collection (Newman et al. (1994) Plant Physiol. 106:1241-1255). It is
 picked from that collection and the insert sequenced completely (SEQ ID
 NO:3). The sequence from that cDNA clone is identical to the sequence
 derived from plasmid rescue from the 8388 line (SEQ ID NO:1), excepting
 that there are 5 silent nucleotide substitutions due to allelic variation
 in the open reading frame of the two sequences. The substitutions are a C
 at base 282 of SEQ ID NO:1 to a G at base 553 of SEQ ID NO:3; a G at base
 1011 of SEQ ID NO:1 to a T at base 1282 of SEQ ID NO:3; a C at base 1188
 of SEQ ID NO:1 to a T at base 1459 of SEQ ID NO:3; C at base 1404 of SEQ
 ID NO:1 to a T at base 1675 of SEQ ID NO:3; a G at base 1413 of SEQ ID
 NO:1 to a T at base 1684 of SEQ ID NO:3. These silent substitutions do not
 effect the polypeptides encoded by SEQ ID NO:1 or SEQ ID NO:3; they are
 identical.
 Example 6b
 Isolation and Identification of 18048 cDNA Coding Region
 A cDNA fragment corresponding to the coding region of the 18048 gene is
 amplified with primers from the putative coding region of this gene (SEQ
 ID NO:5). These primers are designed using the alignments of deduced
 peptides from ORF's in the genomic DNA with the Arl proteins from
 Drosophila, human, rat and yeast. The deduced polypeptide encoded by the
 18048 gene is shown in SEQ ID NO:6.
 Southern blot analysis shows that the 18048 gene is single copy in
 Arabidopsis, and is disrupted by a T-DNA insertion in the mutant line
 examined. In addition, northern blot analysis reveals that the 18048 gene
 from Arabidopsis is expressed in vegetative tissues of young seedlings and
 four-week-old plants. Because the 18048 gene is expressed in vegetative
 tissues, the function of this gene is likely to be essential throughout
 the life cycle, as well as in early embryo development. Therefore,
 chemicals that inhibit 18048-gene function are likely to be lethal when
 applied to plants.
 Example 6c
 Isolation and Identification of 16713 cDNA Coding Region
 A cDNA fragment corresponding to the coding region of the 16713 gene is
 cloned by PCR from the pFL61 (Minet et al. (1992) Plant Journal,
 2:417-422) cDNA library (SEQ ID NO:7). The deduced polypeptide encoded by
 the 16713 gene is shown in SEQ ID NO:8.
 Northern blot analysis reveals that the 16713 gene from Arabidopsis is
 expressed in vegetative tissues of young seedlings and four-week-old
 plants. Because the 16713 gene is expressed in vegetative tissues, the
 function of this gene is likely to be essential throughout the life cycle,
 as well as in early embryo development. Therefore, chemicals that inhibit
 16713-gene function are likely to be lethal when applied to plants.
 Example 7a
 Expression of Recombinant 8388 Protein in Hetcrologous Expression Systems
 The coding region of the protein, corresponding to the cDNA clone SEQ ID
 NO:1, is subcloned into previously described expression vectors, and
 transformed into E. coli using the manufacturer's conditions. Specific
 examples include plasmids such as pBluescript (Stratagene, La Jolla,
 Calif.), the pET vector system (Novagen, Inc., Madison, Wis.) pFLAG
 (International Biotechnologies, Inc., New Haven, Conn.), and pTrcHis
 (Invitrogen, La Jolla, Calif.). E. coli is cultured, and expression of the
 8388 activity is confirmed. Alternatively, eukaryotic expression systems
 such as cultured insect cells infected with specific viruses may be
 preferred. Examples of vectors and insect cell lines are described
 previously. Protein conferring 8388 activity is isolated using standard
 techniiques.
 Example 7b
 Expression of Recombinant 18048 Protein in Heterologous Expression Systems
 The coding region of the protein, corresponding to the cDNA clone SEQ ID
 NO:5, is subcloned into previously described expression vectors, and
 transformed into E. coli using the manufacturer's conditions. Specific
 examples include plasmids such as pBluescript (Stratagene, La Jolla,
 Calif.), the pET vector system (Novagen, Inc., Madison, Wis.) pFLAG
 (International Biotechnologies, Inc., New Haven, Conn.), and pTrcHis
 (Invitrogen, La Jolla, Calif.). E. coli is cultured, and expression of the
 18048 activity is confirmed. Alternatively, eukaryotic expression systems
 such as cultured insect cells infected with specific viruses may be
 preferred. Examples of vectors and insect cell lines are described
 previously. Protein conferring 18048 activity is isolated using standard
 techniques.
 Example 7c
 Expression of Recombinant 16713 Protein in Heterologous Expression Systems
 The coding region of the protein, corresponding to the cDNA clone SEQ ID
 NO:7, is subcloned into previously described expression vectors, and
 transformed into E. coli using the manufacturer's conditions. Specific
 examples include plasmids such as pBluescript (Stratagene, La Jolla,
 Calif.), the pET vector system (Novagen, Inc., Madison, Wis.) pFLAG
 (International Biotechnologies, Inc., New Haven, Conn.), and pTrcHis
 (Invitrogen, La Jolla, Calif.). E. coli is cultured, and expression of the
 16713 activity is confirmed. Alternatively, eukaryotic expression systems
 such as cultured insect cells infected with specific viruses may be
 preferred. Examples of vectors and insect cell lines are described
 previously. Protein conferring 16713 activity is isolated using standard
 techniques.
 Example 7d
 Expression of Recombinant 4144 Protein in Heterologous Expression Systems
 The coding region of the protein, corresponding to the cDNA clone SEQ ID
 NO:21, is subcloned into an appropriate expression vector, and transformed
 into E. coli using the manufacturer's conditions. Specific examples
 include plasmids such as pBluescript (Stratagene, La Jolla, Calif.), pFLAG
 (International Biotechnologies, Inc., New Haven, Conn.), and pTrcHis
 (Invitrogen, La Jolla, Calif.). E. coli is cultured, and expression of the
 4144 activity is confirmed. Protein conferring 4144 activity is isolated
 using standard techniques.
 Example 8a
 In vitro Recombination of 8388 Genes by DNA Shuffling
 The nucleotide sequence shown in SEQ ID NO:1 is amplified by PCR. The
 resulting DNA fragment is digested by DNaseI treatment essentially as
 described (Stemmer et al. (1994) PNAS 91: 10747-10751) and the PCR primers
 are removed from the reaction mixture. A PCR reaction is carried out
 without primers and is followed by a PCR reaction with the primers, both
 as described (Stemmer et al. (1994) PNAS 91: 10747-10751). The resulting
 DNA fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-01) for
 use in bacteria, or into pESC vectors (Stratagene Catalog) for use in
 yeast; and transformed into a bacterial or yeast strain deficient in 8388
 activity by electroporation using the Biorad Gene Pulser and the
 manufacturer's conditions. The transformed bacteria or yeast are grown on
 medium that contains inhibitory concentrations of an inhibitor of 8388
 activity and those colonies that grow in the presence of the inhibitor are
 selected. Colonies that grow in the presence of normally inhibitory
 concentrations of inhibitor are picked and purified by repeated
 restreaking. Their plasmids are purified and the DNA sequences of cDNA
 inserts from plasmids that pass this test are then determined.
 In a similar reaction, PCR-amplified DNA fragments comprising the A.
 thaliana 8388 gene encoding the protein and PCR-amplified DNA fragments
 comprising the 8388 gene from E. coli are recombined in vitro and
 resulting variants with improved tolerance to the inhibitor are recovered
 as described above.
 Example 8b
 In vitro Recombination of 18048 Genes by DNA Shuffling
 The nucleotide sequence shown in SEQ ID NO:5 is amplified by PCR. The
 resulting DNA fragment is digested by DNase I treatment essentially as
 described (Stemmer et al. (1994) PNAS 91: 10747-10751) and the PCR primers
 are removed from the reaction mixture. A PCR reaction is carried out
 without primers and is followed by a PCR reaction with the primers, both
 as described (Stemmer et al. (1994) PNAS 91: 10747-10751). The resulting
 DNA fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-01) for
 use in bacteria, or into pESC vectors (Stratagene Catalog) for use in
 yeast; and transformed into a bacterial or yeast strain deficient in 18048
 activity by electroporation using the Biorad Gene Pulser and the
 manufacturer's conditions. The transformed bacteria or yeast are grown on
 medium that contains inhibitory concentrations of an inhibitor of 18048
 activity and those colonies that grow in the presence of the inhibitor are
 selected. Colonies that grow in the presence of normally inhibitory
 concentrations of inhibitor are picked and purified by repeated
 restreaking. Their plasmids are purified and the DNA sequences of cDNA
 inserts from plasmids that pass this test are then determined.
