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Plant sugar transport proteins

6383776

Abstract

This invention relates to an isolated nucleic acid fragment encoding a sugar transport protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sugar transport protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sugar transport protein in a transformed host cell.


Claims

What is claimed is:

1. An isolated polynucleotide comprising:

(a) a first nucleotide sequence encoding a first polypeptide, wherein the first polypeptide comprises at least 117 amino acid residues, wherein the first polypeptide is a sugar transport protein, and wherein the amino acid sequence of the first polypeptide has 90% identity when compared to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:12,

(b) a second nucleotide sequence encoding a second polypeptide, wherein the second polypeptide comprises at least 228 amino acid residues, wherein the second polypeptide is a sugar transport protein, and wherein the amino acid sequence of the second polypeptide has 90% identity when compared to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:16,

(c) a third nucleotide sequence encoding a third polypeptide, wherein the third polypeptide comprises at least 737 amino acid residues, wherein the third polypeptide is a sugar transport protein, and wherein the amino acid sequence of the third polypeptide has 90% identity when compared to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8, or

(d) a fourth nucleotide sequence comprising the complement of the first, second, or third nucleotide sequence.

2. The polynucleotide of claim 1, wherein the amino acid sequence of the first polypeptide has 95% identity when compared to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:12, wherein the amino acid sequence of the second polypeptide has 95% identity when compared to the amino acid sequence of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:16, and wherein the amino acid sequence of the third polypeptide has 95% identity when compared to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8.

3. The polynucleotide of claim 1 comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15.

4.The polynucleotide of claim 1 wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

5. A chimeric gene comprising the polynucleotide of claim 1 operably linked to a regulatory sequence.

6. A vector comprising the polynucleotide of claim 1.

7. A polynucleotide comprising a nucleotide sequence containing at least 30 nucleotides, wherein the nucleotide sequence is comprised by the polynucleotide of claim 1.

8. A method for transforming a cell comprising transforming a cell with the polynucleotide of claim 1.

9. A cell comprising the chimeric gene of claim 5.

10. A method for producing a plant comprising transforming a plant cell with the chimeric gene of claim 5 and regenerating a plant from the transformed plant cell.

11. A plant comprising the chimeric gene of claim 5.

12. A seed comprising the chimeric gene of claim 5.


Description

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding sugar transport proteins in plants and seeds.

BACKGROUND OF THE INVENTION

Sugar is one form of carbohydrate produced in photosynthesizing cells in most higher plants and is the main form of transported carbon in most annual field crops such as corn, rice, soybeans and wheat. As such its movement and concentration across various plant membranes is critical to plant growth and development. In addition sugar is the main form of carbon that moves into developing seeds of soybeans, rice, corn and wheat. This movement and concentration is accomplished by the action of carrier proteins that act to transport sugar against a concentration gradient often by coupling sugar movement to the opposite vectoral movement of a proton. Specific sugar carrier proteins from these crop plants could be manipulated in efforts to control carbon flux and the timing and extent of sugar transport phenomena (e.g., grain fill duration) that are important factors in crop yield and quality. Accordingly, the availability of nucleic acid sequences encoding all or a portion of sugar transport proteins would facilitate studies to better understand carbon flux and sugar transport in plants, provide genetic tools for the manipulation of sugar transport, and provide a means to control carbohydrate transport and distribution in plant cells.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragments encoding sugar transport proteins. Specifically, this invention concerns an isolated nucleic acid fragment encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein.

An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a sugar transport protein selected from the group consisting of Arabidopsis thaliana-like sugar transport protein and Beta vulgaris-like sugar transport protein.

In another embodiment, the instant invention relates to a chimeric gene encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.

In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method of altering the level of expression of an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein; 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 Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein in the transformed host cell.

An additional embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein.

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 Listing which form a part of this application.

FIGS. 1A-1E show a comparison of the amino acid sequences set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 and 16 with the Arabidopsis thaliana-like sugar transport protein amino acid sequence set forth in SEQ ID NO:29. Amino acid designations in small case letters represent regions that are thought to be Arabidopsis thaliana-like sugar transport protein signatures.

FIG. 2 shows a comparison of the amino acid sequences set forth in SEQ ID NOS:18, 35 20, 22, 24, 26 and 28 with the Beta vulgaris-like sugar transport protein amino acid sequence set forth in SEQ ID NO:30.

