WO1998056935A2 - Plant amino acid biosynthetic enzymes - Google Patents

Abstract

This invention relates to an isolated nucleic acid fragment encoding a plant enzyme that catalyze steps in the biosynthesis of lysine, threonine, methionine, cysteine and isoleucine from aspartate, the enzyme being a member selected from the group consisting of aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the enzyme in a transformed host cell.

Description

TITLE PLANT AMINO ACID BIOSYNTHETIC ENZYMES This application claims the benefit of U.S. Provisional Application No. 60/049406, filed June 12, 1997, and U.S. Provisional Application No. 60/065385. filed November 12, 1997.

FIELD OF THE INVENTION This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in amino acid biosynthesis in plants and seeds. BACKGROUND OF THE INVENTION

Many vertebrates, including man. lack the ability to manufacture a number of amino acids and therefore require these amino acids preformed in the diet. These are called essential amino acids. Human food and animal feed, derived from many grains, are deficient in essential amino acids, such as lysine. the sulfur amino acids methionine and cysteine. threonine and tryptophan. For example, in com (Zea mays L.) lysine is the most limitinε amino acid for the dietary requirements of many animals. Soybean (Glycine max L.) meal is used as an additive to corn-based animal feeds primarily as a lysine supplement. Thus, an increase in the lysine content of either com or soybean would reduce or eliminate the need to supplement mixed grain feeds with lysine produced via fermentation of microbes. Furthermore, in com the sulfur amino acids are the third most limiting amino acids, after lysine and tryptophan. for the dietary requirements of many animals. The use of soybean meal, which is rich in lysine and tryptophan. to supplement com in animal feed is limited by the low sulfur amino acid content of the legume. Thus, an increase in the sulfur amino acid content of either com or soybean would improve the nutritional quality of the mixtures and reduce the need for further supplementation through addition of more expensive methionine. Lysine. threonine. methionine. cysteine and isoleucine are amino acids derived from aspartate. Regulation of the biosynthesis of each member of this family is interconnected (see Figure 1 ). One approach to increasing the nutritional quality of human foods and animal feed is to increase the production and accumulation of specific free amino acids via genetic engineering of this biosynthetic pathway. Alteration of the activity of enzymes in this pathway could lead to altered levels of lysine, threonine. methionine, cysteine and isoleucine. However, few of the genes encoding enzymes that regulate this pathway in plants, especially com and soybeans, are available.

The organization of the pathway leading to biosynthesis of lysine, threonine, methionine, cysteine and isoleucine indicates that over-expression or reduction of expression of genes encoding, inter alia, aspartic semialedhyde dehydrogenase. homoserine kinase, diaminopimelate decarboxylase, cysteine synthase and cystathionine β-lyase in com and soybean could be used to alter levels of these amino acids in human food and animal feed. Accordingly, availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate development of nutritionally improved crop plants.

SUMMARY OF THE INVENTION The instant invention relates to isolated nucleic acid fragments encoding plant enzymes involved in amino acid biosynthesis. Specifically, this invention concerns isolated nucleic acid fragments encoding the following plant enzymes that catalyze steps in the biosynthesis of lysine, threonine, methionine, cysteine and isoleucine from aspartate: aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding the listed plant biosynthetic enzymes.

In another embodiment, the instant invention relates to chimeric genes encoding the amino acid biosynthetic acid enzymes listed above or to chimeric genes that comprise nucleic acid fragments that are complementary to the nucleic acid fragments encoding the enzymes, operably linked to suitable regulatory sequences, wherein expression of the chimeric genes results in production of levels of the encoded enzymes in transformed host cells that are altered (i.e., increased or decreased) from the levels produced in untransformed host cells.

In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a plant amino acid biosynthetic enzyme operably linked to suitable regulatory sequences, the enzyme selected from the group consisting of: aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase. cysteine synthase and cystathionine β-lyase. Expression of the chimeric gene results in production of altered levels of the biosynthetic enzyme in the transformed host cell. The transformed host cells can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method of altering the level of expression of a plant biosynthetic enzyme in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a plant biosynthetic enzyme selected from the group consisting of aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase, operably linked to suitable regulatory sequences; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the biosynthetic enzyme in the transformed host cell.

An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or substantially all of an amino acid sequence encoding a plant aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase.

A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a plant biosynthetic enzyme selected from the group consisting of aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a plant biosynthetic enzyme selected from the group consisting of aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the biosynthetic enzyme in the transformed host cell; (c) optionally purifying the biosynthetic enzyme expressed by the transformed host cell; (d) treating the biosynthetic enzyme with a compound to be tested; and (e) comparing the activity of the biosynthetic enzyme that has been treated with a test compound to the activity of an untreated biosynthetic enzyme, thereby selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS The invention can be more fully understood from the following detailed description and the accompanying drawings and sequence descriptions which form a part of this application.

Figure 1 depicts the biosynthetic pathway for the aspartate family of amino acids. The following abbreviations are used: AK = aspartokinase; ASADH = aspartic semialdehyde dehydrogenase: DHDPS = dihydrodipicolinate synthase; DHDPR = dihydrodipicolinate reductase; DAPEP = diaminopimelate epimerase; DAPDC = diaminopimelate decarboxylase: HDH = homoserine dehydrogenase; HK = homoserine kinase; TS = threonine synthase; TD = threonine deaminase; CγS = cystathionine γ-synthase; CβL = cystathionine β-lyase; MS = methionine synthase; CS = cysteine synthase; and SAMS = S-adenosylmethionine synthase.

Figures 2 through 6 show the amino acid sequence alignments between the known art sequence for aspartic semialdehyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase, and cystathionine β-lyase vs the sequences included in this application. Alignments were performed using the Clustal alogarithm described in Higgins and Sharp (1989) (CABIOS 5:151-153). Identical amino acids are indicated by gray boxes. In cases where the alignment includes more than two sequences only amino acids which are identical in all sequences are indicated. A description of Figures 2 through 6 follows: Figure 2 depicts the amino acid sequence alignment between the aspartic semialdehyde dehydrogenases from rice clone rlr.pk003.dl 1 (SEQ ID NO:2). wheat clone wrl.pk0004.cl 1 (SEQ ID NO:4), soybean clone sfll.pk0122.f9 (SEQ ID NO:6), and Legionella pneumophila (GenBank Accession No. AF034213. SEQ ID NO:7). Figure 3 depicts the amino acid sequence alignments between the diaminopimelate decarboxylases from com clones cen3n.pk0067.a3 (SEQ ID NO:9), crln.pk0103.d8 (SEQ ID NO:l 1), rice clone rl0n.pk0013.b9 (SEQ ID NO:13), soybean clone srl.pk0132.cl (SEQ ID NO:15), wheat clone wlkl.pk0012.c2 (SEQ ID NO:17), and Pseudomonas aeruginosa (GenBank Accession No. 118304, SEQ ID NO:20). Figure 4 depicts the amino acid sequence alignments between the homoserine kinase from com clone crln.pk0009.g4 (SEQ ID NO:22), rice clone rcalc.pk005.k3 (SEQ ID NO:24), soybean clone ses8w,pk0020.b5 (SEQ ID NO:26), wheat clone wlln.pk0065.f2 (SEQ ID NO:28), and Methanococcus jannaschii (GenBank Accession No. U67553, SEQ ID NO:29). Figure 5 depicts the amino acid sequence alignment between the cysteine synthase from soybean clone se3.05h06 (SEQ ID NO:31), and Citrullus lanatus (GenBank Accession No. D28777, SEQ ID NO:32).

Figure 6 depicts the amino acid sequence alignment between the cystathionine β-lyase from com clone cenl .pk0061.d4 (SEQ ID NO:34), rice clone rlrl2.pk0061.d4 (SEQ ID NO:36), soybean clone sfll .pk0012.c4 (SEQ ID NO:38), wheat clone wrl . pk0091.g6 (SEQ ID NO:40), and Arabidopsis thaliana (GenBank Accession No. L40511, SEQ ID NO:41). The following sequence descriptions and sequences listings attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. SEQ ID NO:l is the nucleotide sequence comprising the entire cDNA insert in clone rlr48.pk0003.dl2 encoding a portion of a rice aspartic-semialdehyde dehydrogenase.

SEQ ID NO:2 is the deduced amino acid sequence of a portion of a rice aspartic- semialdehyde dehydrogenase derived from the nucleotide sequence of SEQ ID NO:l.

SEQ ID NO:3 is the nucleotide sequence comprising a portion of the cDNA insert in clone wrl .pk0004.cl 1 encoding a portion of a wheat aspartic-semialdehyde dehydrogenase. SEQ ID NO:4 is the deduced amino acid sequence of a portion of a wheat aspartic- semialdehyde dehydrogenase derived from the nucleotide sequence of SEQ ID NO:3.

SEQ ID NO: 5 is the nucleotide sequence comprising a portion of the cDNA insert in clone sfll.pk0122.f9 encoding a portion of a soybean aspartic-semialdehyde dehydrogenase. SEQ ID NO:6 is the deduced amino acid sequence of a portion of a soybean aspartic- semialdehyde dehydrogenase derived from the nucleotide sequence of SEQ ID NO:5.

SEQ ID NO:7 is the amino acid sequence for a Legionella pneumophila semialdehyde dehydrogenase found in GenBank Accession No. AF034213. SEQ ID NO: 8 is the nucleotide sequence comprising the entire cDNA insert in clone cen3n.pk0067.a3 encoding a portion of a com diaminopimelate decarboxylase.

SEQ ID NO:9 is the deduced amino acid sequence of a portion of a com diaminopimelate decarboxylase derived from the nucleotide sequence of SEQ ID NO:8. SEQ ID NO: 10 is the nucleotide sequence comprising the entire cDNA insert in clone crln.pk0103.d8 encoding a portion of a com diaminopimelate decarboxylase.

SEQ ID NO:l 1 is the deduced amino acid sequence of a portion of a com diaminopimelate decarboxylase derived from the nucleotide sequence of SEQ ID NO: 10.

SEQ ID NO: 12 is the nucleotide sequence comprising the entire cDNA insert in clone rlOn.pkOOl 3.b9 encoding a portion of a rice diaminopimelate decarboxylase.

SEQ ID NO: 13 is the deduced amino acid sequence of a portion of a rice diaminopimelate decarboxylase derived from the nucleotide sequence of SEQ ID NO: 12.

SEQ ID NO: 14 is the nucleotide sequence comprising the entire cDNA insert in clone srl.pk0132.cl encoding a soybean diaminopimelate decarboxylase. SEQ ID NO: 15 is the deduced amino acid sequence of a portion of a soybean diaminopimelate decarboxylase derived from the nucleotide sequence of SEQ ID NO: 14.

SEQ ID NO: 16 is the nucleotide sequence comprising a portion of the cDNA insert in clone wlkl.pk0012.c2 encoding a wheat diaminopimelate decarboxylase.

SEQ ID NO: 17 is the deduced amino acid sequence of a portion of a wheat diaminopimelate decarboxylase derived from the nucleotide sequence of SEQ ID NO: 16.

SEQ ID NO: 18 is the nucleotide sequence comprising a portion of the cDNA insert in clone sdp3c.pk001.ol5 encoding a soybean diaminopimelate decarboxylase.

SEQ ID NO: 19 is the deduced amino acid sequence of a portion of a soybean diaminopimelate decarboxylase derived from the nucleotide sequence of SEQ ID NO: 18. SEQ ID NO:20 is the amino acid sequence of a Pseudomonas aeruginosa diaminopimelate decarboxylase found in GenBank Accession No. 118304.

SEQ ID NO:21 is the nucleotide sequence comprising the entire cDNA insert in clone crln.pk0009.g4 encoding a com homoserine kinase.

SEQ ID NO:22 is the deduced amino acid sequence of a portion of a com homoserine kinase encoded by SEQ ID NO:21.

SEQ ID NO:23 is the nucleotide sequence comprising a portion of the cDNA insert in clone rcalc.pk005.k3 encoding a rice homoserine kinase.

SEQ ID NO:24 is the deduced amino acid sequence of a portion of a rice homoserine kinase encoded by SEQ ID NO:23. SEQ ID NO:25 is the nucleotide sequence comprising the entire cDNA insert in clone ses8w.pk0020.b5 encoding a soybean homoserine kinase.

SEQ ID NO:26 is the deduced amino acid sequence of a soybean homoserine kinase derived from the nucleotide sequence of SEQ ID NO:25. SEQ ID NO:27 is the nucleotide sequence comprising a portion of the cDNA insert in clone wlln.pk0065.f2 encoding a wheat homoserine kinase.

SEQ ID NO:28 is the deduced amino acid sequence of a portion of a wheat homoserine kinase derived from the nucleotide sequence of SEQ ID NO:27. SEQ ID NO:29 is the amino acid sequence of a Methanococcus jannaschii homoserine kinase found in GenBank Accession No. U67553.

SEQ ID NO:30 is the nucleotide sequence comprising the entire cDNA insert in clone se3.05h06 encoding a soybean cysteine synthase.

SEQ ID NO:31 is the deduced amino acid sequence of a soybean cysteine synthase derived from the nucleotide sequence of SEQ ID NO:30.

SEQ ID NO:32 is the amino acid sequence of a Citrullus lanatus cysteine synthase found in GenBank Accession No. D28777.

SEQ ID NO:33 is the nucleotide sequence comprising the entire cDNA insert in clone cenl .pk0061.d4 encoding a com cystathionine β-lyase. SEQ ID NO:34 is the deduced amino acid sequence of a portion of a com cystathionine β-lyase derived from the nucleotide sequence of SEQ ID NO:33.

SEQ ID NO:35 is the nucleotide sequence comprising a portion of the cDNA insert in clone rlrl2.pk0026.gl encoding a rice cystathionine β-lyase.

SEQ ID NO:36 is the deduced amino acid sequence of a portion of a rice cystathionine β-lyase derived from the nucleotide sequence of SEQ ID NO:36.

SEQ ID NO:37 is the nucleotide sequence comprising the entire cDNA insert in clone sfll.pk0012.c4 encoding a soybean cystathionine β-lyase.

SEQ ID NO:38 is the deduced amino acid sequence of an entire soybean cystathionine β-lyase derived from the nucleotide sequence of SEQ ID NO:37. SEQ ID NO:39 is the nucleotide sequence comprising a portion of the cDNA insert in clone wrl .pk0091.g6 encoding a wheat cystathionine β-lyase.

SEQ ID NO:40 is the deduced amino acid sequence of a portion of a wheat cystathionine β-lyase derived from the nucleotide sequence of SEQ ID NO:39.

SEQ ID NO:41 is the amino acid sequence of a Arabidopsis thaliana cystathionine β-lyase found in GenBank Accession No. L40511.

The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research 75:3021-3030 (1985) and in the Biochemical Journal 219 (No. :345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION In the context of this disclosure, a number of terms shall be utilized. As used herein, an "isolated nucleic acid fragment" is a polymer of RNA or DNA that is single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less that the entire coding region of a gene, and by nucleic acid fragments that do not share 100% identity with the gene to be suppressed. Moreover, alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are 90% identical to the identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are 95% identical to the DNA sequence of the nucleic acid fragments reported herein. A "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

"Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the amino acid biosynthetic enzymes as set forth in SEQ ID NOs:2, 4, 6, 9, 1 1 , 13, 15, 17, 19. 22, 24, 26, 28, 31, 34, 36, 38, and 40. The skilled artisan is well aware of the "codon-bias^" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

"Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available. "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. "Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 75:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined. DNA fragments of different lengths may have identical promoter activity.

The "translation leader sequence" refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G.D. (1995) Molecular Biotechnology 5:225). The "3' non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 7:671-680.

"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e.. at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes. The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

"Altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

"Mature" protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA; i.e.. with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

A "chloroplast transit peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. "Chloroplast transit sequence^" refers to a nucleotide sequence that encodes a chloroplast transit peptide. A "signal peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys.700:1627-1632).

"Transformation^" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:211) and particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Pat. No. 4,945,050). Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").

Nucleic acid fragments encoding at least a portion of several plant amino acid biosynthetic enzymes have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. Table 1 lists the amino acid biosynthetic enzymes that are described herein, and the designation of the cDNA clones that comprise the nucleic acid fragments encoding these enzymes. TABLE 1 Amino Acid Biosynthetic Enzymes

Enzyme Clone Plant aspartic semialedhyde dehydrogenase rlr48.pk003.dl2 rice wrl.pk0004.cll wheat sfll.pk0122.f9 soybean diaminopimelate decarboxylase cen3n.pk0067.a3 corn crln.pk0103.d8 corn rl0n.pk0013.b9 rice srl.pk0132.cl soybean wlkl.pk0012.c2 wheat sdp3c.pk001.ol5 soybean homoserine kinase crln.pk0009.g4 corn rcalc.pk005.k3 rice ses8w.pk0020.b5 soybean wlln.pk0065.f2 wheat cysteine synthase se3.05h06 soybean cystathionine β-lyase cenl .pk0061.d4 corn rlrl2.pk0026.gl rice sfll.ρk0012.c4 soybean wrl.pk0091.g6 wheat

The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous enzymes from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other amino acid biosynthetic enzymes, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of or full-length of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., (1988) Proc. Natl. Asoc. Sci. USA §5:8998) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., (1989) Proc. Natl. Asoc. Sci. USA 86:5613; Loh et al., (1989) Science 243:211). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman, M.A. and Martin, G.R., (1989) Techniques 7:165).