 In a similar reaction, PCR-amplified DNA fragments comprising the A.
 thaliana 18048 gene encoding the protein and PCR-amplified DNA fragments
 comprising the 18048 gene from E. coli are recombined in vitro and
 resulting variants with improved tolerance to the inhibitor are recovered
 as described above.
 Example 8c
 In vitro Recombination of 16713 Genes by DNA Shuffling
 The nucleotide sequence shown in SEQ ID NO:7 is amplified by PCR. The
 resulting DNA fragment is digested by DNase I treatment essentially as
 described (Stemmer et al. (1994) PNAS 91: 10747-10751) and the PCR primers
 are removed from the reaction mixture. A PCR reaction is carried out
 without primers and is followed by a PCR reaction with the primers, both
 as described (Stemmer et al. (1994) PNAS 91: 10747-10751). The resulting
 DNA fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-01) for
 use in bacteria, or into pESC vectors (Stratagene Catalog) for use in
 yeast; and transformed into a bacterial or yeast strain deficient in 16713
 activity by electroporation using the Biorad Gene Pulser and the
 manufacturer's conditions. The transformed bacteria or yeast are grown on
 medium that contains inhibitory concentrations of an inhibitor of 16713
 activity and those colonies that grow in the presence of the inhibitor are
 selected. Colonies that grow in the presence of normally inhibitory
 concentrations of inhibitor are picked and purified by repeated
 restreaking. Their plasmids are purified and the DNA sequences of cDNA
 inserts from plasmids that pass this test are then determined.
 In a similar reaction, PCR-amplified DNA fragments comprising the A.
 thaliana 16713 gene encoding the protein and PCR-amplified DNA fragments
 comprising the 16713 gene from E. coli are recombined in vitro and
 resulting variants with improved tolerance to the inhibitor are recovered
 as described above.
 Example 8d
 In vitro Recombination of 4144 Genes by DNA Shuffling
 The nucleotide sequence of SEQ ID NO:21 is amplified by PCR. The resulting
 DNA fragment is digested by DNaseI treatment essentially as described
 (Stemmer et al. (1994) PNAS 91: 10747-10751) and the PCR primers are
 removed from the reaction mixture. A PCR reaction is carried out without
 primers and is followed by a PCR reaction with the primers, both as
 described (Stemmer et al. (1994) PNAS 91: 10747-10751). The resulting DNA
 fragments are cloned into pTRC99a (Pharmacia, Cat no: 27-5007-01) for use
 in bacteria, and transformed into a bacterial strain deficient in 4144
 activity by electroporation using the Biorad Gene Pulser and the
 manufacturer's conditions. The transformed bacteria are grown on medium
 that contains inhibitory concentrations of an inhibitor of 4144 activity
 and those colonies that grow in the presence of the inhibitor are
 selected. Colonies that grow in the presence of normally inhibitory
 concentrations of inhibitor are picked and purified by repeated
 restreaking. Their plasmids are purified and the DNA sequences of cDNA
 inserts from plasmids that pass this test are then determined.
 Alternatively, the DNA fragments are cloned into expression vectors for
 transient or stable transformation into plant cells, which are screened
 for differential survival and/or growth in the presence of an inhibitor of
 4144 activity. In a similar reaction, PCR-amplified DNA fragments
 comprising the Arabidopsis 4144 gene encoding the protein and
 PCR-amplified DNA fragments derived from or comprising another 4144 gene
 are recombined in vitro and resulting
 Example 9a
 In vitro Recombination of 8388 Genes by Staggered Extension Process
 The Arabiclopsis thaliana 8388 gene encoding the 8388 protein and the E.
 coli 8388 homologous gene are each cloned into the polylinker of a
 pBluescript vector. A PCR reaction is carried out essentially as described
 (Zhao et al. (1998) Nature Biotechnology 16: 258-261) using the "reverse
 primer" and the "M13-20 primer" (Stratagene Catalog). Amplified PCR
 fragments are digested with appropriate restriction enzymes and cloned
 into pTRC99a and mutated 8388 genes are screened as described in Example
 8a.
 Example 9b
 In vitro Recombination of 18048 Genes by Staggered Extension Process
 The Arabidopsis thaliana 18048 gene encoding the 18048 protein and the E.
 coli 18048 homologous gene are each cloned into the polylinker of a
 pBluescript vector. A PCR reaction is carried out essentially as described
 (Zhao et al. (1998) Nature Biotechnology 16: 258-261) using the "reverse
 primer" and the "M13-20 primer" (Stratagene Catalog). Arnplified PCR
 fragments are digested with appropriate restriction enzymes and cloned
 into pTRC99a and mutated 18048 genes are screened as described in Example
 8b.
 Example 9c
 In vitro Recombination of 16713 Genes by Staggered Extension Process
 The Arabidopsis thaliana 16713 gene encoding the 16713 protein and the E.
 coli 16713 homologous gene are each cloned into the polylinker of a
 pBluescript vector. A PCR reaction is carried out essentially as described
 (Zhao et al. (1998) Nature Biotechnology 16: 258-261) using the "reverse
 primer" and the "M13-20 primer" (Stratagene Catalog). Amplified PCR
 fragments are digested with appropriate restriction enzymes and cloned
 into pTRC99a and mutated 16713 genes are screened as described in Example
 8c.
 Example 9d
 In vitro Recombination of 4144 Genes by Staggered Extension Process
 The Arabidopsis 4144 gene encoding the 4144 protein and another 4144 gene,
 or homologs thereof, or fragments thereof, are each cloned into the
 polylinker of a pBluescript vector. A PCR reaction is carried out
 essentially as described (Zhao et al. (1998) Nature Biotechnology 16:
 258-261) using the "reverse primer" and the "M13-20 primer" (Stratagene
 Catalog). Amplified PCR fragments are digested with appropriate
 restriction enzymes and cloned into pTRC99a and mutated 4144 genes are
 screened as described in Example 8d.
 Example 10
 In vitro Binding Assays
 Recombinant 8388, 18048, 16713, or 4144 protein is obtained, for example,
 according to Example 7a, 7b, 7c, or 7d, respectively. The protein is
 immobilized on chips appropriate for ligand binding assays using
 techniques which are well known in the art. The protein immobilized on the
 chip is exposed to sample compound in solution according to methods well
 know in the art. While the sample compound is in contact with the
 immobilized protein measurements capable of detecting protein-ligand
 interactions are conducted. Examples of such measurements are SELDI,
 biacore and FCS, described above. Compounds found to bind the protein are
 readily discovered in this fashion and are subjected to further
 characterization.
 Example 11a
 3-Ketoacyl-CoA Thiolase Activity Assay
 The 3-ketoacyl-CoA thiolase activity assay is derived from Olesen et al.
 (1997) FEBS Letters 412, 138-140. The reaction volumes are preferably the
 ones described below, but can be varied depending on the experimental
 requirements. 0.01-1.0.times.10.sup.-3 unit of an enzyme having
 3-ketoacyl-CoA thiolase activity (one unit of activity is defined as the
 amount of enzyme required to produce 1 .mu.mol/min of product) and 10-500
 .mu.M, but preferably 250 .mu.M acetoacetyl-CoA (AcAc-CoA) are mixed in a
 final volume of 20 .mu.L Tris-HCI (pH 7.0-9.0, but preferable 8.5) and
 10-250 .mu.M, but preferably 50 .mu.M CoA. The production of acetyl-CoA is
 determined preferably according to Olesen et al. (1997) FEBS Letters 412,
 138-140 by following the breakage of acetoacetyl-CoA (AcAc-CoA), measured
 by the decrease in absorption of the enol form at 302 nm. Alternatively,
 the formation of new thioester bonds can be measured by detecting
 increases in absorbance at 233 nm.
 A follow-up HPLC assay is described in Antonenkov et al. (1997) J
 Biological Chemistry 272: 26023-26031, which is incorporated herein by
 reference.
 Example 11b
 RNA Helicase Assay
 Assays for RNA helicase are described in the following references. The
 technique of fluorescence polarization is described in Spears et al.
 (1997) Analytical Biochemistry 247: 130-137. The technique of fluorescence
 energy transfer is described in Bjornson et al. (1994) Biochemistry 33:
 14306-14316. The technique of fluorescence energy quenching is described
 in Houston et al. (1994) Proc. Natl. Acad. Sci. USA 91: 5471-5474. The
 technique of time resolved fluorescence energy transfer is described in
 Earnshaw et al. (1999) Journal of Biomolecular Screening 4: 239-248. All
 of the references described in this example are hereby incorporated by
 reference.