The following sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. .sctn.1.821-1.825.

SEQ ID NO:1 is the nucleotide sequence comprising a contig assembled from the DNA inserts in clones p0032.crcba66r, p0097.cqran41r, cr1n.pk0143.h10, p0128.cpict38, p0106.cjlpm67r, cil1c.pk001.f21, p0072.comgi92r, p0114.cimm181r and p0002.cgevb73r encoding a corn Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:2 is the deduced amino acid sequence of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO: 1.

SEQ ID NO:3 is the nucleotide sequence comprising a contig assembled from the cDNA inserts in clones rlr12.pk0013.d11 and rds1c.pk007.n17 encoding a portion of a rice Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:4 is the deduced amino acid sequence of a portion of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence comprising a the entire cDNA insert in clone rls6.pk0003.d5 encoding a portion of a rice Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:6 is the deduced amino acid sequence of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence comprising a contig assembled from the cDNA inserts in clones sgs4c.pk005.c9, sfl1.pk0079.a4 and sdp3c.pk012.i1 encoding a soybean Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:8 is the deduced amino acid sequence of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence comprising a portion of the cDNA insert in clone ss1.pk0022.f1 encoding a portion of a soybean Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:10 is the deduced amino acid sequence of a portion of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide sequence comprising a portion of the cDNA insert in clone wlk8.pk0001.a12 encoding a portion of a wheat Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:12 is the deduced amino acid sequence of a portion of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:11.

SEQ ID NO:13 is the nucleotide sequence comprising a contig assembled from the cDNA inserts in clones wlm96.pk043.e19 and wre1n.pk0062.g6 encoding a portion of a wheat Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:14 is the deduced amino acid sequence of a portion of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:13.

SEQ ID NO:15 is the nucleotide sequence comprising a portion of the cDNA insert in clone wre1n.pk0006.b4 encoding a portion of a wheat Arabidopsis thaliana-like sugar transport protein.

SEQ ID NO:16 is the deduced amino acid sequence of a portion of an Arabidopsis thaliana-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:15.

SEQ ID NO:17 is the nucleotide sequence comprising a portion of the cDNA insert in clone cc1.mn0002.h1 encoding a portion of a corn Beta vulgaris-like sugar transport protein.

SEQ ID NO:18 is the deduced amino acid sequence of a portion of a Beta vulgaris-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:17.

SEQ ID NO:19 is the nucleotide sequence comprising the entire cDNA insert in clone cepe7.pk0018.g3 encoding a corn Beta vulgaris-like sugar transport protein.

SEQ ID NO:20 is the deduced amino acid sequence of a Beta vulgaris-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:19.

SEQ ID NO:21 is the nucleotide sequence comprising a contig assembled from the cDNA inserts in clones rlr6.pk0005.b10, r10n.pk102.p24 and r10n.pk107.p2 encoding a rice Beta vulgaris-like sugar transport protein.

SEQ ID NO:22 is the deduced amino acid sequence of a Beta vulgaris-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:21.

SEQ ID NO:23 is the nucleotide sequence comprising a contig assembled from the cDNA inserts in clones sr1.pk0061.g8, sfl1.pk0058.h12, sgs2c.pk004.o17 and sre.pk0032.h6 encoding a soybean Beta vulgaris-like sugar transport protein.

SEQ ID NO:24 is the deduced amino acid sequence of a Beta vulgaris-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:23.

SEQ ID NO:25 is the nucleotide sequence comprising the entire cDNA insert in clone wlk8.pk0001.a 11 encoding a wheat Beta vulgaris-like sugar transport protein.

SEQ ID NO:26 is the deduced amino acid sequence of a Beta vulgaris-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence comprising the entire cDNA insert in clone wlm1.pk0012.h1 encoding a wheat Beta vulgaris-like sugar transport protein.

SEQ ID NO:28 is the deduced amino acid sequence of a Beta vulgaris-like sugar transport protein derived from the nucleotide sequence of SEQ ID NO:28.

SEQ ID NO:29 is the amino acid sequence of an Arabidopsis thaliana (NCBI Identification No. gi 3080420) sugar transport protein.

SEQ ID NO:30 is the amino acid sequence of a Beta vulgaris (NCBI Identification No. gi 1778093) sugar transport protein.