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lemer, R.A. (1984) Adv. Immunol. 5(5:1 ; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed biosynthetic enzymes are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of free amino acids in those cells. Overexpression of the biosynthetic enzymes of the instant invention may be accomplished by first constructing chimeric genes in which the coding region are operably linked to promoters capable of directing expression of a gene in the desired tissues at the desired stage of development. For reasons of convenience, the chimeric genes may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non- coding sequences encoding transcription termination signals may also be provided. The instant chimeric genes may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric genes can then constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 275:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis. For some applications it may be useful to direct the instant biosynthetic enzyme to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric genes described above may be further supplemented by altering the coding sequences to encode enzymes with appropriate intracellular targeting sequences such as transit sequences (Keegstra, K. (1989) Cell 56:241-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel, N. (1992) Plant Phys.100:1627-1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future. It may also be desirable to reduce or eliminate expression of the genes encoding the instant biosynthetic enzymes in plants for some applications. In order to accomplish this, chimeric genes designed for co-suppression of the instant biosynthetic enzymes can be constructed by linking the genes or gene fragments encoding the enzymes to plant promoter sequences. Alternatively, chimeric genes designed to express antisense RNA for all or part of the instant nucleic acid fragments can be constructed by linking the genes or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

The instant amino acid biosynthetic enzymes (or portions of the enzymes) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the enzymes by methods well known to those skilled in the art. The antibodies are useful for detecting the enzymes in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant amino acid biosynthetic enzymes are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of the instant amino acid biosynthetic enzymes. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the enzymes. An example of a vector for high level expression of the instant amino acid biosynthetic enzymes in a bacterial host is provided (Example 6).

Additionally, the instant plant amino acid biosynthetic enzymes can be used as a targets to facilitate design and/or identification of inhibitors of the enzymes that may be useful as herbicides. This is desirable because the enzymes described herein catalyze various steps in a pathway leading to production of several essential amino acids. Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition of amino acid biosynthesis sufficient to inhibit plant growth. Thus, the instant plant amino acid biosynthetic enzymes could be appropriate for new herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at., (1987) Genomics 7:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein, D. et al., (1980) Am. J. Hum. Genet. 52:314-331).

The production and use of plant gene-derived probes for use in genetic mapping is described in R. Bernatzky, R. and Tanksley, S. D. (1986) Plant Mol. Biol. Reporter 4(1):31-A\. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel, J. D., et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan, M. et al. (1995) Genome Research 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian, H. H. (1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield, V. C. et al. (1993) Genomics 7(5:325-332), allele-specific ligation (Landegren, U. et al. (1988) Science 247:1077-1080), nucleotide extension reactions (Sokolov, B. P. (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter, M. A. et al. (1997) Nature Genetics 7:22-28) and Happy Mapping (Dear, P. H. and Cook, P. R. (1989) Nucleic Acid Res. 77:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer, (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al., (1995) Proc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell 7:75). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant amino acid biosynthetic enzymes. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding an aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase or cystathionine β-lyase can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase or cystathionine β-lyase gene product. EXAMPLES The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

EXAMPLE 1 Composition of cDNA Libraries: Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various co , rice, soybean and wheat tissues were prepared. The characteristics of the libraries are described below.

TABLE 2 cDNA Libraries from Com, Soybean and Other Plant Tissues

Library Tissue Clone cenl Com Endosperm 12 Days After Pollination cenl.pk0061.d4 cen3n Com Endosperm 20 Days After Pollination* cen3n.pk0067.a3 crln Com Root From 7 Day Seedlings* crln.pk0009.g4 crln.pk0103.d8 rcalc Rice Nipponbare callus rcalc.pk005.k3 rlOn Rice Leaf 15 Days After Germination* rl0n.pk0013.b9 rlrl2 Rice Leaf 15 Days After Germination, 12 hours after rlrl2.pk0026.gl infection of strain Magaporthe grisea 4360-R-62 (AVR2- YAMO) rlr48 Rice Leaf 15 Days After Germination 48 hours after rlr48.pk003.dl2 infection of strain Magaporthe grisea 4360-R-62 (AVR2- YAMO) sdp3c Soybean Developing Pods 8-9 mm sdp3c.pk001.ol5 se3 Soybean Embryo 13 Days After Flowering se3.05h06 ses8w Mature Soybean Embryo 8 Weeks After Subculture ses8w.pk0020.b5 sfll Soybean Immature Flower sfll.pk0012.c4 srl Soybean Root From 10 Day Old Seedlings srl.pk0132.cl wlln Wheat Leaf from 7 Day Old Seedling* wlln.pk0065.f2 wlkl Wheat Seedlings 1 hour After Fungicide Treatment** wlkl. kOOl 2x2 wrl Wheat Root From 7 Day Old Seedlings wrl.pk0004.cl l wrl.pk0091.g6

*These libraries were normalized essentially as described in U.S. Pat. No. 5,482,845 **Application of 6-iodo-2-propoxy-3-propyl-4(3/7)-quinazolinone; synthesis and methods of using this compound are described in USSN 08/545,827, incorporated herein by reference. cDNA libraries were prepared in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Conversion of the Uni-ZAP™ XR libraries into plasmid libraries was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells. Amplified insert DNAs or plasmid DNAs were sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams, M. D. et al., (1991) Science 252:1651). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

EXAMPLE 2 Identification and Characterization of cDNA Clones ESTs encoding plant amino acid biosynthetic enzymes were identified by conducting

BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and

DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish. W. and States, D. J. (1993) Nature Genetics 5:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins. The BLASTX search using the nucleotide sequences from clones rlr48.pk0003.dl2 and wrl .pk0004.cl 1 revealed similarity of the protein encoded by the cDNAs to Synechocystis sp. aspartate semialdehyde dehydrogenase (DDJB Accession No. D64006; rlr48.pk0003.dl2 pLog = 44.00; wrl .pk0004.cl 1 pLog = 34.89). The BLASTX search using the entire cDNA inserts in clones rlr48.pk0003.dl2 and wrl.pk0004.cl 1 revealed a higher pLog value vs. the Synechocystis sp protein and similarity of the protein encoded by the cDNAs to Legionella pneumophila aspartate semialdehyde dehydrogenase (GenBank Accession No. AF034213). The BLASTX search using the nucleotide sequence from clone sfll.pk0122.f9 revealed similarity of the protein encoded by the cDNA to Legionella pneumophila. The BLAST results for these sequences is shown in Table 3:

TABLE 3

BLAST Results for Clones Encoding Polypeptides Homologous to Aspartate Semialdehyde Dehydrogenase

BLAST pLog Score

Clone D64006 (Synechocystis sp) AF034213 (L. pneumophila) rlr48.pk0003.dl2 51.00 36.00 wrl.pk0004.cl l 67.96 44.74 sfll.pk0122.f9 6.60

The sequence of the entire cDNA insert in clone rlr48.pk0003.dl2 is set forth in SEQ ID NO: 1 ; the deduced amino acid sequence is set forth in SEQ ID NO:2. The sequence of the entire cDNA insert in clone wrl .pk0004.cl 1 is set forth in SEQ ID NO:3; the deduced amino acid sequence is set forth in SEQ ID NO:4. The sequence of a portion of the cDNA insert from clone sfll.pk0122.f9 is set forth in SEQ ID NO:5; the deduced amino acid sequence is set forth in SEQ ID NO:6. Sequence alignments and BLAST scores and probabilities indicate that the instant nucleic acid fragments encode a portion of a rice (rlr48.pk0003.dl2), wheat (wrl.pk0004.cl 1), and soybean (sfll.pk0122.f9) aspartate semialdehyde dehydrogenase enzyme. These sequences represent the first plant sequences identified for aspartate semialdehyde dehydrogenase. As depicted in Figure 1, the rice and wheat sequences are 94% identical over 204 amino acids and 58% identical to the Legionella pneumophila sequence. The soybean sequence aligns to a more 5' region of the Legionella pneumophila sequence with 44% identity over 54 amino acids.

The BLASTX search using the nucleotide sequences from clones cen3n.pk0067.a3, crln.pk0103.d8. rl0n.pk0013.b9, srl .pk0132.cl, and wlkl.pk0012.c2 revealed similarity of the proteins encoded by the cDNAs to Aquifex aeolicus (GenBank Accession No. AE000728) and Pseudomonas aeruginosa (GenBank Accession No. M23174) diaminopimelate decarboxylase. The BLAST results for each of these ESTs are shown in Table 4:

TABLE 4

BLAST Results for Clones Encoding Polypeptides Homologous to Diaminopimelate Decarboxylase

BLAST pLog Score

Clone AE000728 (A. aeolicus) M23174 (P. aeruginosa) cen3n.pk0067.a3 58.22 56.00 crln.pk0103.d8 75.25 79.12 rl0n.pk0013.b9 46.40 44.00 srl.pk0132.cl 44.70 39.15 wlkl. pkOO 12x2 20.48 19.05

The nucleotide sequence of the entire cDNA insert in clone cen3n.pk0067.a3 was determined and is set forth in SEQ ID NO:8, the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:9. The nucleotide sequence of the entire cDNA insert in clone crln.pk0103.d8 was determined and is set forth in SEQ ID NO:10. the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:l 1. The nucleotide sequence of the entire cDNA insert in clone rl0n.pk0013.b9 was determined and is set forth in SEQ ID NO:12, the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 13. The sequence of the entire cDNA insert in clone srl .pk0132xl was determined and is set forth in SEQ ID NO:14; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:15. The sequence of a portion of the cDNA insert from clone wlkl.pk0012.c2 is set forth in SEQ ID NO: 16, the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 17. The deduced amino acid sequence of cDNA clone srl.pkO 132x1 was then used to query the cDNA sequences obtained in Example 1 using the TFASTA algorithm (Pearson, W. R. (1990) Methods in En∑ymology 755:63-98). An additional cDNA clone, sdpc.pk001.ol5, was identified as sharing homology with srl .pk0132.cl . BLASTX search using the nucleotide sequences from clone sdpc.pk001.ol5 revealed similarity of the proteins encoded by the cDNA to Pseudomonas fluorescens diaminopimelate decarboxylase (EMBO Accession No. Y12268; pLog = 8.66). The sequence of a portion of the cDNA insert in clone sdpc.pk001.ol5 is set forth in SEQ ID NO: 18; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 19. The deduced amino acid sequences show significant sequence identity to the Aquifex aeolicus and Pseudomonas aeruginosa diaminopimelate decarboxylase enzymes. Sequence alignments and BLAST scores and probabilities indicate that the instant nucleic acid fragments encode at least a portion of com (cen3n.pk0067.a3 and crln.pk0103.d8), rice (rl0n.pk0013.b9), soybean (srl.pk0132xl and sdpc.pk001.ol5), and wheat (wlkl.pk0012x2) diaminopimelate decarboxylase enzymes. These are the first plant sequences identified that encode a diaminopimelate decarboxylase enzyme. The alignment shown in Figure 3 indicates that the com sequences (cen3n.pk0067.a3 and crln.pk0103.d8) are 98% identical over 323 amino acids. The full-length com sequence (crln.pk0103.d8) is 90.52% identical to the rice sequence (rl0n.pk0013.b9) over 306 amino acids, 96% identical to the wheat sequence (wlkl. pkOO 12x2) over 73 amino acids, and 78.46% identical to the soybean full insert sequence (srl.pk0132xl) over 260 amino acids. The same co sequence is 35.29% identical to the Pseudomonas aeruginosa diaminopimelate decarboxylase enzyme.

The BLASTX search using the nucleotide sequences from clones crln.pk0009.g4, rcalc.pk005.k3, ses8w.pk0020.b5, and wlln.pk0065.f2 revealed similarity of the protein encoded by the cDNA to Methanococcus jannaschii homoserine kinase enzyme (GenBank Accession No. U67553). The BLAST results for each of these sequences are shown in Table 5:

TABLE 5

BLAST Results for Clones Encoding Polypeptides Homologous to Homoserine Kinase _^^^^

BLAST pLog Score Clone GenBank Accession No U67553 crln.pk0009.g4 19.30 rcalc.pk005.k3 15.21 ses8w.pk0020.b5 35.30 wlln.pk0065.f2 5_68

The nucleotide sequence of the entire cDNA insert in clone crln.pk0009.g4 was determined and is set forth in SEQ ID NO:21; the amino acid sequence deduced from this nucleotide sequence is set forth in SEQ ID NO:22. The sequence of a portion of the cDNA insert from clone rcalc.pk005.k3 is set forth in SEQ ID NO:23, the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:24. The sequence of the entire cDNA insert in clone ses8w.pk0020.b5 was determined and is set forth in SEQ ID NO:25; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:26. The sequence of a portion of the cDNA insert from clone wlln.pk0065.f2 is set forth in SEQ ID NO:27, the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:28. Sequence alignments and BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of a com (crln.pk0009.g4), rice (rcalc.pk005.k3), soybean (ses8w.pk0020.b5), and wheat (wlln.pk0065f2) homoserine kinase enzyme. These sequences represent the first plant sequences encoding homoserine kinase. An alignment of the deduced amino acid sequences with the sequence of the Methanococcus jannaschii homoserine kinase enzyme shown in Figure 4 indicates that the com (crln.pk0009.g4) sequence is 100% identical to the rice sequence (rcalc.pk005.k3) over 22 amino acids. The com sequence is 100% identical to the wheat sequence (wlln.pk0065f2) over 72 amino acids. The com sequence is 60.35% identical to the soybean sequence (ses8w.pk0020.b5) over 179 amino acids. The soybean clone (ses8w.pk0020.b5) contains the sequence for a full-length homoserine kinase and is 34.67% identical to the Methanococcus jannaschii sequence.

The BLASTX search using the nucleotide sequence of clone se3.05h06 revealed similarity of the protein encoded by the cDNA to Citrullus lanatus cysteine synthase (DDJB Accession No. D28777; pLog = 59.06). The sequence of the entire cDNA insert in clone se3.05h06 was determined and is set forth in SEQ ID NO:30; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 31. The entire cDNA insert in clone se3.05h06 was reevaluated by BLAST, yielding an even higher pLog value vs. the Citrullus lanatus cysteine synthase (D28777; pLog = 182.64). Sequence alignments and BLAST scores and probabilities indicate that the instant nucleic acid fragment encodes the entire soybean cysteine synthase enzyme. This is the first soybean EST identified for cysteine synthase. Figure 5 shows the alignment of the amino acid sequence deduced from the clone se3.05h06 and the Citullus lanatus cysteine synthase. The soybean sequence is 42% identical to the Citullus lanatus over 325 amino acids. The BLASTX search using the nucleotide sequences of clones cenl .pk0061.d4, rlrl2.pk0026.gl, sfll.pk0012.c4, and wrl.pk0091.g6 revealed similarity of the proteins encoded by the cDNAs to Arabidopsis thaliana cystathionine β-lyase (GenBank Accession No. L40511). The BLAST results for each of these ESTs are shown in TABLE 6:

TABLE 6

BLAST Results for Clones Encoding Polypeptides Homologous to Cystathionine β-Lyase

BLAST pLog Score Clone GenBank Accession No L40511 cenl.pk0061.d4 50.41 rlrl2.pk0026.gl 39.00 sfll.pk0012x4 33.85 wrl.pk0091.g6 52.52

The sequence of the entire cDNA insert in clone cenl.pk0061.d4 was determined and is set forth in SEQ ID NO:33; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:34. The entire cDNA insert in clone cenl.pk0061.d4 was reevaluated by BLAST, yielding an even higher pLog value vs. the Arabidopsis thaliana (L40511; pLog = 128.54) enzyme. The sequence of a portion of the cDNA insert in clone rlrl2.pk0026.gl is set forth in SEQ ID NO:35; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:36. The sequence of the entire cDNA insert in clone sfll.pk0012x4 was determined and is set forth in SEQ ID NO:37; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:38. The entire cDNA insert in clone sfll.pk0012x4 was reevaluated by BLAST, yielding an even higher pLog value vs. the Arabidopsis thaliana (L40511 ; pLog = 221.92) enzyme. The sequence of a portion of the cDNA insert in clone wrl .pk0091.g6 is set forth in SEQ ID NO:39; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:40. Sequence alignments and BLAST scores and probabilities indicate that the instant nucleic acid fragments encode a portion of a com (cenl.pk0061.d4), rice (rlrl2.pk0026.gl), and wheat (wrl.pk0091.g6) cystathionine β-lyase enzyme. The clone sfll.pk0012x4 encodes an entire soybean cystathionine β-lyase enzyme. These are the first com, rice, soybean, and wheat ESTs identified that encode a cystathionine β-lyase enzyme. Figure 6 shows the alignment of the deduced amino acid sequence from clone cenl.pk0061.d4, rlrl2.pk0026.gl, wrl.pk0091.g6, sfll.pk0012x4 with the Arabidopsis thaliana cystathionine β-lyase sequence. Over 226 amino acids the com sequence (cenl.pk0061.d4) is 88.5% identical to the soybean (sfll.pk0012x4) sequence and 81%) identical to the Arabidopsis thaliana sequence. The rice sequence contains 75 amino acids which are 68% identical to the soybean sequence, and 76% identical to the Arabidopsis thaliana sequence. The wheat sequence contains 42 amino acids which are 66.41% identical to the soybean sequence and 71% identical to the Arabidopsis thaliana sequence. The 465 amino acid soybean sequence (sfll .pkOO 12x4) is 74% identical to the Arabidopsis thaliana sequence.