 Example 12
 Plastid Transformation
 Transformation Vectors
 For expression of a nucleotide sequence encoding a polypcptide having 8388,
 18048, 16713, or 4144 activity encoding in plant plastids, plastid
 transformation vector pPH143 or pPH145 (WO 97/32011) is used; and this
 reference is incorporated herein by reference. The nucleotide sequence is
 inserted into pPH 143 thereby replacing the PROTOX coding sequence. This
 vector is then used for plastid transformation and selection of
 transformants for spectinomycin resistance. Alternatively, the nucleotide
 sequence is inserted in pPH143 so that it replaces the aadH gene. In this
 case, transformants are selected for resistance to PROTOX inhibitors.
 Plastid Transformation
 Seeds of Nicotiana tabacum c.v. `Xanthi nc` are germinated seven per plate
 in a 1" circular array on T agar medium and bombarded 12-14 days after
 sowing with 1 .mu.m tungsten particles (M10, Biorad, Hercules, Calif.)
 coated with DNA from plasmids pPH143 and pPH145 essentially as described
 (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917).
 Bombarded seedlings are incubated on T medium for two days after which
 leaves are excised and placed abaxial side up in bright light (350-500
 tmol photons/m.sup.2 /s) on plates of RMOP medium (Svab, Z., Hajdukiewicz,
 P. and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530)
 containing 500 .mu.g/ml spectinomycin dihydrochloride (Sigma, St. Louis,
 Mo.). Resistant shoots appearing underneath the bleached leaves three to
 eight weeks after bombardment are subdloned onto the same selective
 medium, allowed to form callus, and secondary shoots isolated and
 subcloned. Complete segregation of transformed plastid genome copies
 (homoplasmicity) in independent subclones is assessed by standard
 techniques of Southern blotting (Sambrook et al., (1989) Molecular
 Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
 Harbor). Homoplasmic shoots are rooted aseptically on
 spectinomycin-containing MS/IBA medium (McBride, K. E. et al. (1994) Proc.
 Natl. Acad. Sci. USA 91, 7301-7305) and transferred to the greenhouse.
 The above-disclosed embodiments are illustrative. This disclosure of the
 invention will place one skilled in the art in possession of many
 variations of the invention. All such obvious and foreseeable variations
 are intended to be encompassed by the appended claims.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 26
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 1656
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (1)..(1656)
 &lt;400&gt; SEQUENCE: 1
 atg gcg gca tca act tca acc cga ttc ctt gtt ctg ctc aaa gat ttt 48
 Met Ala Ala Ser Thr Ser Thr Arg Phe Leu Val Leu Leu Lys Asp Phe
 1 5 10 15
 tct gcc ttc aga aag ata tca tgg act tgt gct gca act aat ttt cac 96
 Ser Ala Phe Arg Lys Ile Ser Trp Thr Cys Ala Ala Thr Asn Phe His
 20 25 30
 cgc caa tct cgt ttt tta tgc cat gtt gcg aaa gaa gac ggg tct ctt 144
 Arg Gln Ser Arg Phe Leu Cys His Val Ala Lys Glu Asp Gly Ser Leu
 35 40 45
 act ctt gca agc ctt gat ttg ggg aac aaa cca cgg aaa ttt ggg aag 192
 Thr Leu Ala Ser Leu Asp Leu Gly Asn Lys Pro Arg Lys Phe Gly Lys
 50 55 60
 ggt aag gcg atg aag ctt gag gga agt ttt gtt act gaa atg ggt caa 240
 Gly Lys Ala Met Lys Leu Glu Gly Ser Phe Val Thr Glu Met Gly Gln
 65 70 75 80
 ggt aag gta aga gcg gta aag aac gat aaa atg aaa gtt gtc aag gaa 288
 Gly Lys Val Arg Ala Val Lys Asn Asp Lys Met Lys Val Val Lys Glu
 85 90 95
 aaa aag cca gct gag ata gtg tct cct ttg ttt tct gca aaa tcc ttt 336
 Lys Lys Pro Ala Glu Ile Val Ser Pro Leu Phe Ser Ala Lys Ser Phe
 100 105 110
 gag gag ctt ggc ctc ccg gat tcc ttg tta gac agt ttg gaa aga gaa 384
 Glu Glu Leu Gly Leu Pro Asp Ser Leu Leu Asp Ser Leu Glu Arg Glu
 115 120 125
 ggt ttc tct gtc cca aca gat gtc caa tca gca gct gtc ccg gca ata 432
 Gly Phe Ser Val Pro Thr Asp Val Gln Ser Ala Ala Val Pro Ala Ile
 130 135 140
 atc aaa ggt cac gat gca gtg att cag tct tac aca gga tct ggc aaa 480
 Ile Lys Gly His Asp Ala Val Ile Gln Ser Tyr Thr Gly Ser Gly Lys
 145 150 155 160
 aca tta gct tat ctg ctt cca ata ttg tcc gaa att ggt cct cta gca 528
 Thr Leu Ala Tyr Leu Leu Pro Ile Leu Ser Glu Ile Gly Pro Leu Ala
 165 170 175
 gaa aaa tct aga agt tcg cac agt gaa aat gat aag agg act gag att 576
 Glu Lys Ser Arg Ser Ser His Ser Glu Asn Asp Lys Arg Thr Glu Ile
 180 185 190
 cag gca atg atc gtg gct cca tca aga gaa ctc ggt atg cag ata gta 624
 Gln Ala Met Ile Val Ala Pro Ser Arg Glu Leu Gly Met Gln Ile Val
 195 200 205
 aga gag gta gag aaa ctg ctc gga cct gtt cac cgt aga atg gtt cag 672
 Arg Glu Val Glu Lys Leu Leu Gly Pro Val His Arg Arg Met Val Gln
 210 215 220
 cag ttg gta gga ggt gca aac cga atg agg caa gaa gag gcc ctt aag 720
 Gln Leu Val Gly Gly Ala Asn Arg Met Arg Gln Glu Glu Ala Leu Lys
 225 230 235 240
 aaa aat aaa cct gca att gtt gtt ggc act ccc ggg aga att gca gag 768
 Lys Asn Lys Pro Ala Ile Val Val Gly Thr Pro Gly Arg Ile Ala Glu
 245 250 255
 ata agc aaa ggt gga aaa ttg cac act cat ggg tgt aga ttc ttg gtg 816
 Ile Ser Lys Gly Gly Lys Leu His Thr His Gly Cys Arg Phe Leu Val
 260 265 270
 cta gac gaa gtc gat gag ctt tta tcg ttt aat ttc cga gaa gat atc 864
 Leu Asp Glu Val Asp Glu Leu Leu Ser Phe Asn Phe Arg Glu Asp Ile
 275 280 285
 cat cga ata cta gaa cat gta gga aag aga tct ggg gct ggt cct aaa 912
 His Arg Ile Leu Glu His Val Gly Lys Arg Ser Gly Ala Gly Pro Lys
 290 295 300
 gga gaa gtc gat gaa cgg gct aac cgg cag acc att cta gtc tct gca 960
 Gly Glu Val Asp Glu Arg Ala Asn Arg Gln Thr Ile Leu Val Ser Ala
 305 310 315 320
 act gtg cca ttc tcg gtt atc cga gca gct aaa agc tgg agt cac gag 1008
 Thr Val Pro Phe Ser Val Ile Arg Ala Ala Lys Ser Trp Ser His Glu
 325 330 335
 ccg gtt ctt gtc caa gcc aac aaa gtc act cct ctt gat acc gtt caa 1056
 Pro Val Leu Val Gln Ala Asn Lys Val Thr Pro Leu Asp Thr Val Gln
 340 345 350
 cca tct gca ccg gta atg agc ttg act ccc aca act tct gaa gct gat 1104
 Pro Ser Ala Pro Val Met Ser Leu Thr Pro Thr Thr Ser Glu Ala Asp
 355 360 365
 ggc cag att cag act act att cag agc tta cct cca gct tta aaa cac 1152
 Gly Gln Ile Gln Thr Thr Ile Gln Ser Leu Pro Pro Ala Leu Lys His
 370 375 380
 tat tac tgc atc tca aag cat caa cac aaa gtc gac acg tta agg aga 1200
 Tyr Tyr Cys Ile Ser Lys His Gln His Lys Val Asp Thr Leu Arg Arg
 385 390 395 400
 tgc gtt cac gcc ctc gat gcc caa tcg gtt ata gct ttc atg aac cac 1248
 Cys Val His Ala Leu Asp Ala Gln Ser Val Ile Ala Phe Met Asn His
 405 410 415
 tca agg cag ctc aaa gat gtg gtc tac aaa ctc gaa gct cgt ggt atg 1296
 Ser Arg Gln Leu Lys Asp Val Val Tyr Lys Leu Glu Ala Arg Gly Met
 420 425 430
 aat tca gct gag atg cac gga gat ctc ggg aag cta ggg aga tca aca 1344
 Asn Ser Ala Glu Met His Gly Asp Leu Gly Lys Leu Gly Arg Ser Thr
 435 440 445
 gtt cta aag aag ttc aag aac ggg gaa atc aag gta ctt gtg aca aac 1392
 Val Leu Lys Lys Phe Lys Asn Gly Glu Ile Lys Val Leu Val Thr Asn
 450 455 460
 gag ctc tct gcc cgg ggt ctg gat gtt gcg gaa tgt gat ctg gtg gtg 1440
 Glu Leu Ser Ala Arg Gly Leu Asp Val Ala Glu Cys Asp Leu Val Val
 465 470 475 480
 aat ctt gag ctt cca act gat gcg gtt cac tat gct cat cga gct ggg 1488
 Asn Leu Glu Leu Pro Thr Asp Ala Val His Tyr Ala His Arg Ala Gly
 485 490 495
 aga aca ggg agg ctg gga agg aaa ggg acg gtg gta aca gtg tgc gag 1536
 Arg Thr Gly Arg Leu Gly Arg Lys Gly Thr Val Val Thr Val Cys Glu
 500 505 510
 gaa tca caa gtg ttt ata gtg aag aag atg gag aag cag ctt ggt ttg 1584
 Glu Ser Gln Val Phe Ile Val Lys Lys Met Glu Lys Gln Leu Gly Leu
 515 520 525
 cct ttc ttg tat tgt gag ttt gtt gat gga gag ctt gtt gtc act gag 1632
 Pro Phe Leu Tyr Cys Glu Phe Val Asp Gly Glu Leu Val Val Thr Glu
 530 535 540
 gaa gat aaa gct att ata agg tga 1656
 Glu Asp Lys Ala Ile Ile Arg
 545 550
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 551
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;400&gt; SEQUENCE: 2
 Met Ala Ala Ser Thr Ser Thr Arg Phe Leu Val Leu Leu Lys Asp Phe
 1 5 10 15
 Ser Ala Phe Arg Lys Ile Ser Trp Thr Cys Ala Ala Thr Asn Phe His
 20 25 30
 Arg Gln Ser Arg Phe Leu Cys His Val Ala Lys Glu Asp Gly Ser Leu
 35 40 45
 Thr Leu Ala Ser Leu Asp Leu Gly Asn Lys Pro Arg Lys Phe Gly Lys
 50 55 60
 Gly Lys Ala Met Lys Leu Glu Gly Ser Phe Val Thr Glu Met Gly Gln
 65 70 75 80
 Gly Lys Val Arg Ala Val Lys Asn Asp Lys Met Lys Val Val Lys Glu
 85 90 95
 Lys Lys Pro Ala Glu Ile Val Ser Pro Leu Phe Ser Ala Lys Ser Phe
 100 105 110
 Glu Glu Leu Gly Leu Pro Asp Ser Leu Leu Asp Ser Leu Glu Arg Glu
 115 120 125
 Gly Phe Ser Val Pro Thr Asp Val Gln Ser Ala Ala Val Pro Ala Ile
 130 135 140
 Ile Lys Gly His Asp Ala Val Ile Gln Ser Tyr Thr Gly Ser Gly Lys
 145 150 155 160
 Thr Leu Ala Tyr Leu Leu Pro Ile Leu Ser Glu Ile Gly Pro Leu Ala
 165 170 175
 Glu Lys Ser Arg Ser Ser His Ser Glu Asn Asp Lys Arg Thr Glu Ile
 180 185 190
 Gln Ala Met Ile Val Ala Pro Ser Arg Glu Leu Gly Met Gln Ile Val
 195 200 205
 Arg Glu Val Glu Lys Leu Leu Gly Pro Val His Arg Arg Met Val Gln
 210 215 220
 Gln Leu Val Gly Gly Ala Asn Arg Met Arg Gln Glu Glu Ala Leu Lys
 225 230 235 240
 Lys Asn Lys Pro Ala Ile Val Val Gly Thr Pro Gly Arg Ile Ala Glu
 245 250 255
 Ile Ser Lys Gly Gly Lys Leu His Thr His Gly Cys Arg Phe Leu Val
 260 265 270
 Leu Asp Glu Val Asp Glu Leu Leu Ser Phe Asn Phe Arg Glu Asp Ile
 275 280 285
 His Arg Ile Leu Glu His Val Gly Lys Arg Ser Gly Ala Gly Pro Lys
 290 295 300
 Gly Glu Val Asp Glu Arg Ala Asn Arg Gln Thr Ile Leu Val Ser Ala
 305 310 315 320
 Thr Val Pro Phe Ser Val Ile Arg Ala Ala Lys Ser Trp Ser His Glu
 325 330 335
 Pro Val Leu Val Gln Ala Asn Lys Val Thr Pro Leu Asp Thr Val Gln
 340 345 350
 Pro Ser Ala Pro Val Met Ser Leu Thr Pro Thr Thr Ser Glu Ala Asp
 355 360 365
 Gly Gln Ile Gln Thr Thr Ile Gln Ser Leu Pro Pro Ala Leu Lys His
 370 375 380
 Tyr Tyr Cys Ile Ser Lys His Gln His Lys Val Asp Thr Leu Arg Arg
 385 390 395 400
 Cys Val His Ala Leu Asp Ala Gln Ser Val Ile Ala Phe Met Asn His
 405 410 415
 Ser Arg Gln Leu Lys Asp Val Val Tyr Lys Leu Glu Ala Arg Gly Met
 420 425 430
 Asn Ser Ala Glu Met His Gly Asp Leu Gly Lys Leu Gly Arg Ser Thr
 435 440 445
 Val Leu Lys Lys Phe Lys Asn Gly Glu Ile Lys Val Leu Val Thr Asn
 450 455 460
 Glu Leu Ser Ala Arg Gly Leu Asp Val Ala Glu Cys Asp Leu Val Val
 465 470 475 480
 Asn Leu Glu Leu Pro Thr Asp Ala Val His Tyr Ala His Arg Ala Gly
 485 490 495
 Arg Thr Gly Arg Leu Gly Arg Lys Gly Thr Val Val Thr Val Cys Glu
 500 505 510
 Glu Ser Gln Val Phe Ile Val Lys Lys Met Glu Lys Gln Leu Gly Leu
 515 520 525
 Pro Phe Leu Tyr Cys Glu Phe Val Asp Gly Glu Leu Val Val Thr Glu
 530 535 540
 Glu Asp Lys Ala Ile Ile Arg
 545 550
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 1997
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: 5'UTR
 &lt;222&gt; LOCATION: (1)..(271)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (272)..(1927)
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: 3'UTR
 &lt;222&gt; LOCATION: (1928)..(1997)
 &lt;400&gt; SEQUENCE: 3
 attttttgag tcggaacctg aagtatttta gtccgtttgt gataaagaaa accgagactg 60
 taccggttta tcttcagacc cggttgtttg tccggtttgg taaaattaga acctaacctt 120
 tttatccaga actggagact ttggaagaac tgtagaagtg ttgttctctt cgtatcgtcc 180
 tcaatcctca tggagactat tatcaggctg ttttgagcaa acgctgtgat aaagaggctt 240
 tctttcttgc tagcaagtac acacgagtga c atg gcg gca tca act tca acc 292
 Met Ala Ala Ser Thr Ser Thr
 1 5
 cga ttc ctt gtt ctg ctc aaa gat ttt tct gcc ttc aga aag ata tca 340
 Arg Phe Leu Val Leu Leu Lys Asp Phe Ser Ala Phe Arg Lys Ile Ser
 10 15 20
 tgg act tgt gct gca act aat ttt cac cgc caa tct cgt ttt tta tgc 388
 Trp Thr Cys Ala Ala Thr Asn Phe His Arg Gln Ser Arg Phe Leu Cys
 25 30 35
 cat gtt gcg aaa gaa gac ggg tct ctt act ctt gca agc ctt gat ttg 436
 His Val Ala Lys Glu Asp Gly Ser Leu Thr Leu Ala Ser Leu Asp Leu
 40 45 50 55
 ggg aac aaa cca cgg aaa ttt ggg aag ggt aag gcg atg aag ctt gag 484
 Gly Asn Lys Pro Arg Lys Phe Gly Lys Gly Lys Ala Met Lys Leu Glu
 60 65 70
 gga agt ttt gtt act gaa atg ggt caa ggt aag gta aga gcg gta aag 532