EXAMPLE 3 Expression of Chimeric Genes in Monocot Cells A chimeric gene comprising a cDNA encoding an amino acid biosynthetic enzyme in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constmcted. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes Ncol and Smal and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb Ncol-Smal fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard,

Manassas, VA 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XLl-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U. S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding a plant amino acid biosynthetic enzyme, and the 10 kD zein 3' region.

The chimeric gene described above can then be introduced into com cells by the following procedure. Immature com embryos can be dissected from developing caryopses derived from crosses of the inbred com lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27°C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al., (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the com tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm. For bombardment, the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate- supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., (1990) Bio/Technology 5:833-839).

EXAMPLE 4 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261 :9228-9238) can be used for expression of the instant amino acid biosynthetic enzymes in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Neo I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites. The cDNA fragment of this gene may be generated by polymerase chain reaction

(PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

Soybean embroys may then be transformed with the expression vector comprising sequences encoding a plant amino acid biosynthetic enzyme. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26°C on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below. Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day /night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium. Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Kline et al. (1987) Nature (London) 527:70, U.S. Patent No. 4,945,050). A Du Pont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Vims (Odell et al. (1985) Nature 575:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5' region, the fragment encoding the biosynthetic enzyme and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two- week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos. EXAMPLE 5 Analysis of Amino Acid Content of the Seeds of Transformed Plants To analyze for expression of the chimeric genes in seeds and for the consequences of expression on the amino acid content in the seeds, a seed meal can be prepared by any of a number of suitable methods known to those skilled in the art. The seed meal can be partially or completely defatted, via hexane extraction for example, if desired. Protein extracts can be prepared from the meal and analyzed for enzyme activity. Alternatively the presence of any of the expressed enzymes can be tested for immunologically by methods well-known to those skilled in the art. To measure free amino acid composition of the seeds, free amino acids can be extracted from the meal and analyzed by methods known to those skilled in the art (Bieleski et al. (1966) Anal. Biochem. 7:278-293). Amino acid composition can then be determined using any commercially available amino acid analyzer. To measure total amino acid composition of the seeds, meal containing both protein-bound and free amino acids can be acid hydrolyzed to release the protein-bound amino acids and the composition can then be determined using any commercially available amino acid analyzer. Seeds expressing the instant amino acid biosynthetic enzymes and with altered lysine, threonine, methionine, cysteine and/or isoleucine content than the wild type seeds can thus be identified and propagated.

To measure free amino acid composition of the seeds, free amino acids can be extracted from 8-10 milligrams of the seed meal in 1.0 mL of methanol/chloroform/water mixed in ratio of 12v/5v/3v (MCW) at room temperature. The mixture can be vortexed and then centrifuged in an eppendorf microcentrifuge for about 3 min; approximately 0.8 mL of supernatant is then decanted. To this supernatant, 0.2 mL of chloroform is added followed by 0.3 mL of water. The mixture is then vortexed and centrifuged in an eppendorf microcentrifuge for about 3 min. The upper aqueous phase, approximately 1.0 mL, can then be removed and dried down in a Savant Speed Vac Concentrator. The samples are then hydrolyzed in 6N hydrochloric acid, 0.4% β-mercaptoethanol under nitrogen for 24 h at 110-120°C. Ten percent of the sample can then be analyzed using a Beckman Model 6300 amino acid analyzer using post-column ninhydrin detection. Relative free amino acid levels in the seeds are then compared as ratios of lysine, threonine, methionine, cysteine and/or isoleucine to leucine, thus using leucine as an internal standard.

EXAMPLE 6 Expression of Chimeric Genes in Microbial Cells The cDNAs encoding the instant plant amino acid biosynthetic enzymes can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 5(5:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Neo I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the enzyme. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the enzyme are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 759:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight. EXAMPLE 7

Evaluating Compounds for Their Ability to Inhibit the Activity of a Plant Amino Acid Biosynthetic Enzyme The plant amino acid biosynthetic enzymes described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 6, or expression in eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines. The instant enzymes may be expressed separately as mature proteins, or may be co- expressed in E. coli or another suitable expression background. In addition, whether expressed separately or in combination, the instant enzymes may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase ("GST"), thioredoxin ("Trx"), maltose binding protein, and C- and or N-terminal hexahistidine polypeptide ("(His)₆"). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzymes. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the biosynthetic enzyme. Purification of the instant enzymes, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to. homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the enzymes are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, an enzyme may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)g peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage of the thioredoxin fusion protein and the biosynthetic enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin.

Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation of the plant amino acid biosynthetic enzymes disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. Examples of assays for many of these enzymes can be found in Methods in Enzymology Vol. V, (Colowick and Kaplan eds.) Academic Press, New York or Methods in Enzymology Vol. XVII, (Tabor and Tabor eds.) Academic Press, New York. Specific examples may be found in the following references, each of which is incorporated herein by reference: aspartic semialdehyde dehydrogenase may be assayed as described in Black et al. (1955) J. Biol. Chem. 275:39-50, or Cremer et al. (1988) J. Gen. Microbiol. 754:3221-3229; diaminopimelate decarboxylase may be assayed as described in Work (1962) in Methods in Enzymology Vol. V, (Colowick and Kaplan eds.) 864-870, Academic Press, New York or Cremer et al. (1988) J Gen. Microbiol. 134:3221-3229; homoserine kinase may be assayed as described in Aarnes (1976) Plant Sci. Lett. 7:187-194; cysteine synthase may be assayed as described in Thompson et al. (1968) Biochem. Biophys. Res. Commun. 31: 281-286 or BertagnoUi et al. (1977) Plant Physiol. (50:115-121; and cystathionine β-lyase may be assayed as described in Giovanelli et al. (1971) Biochim. Biophys. Acta 227:654-670 or Droux et al. (1995) Arch. Biochem Biophys. 57(5:585-595.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY

(B) STREET: 1007 MARKET STREET

(C) CITY: WILMINGTON

(D) STATE: DELAWARE

(E) COUNTRY: USA

(F) ZIP: 19898

(G) TELEPHONE: 302-992-4926 (H) TELEFAX: 302-773-0164 (I) TELEX: 6717325

(ii) TITLE OF INVENTION: PLANT AMINO ACID BIOSYNTHETIC ENZYMES

(iii) NUMBER OF SEQUENCES: 41

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: DISKETTE, 3.50 INCH

(B) COMPUTER: IBM PC COMPATIBLE

(C) OPERATING SYSTEM: MICROSOFT WORD FOR WINDOWS 95

(D) SOFTWARE: MICROSOFT WORD VERSION 7.0A

(v) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/049,406

(B) FILING DATE: JUNE 12, 1997

(vii) ATTORNEY/AGENT INFORMATION:

(A) NAME: MAJARIAN, WILLIAM R.

(B) REGISTRATION NUMBER: 41,173

(C) REFERENCE/DOCKET NUMBER: BB-1116

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 826 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: rlr48.pk003.dl2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TGGTACCGCC ACGCCAAGGT GGTAAGGATG GTTGTCAGCA CTTACCAAGC AGCAAGTGGT 60

GCTGGGGCTG CGGCCATGGA AGAACTCAAA CTTCAAACTC AAGAGGTCTT GGCGGGGAAA 120

GCACCAACAT GCAACATTTT CAGTCAGCAG TATGCTTTTA ATATATTTTC ACATAATGCA 180

CCAATTGTTG AAAATGGGTA CAATGAGGAG GAGATGAAGA TGGTGAAGGA GACCAGAAAA 240

ATCTGGAATG ATAAAGATGT GAAGGTAACT GCAACCTGCA TACGAGTTCC TGTGATGCGT 300

GCACATGCTG AAAGTGTGAA TCTACAGTTT GAAAAGCCAC TTGATGAGGA TACTGCAAGG 360

GAAATCTTGA GGGCAGCTGA AGGTGTTACC ATTATTGATG ACCGTGCTTC CAATCGCTTC 420

CCCACACCTC TTGAGGTATC GGATAAAGAT GATGTAGCAG TGGGTAGAAT TCGTCAGGAT 480

TTGTCGCAAG ATGATAACAA AGGGCTGGAC ATATTTGTTT GTGGAGATCA AATACGTAAA 540

GGTGCTGCAC TCAATGCTGT GCAGATTGCT GAAATGCTAC TCAAGTGATT TTCTTTTCTG 600

TACCTTTCTC TCCTTGCCCC TCTTTGCTCT AGTCATTGTT TGACGGATGT ACTCTGGTTA 660

GTATGAGATC AATTTTGATC ATCTTTTGTA ATCTATATTC CTAGTGAAAT AAATGTAAAA 720

CGGTTTTGCT CTATCTTCTG CACAAGTGTA GAAGAAATCT GAAATTGGGA AATTGGAGTG 780

TGGCCCTTGT TCAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAA 826

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 195 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: rlr48.pk003.dl2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Trp Tyr Arg His Ala Lys Val Val Arg Met Val Val Ser Thr Tyr Gin 1 5 10 15

Ala Ala Ser Gly Ala Gly Ala Ala Ala Met Glu Glu Leu Lys Leu Gin 20 25 30

Thr Gin Glu Val Leu Ala Gly Lys Ala Pro Thr Cys Asn lie Phe Ser 35 40 45 Gin Gin Tyr Ala Phe Asn lie Phe Ser His Asn Ala Pro lie Val Glu 50 55 60

Asn Gly Tyr Asn Glu Glu Glu Met Lys Met Val Lys Glu Thr Arg Lys 65 70 75 80 lie Trp Asn Asp Lys Asp Val Lys Val Thr Ala Thr Cys lie Arg Val 85 90 95

Pro Val Met Arg Ala His Ala Glu Ser Val Asn Leu Gin Phe Glu Lys 100 105 110

Pro Leu Asp Glu Asp Thr Ala Arg Glu lie Leu Arg Ala Ala Glu Gly 115 120 125

Val Thr lie lie Asp ASD Arg Ala Ser Asn Arg Phe Pro Thr Pro Leu 130 135 140

Glu Val Ser Asp Lys ASD Asp Val Ala Val Gly Arg lie Arg Gin Asp 145 150 155 160

Leu Ser Gin Asp Asp Asn Lys Gly Leu Asp lie Phe Val Cys Gly ASD 165 170 175

Gin lie Arg Lys Gly Ala Ala Leu Asn Ala Val Gin He Ala Glu Met 180 185 190

Leu Leu Lys 195

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 875 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: wr 1. pk0004. ell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CCTCATGGCT GTCACGCCGC TGCATCGCCA CGCCAAGGTG AAAAGGATGG TTGTCAGCAC 60

ATACCAAGCA GCAAGTGGTG CTGGTGCTGC AGCCATGGAA GAACTCAAAC TTCAGACTCG 120

AGAGGTCTTG GAAGGAAAGC CACCAACCTG TAACATTTTC AGTCAACAGT ATGCTTTTAA 180

TATATTTTCG CATAATGCAC CTATTGTTGA AAATGGCTAT AATGAGGAAG AGATGAAAAT 240

GGTGAAGGAG ACCAGAAAAA TCTGGAATGA CAAGGATGTA AGAGTAACTG CAACTTGTAT 300

ACGGGTTCCT ACGATGCGCG CGCATGCCGA AAGCGTGAAT CTACAGTTTG AAAAGCCACT 360

TGATGAGGAC ACTGCCAGAG AAATCTTGAG GGCAGCTCCT GGTGTTACCA TTAGTGACGA 420

CCGTGCTGCC AACCGCTTCC CTACACCACT GGAGGTATCG GATAAAGATG ACGTATCAGT 480

TGGTAGGATT CGCCAGGACT TGTCACAAGA TGATAACAGA GGGTTGGAGT TATTTGTCTG 540

TGGAGACCAG ATACGTAAAG GCGCCGCGCT GAACGCTGTG CAGATTGCTG AAATGCTACT 600 GAAGTGACCG CCTTTTTACC ATTGTCTCAT GTGCCACGTT GCTCTATCCA TTGATGGATT 660

GATGTACTCT AGTCACTTTC AACCCAGTTT TGGTCGTCGT CTTTTTTGTA ATCTGTCAAC 720

CTAGCAGAAG AAGTGTAAGA CGGGCTTTAG TCATCTGTTG CACACAAAAG TGCAGCCACA 780

AGTTTAGAAA AGGAGGGTTT TCACTTGTTC GGATTTTGCC TTAGGTTGGA CTTTGTTGCA 840

AGTTGTCGTT TGTTTCTTGA AAGCTGGTCT GCTGT 875 (2) INFORMATION FOR SEQ ID NO: 4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 201 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v i) IMMEDIATE SOURCE:

(B) CLONE: wrl .pk0004. ell

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : :

Leu Met Ala Val Thr Pro Leu His Arg His Ala Lys Val Lys Arg Met 1 5 10 15

Val Val Ser Thr Tyr Gin Ala Ala Ser Gly Ala Gly Ala Ala Ala Met 20 25 30

Glu Glu Leu Lys Leu Gin Thr Arg Glu Val Leu Glu Gly Lys Pro Pro 35 40 45

Thr Cys Asn He Phe Ser Gin Gin Tyr Ala Phe Asn He Phe Ser H s 50 55 60

Asn Ala Pro He Val Glu Asn Gly Tyr Asn Glu Glu Glu Met Lys Met 65 70 75 80

Val Lys Glu Thr Arg Lys He Trp Asn Asp Lys Asp Val Arg Val Thr 85 90 95

Ala Thr Cys He Arg Val Pro Thr Met Arg Ala His Ala Glu Ser Va_^ 100 105 110

Asn Leu Gin Phe Glu Lys Pro Leu Asp Glu Asp Thr Ala Arg Glu He 115 120 125

Leu Arg Ala Ala Pro Gly Val Thr He Ser Asp Asp Arg Ala Ala Asn 130 135 140

Arg Phe Pro Thr Pro Leu Glu Val Ser Asp Lys Asp Asp Val Ser Val 145 150 155 160

Gly Arg He Arg Gin Asp Leu Ser Gin Asp Asp Asn Arg Gly Leu Glu 165 170 175

Leu Phe Val Cys Gly Asp Gin He Arg Lys Gly Ala Ala Leu Asn Ala 180 185 190

Val Gin He Ala Glu Met Leu Leu Lys 195 200 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 457 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: sf11. pk0122. f9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GTCTGTTTTA AAATCCAACA CTTAATCTCT CTCTTCGCAG CCTAAAATCC CAATGGCTTC 60

ACTCTCTGTT TTGCGCCACA ACCACCTCTT CTCGGGCCCC CTCCCGGCCC GCCCCAAGCC 120

CACCTCCTCC TCCTCCTCCA GGATCCGAAT GTCCCTCCGC GAGAACGGCC CCTCCATCGC 180

CGTCGTGGGC GTCACCGGCG CCGTCGGCCA NGAGTTCCTC TCCGTCCTCT CCGACCGCGA 240

CTTCCCCTAC CGCTCCATTC ATATGCTGGC TTCCAAGCGC TCCGCTGGAC GCCGCATC.-.C 300

CTTCGAGGAC AGGGACTACN TCTTCAGGAG CTCACGCCGG AGAGTTCGAC GG GTCG CA 360

TCGCGCTCTT CAGCGCNGGG GGTCCATCAA NNAAGCATTC GGACCATCGN CGTAAATCGN 420

GGGACGGNCG TNGNCAANAT ANCTCCGGTT NCCTTTG 457

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 86 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: sf11. pk0122. f9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Ala Ser Leu Ser Val Leu Arg His Asn His Leu Phe Ser Gly Pro 1 5 10 15

Leu Pro Ala Arg Pro Lys Pro Thr Ser Ser Ser Ser Ser Arg He Arg 20 25 30

Met Ser Leu Arg Glu Asn Gly Pro Ser He Ala Val Val Gly Val Thr 35 40 45

Gly Ala Val Gly Gin Glu Phe Leu Ser Val Leu Ser Asp Arg Asp Phe 50 55 60

Pro Tyr Arg Ser He His Met Leu Ala Ser Lys Arg Ser Ala Gly Arg 65 70 75 80

Arg He Thr Phe Glu Asp 85 (2) INFORMATION FOR SEQ ID NO: 7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 160 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Legionella pneumophila

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Ser Arg His Leu Asn Val Ala He Val Gly Ala Thr Gly Ala Val 1 5 10 15

Gly Glu Thr Phe Leu Thr Val Leu Glu Glu Arg Asn Phe Pro He Lys 20 25 30

Ser Leu Tyr Pro Leu Ala Ser Ser Arg Ser Val Gly Lys Thr Val Tr.r 35 40 45

Phe Arg ASD Gin Glu Leu ASD Val Leu Asp Leu Ala G__.u Phe _»sp Pr.e 50 ^" 55 60

Ser Lys Val Asp Leu Ala Leu Phe Ser Ala Gly Gly Ala Val Ser L^s 65 70 75 80

Glu Tyr Ala Pro Lys Ala Val Ala Ala Gly Cys Val Val Val Asp Asn 85 90 95

Thr Ser Cys Phe Arg Tyr Glu Asp Asp He Pro Leu Val Val Pro Gly 100 105 110

Ser Glu Ser Ser Ser Asn Arg Asp Tyr Thr Lys Arg Gly He He Ala 115 120 125

Asn Pro Asn Cys Ser Thr He Gin Met Val Val Ala Leu Lys Pro He 130 135 140

Tyr Asp Ala Val Gly He Ser Arg He Asn Val Ala Thr Tyr Gin Ser 145 150 155 160

(2) INFORMATION FOR SEQ ID NO: 8:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1054 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: cen3n . pk0067. a3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

ATTTAACGGA AATGGGAAGA CACTCGAACA TCTTAAATTA GCTGCTGAGA GTGGAGTATT 60

TGTAAATGTG GATAGCGAAT TTGATTTGGA GAATATTGTC AGAGCTGCAA GAGCTACTGG 120

AAAGAAAGTG CCTGTTTTGC TTCGAATAAA TCCAGATGTG GATCCGCAGG TACATCCTTA 180 TGTTGCCACG GGAAATAAAA CGTCTAAATT TGGGATCCGC AATGAGAAAT TGCAATGGTT 240

TTTGGACTCT ATCAAGTCAT ACCCGAATGA AATCAAACTC GTTGGTGTTC ATTGCCATCT 300

GGGATCTACT ATTACAAAGG TTGATATATT CAGAGATGCT GCAGTTCTTA TGCTGAATTA 360

TGTCGATGAA ATTCGAGCAC AAGGTTTTAA GTTGGAGTAC CTGAATATCG GAGGTGGTTT 420

GGGAATAGAT TACCATCATA CCGATGCAGT CTTACCTACA CCTATGGATC TCATCAACAC 480

TGTGCGAGAA TTAGTTCTCT CTCAAGATCT CACTCTTATT ATTGAACCCG GAAGATCCTT 540

GATTGCTAAT ACTTGCTGCT TCGTCAATAG AGTAACTGGT GTTAAATCTA ATGGTACAAA 600

GAATTTCATT GTTGTTGATG GCAGCATGGC AGAACTCATC AGACCTAGTC TGTATGGAGC 660

ATACCAGCAT ATCGAACTGG TCTCTCCCCC CACTCCTGGT GCTGAAGCAG CGACCTTCGA 720

TATTGTTGGA CCAGTTTGTG AGTCTGCAGA TTTCCTTGGA AAAGATAGGG AACTTCCAAC 780

ACCTGATGAG GGAGCTGGAC TGGTTGTTCA TGATGCAGGT GCCTACTGCA TGAGCATGGC 840

TTCCACCTAC AACCTGAAGT TGAGGCCACC GGAATACTGG GTGGAAGCGG ACGGTTCGAT 900

CGTTAAGATC AGGCATGGAG AGAAGCTTGA TGACTACATG AAGTTCTTTG ATGGTCTTCC 960

TGCTTAGATG TTTATTATCT GCGACTGCTA CGGACGATGT TTTCTTGGGG ATAATTGGAT 1020

TTTCTTTGTC AAAAAAAAAA AAAAAAAAAA AAAA 1054 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 321 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: cen3n . pk0067. a3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Phe Asn Gly Asn Gly Lys Thr Leu Glu His Leu Lys Leu Ala Ala Glu 1 5 10 15

Ser Gly Val Phe Val Asn Val Asp Ser Glu Phe Asp Leu Glu Asn He 20 25 30

Val Arg Ala Ala Arg Ala Thr Gly Lys Lys Val Pro Val Leu Leu Arg 35 40 45

He Asn Pro Asp Val Asp Pro Gin Val His Pro Tyr Val Ala Thr Gly 50 55 60

Asn Lys Thr Ser Lys Phe Gly He Arg Asn Glu Lys Leu Gin Trp Phe 65 70 75 80

Leu Asp Ser He Lys Ser Tyr Pro Asn Glu He Lys Leu Val Gly Val 85 90 95

His Cys His Leu Gly Ser Thr He Thr Lys Val Asp He Phe Arg Asp 100 105 110 Ala Ala Val Leu Met Leu Asn Tyr Val Asp Glu He Arg Ala Gin Gly 115 120 125

Phe Lys Leu Glu Tyr Leu Asn He Gly Gly Gly Leu Gly He Asp Tyr 130 135 140

His His Thr Asp Ala Val Leu Pro Thr Pro Met Asp Leu He Asn Thr 145 150 155 160

Val Arg Glu Leu Val Leu Ser Gin Asp Leu Thr Leu He He Glu Pro 165 170 175

Gly Arg Ser Leu He Ala Asn Thr Cys Cys Phe Val Asn Arg Val Thr 180 185 190

Gly Val Lys Ser Asn Gly Thr Lys Asn Phe He Val Val Asp Gly Ser 195 200 205

Met Ala Glu Leu He Arg Pro Ser Leu Tyr Gly Ala Tyr Gin His He 210 215 220

Glu Leu Val Ser Pro Pro Thr Pro Gly Ala Glu Ala Ala Thr Phe Asp 225 230 235 240

He Val Gly Pro Val Cys Glu Ser Ala Asp Phe Leu Gly Lys Asp Arg 245 250 255

Glu Leu Pro Thr Pro Asp Glu Gly Ala Gly Leu Val Val His Asp Ala 260 265 270

Gly Ala Tyr Cys Met Ser Met Ala Ser Thr Tyr Asn Leu Lys Leu Arg 275 280 285

Pro Pro Glu Tyr Trp Val Glu Ala Asp Gly Ser He Val Lys He Arg 290 295 300

His Gly Glu Lys Leu Asp Asp Tyr Met Lys Phe Phe Asp Gly Leu Pro 305 310 315 320

Ala

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1813 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: crln.pk0103. d8

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CGCTTCCTGG AAGGCTGGAA CAGAAAGAAC CCTAAACCCT AGCAATGGCG GCGGCGAACC 60

TGCTGTCGCG CTCCCTTCTC CCCACCCCAA ACACTATCCG AACGAGCCAC CCCACCCCGC 120

GGAGCCCAGC CGTCGTCTCC TTCCCCCGCC GCCGTGCCCG CCTGTCCGTG TGCGCCTCCG 180

TCTCCATGGC CTCCCCGTCC CCACCGCCAC AGCCCGCGGC GGCCGGCGTG CCGAAGCACT 240

GCTTCCGGCG CGGCGCCGAC GGCTACCTGT ACTGCGAGGG AGTGAGGGTG GAAGACGCGA 300 TGGCGGCTGC CGAGCGCAGC CCCTTCTATC TCTACAGCAA GCTTCAGATC CTCCGCAACT 360

TCGCCGCTTA CCGCGACGCT CTCCAGGGGC TCCGCTCCAT CGTCGGGTAT GCCGTGAAGG 420

CCAACAATAA CCTCCCCGTG CTACGCGTCC TGCGTGAGCT TGGCTGCGGC GCCGTCCTCG 480

TCAGCGGCAA CGAGCTCCGA CTCGCCCTCC AGGCGGGATT CGACCCCGCC AGGTGTATAT 540

TTAACGGAAA TGGGAAGACA CTCGAAGATC TTAAATTGGC TGCTGAGAGT GGAGTATTTG 600

TAAATGTGGA TAGTGAATTT GATTTAGAGA ATATTGTCAG AGCTGCAAGA GCTACTGGAA 660

AGAAAGTGCC TGTTTTACTT AGAATAAATC CAGATGTGGA TCCACAGGTA CATCCATATG 720

TTGCCACGGG AAATAAAACA TCCAAATTCG GGATCCGCAA TGAGAAATTG CAATGGTTTT 780

TGAACTCTAT CAAGTCATAC TCGAATGAAA TCAAACTCGT TGGTGTTCAT TGCCATCTGG 840

GATCTACTAT TACAAAGGTT GATATATTCA GAGATGCTGC AGTGCTTATG GTGAATTATG 900

TCGATGAAAT TCGAGCACAA GGTTTTAAGT TGGAGTACCT GAATATTGGA GGTGGTTTGG 960

GAATAGATTA CCATCATACC GATGCAGTCT TACCTACACC TATGGATCTC ATCAACACT3 1020

TACGAGAATT AGTTCTCTCT CAAGATCTTA CTCTTATTAT TGAACCTGGA AGATCCTT3A 1080

TTGCTAATAC TTGCTGCTTC GTCAATAGAG TAACTGGTGT TAAATCTAAT GGTACAAAGA 1140

ATTTCATTGT TGTTGATGGC AGCATGGCAG AACTCATCAG ACCTAGCCTG TATGGAGCAT 1200

ATCAGCATAT CGAATTGGTC TCTCCCCCCA CTCCTGGTGC TGAAGTAGCG ACCTTCGATA 1260

TTGTTGGGCC AGTTTGTGAG TCTGCAGATT TCCTTGGAAA AGATAGGGAA CTTCCAACAC 1320

CTGATGAGGG AGCTGGACTG GTTGTTCATG ATGCAGGTGC CTACTGCATG AGCATGGCTT 1380

CCACCTACAA CCTGAAGTTG AGGCCGCCAG AGTACTGGGT TGAAGAGGAT GGTTCGATTG 1440

TTAAGATCAG GCATGAAGAG AAGCTCGATG ACTACATGAA GTTCTTTGAT GGTCTTCCTG 1500

CTTAGATGTT TATTTGTGAC TGCTAGGGGC GATGTTTTCT TGGAGATAAT TGAATTT7TC 1560

TTTGTCAAGC TCATTTTGCT TTCTTGTGGT TGTTATGGAA TGTTACTGGA TACTGGATAG 1620

TTAGTTCGGC CTGTAGGCGT ATCCTCCTGA ACTTACCTCT CATTGCTGTT AGTTTTGGCA 1680

CCAAGTTTGT TCCCAATTGC TATTTACGGA AGTTATTGCA TAAAGGGCTG TTTGGTTGTA 1740

ATCTTCCCGT AAGAATAAGA TGCATGTTTT TGAGTTAAAA AAGGGGGGGC CCGGTACCCA 1800

ATTCGCCCTA TAG 1813 (2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 486 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: crln.pk0103. d8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Ala Ala Ala Asn Leu Leu Ser Arg Ser Leu Leu Pro Thr Pro Asn 1 5 10 15

Thr He Arg Thr Ser His Pro Thr Pro Arg Ser Pro Ala Val Val Ser 20 25 30

Phe Pro Arg Arg Arg Ala Arg Leu Ser Val Cys Ala Ser Val Ser Met 35 40 45

Ala Ser Pro Ser Pro Pro Pro Gin Pro Ala Ala Ala Gly Val Pro Lys 50 55 60

His Cys Phe Arg Arg Gly Ala Asp Gly Tyr Leu Tyr Cys Glu Gly Val 65 70 75 80

Arg Val Glu Asp Ala Met Ala Ala Ala Glu Arg Ser Pro Phe Tyr Leu 85 90 95

Tyr Ser Lys Leu Gin He Leu Arg Asn Phe Ala Ala Tyr Arg Asp Ala 100 105 110

Leu Gin Gly Leu Arg Ser He Val Gly Tyr Ala Val Lys Ala Asn Asn 115 120 125

Asn Leu Pro Val Leu Arg Val Leu Arg Glu Leu Gly Cys Gly Ala Val 130 135 140

Leu Val Ser Gly Asn Glu Leu Arg Leu Ala Leu Gin Ala Gly Phe ASD 145 150 155 160

Pro Ala Arg Cys He Phe Asn Gly Asn Gly Lys Thr Leu Glu Asp Leu 165 ^* 170 175

Lys Leu Ala Ala Glu Ser Gly Val Phe Val Asn Val Asp Ser Glu Phe 180 185 190

Asp Leu Glu Asn He Val Arg Ala Ala Arg Ala Thr Gly Lys Lys Val 195 200 205

Pro Val Leu Leu Arα He Asn Pro Asp Val Asp Pro Gin Val His Pro 210 ^' 215 ^* 220

Tyr Val Ala Thr Gly Asn Lys Thr Ser Lys Phe Gly He Arg Asn Glu 225 230 235 240

Lys Leu Gin Trp Phe Leu Asn Ser He Lys Ser Tyr Ser Asn Glu He 245 250 255

Lys Leu Val Gly Val His Cys His Leu Gly Ser Thr He Thr Lys Val 260 265 270

Asp He Phe Arg Asp Ala Ala Val Leu Met Val Asn Tyr Val Asp Glu 275 280 285

He Arg Ala Gin Gly Phe Lys Leu Glu Tyr Leu Asn He Gly Gly Gly 290 295 300

Leu Gly He Asp Tyr His His Thr Asp Ala Val Leu Pro Thr Pro Met 305 310 315 320

Asp Leu He Asn Thr Val Arg Glu Leu Val Leu Ser Gin Asp Leu Thr 325 330 335 Leu He He Glu Pro Gly Arg Ser Leu He Ala Asn Thr Cys Cys Phe 340 345 350

Val Asn Arg Val Thr Gly Val Lys Ser Asn Gly Thr Lys Asn Phe He 355 360 365

Val Val Asp Gly Ser Met Ala Glu Leu He Arg Pro Ser Leu Tyr Gly 370 375 380

Ala Tyr Gin His He Glu Leu Val Ser Pro Pro Thr Pro Gly Ala Glu 385 390 395 400

Val Ala Thr Phe Asp He Val Gly Pro Val Cys Glu Ser Ala Asp Phe 405 410 415

Leu Gly Lys Asp Arg Glu Leu Pro Thr Pro Asp Glu Gly Ala Gly Leu 420 425 430

Val Val His Asp Ala Gly Ala Tyr Cys Met Ser Met Ala Ser Thr Tyr 435 440 445

Asn Leu Lys Leu Arg Pro Pro Glu Tyr Trp Val Glu Glu Asp Gly Ser 450 455 460

He Val Lys He Arg his Glu Glu Lys Leu Asp Asp Tyr Met Lys Phe 465 470 475 480

Phe Asp Gly Leu Pro Ala 485

(2) INFORMATION FOR SEQ ID NO: 12:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1116 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: rlOn. pk0013. 9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CTTACACGGA GTGTTTGTAA ACATAGACAG TGAATTTGAT TTGGAGAATA TTGTCACTGC 60

TGCGAGAGTT GCTGGGAAGA AAGTCCCTGT TTTGCTCAGG ATAAACCCAG ATGTGGATCC 120

ACAGGTCCAT CCTTATGTTG CGACTGGAAA CAAAACCTCC AAATTTGGTA TCCGTAATGA 180

GAAACTACAA TGGTTCTTAG ACTCTATCAA GTCATACTCA AATGATATCA CACTGGTGGG 240

TGTTCATTGT CATCTGGGAT CTACCATTAC AAAGGTCGAT ATATTTAGAG ATGCGGCAGG 300

TCTTATGGTG AATTATGTTG ATGAAATTCG AGCACAAGGT TTTGAACTGG AATATCTCAA 360

TATTGGCGGT GGCCTGGGCA TAGWTTATCA CCACACGGAT GCAGTCTTGC CTACACCTAT 420

GGGACCTCAT CAACACTGTG CCGAAGAATT AGTTCTGTCA CGAGATCTTA CACTCATCAT 480

TGAACCTGGG AGATCCCTCA TAGCTAACAC TTGCTGCTTC GTCAATAGGG TCACTGGTGT 540

TAAATCTAAT GGTACAAAGA ATTTCATTGT AGTTGATGGC AGCATGGCAG AGCTTATCAG 600

ACCAAGTCTA TATGGAGCAT ACCAGCATAT CGAACTGGTT TCTCCTTCCC CAGATGCAGA 660 AGTAGCAACA TTCGATATTG TTGGACCAGT TTGTGAATCT GCAGATTTCC TTGGCAAAGA 720

CAGGGAACTT CCAACACCTG ATAAGGGAGC TGGTTTGGTG GTTCATGACG CAGGAGCCTA 780

CTGCATGAGC ATGGCTTCAA CCTACAACTT GAAGTTGCGA CCACCTGAAT ATTGGGTAGA 840

AGATGATGGG TCCATTGCTA AGATTCGGCG TGGAGAGTCA TTTGATGACT ACATGAAGTT 900

CTTTGATAAT CTCTCTGCCT AACTCGTTTT CCTGCAATTG TAATAAGATT TTTCTCTTGT 960

TATGTGTGGC TGTATCAGGA TTCGGATTGA TAGCGCAGTA CAGTTTGCTG TAGAATCGGT 1020

ATTTTTTTTT ATTGTACTGT GATGTCGGTA CCTTATTTTA TCCAAAGATT TTTGGCAAAT 1080

TTTGCTACAG GACACTTAAA AAAAAAAAAA AAAAAA 1116 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: rlOn. pk0013.b9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Leu His Gly Val Phe Val Asn He Asp Ser Glu Phe Asp Leu Glu Asn 1 5 10 15