 Gly Ser Phe Val Thr Glu Met Gly Gln Gly Lys Val Arg Ala Val Lys
 75 80 85
 aac gat aaa atg aaa gtt gtg aag gaa aaa aag cca gct gag ata gtg 580
 Asn Asp Lys Met Lys Val Val Lys Glu Lys Lys Pro Ala Glu Ile Val
 90 95 100
 tct cct ttg ttt tct gca aaa tcc ttt gag gag ctt ggc ctc ccg gat 628
 Ser Pro Leu Phe Ser Ala Lys Ser Phe Glu Glu Leu Gly Leu Pro Asp
 105 110 115
 tcc ttg tta gac agt ttg gaa aga gaa ggt ttc tct gtc cca aca gat 676
 Ser Leu Leu Asp Ser Leu Glu Arg Glu Gly Phe Ser Val Pro Thr Asp
 120 125 130 135
 gtc caa tca gca gct gtc ccg gca ata atc aaa ggt cac gat gca gtg 724
 Val Gln Ser Ala Ala Val Pro Ala Ile Ile Lys Gly His Asp Ala Val
 140 145 150
 att cag tct tac aca gga tct ggc aaa aca tta gct tat ctg ctt cca 772
 Ile Gln Ser Tyr Thr Gly Ser Gly Lys Thr Leu Ala Tyr Leu Leu Pro
 155 160 165
 ata ttg tcc gaa att ggt cct cta gca gaa aaa tct aga agt tcg cac 820
 Ile Leu Ser Glu Ile Gly Pro Leu Ala Glu Lys Ser Arg Ser Ser His
 170 175 180
 agt gaa aat gat aag agg act gag att cag gca atg atc gtg gct cca 868
 Ser Glu Asn Asp Lys Arg Thr Glu Ile Gln Ala Met Ile Val Ala Pro
 185 190 195
 tca aga gaa ctc ggt atg cag ata gta aga gag gta gag aaa ctg ctc 916
 Ser Arg Glu Leu Gly Met Gln Ile Val Arg Glu Val Glu Lys Leu Leu
 200 205 210 215
 gga cct gtt cac cgt aga atg gtt cag cag ttg gta gga ggt gca aac 964
 Gly Pro Val His Arg Arg Met Val Gln Gln Leu Val Gly Gly Ala Asn
 220 225 230
 cga atg agg caa gaa gag gcc ctt aag aaa aat aaa cct gca att gtt 1012
 Arg Met Arg Gln Glu Glu Ala Leu Lys Lys Asn Lys Pro Ala Ile Val
 235 240 245
 gtt ggc act ccc ggg aga att gca gag ata agc aaa ggt gga aaa ttg 1060
 Val Gly Thr Pro Gly Arg Ile Ala Glu Ile Ser Lys Gly Gly Lys Leu
 250 255 260
 cac act cat ggg tgt aga ttc ttg gtg cta gac gaa gtc gat gag ctt 1108
 His Thr His Gly Cys Arg Phe Leu Val Leu Asp Glu Val Asp Glu Leu
 265 270 275
 tta tcg ttt aat ttc cga gaa gat atc cat cga ata cta gaa cat gta 1156
 Leu Ser Phe Asn Phe Arg Glu Asp Ile His Arg Ile Leu Glu His Val
 280 285 290 295
 gga aag aga tct ggg gct ggt cct aaa gga gaa gtc gat gaa cgg gct 1204
 Gly Lys Arg Ser Gly Ala Gly Pro Lys Gly Glu Val Asp Glu Arg Ala
 300 305 310
 aac cgg cag acc att cta gtc tct gca act gtg cca ttc tcg gtt atc 1252
 Asn Arg Gln Thr Ile Leu Val Ser Ala Thr Val Pro Phe Ser Val Ile
 315 320 325
 cga gca gct aaa agc tgg agt cac gag cct gtt ctt gtc caa gcc aac 1300
 Arg Ala Ala Lys Ser Trp Ser His Glu Pro Val Leu Val Gln Ala Asn
 330 335 340
 aaa gtc act cct ctt gat acc gtt caa cca tct gca ccg gta atg agc 1348
 Lys Val Thr Pro Leu Asp Thr Val Gln Pro Ser Ala Pro Val Met Ser
 345 350 355
 ttg act ccc aca act tct gaa gct gat ggc cag att cag act act att 1396
 Leu Thr Pro Thr Thr Ser Glu Ala Asp Gly Gln Ile Gln Thr Thr Ile
 360 365 370 375
 cag agc tta cct cca gct tta aaa cac tat tac tgc atc tca aag cat 1444
 Gln Ser Leu Pro Pro Ala Leu Lys His Tyr Tyr Cys Ile Ser Lys His
 380 385 390
 caa cac aaa gtc gat acg tta agg aga tgc gtt cac gcc ctc gat gcc 1492
 Gln His Lys Val Asp Thr Leu Arg Arg Cys Val His Ala Leu Asp Ala
 395 400 405
 caa tcg gtt ata gct ttc atg aac cac tca agg cag ctc aaa gat gtg 1540
 Gln Ser Val Ile Ala Phe Met Asn His Ser Arg Gln Leu Lys Asp Val
 410 415 420
 gtc tac aaa ctc gaa gct cgt ggt atg aat tca gct gag atg cac gga 1588
 Val Tyr Lys Leu Glu Ala Arg Gly Met Asn Ser Ala Glu Met His Gly
 425 430 435
 gat ctc ggg aag cta ggg aga tca aca gtt cta aag aag ttc aag aac 1636
 Asp Leu Gly Lys Leu Gly Arg Ser Thr Val Leu Lys Lys Phe Lys Asn
 440 445 450 455
 ggg gaa atc aag gta ctt gtg aca aac gag ctc tct gct cgg ggt ctt 1684
 Gly Glu Ile Lys Val Leu Val Thr Asn Glu Leu Ser Ala Arg Gly Leu
 460 465 470
 gat gtt gcg gaa tgt gat ctg gtg gtg aat ctt gag ctt cca act gat 1732
 Asp Val Ala Glu Cys Asp Leu Val Val Asn Leu Glu Leu Pro Thr Asp
 475 480 485
 gcg gtt cac tat gct cat cga gct ggg aga aca ggg agg ctg gga agg 1780
 Ala Val His Tyr Ala His Arg Ala Gly Arg Thr Gly Arg Leu Gly Arg
 490 495 500
 aaa ggg acg gtg gta aca gtg tgc gag gaa tca caa gtg ttt ata gtg 1828
 Lys Gly Thr Val Val Thr Val Cys Glu Glu Ser Gln Val Phe Ile Val
 505 510 515
 aag aag atg gag aag cag ctt ggt ttg cct ttc ttg tat tgt gag ttt 1876
 Lys Lys Met Glu Lys Gln Leu Gly Leu Pro Phe Leu Tyr Cys Glu Phe
 520 525 530 535
 gtt gat gga gag ctt gtt gtc act gag gaa gat aaa gct att ata agg 1924
 Val Asp Gly Glu Leu Val Val Thr Glu Glu Asp Lys Ala Ile Ile Arg
 540 545 550
 tga aaatctaaag atgtaatttt cagatactat tattactatt gaaaattcag 1977
 agtcaaaaaa aaaaaaaaaa 1997
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 551
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;400&gt; SEQUENCE: 4
 Met Ala Ala Ser Thr Ser Thr Arg Phe Leu Val Leu Leu Lys Asp Phe
 1 5 10 15
 Ser Ala Phe Arg Lys Ile Ser Trp Thr Cys Ala Ala Thr Asn Phe His
 20 25 30
 Arg Gln Ser Arg Phe Leu Cys His Val Ala Lys Glu Asp Gly Ser Leu
 35 40 45
 Thr Leu Ala Ser Leu Asp Leu Gly Asn Lys Pro Arg Lys Phe Gly Lys
 50 55 60
 Gly Lys Ala Met Lys Leu Glu Gly Ser Phe Val Thr Glu Met Gly Gln
 65 70 75 80
 Gly Lys Val Arg Ala Val Lys Asn Asp Lys Met Lys Val Val Lys Glu
 85 90 95
 Lys Lys Pro Ala Glu Ile Val Ser Pro Leu Phe Ser Ala Lys Ser Phe
 100 105 110
 Glu Glu Leu Gly Leu Pro Asp Ser Leu Leu Asp Ser Leu Glu Arg Glu
 115 120 125
 Gly Phe Ser Val Pro Thr Asp Val Gln Ser Ala Ala Val Pro Ala Ile
 130 135 140
 Ile Lys Gly His Asp Ala Val Ile Gln Ser Tyr Thr Gly Ser Gly Lys
 145 150 155 160
 Thr Leu Ala Tyr Leu Leu Pro Ile Leu Ser Glu Ile Gly Pro Leu Ala
 165 170 175
 Glu Lys Ser Arg Ser Ser His Ser Glu Asn Asp Lys Arg Thr Glu Ile
 180 185 190
 Gln Ala Met Ile Val Ala Pro Ser Arg Glu Leu Gly Met Gln Ile Val
 195 200 205
 Arg Glu Val Glu Lys Leu Leu Gly Pro Val His Arg Arg Met Val Gln
 210 215 220
 Gln Leu Val Gly Gly Ala Asn Arg Met Arg Gln Glu Glu Ala Leu Lys
 225 230 235 240
 Lys Asn Lys Pro Ala Ile Val Val Gly Thr Pro Gly Arg Ile Ala Glu
 245 250 255
 Ile Ser Lys Gly Gly Lys Leu His Thr His Gly Cys Arg Phe Leu Val
 260 265 270