He Val Thr Ala Ala Arg Val Ala Gly Lys Lys Val Pro Val Leu Leu 20 25 30

Arg He Asn Pro Asp Val Asp Pro Gin Val His Pro Tyr Val Ala Thr 35 40 45

Gly Asn L^s Thr Ser Lys Phe Gly He Arg Asn Glu Lys Leu Gin Trp 50 55 60

Phe Leu Asp Ser He Lys Ser Tyr Ser Asn Asp He Thr Leu Val Gly 65 70 75 80

Val His Cys His Leu Gly Ser Thr He Thr Lys Val Asp He Phe Arg 85 90 95

Asp Ala Ala Gly Leu Met Val Asn Tyr Val Asp Glu He Arg Ala Gin 100 105 110

Gly Phe Glu Leu Glu Tyr Leu Asn He Gly Gly Gly Leu Gly He Xaa 115 120 125

Tyr His His Thr Asp Ala Val Leu Pro Thr Pro Met Gly Pro His Gin 130 135 140

His Cys Ala Glu Glu Leu Val Leu Ser Arg Asp Leu Thr Leu He He 145 150 155 160

Glu Pro Gly Arg Ser Leu He Ala Asn Thr Cys Cys Phe Val Asn Arg 165 170 175 Val Thr Gly Val Lys Ser Asn Gly Thr Lys Asn Phe He Val Val ASD 180 185 190

Gly Ser Met Ala Glu Leu He Arg Pro Ser Leu Tyr Gly Ala Tyr Glr. 195 200 205

His He Glu Leu Val Ser Pro Ser Pro Asp Ala Glu Val Ala Thr Phe 210 215 220

Asp He Val Gly Pro Val Cys Glu Ser Ala Asp Phe Leu Gly Lys Asp 225 230 235 240

Arg Glu Leu Pro Thr Pro ASD Lys Gly Ala Gly Leu Val Val His ASD 245 250 255

Ala Gly Ala Tyr Cys Met Ser Met Ala Ser Thr Tyr Asn Leu Lys Leu 260 265 270

Arg Pro Pro Glu Tyr Trp Val Glu Asp Asp Gly Ser He Ala Lys He 275 280 285

Arg Arg Gly Glu Ser Phe Asp Asp Tyr Met Lys Phe Phe Asp Asn Leu 290 295 300

Ser Ala 305

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 968 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: srl . pk0132. cl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GTTGCCACTG GGAATAAGAA CTCTAAATTT GGCATTAGAA ATGAGAAGCT GCAGTGCTTT 60

TTAGATGCAG TGAAGGAACA TCCTAATGAG CTCAAACTTG TAGGGGCCCA CTGCCATCTT 120

GGTTCAACAA TTACCAAGGT TGACATTTTC AGGGATGCAG CCACCATTAT GATCAACTAC 180

ATTGACCAAA TCCGAGATCA GGGTTTTGAA GTTGATTACT TAAATATTGG TGGAGGACTT 240

GGGATAGATT ATTATCATTC TGGTGCCATC CTTCCTACAC CTAGAGATCT CATTGACACT 300

GTACGAGATC TTGTTATTTC ACGTGGTCTT AATCTCATCA TTGAACCAGG AAGATCACTC 360

ATTGCAAACA CGTGTTGCTT AGTTAACCGG GTGACAGGTG TTAAAACTAA TGGATCT.AAA 420

AACTTCATTG TAATTGATGG AAGTATGGCT GAACTTATCC GCCCTAGTCT TTATGATGCT 480

TACCAGCATA TAGAGCTGGT TTCCCCTGCC CCGTCAAATG CTGAAACAGA AACTTTTGAT 540

GTGGTTGGCC CTGTCTGTGA GTCTGCAGAT TTCTTAGGAA AAGGAAGAGA ACTTCCTACT 600

CCAGCCAAGG GTACTGGTTT GGTTGTTCAT GATGCTGGTG CTTATTGCAT GAGCATGGCA 660

TCAACCTACA ATCTAAAGAT GCGGCCTCCT GAGTATTGGG TTGAAGATGA TGGATCA3TG 720 AGCAAAATAA GACATGGAGA GACTTTTGAA GACCACATTC GGTTTTTTGA GGGGCTTTGA 780

GCTAATAATT TATCTTGTAG GAAAGAAGGC TGGAGAATTG TTATGTACTT GGAGTTTGAA 840

TCTTTCCTCG TCAATGAATG CATGACTCTT GTAGTTCTGT TTCTTCCGTT CTAATTGAAT 900

GTTGACTCCC ATGACAGGAA CAGAGAATAA AGTTGATTTC AGTTAGATTT AAAAAAAAAA 960

AAAAAAAA 968 (2) INFORMATION FOR SEQ ID NO: 15:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 259 ammo acids

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide

(vii, IMMEDIATE SOURCE:

(B) CLONE: srl . pk0132. cl

(x ¹ SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Val Ala Tr.r Gly Asn Lys Asn Ser Lys Phe Gly He Arg Asn Glu Lys 1 5 10 15

Leu Gin Cys Pne Leu Asp Ala Val Lys Glu His Pro Asn G_J Leu Lys 20 ^' 25 30

Leu Val G v Ala His Cys His Leu Gly Ser Thr He Thr Lys Val Asp 35 40 45

He Phe Arα Asp Ala Ala Thr He Met He Asn Tyr He ASD Gin He 50 55 60

Arg Asp G_n Gly Phe Glu Val Asp Tyr Leu Asn He Gly Gly Gly Leu 65 70 75 80

Gly He ASD Tyr Tyr His Ser Gly Ala He Leu Pro Thr Pro Arg Asp 85 90 95

Leu He ASD Tnr Val Arg Asp Leu Val He Ser Arg Gly Leu Asn Leu 100 ^* 105 110

He He Gl_- Pro Gly Arg Ser Leu He Ala Asn Thr Cys Cys Leu Val 115 120 125

Asn Arg Val Thr Gly Val Lys Thr Asn Gly Ser Lys Asn Phe He Val 130 135 140

He Asp Gly Ser Met Ala Glu Leu He Arg Pro Ser Leu Tyr Asp Ala 145 150 155 160

Tyr Gin His He Glu Leu Val Ser Pro Ala Pro Ser Asn Ala Glu Thr 165 170 175

Glu Thr Phe Asp Val Val Gly Pro Val Cys Glu Ser Ala Asp Phe Leu 180 185 190

Gly Lys Glv Arg Glu Leu Pro Thr Pro Ala Lys Gly Thr Gly Leu Val 195 200 205

Val His ASD Ala Gly Ala Tyr Cys Met Ser Met Ala Ser Thr Tyr Asn 210 215 220 Leu Lys Met Arg Pro Pro Glu Tyr Trp Val Glu Asp Asp Gly Ser Val 225 230 235 240

Ser Lys He Arg His Gly Glu Thr Phe Glu Asp His He Arg Phe Phe 245 250 255

Glu Gly Leu

(2) INFORMATION FOR SEQ ID NO: 16:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 676 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: wlkl . pk0012. c2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TTTGAGTTGG AGTACCTGAA TATTGGAGGT GGTTTGGGGA TAGACTACCA CCACACTGGT 60

GCAGTCTTGC CTACACCTAT GGATCTTATC AACACTGTCC GGGAATTGGT CCTCTCACGG 120

GATCTTACTC TCATTATTGA ACCTGGAAGA TCCCTGATCG CCAATACTTG CTGCTTCGTC 180

AATAAGGTCA CTGGTGTAAA ATCGAATGGC ACGAAGAATT TCATTGTAGT TGATGGCAGC 240

ATGGCCGAGC TCATCAGGCC TAGTCTATAT GGAGCATATC AGCATATAGA ACTAGTTCTC 300

CCTCTCCAAG GTGCAGAAGT AGCAACCTTC CGATATTGTT GGGGCCAGTC TGCGAATCTG 360

CAGATTCCTT GGNAAAGACA AGGAGTTCCA ACACCTGACA AGGGANCTGG TTTGGGTGTC 420

CACGACGCAN GANCTACTGC ATGAGCATGG CTTCNACCTA CAACCTGAAG ATGAGGCAAC 480

CGAGTATTGG GTANAGGACA TGGNCCATGT AAGATAAGCA CGGGGAAACA TTGACGACAC 540

ATGAGTCTTG ATNGCTCCGC CAGGCCTTTA CTGGTTGGNA ACNAGCTTCA TTGTNNCCAC 600

CGTGGAATCT GGGAACATCN TGTTGTAGTG GCACCACANA GGGNTTTTGN GACAATCACA 660

NTAGATGAGA TTNTGG 676

(2) INFORMATION FOR SEQ ID NO: 17:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 73 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: wlkl .pk0012. c2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Pro Thr Pro Met Asp Leu He Asn Thr Val Arg Glu Leu Val Leu Ser 1 5 10 15 Arg Asp Leu Thr Leu He He Glu Pro Gly Arg Ser Leu He Ala Asn 20 25 30

Thr Cys Cys Phe Val Asn Lys Val Thr Gly Val Lys Ser Asn Gly Thr 35 40 45

Lys Asn Phe He Val Val Asp Gly Ser Met Ala Glu Leu He Arg Pro 50 55 60

Ser Leu Tyr Gly Ala Tyr Gin His He 65 70

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 544 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii, IMMEDIATE SOURCE:

(B) CLONE: sdp3c.pk001.ol5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

TTGCAACACA CATTGTCTTG TCGGCAAAAT CTTCCACCAA CAACACACAG CCATGGCAGG 60

CTCAAACATT CTTTCTCACT CTCCTTCCCT TCCCAAAACC TACAGCCACT CCTTAAACCA 120

AAACGCGTTA TCCCAAAAGC TTTTTTTTCT GCCCCTCAAA TTCAAAGCCA CCACAAAACC 180

ACGTGCTCTC AGAGCGGTTC TCTCGCAGAA CGCTGTCAAA ACCTCGGTGG AGGACACAAA 240

GAACGCTCAT TTTCAGCACT GTTTCACCAA ATCCGAAGAT GGGTATCTGT ACTGTGAGGG 300

CCTCAAGGTG CATGACATCA TGGAATCTGT TGAGAGAAGA CCTTTCTATT TGTACAGCAA 360

GCCCCAGATA ACTAGGAATG TTGAAGCCTA CAAGGATGCA TTGGAAGGGT TGAACTCCAT 420

AATTGGTTAT GCCATTAAGG CCAATAATAA CTTGAAGATT TTGGNACATT TGAGGCACTT 480

GGGTTGTGGT GCTGTGCTTG TTAGTGGGAA TGAGCTGAAG TTGNTCTTCG AGCTGGNTTT 540

GTTC 544

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 62 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: sdp3c.pk001.ol5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Arg Arg Pro Phe Tyr Leu Tyr Ser Lys Pro Gin He Thr Arg Asn Val 1 5 10 15 Glu Ala Tyr Lys Asp Ala Leu Glu Gly Leu Asn Ser He He Gly Tyr 20 25 30

Ala He Lys Ala Asn Asn Asn Leu Lys He Leu Xaa His Leu Arg His 35 40 45

Leu Gly Cys Gly Ala Val Leu Val Ser Gly Asn Glu Leu Lys 50 55 60

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 371 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pseudomonas aeruginosa

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Met Lys Arg Val Gly Leu He Gly Trp Arg Gly Met Val Gly Ser Val 1 5 10 15

Leu He Gin Arg Met Leu Glu Glu Arg Asp Phe Asp Leu He Glu Pro 20 25 30

Val Phe Phe Thr Thr Ser Asn Val Gly Ala Gin Ala Pro Glu Val ASD 35 40 45

Lys Asp He Ala Pro Leu Lys Asp Ala Tyr Ser He Asp Glu Leu Lys 50 55 60

Thr Leu Asp Val He Leu Thr Cys Gin Gly Gly Asp Tyr Thr Ser Glu 65 70 75 80

Val Phe Pro Lys Leu Arg Glu Ala Gly Trp Gin Gly Tyr Trp He ASD 85 90 95

Ala Ala Ser Ser Leu Arg Met Glu Asp Asp Ala Val He Val Leu ASD 100 105 110

Pro Val Asn Arg Lys Val He Asp Gin Ala Leu Asp Ala Gly Thr Arg 115 120 125

Asn Tyr He Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ala Leu 130 135 140

Gly Gly Leu Phe Asp Ala Gly Leu Val Glu Trp Met Ser Ala Met Thr 145 150 155 160

Tyr Gin Ala Ala Ser Gly Ala Gly Ala Gin Asn Met Arg Asp Leu Leu 165 170 175

Lys Gin Met Gly Ala Ala His Ala Ser Val Ala Asp Asp Leu Ala Asn 180 185 190

Pro Ala Ser Ala He Leu Asp He Asp Arg Lys Val Ala Glu Thr Leu 195 200 205

Arg Ser Glu Ala Phe Pro Thr Glu His Phe Gly Ala Pro Leu Gly Gly 210 215 220 Ser Leu He Pro Trp He Asp Lys Glu Leu Ser Gin Arg Arg Gin Ser 225 230 235 240

Arg Glu Glu Trp Lys Ala Gin Ala Glu Thr Asn Lys He Leu Ala Arg 245 250 255

Phe Lys Asn Pro He Pro Val Asp Gly He Cys Val Arg Val Gly Ala 260 265 270

Met Arg Cys His Ser Gin Ala Leu Thr He Lys Leu Asn Lys Asp Val 275 280 285

Pro Leu Thr Asp He Glu Gly Leu He Arg Gin His Asn Pro Trp Val 290 295 300

Lys Leu Val Pro Asn His Arg Glu Val Ser Val Arg Glu Leu Thr Pro 305 310 315 320

Ala Ala Val Thr Gly Thr Leu Ser Val Pro Val Gly Arg Leu Arg Lys 325 330 335

Leu Asn Met Val Ser Gin Tyr Leu Gly Ala Phe Thr Val Gly Asp Gin 340 345 350

Leu Leu Tro Gly Ala Ala Glu Pro Leu Arg Arg Met Lej Arg He Leu 355 360 365

Leu Glu Arg 370

(2) INFORMATION FOR SEQ ID NO: 21:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 788 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: crln . pk0009. g4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

CGACAACATC GCCCCCGCCA TCCTCGGCGG CTTCGTCCTC GTCCGCAGCT ACGACCCCTT 60

TCACCTCGTC CCGCTTTCCT TCCCGCCAGC GCTCCGCCTC CACTTCGTCC TGGTCACCCC 120

CGACTTCGAG GCGCCCACGA GCAAGATGCG CGCCGCGCTG CCCAGGCAGG TCGACGTCCA 180

GCAGCACGTG CGCAACTCCA GCCAGGCAGC GGCGCTCGTG GCGGCGGTGC TGCAGGGGGA 240

CGCGGGCCTC ATCGGCTCCG CGATGTCGTC CGACGGCATC GTGGAGCCCA CCAGGGCACC 300

CCTCATACCT GGCATGGCGG CCGTAAAGGC GGCGGCCCTG CAAGCTGGAG CGCTGGGCTG 360

CACAATTAGC GGCGCGGGCC CCACAGTGGT GGCCGTCATC CAAGGGGAGG AAAGGGGGGA 420

GGAGGTTGCC CGCAAGATGG TGGACGCGTT CTGGAGCGCA GGCAAGCTCA AGGCGACAGC 480

AACCGTCGCG CAGCTCGATA CCCTTGGTGC CAGGGTCATC GCCACGTCAT CCTTGAACTA 540

GCAAAAGATT CGGAAAGTGG TACTGCAATT GTATCACCAA ACAAGGAAGA ATGAAGGGGA 600

ACCCCATGGA TTTGTATGTT TTCTCTTCTT TCTTGCATCT TTAGGTGGTT AATTGGCTTT 660 GGAATAAATG AGATGGAGGA CATCGCTAGA ACAATTCTGT TCCGTGGGCT GTAATTTCAA 720

TTTGGGCTGG TTTCTTTATC ATGCCATGGA TAATTATGAA TAAATTTGAG GTAGTTTGTT 780

AAAAAAAA 788 (2) INFORMATION FOR SEQ ID NO: 22:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 179 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: crln.pk0009. g4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Asp Asn He Ala Pro Ala He Leu Gly Gly Phe Val Leu Val Arg Ser 1 5 10 15