 Leu Asp Glu Val Asp Glu Leu Leu Ser Phe Asn Phe Arg Glu Asp Ile
 275 280 285
 His Arg Ile Leu Glu His Val Gly Lys Arg Ser Gly Ala Gly Pro Lys
 290 295 300
 Gly Glu Val Asp Glu Arg Ala Asn Arg Gln Thr Ile Leu Val Ser Ala
 305 310 315 320
 Thr Val Pro Phe Ser Val Ile Arg Ala Ala Lys Ser Trp Ser His Glu
 325 330 335
 Pro Val Leu Val Gln Ala Asn Lys Val Thr Pro Leu Asp Thr Val Gln
 340 345 350
 Pro Ser Ala Pro Val Met Ser Leu Thr Pro Thr Thr Ser Glu Ala Asp
 355 360 365
 Gly Gln Ile Gln Thr Thr Ile Gln Ser Leu Pro Pro Ala Leu Lys His
 370 375 380
 Tyr Tyr Cys Ile Ser Lys His Gln His Lys Val Asp Thr Leu Arg Arg
 385 390 395 400
 Cys Val His Ala Leu Asp Ala Gln Ser Val Ile Ala Phe Met Asn His
 405 410 415
 Ser Arg Gln Leu Lys Asp Val Val Tyr Lys Leu Glu Ala Arg Gly Met
 420 425 430
 Asn Ser Ala Glu Met His Gly Asp Leu Gly Lys Leu Gly Arg Ser Thr
 435 440 445
 Val Leu Lys Lys Phe Lys Asn Gly Glu Ile Lys Val Leu Val Thr Asn
 450 455 460
 Glu Leu Ser Ala Arg Gly Leu Asp Val Ala Glu Cys Asp Leu Val Val
 465 470 475 480
 Asn Leu Glu Leu Pro Thr Asp Ala Val His Tyr Ala His Arg Ala Gly
 485 490 495
 Arg Thr Gly Arg Leu Gly Arg Lys Gly Thr Val Val Thr Val Cys Glu
 500 505 510
 Glu Ser Gln Val Phe Ile Val Lys Lys Met Glu Lys Gln Leu Gly Leu
 515 520 525
 Pro Phe Leu Tyr Cys Glu Phe Val Asp Gly Glu Leu Val Val Thr Glu
 530 535 540
 Glu Asp Lys Ala Ile Ile Arg
 545 550
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 558
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (1)..(558)
 &lt;400&gt; SEQUENCE: 5
 atg gga ctg tta agc ata atc cgg aag atc aag aag aaa gag aag gag 48
 Met Gly Leu Leu Ser Ile Ile Arg Lys Ile Lys Lys Lys Glu Lys Glu
 1 5 10 15
 atg cgt att ctt atg gtt gga ctt gat aat tct ggg aag acg acg att 96
 Met Arg Ile Leu Met Val Gly Leu Asp Asn Ser Gly Lys Thr Thr Ile
 20 25 30
 gtt ctg aaa ata aac gga gaa gac aca agt gtg att agt cca act ctt 144
 Val Leu Lys Ile Asn Gly Glu Asp Thr Ser Val Ile Ser Pro Thr Leu
 35 40 45
 gga ttc aac atc aaa acc att atc tac caa aag tat acg cta aat ata 192
 Gly Phe Asn Ile Lys Thr Ile Ile Tyr Gln Lys Tyr Thr Leu Asn Ile
 50 55 60
 tgg gat gtt ggt ggg caa aag act ata aga tcg tat tgg agg aat tac 240
 Trp Asp Val Gly Gly Gln Lys Thr Ile Arg Ser Tyr Trp Arg Asn Tyr
 65 70 75 80
 ttt gag cag act gat ggt ttg gtt tgg gtg gtt gat agt tct gat ctt 288
 Phe Glu Gln Thr Asp Gly Leu Val Trp Val Val Asp Ser Ser Asp Leu
 85 90 95
 agg agg tta gat gat tgc aag atg gaa ctt gac aat ctc ttg aaa gaa 336
 Arg Arg Leu Asp Asp Cys Lys Met Glu Leu Asp Asn Leu Leu Lys Glu
 100 105 110
 gag agg cta gct ggt tca tct ttg ctg ata cta gca aat aag cag gat 384
 Glu Arg Leu Ala Gly Ser Ser Leu Leu Ile Leu Ala Asn Lys Gln Asp
 115 120 125
 att caa ggt gca cta aca cct gat gaa att ggc aag gtg cta aac tta 432
 Ile Gln Gly Ala Leu Thr Pro Asp Glu Ile Gly Lys Val Leu Asn Leu
 130 135 140
 gag tcc atg gat aaa agc cgg cac tgg aag ata gtg ggt tgc agc gca 480
 Glu Ser Met Asp Lys Ser Arg His Trp Lys Ile Val Gly Cys Ser Ala
 145 150 155 160
 tac acg ggt gaa ggt ttg ttg gaa gga ttc gat tgg ttg gtt caa gac 528
 Tyr Thr Gly Glu Gly Leu Leu Glu Gly Phe Asp Trp Leu Val Gln Asp
 165 170 175
 att gcc tcc agg att tac atg ctt gac taa 558
 Ile Ala Ser Arg Ile Tyr Met Leu Asp
 180 185
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 185
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;400&gt; SEQUENCE: 6
 Met Gly Leu Leu Ser Ile Ile Arg Lys Ile Lys Lys Lys Glu Lys Glu
 1 5 10 15
 Met Arg Ile Leu Met Val Gly Leu Asp Asn Ser Gly Lys Thr Thr Ile
 20 25 30
 Val Leu Lys Ile Asn Gly Glu Asp Thr Ser Val Ile Ser Pro Thr Leu
 35 40 45
 Gly Phe Asn Ile Lys Thr Ile Ile Tyr Gln Lys Tyr Thr Leu Asn Ile
 50 55 60
 Trp Asp Val Gly Gly Gln Lys Thr Ile Arg Ser Tyr Trp Arg Asn Tyr
 65 70 75 80
 Phe Glu Gln Thr Asp Gly Leu Val Trp Val Val Asp Ser Ser Asp Leu
 85 90 95
 Arg Arg Leu Asp Asp Cys Lys Met Glu Leu Asp Asn Leu Leu Lys Glu
 100 105 110
 Glu Arg Leu Ala Gly Ser Ser Leu Leu Ile Leu Ala Asn Lys Gln Asp
 115 120 125
 Ile Gln Gly Ala Leu Thr Pro Asp Glu Ile Gly Lys Val Leu Asn Leu
 130 135 140
 Glu Ser Met Asp Lys Ser Arg His Trp Lys Ile Val Gly Cys Ser Ala
 145 150 155 160
 Tyr Thr Gly Glu Gly Leu Leu Glu Gly Phe Asp Trp Leu Val Gln Asp
 165 170 175
 Ile Ala Ser Arg Ile Tyr Met Leu Asp
 180 185
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 1212
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (1)..(1212)
 &lt;400&gt; SEQUENCE: 7
 atg gcc cat aca tca gaa tct gtg aat cct aga gat gtt tgc att gtg 48
 Met Ala His Thr Ser Glu Ser Val Asn Pro Arg Asp Val Cys Ile Val
 1 5 10 15
 ggt gtt gca cgt act cca atg ggt ggc ttt ctc gga tct ctt tca tct 96
 Gly Val Ala Arg Thr Pro Met Gly Gly Phe Leu Gly Ser Leu Ser Ser
 20 25 30
 tta cct gcc aca aag ctt gga tct tta gct att gca gct gct ttg aag 144
 Leu Pro Ala Thr Lys Leu Gly Ser Leu Ala Ile Ala Ala Ala Leu Lys
 35 40 45
 aga gca aat gtt gat cca gct ctt gtt caa gaa gtt gtc ttt ggc aat 192
 Arg Ala Asn Val Asp Pro Ala Leu Val Gln Glu Val Val Phe Gly Asn
 50 55 60
 gtt ctt agt gct aat ttg ggt caa gct cct gct cgt caa gct gct tta 240
 Val Leu Ser Ala Asn Leu Gly Gln Ala Pro Ala Arg Gln Ala Ala Leu
 65 70 75 80
 ggt gca gga atc cct aac tct gtt atc tgt act aca gtt aac aag gtt 288
 Gly Ala Gly Ile Pro Asn Ser Val Ile Cys Thr Thr Val Asn Lys Val
 85 90 95
 tgt gca tca ggc atg aaa gcg gta atg att gct gct caa agt atc cag 336
 Cys Ala Ser Gly Met Lys Ala Val Met Ile Ala Ala Gln Ser Ile Gln
 100 105 110
 tta ggg atc aat gat gta gtt gtg gcg ggt ggt atg gaa agc atg tct 384
 Leu Gly Ile Asn Asp Val Val Val Ala Gly Gly Met Glu Ser Met Ser
 115 120 125
 aat aca cca aaa tat ttg gca gaa gca agg aag gga tct cgt ttt ggt 432
 Asn Thr Pro Lys Tyr Leu Ala Glu Ala Arg Lys Gly Ser Arg Phe Gly
 130 135 140
 cat gat tct tta gta gat gga atg ttg aag gat gga cta tgg gat gtc 480
 His Asp Ser Leu Val Asp Gly Met Leu Lys Asp Gly Leu Trp Asp Val
 145 150 155 160
 tat aac gac tgt ggg atg gga agc tgt gca gaa tta tgc gct gag aag 528
 Tyr Asn Asp Cys Gly Met Gly Ser Cys Ala Glu Leu Cys Ala Glu Lys