Tyr Asp Pro Phe His Leu Val Pro Leu Ser Phe Pro Pro Ala Leu Arg 20 25 30

Leu His Phe Val Leu Val Thr Pro Asp Phe Glu Ala Pro Thr Ser Lys 35 40 45

Met Arg Ala Ala Leu Pro Arg Gin Val Asp Val Gin Gin His Val Arg 50 55 60

Asn Ser Ser Gin Ala Ala Ala Leu Val Ala Ala Val Leu Gin Gly ASD 65 70 75 80^"

Ala Gly Leu He Gly Ser Ala Met Ser Ser Asp Glv He Val Glu Pro 85 90 95

Thr Arg Ala Pro Leu He Pro Gly Met Ala Ala Val Lys Ala Ala Ala 100 105 110

Leu Gin Ala Gly Ala Leu Gly Cys Thr He Ser Gly Ala Gly Pro T-r 115 120 125

Val Val Ala Val He Gin Gly Glu Glu Arg Gly Glu Glu Val Ala Arg 130 135 140

Lys Met Val Asp Ala Phe Trp Ser Ala Gly Lys Leu Lys Ala Thr Ala 145 150 155 160

Thr Val Ala Gin Leu ASD Thr Leu Gly Ala Arg Val He Ala Thr Ser 165 170 175

Ser Leu Asn

(2) INFORMATION FOR SEQ ID NO:23:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 601 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: rcall .pk005. k3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GTCGCCGCCA TCGCTGCCCT TCGCGCCCTC GATGTCAAGT CCCACGCCGT CTCCATCCAC 60

CTCACCAAGG GCCTCCCCCT CGGCTCCGGC CTCGGCTCCT CCGCCGCCTC CGCCGCCGCC 120

GCTGCCAAGG CCGTTGACGC CCTCTTCGGC TCCCTCCTAC ACCAAGATGA CCTCGTCCTC 180

GCGGGCCTCG AGTCCGAGAA AGCCGTCAGT GGCTTCCACG CCGACAACAT CGCCCCGGCC 240

ATCCTCGGCG GCTTCGTCCT CGTCCGCAGC TACGACCCCT TCCACCTCAT CCCGCTCTCC 300

TCCCCACCTG CCCTCCGCCT CCACTTCGTC CTCGTCACGC CCGACTTCGA GGCGCCCACC 360

AAGCAAGATG CGTGCCGCGC TGCCCAAACA GGTGGCCGTC CACCAAGCAC GTCCGCAACT 420

CCAGCCAAGC GG CGCGCTT GTCGCCGCTG TGCTGCAAGG GGACGCCACC CTCATCGGCT 480

CCGCAATGTC CTCCGACGGC ATCGTGGAGC CAACAAGGCG CCGCTGATTC TGGATGGCTG 540

CGGTCAAAGG CGCCGGCTTG GAACTGGGGG AATTGGCTGC ACATCAGTGG AGAAGGCAAN 600

T 601

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 82 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: rcalc . pk005. k3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Val Ser He His Leu Thr Lys Gly Leu Pro Leu Gly Ser Gly Leu Gly 1 5 10 15

Ser Ser Ala Ala Ser Ala Ala Ala Ala Ala Lys Ala Val Asp Ala Leu 20 25 30

Phe Gly Ser Leu Leu His Gin Asp Asp Leu Val Leu Ala Gly Leu Glu 35 40 45

Ser Glu Lys Ala Val Ser Gly Xaa Xaa His Ala Asp Asn He Ala Pro 50 55 60

Ala He Leu Gly Gly Phe Val Leu Val Arg Ser Tyr Asp Pro Phe His 65 70 75 80

Leu He (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1543 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: ses8w.pk0020.b5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

GAAGAGAGAC AAACCAGCAA GAGTGGAGAT GGCGACGTCG ACGTGCTTCC TGTGTCC3TC 60

TACGGCGAGT TTGAAAGGCA GGGCCAGATT CAGAATCAGA ATCAGATGCA GCAGCAGCGT 120

GTCGGTCAAT ATTCGAAGGG AGCCCGAACC TGTAACGACG CTGGTGAAAG CGTTTGCTCC 180

CGCCACGGTG GCGAATCTAG GTCCAGGCTT CGACTTCCTA GGCTGCGCCG TGGACGGACT 240

CGGAGACATT GTGTCGGTGA AGGTTGACCC ACAGGTTCAC CCTGGCGAGA TATGCAT.-.TC 300

CGACATCAGC GGCCACGCCC CAAACAAGCT CAGCAAAAAC CCTCTCTGGA ACTGCGCCGG 360

CATCGCCGCC ATTGAAGTCA TGAAAATGCT CTCCATTCGA TCCGTCGGCC TCTCCCTCTC 420

CCTGGAGAAG GGCCTGCCTT TGGGAAGCGG TCTGGGATCC AGCGCCGCCA GCGCCGCCGC 480

GGCCGCCGTG GCGGTGAACG AGCTGTTTGG GAAGAAATTA AGCGTGGAGG AGCTGGTTCT 540

GGCATCACTG AAATCGGAAG AGAAGGTGTC GGGGTATCAC GCGGACAACG TGGCGCCATC 600

GATAATGGGG GGTTTTGTGC TGATCGGGAG CTACTCGCCG CTGGAGTTGA TGCCGTTGAA 660

GTTTCCGGCA GAGAAGGAGC TGTATTTCGT GCTGGTGACG CCTGAGTTCG AGGCCCCGAC 720

GAAGAAGATG CGGGCAGCGC TGCCTACGGA GATCGGGATG CCGCACCACG TGTGGAACTG 780

CAGCCAGGCA GGTGCTCTGG TGGCGTCGGT GCTGCAGGGC GACGTGGTTG GGTTGGC-C-AA 840

GGCATTG CC TCTGACAAGA TCGTTGAGCC AAGGCGTGCC CCCTTGATTC CTGGCATGGA 900

GGCTGTCAAG AGGGCTGCCA TTCAGGCCGG TGCTTTTGGC TGTACCATCA GCGGCGCCGG 960

CCCTACCGCC GTCGCCGTCA TTGACGACGA GCAAACTGGA CACCTCATTG CCAAACACAT 1020

GATTGACGCT TTTCTCCATG TTGGCAATTT GAAGGCTTCT GCAAATGTCA AGCAGCTTGA 1080

TCGCCTTGGT GCTAGACGCA TTCCAAATTG AACCTTCTCT TCTCTATCTC TATGAGAGGC 1140

TTGTAGATTT CAAGAACCGG ATTTCTTCCA ACTTGCTCGT AACACTCTAA GTGCTGACCG 1200

GTCACATGTA TTTGAAATTT GATCTGATCA ATGAAGCAGC ATTCTAGTGT GGAGGTCTGA 1260

ATAACAAGAG AAACATTAAA CCCAAGCTGG GAGCTCTGTT TGGGTGGTGG AAATTTAAAT 1320

AGATGAATAA TTATGAAAGA CCTAGATCAG GTCAGTGTTA TGGTGAACTC TGAAGCATGT 1380

TTTAGATTTT CTTTGCTTTG TTTTTATCAT ATTTTTATCT TGCTACTTGA GTTGACAAAG 1440

CTCAAAAAGA AGTCATTTTT AGTATTTTCT TGTTTCATTA TGCTAGTTAA TCTTAGCTTT 1500

TGAATAGCAT GTATTGTTCC TTAAAAAAAA AAAAAAAAAA AAA 1543 (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 483 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE: (B) CLONE: ses8w. pk0020.b5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Met Ala Thr Ser Thr Cys Phe Leu Cys Pro Ser Thr Ala Ser Leu Lys 1 5 10 15

Gly Arg Ala Arg Phe Arg He Arg He Arg Cys Ser Ser Ser Val Ser 20 25 30

Val Asn He Arg Arg Glu Pro Glu Pro Val Thr Thr Leu Val Lys Ala 35 40 45

Phe Ala Pro Ala Thr Val Ala Asn Leu Gly Pro Gly Phe Asp Phe Leu 50 55 60

Gly Cys Ala Val Asp Gly Leu Gly Asp He Val Ser Val Lys Val Asp 65 70 75 80

Pro Gin Val His Pro Gly Glu He Cys He Ser Asp He Ser Gly His 85 90 95

Ala Pro Asn Lys Leu Ser Lys Asn Pro Leu Trp Asn Cys Ala Gly He 100 105 110

Ala Ala He Glu Val Met Lys Met Leu Ser He Arg Ser Val Gly Leu 115 120 125

Ser Leu Ser Leu Glu Lys Gly Leu Pro Leu Gly Ser Gly Leu Gly Ser 130 135 140

Ser Ala Ala Ser Ala Ala Ala Ala Ala Val Ala Val Asn Glu Leu Phe 145 150 155 160

Gly Lys Lys Leu Ser Val Glu Glu Leu Val Leu Ala Ser Leu Lys Ser 165 170 175

Glu Glu Lys Val Ser Gly Tyr His Ala Asp Asn Val Ala Pro Ser He 180 185 190

Met Gly Gly Phe Val Leu He Gly Ser Tyr Ser Pro Leu Glu Leu Met 195 200 205

Pro Leu Lys Phe Pro Ala Glu Lys Glu Leu Tyr Phe Val Leu Val Thr 210 215 220

Pro Glu Phe Glu Ala Pro Thr Lys Lys Met Arg Ala Ala Leu Pro Thr 225 230 235 240

Glu He Gly Met Pro His His Val Trp Asn Cys Ser Gin Ala Gly Ala 245 250 255

Leu Val Ala Ser Val Leu Gin Gly Asp Val Val Gly Leu Gly Lys Ala 260 265 270 Leu Ser Ser Asp Lys He Val Glu Pro Arg Arg Ala Pro Leu He Pro 275 280 285

Gly Met Glu Ala Val Lys Arg Ala Ala He Gin Ala Gly Ala Phe Gly 290 295 300

Cys Thr He Ser Gly Ala Gly Pro Thr Ala Val Ala Val He Asp Asp 305 310 315 320

Glu Gin Thr Gly His Leu He Ala Lys His Met He Asp Ala Phe Leu 325 330 335

His Val Gly Asn Leu Lys Ala Ser Ala Asn Val Lys Gin Leu Asp Arg 340 345 350

Leu Gly Ala Arg Arg He Pro Asn Thr Phe Ser Ser Leu Ser Leu Glu 355 360 365

Ala Cys Arg Phe Gin Glu Pro Asp Phe Phe Gin Leu Ala Arg Asn Thr 370 375 380

Leu Ser Ala Asp Arg Ser His Val Phe Glu He Ser Asp Gin Ser Ser 385 390 395 400

He Leu Val Trp Arg Ser Glu Gin Glu Lys His Thr Gin Ala Gly Ser 405 410 415

Ser Val Trp Val Val Glu He He Asp Glu Leu Lys Thr He Arg Ser 420 425 430

Val Leu Trp Thr Leu Lys His Val Leu Asp Phe Leu Cys Phe Val Phe 435 440 445

He He Phe Leu Ser Cys Tyr Leu Ser Gin Ser Ser Lys Arg Ser His 450 455 460

Phe Tyr Phe Leu Val Ser Leu Cys Leu He Leu Ala Phe Glu His Val 465 470 475 480

Leu Phe Leu

(2) INFORMATION FOR SEQ ID NO:27:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 438 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: wlln.pk0065. f2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

CTCGAGTCGG AGAAGGCCGT CAGCGGCTTC CACGCCGACA ACATCGCCCC CGCCATCCTC 60

GGCGGCTTCG TCCTCGTCCG CAGCTACGAC CCCTTTCACC TCGTCCCGCT TTCCTTCCCG 120

CCAGCGCTCC GCCTCCACTT CGTCCTGGTC ACCCCCGACT TCGAGGCGCC CACGAGCAAG 180

ATGCGCGCCG CGCTGCCCAG GCAGGTCGAC GTCCAGCAGC ACGTGCGCAA CTCCAGCCAG 240 GCAGCGGCGC TCCGTGGCGG CGGTGCTGCA NGGGGACGCC GGGCTCATCG GTCCGCGATT 300

TCTCCGACGG GCATCGTGGA CCCACCAAGG AACCCTCATA CCTGGCATGG CGGCCGTAAA 360

GGCGGCGGCC TGCAACTGGA CGCTGGGTGC ACATTAACGG GCGGGCCCAC ATGGTGGCTC 420

NCAGNGAAGA GAGGGGAG 438 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: wlln. pk0065. f2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Leu Glu Ser Glu Lys Ala Val Ser Gly Phe His Ala Asp Asn He Ala 1 5 10 15

Pro Ala He Leu Gly Gly Phe Val Leu Val Arg Ser Tyr Asp Pro Phe 20 25 30

His Leu Val Pro Leu Ser Phe Pro Pro Ala Leu Arg Leu His Phe Val 35 40 45

Leu Val Thr Pro Asp Phe Glu Ala Pro Thr Ser Lys Met Arg Ala Ala 50 55 60

Leu Pro Arg Gin Val Asp Val Gin Gin His Val Arg Asn Ser Ser Gin 65 70 75 80

Ala Ala Ala Leu

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 300 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Methanococcus jannashii

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Met Arg Glu He Met Lys Val Arg Val Lys Ala Pro Cys Thr Ser Ala 1 5 10 15

Asn Leu Gly Val Gly Phe Asp Val Phe Gly Leu Cys Leu Lys Glu Pro 20 25 30

Tyr Asp Val He Glu Val Glu Ala He Asp Asp Lys Glu He He He 35 40 45 Glu Val AS_D Asp Lys Asn He Pro Thr Asp Pro Asp Lys Asn Val Ala 50 ^' 55 60

Gly He Val Ala Lys Lys Met He Asp Asp Phe Asn He Gly Lys Gly 65 70 75 80

Val Lys He Thr He Lys Lys Gly Val Lys Ala Gly Ser Gly Leu Gly 85 90 95

Ser Ser Ala Ala Ser Ser Ala Gly Thr Ala Tyr Ala He Asn Glu Leu 100 105 110

Phe Lys Leu Asn Leu Asp Lys Leu Lys Leu Val Asp Tyr Ala Ser Tyr 115 120 125

Gly Glu Leu Ala Ser Ser Gly Ala Lys His Ala Asp Asn Val Ala Pro 130 135 140

Ala He Phe Glv Gly Phe Thr Met Val Thr Asn Tyr Glu Pro Leu Glu 145 150 155 160

Val Leu His He Pro He Asp Pne Lys Leu Asp He Leu He Ala He 165 170 175

Pro Asn He Ser He Asn Thr Lys Glu Ala Arg Glu He Leu Pro Lys 180 ^' 185 190

Ala Val Gly Leu Lys Asp Leu Val Asn Asn Val Gly Lys Ala Cys Gly 195 200 205

Met Val Tyr Ala Leu Tyr Asn Lys Asp Lys Ser Leu Phe Gly Arg Tyr 210 215 220

Met Met Ser Asp Lys Val He Glu Pro Val Arg Gly Lys Leu He Pro 225 230 235 240

Asn Tyr Phe Lys He Lys Glu Glu Val Lys Asp Lys Val Tyr Gly He 245 250 255

Thr He Ser Gly Ser Gly Pro Ser He He Ala Phe Pro Lys Glu Glu 260 265 270

Phe He Asp Glu Val Glu Asn He Leu Arg Asp Tyr Tyr Glu Asn Tr.r 275 280 285

He Arg Thr Glu Val Gly Lys Gly Val Glu Val Val 290 295 300

(2) INFORMATION FOR SEQ ID NO: 30:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1362 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: se3.05h06

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

ACTTTGTAGT TCGTAGATAG CCGATGTGCT TGTCTTAGTG TGTCAGTCAT TCCTGTTCCT 60

CAAGTCAAGC TTTGTAGTGA GCAGATATAA TGGCTGTTGA AAGGTCCGGA ATTGCCAAAG 120 ATGTTACGGA ATTGATTGGT AAAACCCCAT TAGTATATCT AAATAAACTT GCGGATGGTT 180

GTGTTGCCCG GGTTGCTGCT AAACTGGAGT TGATGGAGCC ATGCTCTAGT GTGAAGGACA 240

GGATTGGGTA TAGTATGATT GCTGATGCAG AAGAGAAGGG ACTTATCACA CCTGGAAAGA 300

GTGTCCTCAT TGAGCCAACA AGTGGTAATA CTGGCATTGG ATTAGCCTTC ATGGCAGCAG 360

CCAGGGGTTA CAAGCTCATA ATTACAATGC CTGCTTCTAT GAGTCTTGAG AGAAGAATCA 420

TTCTATTAGC TTTTGGAGCT GAGTTGGTTC TGACAGATCC TGCTAAGGGA ATGAAAGGTG 480

CTGTTCAGAA GGCTGAAGAG ATATTGGCTA AGACGCCCAA TGCCTACATA CTTCAACAAT 540

TTGAAAACCC TGCCAATCCC AAGGTTCATT ATGAAACCAC TGGTCCAGAG ATATGGAAAG 600

GCTCCGATGG GAAAATTGAT GCATTTGTTT CTGGGATAGG CACTGGTGGT ACAATAACAG 660

GTGCTGGAAA ATATCTTAAA GAGCAGAATC CGAATATAAA GCTGATTGGT GTGGAACCAG 720

TTGAAAGTCC AGTGCTCTCA GGAGGAAAGC CTGGTCCACA CAAGATTCAA GGGATTGGTG 780

CTGGTTTTAT CCCTGGTGTC TTGGAAGTCA ATCTTCTTGA TGAAGTTGTT CAAATATCAA 840

GTGATGAAGC AATAGAAACT GCAAAGCTTC TTGCGCTTAA AGAAGGCCTA TTTGTGGGAA 900

TATCTTCCGG AGCTGCAGCT GCTGCTGCTT TTCAGATTGC AAAAAGACCA GAAAATGCCG 960

GGAAGCTTAT TGTTGCCGTT TTTCCCAGCT TCGGGGAGAG GTACCTGTCC TCCGTGCTAT 1020

TTGAGTCAGT GAGACGCGAA GCTGAAAGCA TGACTTTTGA GCCCTGAATT CCCGTTTAAG 1080

GCTCTCACTA CTGAATTTTC TTGTTACTTG TACCAGGCTT TAACTAGATT GTTAGAGTAC 1140

TACTGTTTGT GACTCTGACT CTAAAATAAA ACTTGCTCCA AAAGACTAGT TTTTCTTGAT 1200

GCCCCTGGAG CGATAATTTT GTGCCTGCAA CATTAAAAAG TATTCAAAGT TGCTTATAAG 1260

TAACATGTTT CATCTTTTGT TGTTGTTGAG ACGAACACGG ATGAGGTCAT AATACTATGT 1320

TTCTGATTTC CTTTGGTAGG GAAAAAAAAA AAAAAAAAAA AA 1362 (2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 325 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: se3.05h06

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Met Ala Val Glu Arg Ser Gly He Ala Lys Asp Val Thr Glu Leu He 1 5 10 15

Gly Lys Thr Pro Leu Val Tyr Leu Asn Lys Leu Ala Asp Gly Cys Val 20 25 30

Ala Arg Val Ala Ala Lys Leu Glu Leu Met Glu Pro Cys Ser Ser Val 35 40 45 Lys Asp Arg He Gly Tyr Ser Met He Ala Asp Ala Glu Glu Lys Glv 50 55 60

Leu He Thr Pro Gly Lys Ser Val Leu He Glu Pro Thr Ser Gly Asn 65 70 75 80

Thr Gly He Gly Leu Ala Phe Met Ala Ala Ala Arg Gly Tyr Lys Leu 85 90 95

He He Thr Met Pro Ala Ser Met Ser Leu Glu Arg Arg He He Leu 100 105 110

Leu Ala Phe Gly Ala Glu Leu Val Leu Thr Asp Pro Ala Lys Gly Met 115 120 125

Lys Gly Ala Val Gin Lys Ala Glu Glu He Leu Ala Lys Thr Pro Asn 130 135 140

Ala Tyr He Leu Gin Gin Phe Glu Asn Pro Ala Asn Pro Lys Val His 145 150 155 160

Tyr Glu Thr Thr Gly Pro Glu He Trp Lys Gly Ser Asp Gly Lys He 165 170 175

Asp Ala Phe Val Ser Gly He Gly Thr Gly Gly Thr He Thr Gly Ala 180 185 190

Gly Lys Tyr Leu Lys Glu Gin Asn Pro Asn He Lys Leu He Gly Val 195 200 205

Glu Pro Val Glu Ser Pro Val Leu Ser Gly Gly Lys Pro Gly Pro His 210 215 220

Lys He Gin Gly He Gly Ala Gly Phe He Pro Gly Val Leu Glu Val 225 230 235 240

Asn Leu Leu Asp Glu Val Val Gin He Ser Ser Asp Glu Ala He Glu 245 250 255

Thr Ala Lys Leu Leu Ala Leu Lys Glu Gly Leu Phe Val Gly He Ser 260 265 270

Ser Gly Ala Ala Ala Ala Ala Ala Phe Gin He Ala Lys Arg Pro Glu 275 280 285

Asn Ala Gly Lys Leu He Val Ala Val Phe Pro Ser Phe Gly Glu Arg 290 295 300

Tyr Leu Ser Ser Val Leu Phe Glu Ser Val Arg Arg Glu Ala Glu Ser 305 310 315 320

Met Thr Phe Glu Pro 325

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 325 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:

(A) ORGANISM: Citrullus lanatus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Met Ala Asp Ala Lys Ser Thr He Ala Lys Asp Val Thr Glu Leu He 1 5 10 15

Gly Asn Thr Pro Leu Val Tyr Leu Asn Arg Val Val Asp Gly Cys Val 20 25 30

Ala Arg Val Ala Ala Lys Leu Glu Met Met Glu Pro Cys Ser Ser Val 35 40 45

Lys Asp Arα He Gly Tyr Ser Met He Ser Asp Ala Glu Asn Lys Gly 50 55 60

Leu He Thr Pro Gly Glu Ser Val Leu He Glu Pro Thr Ser Gly Asn 65 70 75 80

Thr Gly He Gly Leu Ala Phe He Ala Ala Ala Lys Gly Tyr Arg Leu 85 90 95

He He Cys Met Pro Ala Ser Met Ser Leu Glu Arg Arg Thr He Leu 100 105 110

Arg Ala Phe Gly Ala Glu Leu Val Leu Thr Asp Pro Ala Arg Gly Met 115 120 125

Lys Gly Ala Val Gin Lys Ala Glu Glu He Lys Ala Lys Thr Pro Asn 130 135 140

Ser Tyr He Leu Gin Gin Phe Glu Asn Pro Ala Asn Pro Lys He His 145 150 155 160

Tyr Glu Thr Thr Gly Pro Glu He Trp Arg Gly Ser Gly Gly Lys He 165 170 175

Asp Ala Leu Val Ser Gly He Gly Thr Gly Gly Thr Val Thr Gly Ala 180 185 190

Gly Lys Tyr Leu Lys Glu Gin Asn Pro Asn He Lys Leu Tyr Gly Val 195 200 205

Glu Pro Val Glu Ser Ala He Leu Ser Gly Gly Lys Pro Gly Pro His 210 215 220

Lys He Gin Gly He Gly Ala Gly Phe He Pro Gly Val Leu Asp Val 225 230 235 240

Asn Leu Leu Asp Glu Val He Gin Val Ser Ser Glu Glu Ser He Glu 245 250 255

Thr Ala Lys Leu Leu Ala Leu Lys Glu Gly Leu Leu Val Gly He Ser 260 265 270

Ser Gly Ala Ala Ala Ala Ala Ala He Arg He Ala Lys Arg Pro Glu 275 280 285

Asn Ala Gly Lys Leu He Val Ala Val Phe Pro Ser Phe Gly Glu Arg 290 295 300

Tyr Leu Ser Thr Val Leu Phe Glu Ser Val Lys Arg Glu Thr Glu Asn 305 310 315 320

Met Val Phe Glu Pro 325 (2) INFORMATION FOR SEQ ID NO: 33:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 547 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: cenl .pk0061. d4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

GCCTTATGGC TAAGCTTGAG AAGGCGGATC AGGCATTCTG CTTCACCAGT GGGATGGCAG 60

CACTAGCTGC AGTAACACAC CTCCTTAAGT CTGGACAAGA AATAGTTGCT GGAGAGGACA 120

TATATGGTGG CTCAGACCGT CTGCTCTCAC AAGTTGCCCC GAGACATGGG ATTGTAGTAA 180

AACGAATTGA TACAACCAAA ATTAGTGAGG TAACTTCTGC AATTGGGGCC TTGGACTAAA 240

CTAAGTATGG CTTTGAAAAN CCCACCATCC CCGTCCTACA AATTACTGGA TATAAAGAAA 300

ATAGCNAGAG ATAGTCATTA CAATGGGGCT CCTTGTTTTA AGTAGACAAC AGCACATGTC 360

TCCCTGTGCT CTCCCNGTCC TCNTAAAACT TTGGGCCAAA TATNGGTTTG CACCCCAAGC 420

AACCAATTTA TNCTGGGCAT AGCGTNCTTA TGGCNNGGAT CCTTGCCGGG AAGGGGTGAA 480

AGCACTTGGC TAAAGAGATG CATTCCTCNA AAANCTGAAG GNTAAGTTTG GACATTNGAT 540

GCCGGTT 547

(2) INFORMATION FOR SEQ ID NO: 34:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 223 ammo aciαs

(B) TYPE: ammo acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: cenl .pk0061. d4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

He Ala His Ser His Gly Ala Leu Val Leu Val Asp Asn Ser He Met 1 5 10 15

Ser Pro Val Leu Ser Arg Pro He Glu Leu Gly Ala Asp He Val Met 20 25 30

His Ser Ala Thr Lys Phe He Ala Gly His Ser Asp Leu Met Ala Gly 35 40 45

He Leu Ala Val Lys Gly Glu Ser Leu Ala Lys Glu Val Gly Phe Leu 50 55 60

Gin Asn Ala Glu Gly Ser Gly Leu Ala Pro Phe Asp Cys Trp Leu Cys 65 70 75 80 Leu Arg Gly He Lys Thr Met Ala Leu Arg Val Glu Lys Gin Gin Ala 85 90 95

Asn Ala Gin Lys He Ala Glu Phe Leu Ala Ser His Pro Arg Val Lys 100 105 110

Gin Val Asn Tyr Ala Gly Leu Pro Asp His Pro Gly Arg Ala Leu His 115 120 125

Tyr Ser Gin Ala Lys Gly Ala Gly Ser Val Leu Ser Phe Leu Thr Gly 130 135 140

Ser Leu Ala Leu Ser Lys His Val Val Glu Thr Thr Lys Tyr Phe Ser 145 150 155 160

Val Thr Val Ser Phe Gly Ser Val Lys Ser Leu He Ser Leu Pro Cys 165 170 175

Phe Met Ser His Ala Ser He Pro Ala Ser Val Arg Glu Glu Arg Gly 180 185 190

Leu Thr ASD Asp Leu Val Arg He Ser Val Gly He Glu Asp Val Glu 195 200 205

Asp Leu He Ala Asp Leu Asp Arg Ala Leu Arg Thr Gly Pro Val 210 ^* 215 220

(2) INFORMATION FOR SEQ ID NO: 35:

(i; SEQUENCE CHARACTERISTICS:

(A) LENGTH: 547 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii; IMMEDIATE SOURCE:

(B) CLONE: rlrl2. pk0026. gl

(xi SEQUENCE DESCRIPTION: SEQ ID NO: 35:

GCCTTATGGC 7AAGCTTGAG AAGGCGGATC AGGCATTCTG CTTCACCAGT GGGATGGCAG 60

CACTAGCTGC AGTAACACAC CTCCTTAAGT CTGGACAAGA AATAGTTGCT GGAGAGGACA 120

TATATGGTGG CTCAGACCGT CTGCTCTCAC AAGTTGCCCC GAGACATGGG ATTGTAGTAA 180

AACGAATTGA TACAACCAAA ATTAGTGAGG TAACTTCTGC AATTGGGGCC TTGGACTAAA 240

CTAAGTATGG CTTTGAAAAN CCCACCATCC CCGTCCTACA AATTACTGGA TATAAAGAAA 300

ATAGCNAGAG ATAGTCATTA CAATGGGGCT CCTTGTTTTA AGTAGACAAC AGCACATGTC 360

TCCCTGTGCT CTCCCNGTCC TCNTAAAACT TTGGGCCAAA TATNGGTTTG CACCCCAAGC 420

AACCAATTTA TNCTGGGCAT AGCGTNCTTA TGGCNNGGAT CCTTGCCGGG AAGGGGTGAA 480

AGCACTTGGC TAAAGAGATG CATTCCTCNA AAANCTGAAG GNTAAGTTTG GACATTNGAT 540

GCCGGTT 547 (2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 75 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: rlrl2.pk0026. gl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Leu Met Ala Lys Leu Glu Lys Ala Asp Gin Ala Phe Cys Phe Thr Ser 1 5 10 15

Gly Met Ala Ala Leu Ala Ala Val Thr His Leu Leu Lys Ser Gly Gin 20 25 30

Glu He Val Ala Gly Glu Asp He Tyr Gly Gly Ser Asp Arg Leu Leu 35 40 45

Ser Gin Val Ala Pro Arg His Gly He Val Val Lys Arg He Asp Thr 50 55 60

Thr Lys He Ser Glu Val Thr Ser Ala He Gly 65 70 75

(2) INFORMATION FOR SEQ ID NO: 37:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1733 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: sf11. pk0012. c4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

CAAAGACGGC ATTGAAGTTG AACAATCCAT CACTAACACA AGCGCAGACA ACAACATAAC 60

CCTGCTCCAA ACACATCAAT TTCAATAATG TTTTCTTCTG CAATTTCTCA GAAGCCCTTC 120

CTTCAGTCCC TCGTCATTGA TCGTTACGCT CAGAGCACAA CTGCTGCAAC CAGGTGGGAG 180

TGCTTGGGGT TTAACAAGTC AGAAAATTTC AGTACCAAGA GAGTGTTGCG TGCAGAGGGG 240

TTCAAGTTGA ATTGCTTGGT TGAAAATAGA GAGATGGAAG TGGAGTCATC ATCATCATCT 300

TTGGTGGATG ATGCTGCCAT GAGCTTAAGT GAAGAGGATT TAGGGGAGCC TAGTATTTCA 360

ACAATGGTGA TGAATTTCGA GAGTAAGTTT GATCCTTTTG GAGCAATTAG TACCCCGCTT 420

TACCAAACGG CTACTTTTAA GCAGCCTTCT GCAATAGAAA ATGGTCCCTA TGACTATACC 480

AGAAGTGGAA ATCCTACTCG TGATGCTTTA GAAAGTTTAC TAGCAAAGCT TGATAAAGCA 540

GATAGAGCCC TGTGCTTCAC CAGTGGAATG GCTGCTTTGA GTGCTGTTGT TCGTCTTGTT 600

GGAACTGGTG AGGAAATTGT CACCGGAGAT GATGTATATG GTGGCTCAGA TAGGTTGCTG 660 TCTCAAGTAG TTCCAAGGAC TGGAATTGTG GTGAAACGGG TAAATACATG TGATCTAGAT 720

GAGGTTGCTG CTGCCATTGG ACTCAGGACT AAGCTTGTGT GGCTTGAGAG TCCAACCAAT 780

CCTCGGCTTC AAATTTCTGA TATTCGAAAA ATATCAGAGA TGGCTCATTC ACATGGTGCT 840 CTTGTGTTAG TGGACAATAG TATAATGTCA CCTGTGTTGT CTCAGCCATT GGAACTTGGA 900 GCAGATATTG TCATGCACTC AGCTACAAAA TTTATTGCTG GACATAGTGA CATTATGGCT 960

GGTGTGCTTG CTGTGAAGGG TGAAAAGTTG GGAAAGGAAA TGTATTTCTT GCAAAATGCA 1020

GAGGGTTCAG GCTTAGCACC ATTTGACTGT TGGCTTTGTT TGCGAGGAAT CAAGACAATG 1080

GCCCTGCGAA TTGAAAAGCA ACAGGATAAC GCACAGAAGA TTGCAGAGTT CCTTGCCTCC 1140

CATCCTCGAG T3AAGGAAGT GAATTATGCT GGCTTGCCTG GTCATCCTGG TCGTGATTTA 1200

CACTATTCTC A3GCAAAGGG TGCAGGATCT GTGCTTAGCT TCTTGACTGG TTCATTGGCA 1260

CTTTCAAAGC ATATTGTTGA AACTACCAAA TACTTCAGTA TAACCGTCAG CTTTGGGAGT 1320

GTGAAGTCCC 7CATTAGCAT GCCATGCTTT ATGTCACATG CAAGCATACC TGCTGCAGTT 1380

CGCGAGGCCA GAGGTTTAAC TGAAGATCTT GTACGAATAT CTGTGGGAAT TGAGGATGTG 1440

AATGATCTCA TTGCTGATCT TGGCAATGCA CTTAGAACTG GACCTCTTTA ATGTCTTCTC 1500

CACCCCCCCA C3CAAAAAGA AAAAAATTCA TCCTTAAGAA GTTGGATTAG CATGTTGAGG 1560

ATTTGGGAGC A7TGCTATCC TGTCTTTGGA TTCTTGAGAG TGGAAACTTG AAGTGTTGCT 1620

TATGTGCATG 7AATAAAATC AATATTTCCT GTAATTTTGT TGTAACAATT GTTATCCTTA 1680

CCTTGCAATA 7CATGTCATA CAAGTTACTA TTGAAAAAAA AAAAAAAAAA AAA 1733 (2) INFORMATION FOR SEQ ID NO: 38:

(i; SEQUENCE CHARACTERISTICS:

(A) LENGTH: 467 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(ii, MOLECULE TYPE: peptide

(vii, IMMEDIATE SOURCE:

(B) CLONE: sf11.pk0012. c4

(xi; SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Met Phe Ser Ser Ala He Ser Gin Lys Pro Phe Leu Gin Ser Leu Val 1 5 10 15