 165 170 175
 ttt cag att aca agg gag cag caa gat gac tat gca gtt cag agt ttt 576
 Phe Gln Ile Thr Arg Glu Gln Gln Asp Asp Tyr Ala Val Gln Ser Phe
 180 185 190
 gag cgt ggt att gct gcc cag gaa gct ggc gcc ttc aca tgg gaa atc 624
 Glu Arg Gly Ile Ala Ala Gln Glu Ala Gly Ala Phe Thr Trp Glu Ile
 195 200 205
 gtc ccg gtt gaa gtt tct gga gga aga ggt agg cca tca acc att gtt 672
 Val Pro Val Glu Val Ser Gly Gly Arg Gly Arg Pro Ser Thr Ile Val
 210 215 220
 gac aag gac gaa ggt ctt ggg aag ttt gat gct gca aaa ttg agg aaa 720
 Asp Lys Asp Glu Gly Leu Gly Lys Phe Asp Ala Ala Lys Leu Arg Lys
 225 230 235 240
 ctc cgt cct agt ttc aaa gag aat gga ggg act gtt aca gct gga aat 768
 Leu Arg Pro Ser Phe Lys Glu Asn Gly Gly Thr Val Thr Ala Gly Asn
 245 250 255
 gcg tct agc ata agt gat ggt gca gct gcc ctt gtc cta gtg agc gga 816
 Ala Ser Ser Ile Ser Asp Gly Ala Ala Ala Leu Val Leu Val Ser Gly
 260 265 270
 gag aag gct ctt cag cta gga ctt cta gta tta gca aaa att aaa ggg 864
 Glu Lys Ala Leu Gln Leu Gly Leu Leu Val Leu Ala Lys Ile Lys Gly
 275 280 285
 tat ggt gac gca gct cag gaa cca gag ttt ttc act act gct cct gct 912
 Tyr Gly Asp Ala Ala Gln Glu Pro Glu Phe Phe Thr Thr Ala Pro Ala
 290 295 300
 ctt gct ata cca aaa gcc att gca cat gct ggt ttg gaa tct tct caa 960
 Leu Ala Ile Pro Lys Ala Ile Ala His Ala Gly Leu Glu Ser Ser Gln
 305 310 315 320
 gtt gat tac tat gag atc aat gaa gca ttt gca gtt gta gca ctt gca 1008
 Val Asp Tyr Tyr Glu Ile Asn Glu Ala Phe Ala Val Val Ala Leu Ala
 325 330 335
 aat caa aag cta ctc ggg att gct cca gag aaa gtg aac gta aat gga 1056
 Asn Gln Lys Leu Leu Gly Ile Ala Pro Glu Lys Val Asn Val Asn Gly
 340 345 350
 gga gct gtc tcc tta gga cac cct cta ggc tgc agt ggc gcc cgt att 1104
 Gly Ala Val Ser Leu Gly His Pro Leu Gly Cys Ser Gly Ala Arg Ile
 355 360 365
 cta atc acg ttg ctt ggg ata cta aag aag aga aac gga aag tac ggt 1152
 Leu Ile Thr Leu Leu Gly Ile Leu Lys Lys Arg Asn Gly Lys Tyr Gly
 370 375 380
 gtg gga gga gtg tgc aac gga gga gga ggt gct tct gct cta gtt ctt 1200
 Val Gly Gly Val Cys Asn Gly Gly Gly Gly Ala Ser Ala Leu Val Leu
 385 390 395 400
 gag ctc ctt tga 1212
 Glu Leu Leu
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 403
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;400&gt; SEQUENCE: 8
 Met Ala His Thr Ser Glu Ser Val Asn Pro Arg Asp Val Cys Ile Val
 1 5 10 15
 Gly Val Ala Arg Thr Pro Met Gly Gly Phe Leu Gly Ser Leu Ser Ser
 20 25 30
 Leu Pro Ala Thr Lys Leu Gly Ser Leu Ala Ile Ala Ala Ala Leu Lys
 35 40 45
 Arg Ala Asn Val Asp Pro Ala Leu Val Gln Glu Val Val Phe Gly Asn
 50 55 60
 Val Leu Ser Ala Asn Leu Gly Gln Ala Pro Ala Arg Gln Ala Ala Leu
 65 70 75 80
 Gly Ala Gly Ile Pro Asn Ser Val Ile Cys Thr Thr Val Asn Lys Val
 85 90 95
 Cys Ala Ser Gly Met Lys Ala Val Met Ile Ala Ala Gln Ser Ile Gln
 100 105 110
 Leu Gly Ile Asn Asp Val Val Val Ala Gly Gly Met Glu Ser Met Ser
 115 120 125
 Asn Thr Pro Lys Tyr Leu Ala Glu Ala Arg Lys Gly Ser Arg Phe Gly
 130 135 140
 His Asp Ser Leu Val Asp Gly Met Leu Lys Asp Gly Leu Trp Asp Val
 145 150 155 160
 Tyr Asn Asp Cys Gly Met Gly Ser Cys Ala Glu Leu Cys Ala Glu Lys
 165 170 175
 Phe Gln Ile Thr Arg Glu Gln Gln Asp Asp Tyr Ala Val Gln Ser Phe
 180 185 190
 Glu Arg Gly Ile Ala Ala Gln Glu Ala Gly Ala Phe Thr Trp Glu Ile
 195 200 205
 Val Pro Val Glu Val Ser Gly Gly Arg Gly Arg Pro Ser Thr Ile Val
 210 215 220
 Asp Lys Asp Glu Gly Leu Gly Lys Phe Asp Ala Ala Lys Leu Arg Lys
 225 230 235 240
 Leu Arg Pro Ser Phe Lys Glu Asn Gly Gly Thr Val Thr Ala Gly Asn
 245 250 255
 Ala Ser Ser Ile Ser Asp Gly Ala Ala Ala Leu Val Leu Val Ser Gly
 260 265 270
 Glu Lys Ala Leu Gln Leu Gly Leu Leu Val Leu Ala Lys Ile Lys Gly
 275 280 285
 Tyr Gly Asp Ala Ala Gln Glu Pro Glu Phe Phe Thr Thr Ala Pro Ala
 290 295 300
 Leu Ala Ile Pro Lys Ala Ile Ala His Ala Gly Leu Glu Ser Ser Gln
 305 310 315 320
 Val Asp Tyr Tyr Glu Ile Asn Glu Ala Phe Ala Val Val Ala Leu Ala
 325 330 335
 Asn Gln Lys Leu Leu Gly Ile Ala Pro Glu Lys Val Asn Val Asn Gly
 340 345 350
 Gly Ala Val Ser Leu Gly His Pro Leu Gly Cys Ser Gly Ala Arg Ile
 355 360 365
 Leu Ile Thr Leu Leu Gly Ile Leu Lys Lys Arg Asn Gly Lys Tyr Gly
 370 375 380
 Val Gly Gly Val Cys Asn Gly Gly Gly Gly Ala Ser Ala Leu Val Leu
 385 390 395 400
 Glu Leu Leu
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 9
 ngtcgaswga nawgaa 16
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 10
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 10
 tgwgnagsan casaga 16
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 11
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 11
 agwgnagwan cawagg 16
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 12
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 12
 sttgntastn ctntgc 16
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 13
 &lt;211&gt; LENGTH: 15
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 13
 ntcgastwts gwgtt 15
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 14
 &lt;211&gt; LENGTH: 16
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 14
 wgtgnagwan canaga 16
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 15
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 15
 attaggcacc ccaggcttta cactttatg 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 16
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 16
 gtatgttgtg tggaattgtg agcggataac 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 17
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 17
 taacaatttc acacaggaaa cagctatgac 30
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 18
 &lt;211&gt; LENGTH: 34
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 18
 tagcatctga atttcataac caatctcgat acac 34
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 19
 &lt;211&gt; LENGTH: 34
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 19
 gcttcctatt atatcttccc aaattaccaa taca 34
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 20
 &lt;211&gt; LENGTH: 34
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 20
 gccttttcag aaatggataa atagccttgc ttcc 34
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 21
 &lt;211&gt; LENGTH: 705
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (1)..(705)
 &lt;400&gt; SEQUENCE: 21
 atg gcg tct ctt caa caa act cta ttc tct ctt caa tcc aaa ctc cca 48
 Met Ala Ser Leu Gln Gln Thr Leu Phe Ser Leu Gln Ser Lys Leu Pro
 1 5 10 15
 cca tcc tcc ttc caa atc gcc aga tct ctc cca ctc cga aaa acc ttc 96
 Pro Ser Ser Phe Gln Ile Ala Arg Ser Leu Pro Leu Arg Lys Thr Phe
 20 25 30
 cca atc cga atc aac aac ggt gga aac gcc gcc gga gca aga atg tca 144
 Pro Ile Arg Ile Asn Asn Gly Gly Asn Ala Ala Gly Ala Arg Met Ser
 35 40 45
 gcc acc gca gca tca agc tac gcg atg gca tta gca gac gtc gcg aaa 192
 Ala Thr Ala Ala Ser Ser Tyr Ala Met Ala Leu Ala Asp Val Ala Lys
 50 55 60
 aga aac gac aca atg gaa tta aca gtc aca gac atc gag aag ctc gaa 240
 Arg Asn Asp Thr Met Glu Leu Thr Val Thr Asp Ile Glu Lys Leu Glu
 65 70 75 80
 caa gtc ttc tca gat cca caa gta cta aac ttc ttc gcg aat cca aca 288
 Gln Val Phe Ser Asp Pro Gln Val Leu Asn Phe Phe Ala Asn Pro Thr
 85 90 95
 atc acc gtc gag aag aaa cgt caa gtc atc gac gac ata gtg aaa tcg 336
 Ile Thr Val Glu Lys Lys Arg Gln Val Ile Asp Asp Ile Val Lys Ser
 100 105 110
 tcg tct ctt caa tct cac aca tct aac ttc ctc aac gtc ctc gtc gac 384
 Ser Ser Leu Gln Ser His Thr Ser Asn Phe Leu Asn Val Leu Val Asp
 115 120 125
 gcg aat cgg atc aat atc gtg acg gag atc gtt aag gag ttt gag ttg 432
 Ala Asn Arg Ile Asn Ile Val Thr Glu Ile Val Lys Glu Phe Glu Leu
 130 135 140
 gtt tac aat aag cta acg gat aca caa ttg gcg gag gtt agg tcg gtg 480
 Val Tyr Asn Lys Leu Thr Asp Thr Gln Leu Ala Glu Val Arg Ser Val
 145 150 155 160
 gtg aaa ttg gaa gcg ccg caa tta gct cag att gcg aaa cag gtt cag 528
 Val Lys Leu Glu Ala Pro Gln Leu Ala Gln Ile Ala Lys Gln Val Gln
 165 170 175
 aag tta acc gga gct aag aat gtt cgg gtt aag acg gtt att gat gcg 576
 Lys Leu Thr Gly Ala Lys Asn Val Arg Val Lys Thr Val Ile Asp Ala
 180 185 190
 agt ctt gtg gct ggt ttt acg att cgg tat ggt gaa tcc ggt tcg aag 624
 Ser Leu Val Ala Gly Phe Thr Ile Arg Tyr Gly Glu Ser Gly Ser Lys
 195 200 205
 ctt att gat atg agt gtg aag aaa cag ctt gaa gat att gct tct cag 672
 Leu Ile Asp Met Ser Val Lys Lys Gln Leu Glu Asp Ile Ala Ser Gln
 210 215 220
 ctt gaa ctt ggt gag att caa tta gct act tga 705
 Leu Glu Leu Gly Glu Ile Gln Leu Ala Thr
 225 230 235
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 22
 &lt;211&gt; LENGTH: 234
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;400&gt; SEQUENCE: 22
 Met Ala Ser Leu Gln Gln Thr Leu Phe Ser Leu Gln Ser Lys Leu Pro
 1 5 10 15
 Pro Ser Ser Phe Gln Ile Ala Arg Ser Leu Pro Leu Arg Lys Thr Phe
 20 25 30
 Pro Ile Arg Ile Asn Asn Gly Gly Asn Ala Ala Gly Ala Arg Met Ser
 35 40 45
 Ala Thr Ala Ala Ser Ser Tyr Ala Met Ala Leu Ala Asp Val Ala Lys
 50 55 60
 Arg Asn Asp Thr Met Glu Leu Thr Val Thr Asp Ile Glu Lys Leu Glu
 65 70 75 80
 Gln Val Phe Ser Asp Pro Gln Val Leu Asn Phe Phe Ala Asn Pro Thr
 85 90 95
 Ile Thr Val Glu Lys Lys Arg Gln Val Ile Asp Asp Ile Val Lys Ser
 100 105 110
 Ser Ser Leu Gln Ser His Thr Ser Asn Phe Leu Asn Val Leu Val Asp
 115 120 125
 Ala Asn Arg Ile Asn Ile Val Thr Glu Ile Val Lys Glu Phe Glu Leu
 130 135 140
 Val Tyr Asn Lys Leu Thr Asp Thr Gln Leu Ala Glu Val Arg Ser Val
 145 150 155 160
 Val Lys Leu Glu Ala Pro Gln Leu Ala Gln Ile Ala Lys Gln Val Gln
 165 170 175
 Lys Leu Thr Gly Ala Lys Asn Val Arg Val Lys Thr Val Ile Asp Ala
 180 185 190
 Ser Leu Val Ala Gly Phe Thr Ile Arg Tyr Gly Glu Ser Gly Ser Lys
 195 200 205
 Leu Ile Asp Met Ser Val Lys Lys Gln Leu Glu Asp Ile Ala Ser Gln
 210 215 220
 Leu Glu Leu Gly Glu Ile Gln Leu Ala Thr
 225 230
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 23
 &lt;211&gt; LENGTH: 1011
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;400&gt; SEQUENCE: 23
 aaccacaaat ctctctttct ctcaaactct ctcaacaaca acaatggcgt ctcttcaaca 60
 aactctattc tctcttcaat ccaaactccc accatcctcc ttccaaatcg ccagatctct 120
 cccactccga aaaaccttcc caatccgaat caacaacggt ggaaacgccg ccggagcaag 180
 aatgtcagcc accgcagcat caagctacgc gatggcatta gcagacgtcg cgaaaagaaa 240
 cgacacaatg gaattaacag tcacagacat cgagaagctc gaacaagtct tctcagatcc 300
 acaagtacta aacttcttcg cgaatccaac aatcaccgtc gagaagaaac gtcaagtcat 360
 cgacgacata gtgaaatcgt cgtctcttca atctcacaca tctaacttcc tcaacgtcct 420
 cgtcgacgcg aatcggatca atatcgtgac ggagatcgtt aaggagtttg agttggttta 480
 caataagcta acggatacac aattggcgga ggttaggtcg gtggtgaaat tggaagcgcc 540
 gcaattagct cagattgcga aacaggttca gaagttaacc ggagctaaga atgttcgggt 600
 taagacggtt attgatgcga gtcttgtggc tggttttacg attcggtatg gtgaatccgg 660
 ttcgaagctt attgatatga gtgtgaagaa acagcttgaa gatattgctt ctcagcttga 720
 acttggtgag attcaattag ctacttgaga tttgggaaaa attgtataag agaaaaattt 780
 gagaatcttt tttttttgtg caagtttaat tttttttctc ctcatcttct ttctctatta 840
 atcaatcata taatatacag tactgatgat ataataatga ttctgagttt attatctttg 900
 taattgttaa atttagtgaa ttcgaaaacg aattcgaata gtatgtttgc ggattatgcg 960
 ttttggggaa tggttttact gttaaattgc ggttaatctc ggttgaatag a 1011
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 24
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: 5'UTR
 &lt;222&gt; LOCATION: (1)..(21)
 &lt;400&gt; SEQUENCE: 24
 caaactctct caacaacaac a 21
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 25
 &lt;211&gt; LENGTH: 192
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Arabidopsis thaliana
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: 3'UTR
 &lt;222&gt; LOCATION: (1)..(192)
 &lt;400&gt; SEQUENCE: 25
 gatttgggaa aaattgtata agagaaaaat ttgagaatct tttttttttg tgcaagttta 60
 attttttttc tcctcatctt ctttctctat taatcaatca tataatatac agtactgatg 120
 atataataat gattctgagt ttattatctt tgtaattgtt aaatttagtg aattcgaaaa 180
 cgaattcgaa ta 192
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 26
 &lt;211&gt; LENGTH: 20
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence
 oligonucleotide
 &lt;400&gt; SEQUENCE: 26
 gcggacatct acatttttga 20