He Asp Arg Tyr Ala Gin Ser Thr Thr Ala Ala Thr Arg Trp Glu Cys 20 25 30

Leu Gly Phe Asn Lys Ser Glu Asn Phe Ser Thr Lys Arg Val Leu Arg 35 40 45

Ala Glu Gly Phe Lys Leu Asn Cys Leu Val Glu Asn Arg Glu Met Glu 50 55 60

Val Glu Ser Ser Ser Ser Ser Leu Val Asp Asp Ala Ala Met Ser Leu 65 70 75 80 Ser Glu Glu Asp Leu Gly Glu Pro Ser He Ser Thr Met Val Met Asn 85 90 95

Phe Glu Ser Lys Phe Asp Pro Phe Gly Ala He Ser Thr Pro Leu Tyr 100 105 110

Gin Thr Ala Thr Phe Lys Gin Pro Ser Ala He Glu Asn Gly Pro Tvr 115 120 125

Asp Tyr Thr Arg Ser Gly Asn Pro Thr Arg Asp Ala Leu Glu Ser Leu 130 135 140

Leu Ala Lys Leu Asp Lys Ala Asp Arg Ala Leu Cys Phe Thr Ser Gly 145 150 155 160

Met Ala Ala Leu Ser Ala Val Val Arg Leu Val Gly Thr Gly Glu Glu 165 170 175

He Val Thr Gly Asp Asp Val Tyr Gly Gly Ser Asp Arg Leu Leu Ser 180 185 190

Gin Val Val Pro Arg Thr Gly He Val Val Lys Arg Val Asn Thr Cys 195 200 205

Asp Leu ASD Glu Val Ala Ala Ala He Gly Leu Arg Thr Lys Leu Val 210 ^" 215 220

Trp Leu Glu Ser Pro Thr Asn Pro Arg Leu Gin He Ser Asp He Arg 225 230 235 240

Lys He Ser Glu Met Ala His Ser His Gly Ala Leu Val Leu Val Asp 245 250 255

Asn Ser He Met Ser Pro Val Leu Ser Gin Pro Leu Glu Leu Gly Ala 260 265 270

Asp He Val Met His Ser Ala Thr Lys Phe He Ala Gly His Ser Asp 275 280 285

He Met Ala Gly Val Leu Ala Val Lys Gly Glu Lys Leu Gly Lys Glu 290 295 300

Met Tyr Phe Leu Gin Asn Ala Glu Gly Ser Gly Leu Ala Pro Phe Asp 305 310 315 320

Cys Tro Leu Cys Leu Arg Gly He Lys Thr Met Ala Leu Arg He Glu 325 330 335

Lys Gin Gin Asp Asn Ala Gin Lys He Ala Glu Phe Leu Ala Ser His 340 345 350

Pro Arg Val Lys Glu Val Asn Tyr Ala Gly Leu Pro Gly His Pro Gly 355 360 365

Arg Asp Leu His Tyr Ser Gin Ala Lys Gly Ala Gly Ser Val Leu Ser 370 375 380

Phe Leu Thr Gly Ser Leu Ala Leu Ser Lys His He Val Glu Thr Thr 385 390 395 400

Lys Tyr Phe Ser He Thr Val Ser Phe Gly Ser Val Lys Ser Leu He 405 410 415

Ser Met Pro Cys Phe Met Ser His Ala Ser He Pro Ala Ala Val Arg 420 425 430 Glu Ala Arg Gly Leu Thr Glu Asp Leu Val Arg He Ser Val Gly He 435 440 445

Glu Asp Val Asn Asp Leu He Ala Asp Leu Gly Asn Ala Leu Arg Thr 450 455 460

Gly Pro Leu 465

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 637 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:

(B) CLONE: wrl . pk0091. g6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

AGCGTGGCCA CGATACTGAC CAGCTTCGAG AACTCGTTCG ACAAGTATGG GGCTCTCAGC 60

ACGCCGCTGT ACCAGACGGC CACCTTCAAG CAGCCTTCAG CAACCGTTAA TGGAGCTTAT 120

GATTATACTA GAAGTGGCAA CCCTACTCGT GATGTTCTCC AGAGCCTTAT GGCTAAGCTC 180

GAGAAGGCAG ACCAAGCATT CTGCTTCACT AGTGGGATGG CATCACTGGG CTGCAGTAAC 240

ACACCTCCTT CAGGCTGGAC AAGAAATAGT TGCTGGAGAG GACATATATG GTGGTCTGAT 300

CGTCTGCTCT CACAAGTTGT CCCAAGAAAT GGAATTGTAG TAAAACGGGT CGATACAACT 360

AAAATTAACG ACGTGACTGC TGCATCGGAC CCTTGACTAN ACTAGTTTGG TTGAAANCCA 420

CAATCCTCGT CAACAATTAC TGTATAAGAA ATCTCAGGGA TACTCATCCA TGGGGACTGG 480

TTTGGNGGCA ANNTTCATGT CCCANGGCTA CCTGGCCNAT AAANTGGGGN ANTATGGGAG 540

CATCAGTACA AATTATNCTG GCNATGTCTA GGTGGATCTC NTAAGGGGAA NTTGGNAGGA 600

TTCTTCAAAA CCTAGTNGGT TGACTTATGT GGTTGTT 637

(2) INFORMATION FOR SEQ ID NO: 40:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 131 ammo acids

(C) STRANDEDNESS not relevant

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: wrl .pk0091. g6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

Ser Val Ala Thr He Leu Thr Ser Phe Glu Asn Ser Phe Asp Lys Tyr 1 5 10 15 Gly Ala Leu Ser Thr Pro Leu Tyr Gin Thr Ala Thr Phe Lys Gin Pro 20 25 30

Ser Ala Thr Val Asn Gly Ala Tyr Asp Tyr Thr Arg Ser Gly Asn Pro 35 40 45

Thr Arg Asp Val Leu Gin Ser Leu Met Ala Lys Leu Glu Lys Ala Asp 50 55 60

Gin Ala Phe Cys Phe Thr Ser Gly Met Ala Ser Leu Xaa Ala Val Thr 65 70 75 80

His Leu Leu Gin Ala Gly Gin Glu He Val Ala Gly Glu Asp He Tyr 85 90 95

Gly Gly Xaa Asp Arg Leu Leu Ser Gin Val Val Pro Arg Asn Gly He 100 105 110

Val Val Lys Arg Val Asp Thr Thr Lys He Asn Asp Val Thr Ala Ala 115 120 125

Ser Asp Pro 130

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 464 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Arabidopsis thaliana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Thr Ser Ser Leu Ser Leu His Ser Ser Phe Val Pro Ser Phe Ala 1 5 10 15

Asp Leu Ser Asp Arg Gly Leu He Ser Lys Asn Ser Pro Thr Ser Val 20 25 30

Ser He Ser Lys Val Pro Thr Trp Glu Lys Lys Gin He Ser Asn Arg 35 40 45

Asn Ser Phe Lys Leu Asn Cys Val Met Glu Lys Ser Val Asp Gly Gin 50 55 60

Thr His Ser Thr Val Asn Asn Thr Thr Asp Ser Leu Asn Thr Met Asn 65 70 75 80

He Lys Glu Glu Ala Ser Val Ser Thr Leu Leu Val Asn Leu Asp Asn 85 90 95

Lys Phe Asp Pro Phe Asp Ala Met Ser Thr Pro Leu Tyr Gin Thr Ala 100 105 110

Thr Phe Lys Gin Pro Ser Ala He Glu Asn Gly Pro Tyr Asp Tyr Thr 115 120 125

Arg Ser Gly Asn Pro Thr Arg Asp Ala Leu Glu Ser Leu Leu Ala Lys 130 135 140 Leu Asp Lys Ala Asp Arg Ala Phe Cys Phe Thr Ser Gly Met Ala Ala 145 150 155 160

Leu Ser Ala Val Thr His Leu He Lys Asn Gly Glu Glu He Val Ala 165 170 175

Gly ASD Asp Val Tyr Gly Gly Ser Asp Arg Leu Leu Ser Gin Val Val 180 185 190

Pro Arg Ser Gly Val Val Val Lys Arg Val Asn Thr Thr Lys Leu Asp 195 200 205

Glu Val Ala Ala Ala He Gly Pro Gin Thr Lys Leu Val Trp Leu Glu 210 215 220

Ser Pro Thr Asn Pro Arg Gin Gin He Ser Asp He Arg Lys He Ser 225 230 235 240

Glu Met Ala His Ala Gin Gly Ala Leu Val Leu Val Asp Asn Ser He 245 250 255

Met Ser Pro Val Leu Ser Arg Pro Leu Glu Leu Gly Ala Asp He Val 260 265 270

Met His Ser Ala Thr Lys Phe He Ala Gly His Ser Asp Val Met Ala 275 280 285

Gly Val Leu Ala Val Lys Gly Glu Lys Leu Ala Lys Glu Val Tyr Phe 290 295 300

Leu Gin Asn Ser Glu Gly Ser Gly Leu Ala Pro Phe Asp Cys Trp Leu 305 310 315 320

Cys Leu Arg Gly He Lys Thr Met Ala Leu Arg He Glu Lys Gin Gin 325 330 335

Glu Asn Ala Arg Lys He Ala Met Tyr Leu Ser Ser His Pro Arg Val 340 345 350

Lys Lys Val Tyr Tyr Ala Gly Leu Pro Asp His Pro Gly His His Leu 355 360 365

His Phe Ser Gin Ala Lys Gly Ala Gly Ser Val Phe Ser Phe He Thr 370 375 380

Gly Ser Val Ala Leu Ser Lys His Leu Val Glu Thr Thr Lys Tyr Phe 385 390 395 400

Ser He Ala Val Ser Phe Gly Ser Val Lys Ser Leu He Ser Met Pro 405 410 415

Cys Phe Met Ser His Ala Ser He Pro Ala Glu Val Arg Glu Ala Arg 420 425 430

Gly Leu Thr Glu Asp Leu Val Arg He Ser Ala Gly He Glu Asp Val 435 440 445

Asp ASD Leu He Ser Asp Leu Asp He Ala Phe Lys Thr Phe Pro Leu 450 455 460

Claims

CLAIMS What is claimed is:

1. An isolated nucleic acid fragment encoding a plant aspartic semialedhyde dehydrogenase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:2, 4, and 6;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:2, 4, and 6; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

2. The isolated nucleic acid fragment of Claim 1 wherein the nucleotide sequence of the fragment comprises a member selected from the group consisting of SEQ ID NOs:l, 3, and 5.

3. A chimeric gene comprising the nucleic acid fragment of Claim 1 operably linked to suitable regulatory sequences.

4. A transformed host cell comprising the chimeric gene of Claim 3.

5. A plant aspartic semialedhyde dehydrogenase polypeptide comprising an amino acid sequence set forth in a member selected from the group consisting of SEQ ID

NOs:2, 4, and 6.

6. An isolated nucleic acid fragment encoding a plant diaminopimelate decarboxylase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:9, 11, 13, 15, 17, and 19;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:9, 11, 13, 15, 17, and 19; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

7. The isolated nucleic acid fragment of Claim 6 wherein the nucleotide sequence of the fragment comprises a member selected from the group consisting of SEQ ID NOs:8, 10, 12, 14, 16, and 18.

8. A chimeric gene comprising the nucleic acid fragment of Claim 6 operably linked to suitable regulatory sequences.

9. A transformed host cell comprising the chimeric gene of Claim 8.

10. A plant diaminopimelate decarboxylase polypeptide comprising an amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 9, 11, 13, 15, 17, and 19.

11. An isolated nucleic acid fragment encoding a plant homoserine kinase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:22, 24, 26, and 28;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:22, 24, 26,and 28; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

12. The isolated nucleic acid fragment of Claim 1 1 wherein the nucleotide sequence of the fragment comprises a member selected from the group consisting of SEQ ID NOs:21, 23, 25, and 27.

13. A chimeric gene comprising the nucleic acid fragment of Claim 11 operably linked to suitable regulatory sequences.

14. A transformed host cell comprising the chimeric gene of Claim 13.

15. A plant homoserine kinase polypeptide comprising an amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs:22, 24, 26, and 28.

16. An isolated nucleic acid fragment encoding a plant cysteine synthase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:31 ;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:31; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

17. The isolated nucleic acid fragment of Claim 16 wherein the nucleotide sequence of the fragment comprises SEQ ID NO:30.

18. A chimeric gene comprising the nucleic acid fragment of Claim 16 operably linked to suitable regulatory sequences.

19. A transformed host cell comprising the chimeric gene of Claim 18.

20. A cysteine synthase polypeptide comprising an amino acid sequence set forth in SEQ ID NO:31.

21. An isolated nucleic acid fragment encoding a corn cystathionine β-lyase comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:34;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:34; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

22. The isolated nucleic acid fragment of Claim 21 wherein the nucleotide sequence of the fragment comprises SEQ ID NO:33.

23. A chimeric gene comprising the nucleic acid fragment of Claim 21 operably linked to suitable regulatory sequences.

24. A transformed host cell comprising the chimeric gene of Claim 23.

25. A cystathionine β-lyase polypeptide comprising an amino acid sequence set forth in SEQ ID NO:34.

26. An isolated nucleic acid fragment encoding a rice cystathionine β-lyase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:36;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:36; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

27. The isolated nucleic acid fragment of Claim 26 wherein the nucleotide sequence of the fragment comprises SEQ ID NO:35.

28. A chimeric gene comprising the nucleic acid fragment of Claim 26 operably linked to suitable regulatory sequences.

29. A transformed host cell comprising the chimeric gene of Claim 28.

30. A cystathionine β-lyase polypeptide comprising an amino acid sequence set forth in SEQ ID NO:36.

31. An isolated nucleic acid fragment encoding a soybean cystathionine β-lyase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:38;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:38; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

32. The isolated nucleic acid fragment of Claim 31 wherein the nucleotide sequence of the fragment comprises SEQ ID NO:37.

33. A chimeric gene comprising the nucleic acid fragment of Claim 31 operably linked to suitable regulatory sequences.

34. A transformed host cell comprising the chimeric gene of Claim 33.

35. A cystathionine β-lyase polypeptide comprising an amino acid sequence set forth in SEQ ID NO:38.

36. An isolated nucleic acid fragment encoding a wheat cystathionine β-lyase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:40; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:40; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

37. The isolated nucleic acid fragment of Claim 36 wherein the nucleotide sequence of the fragment comprises SEQ ID NO:39.

38. A chimeric gene comprising the nucleic acid fragment of Claim 36 operably linked to suitable regulatory sequences.

39. A transformed host cell comprising the chimeric gene of Claim 38.

40. A cystathionine β-lyase polypeptide comprising an amino acid sequence set forth in SEQ ID NO:40.

41. A method of altering the level of expression of a plant amino acid biosynthetic enzyme in a host cell comprising:

(a) transforming a host cell with the chimeric gene of any of Claims 3, 8, 13, 18, 23, 28, 33, and 38; and (b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of a plant amino acid biosynthetic enzyme in the transformed host cell.

42. A method of obtaining a nucleic acid fragment encoding all or substantially all of the amino acid sequence encoding a plant amino acid biosynthetic enzyme comprising:

(a) probing a cDNA or genomic library with the nucleic acid fragment of any of Claims 1, 6, 11, 16, 21, 26, 31, and 36;

(b) identifying a DNA clone that hybridizes with the nucleic acid fragment of any of Claims 1, 6, 11, 16, 21, 26, 31, and 36; (c) isolating the DNA clone identified in step (b); and

(d) sequencing the cDNA or genomic fragment that comprises the clone isolated in step (c) wherein the sequenced nucleic acid fragment encodes all or substantially all of the amino acid sequence encoding a plant amino acid biosynthetic enzyme.

43. A method of obtaining a nucleic acid fragment encoding a portion of an amino acid sequence encoding a plant amino acid biosynthetic enzyme comprising:

(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in any of SEQ ID NOs:l, 3, 5, 8, 10, 12, 14, 16, 18, 21, 23, 25, 27, 30, 33, 35, 37, and 39; and

(b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector wherein the amplified nucleic acid fragment encodes a portion of an amino acid sequence encoding a plant amino acid biosynthetic enzyme.

44. The product of the method of Claim 42.

45. The product of the method of Claim 43.

46. A method for evaluating at least one compound for its ability to inhibit the activity of a plant biosynthetic enzyme selected from the group consisting of aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase and cysteine synthase, the method comprising the steps of:

(a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a plant biosynthetic enzyme selected from the group consisting of aspartic semialedhyde dehydrogenase, diaminopimelate decarboxylase, homoserine kinase, cysteine synthase and cystathionine β-lyase, operably linked to suitable regulatory sequences;

(b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the biosynthetic enzyme encoded by the operably linked nucleic acid fragment in the transformed host cell;

(c) optionally purifying the biosynthetic enzyme expressed by the transformed host cell;

(d) treating the biosynthetic enzyme with a compound to be tested; and

(e) comparing the activity of the biosynthetic enzyme that has been treated with a test compound to the activity of an untreated biosynthetic enzyme, thereby selecting compounds with potential for inhibitory activity.