The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. .sctn.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, "contig" refers to an assemblage of overlapping nucleic acid sequences to form one contiguous nucleotide sequence. For example, several DNA sequences can be compared and aligned to identify common or overlapping regions. The individual sequences can then be assembled into a single contiguous nucleotide sequence.

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 than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a 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, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize, under stringent conditions (0.1.times.SSC, 0.1% SDS, 65.degree. C.), with the nucleic acid fragments disclosed herein.

Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent similarity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Preferred are those nucleic acid fragments whose nucleotide sequences encode amino acid sequences that are 90% similar to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are 95% similar to the amino acid sequences reported herein. Sequence alignments and percent similarity calculations were performed using the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins, D. G. and Sharp, P. M. (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10) (hereafter, Clustal algorithm). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

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 of ten 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 Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins as set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. 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 15:1-82. 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 3:225).

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 1:671-680.

"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, incorporated herein by reference). 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 sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but 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, incorporated herein by reference). "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. 100:1627-1632).

"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. 143:277) and particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).

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 sugar transport proteins 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 proteins that are described herein, and the designation of the cDNA clones that comprise the nucleic acid fragments encoding these proteins. 

                             TABLE 1
                     Sugar Transport Proteins
    Enzyme                             Clone           Plant
    Sugar Transport Protein (Arabidopsis-like) p0032.crcba66r  Corn
                                       p0097.cqran41r  Corn
                                       crln.pk0143.h10 Corn
                                       p0128.cpict38   Corn
                                       p0106.cjlpm67r  Corn
                                       ci1lc.pk001.f21 Corn
                                       p0072.comgi92r  Corn
                                       p0114.cimm181r  Corn
                                       p0002.cgevb73r  Corn
                                       rdslc.pk007.n17 Rice
                                       rlr12.pk0013.d11 Rice
                                       rls6.pk0003.d5  Rice
                                       sgs4c.pk005.c9  Soybean
                                       sfl1.pk0079.a4  Soybean
                                       sdp3c.pk012.i1  Soybean
                                       ss1.pk0022.f1   Soybean
                                       wlk8.pk0001.a12 Wheat
                                       wlm96.pk043.e19 Wheat
                                       wre1n.pk0062.g6 Wheat
                                       wre1n.pk0006.b4 Wheat
    Sugar Transport Protein            cc1.mn0002.h1   Corn
    (Beta vulgaris-like)               cepe7.pk0018.g3 Corn
                                       rlr6.pk0005.b10 Rice
                                       rl0n.pk102.p24  Rice
                                       rl0n.pk107.p2   Rice
                                       sr1.pk0061.g8   Soybean
                                       sfl1.pk0058.h12 Soybean
                                       sgs2c.pk004.o17 Soybean
                                       sre.pk0032.h6   Soybean
                                       wlk8.pk0001.a11 Wheat
                                       wlml.pk0012.h1  Wheat


The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins 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 Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins, 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 primer 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 or all 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 fall 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 MnRNA 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) PNAS USA 85:8998) 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) PNAS USA 86:5673; Loh et al., (1989) Science 243:217). 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 1:165).

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of 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. 36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins 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 sugar transport in those cells.

Overexpression of the Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of 35 development. For reasons of convenience, the chimeric gene 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 gene may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene 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) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), 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 sugar transport proteins to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by altering the coding sequence to encode Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins with appropriate intracellular targeting sequences such as transit sequences (Keegstra, K. (1989) Cell 56:247-253), 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 genes encoding Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant sugar transport proteins can be constructed by linking a gene or gene fragment encoding an Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene 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 Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant sugar transport proteins 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 a chimeric gene for production of the instant Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded sugar transport protein. An example of a vector for high level expression of the instant Arabidopsis thaliana-like sugar transport proteins or Beta vulgaris-like sugar transport proteins in a bacterial host is provided (Example 7).

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 al., (1987) Genomics 1:174-181) 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. 32:314-331).

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):37-41. 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 16:325-332), allele-specific ligation (Landegren, U. et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov, B. P. (1990) Nucleic Acid Res. 18:3671), 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. 17:6795-6807). 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 Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein. 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 Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the Arabidopsis thaliana-like sugar transport protein or Beta vulgaris-like sugar transport protein 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 corn, rice, soybean and wheat tissues were prepared. The characteristics of the libraries are described below. 

                                   TABLE 2
              cDNA Libraries from Corn, Rice, Soybean and Wheat
    Library Tissue                                          Clone
    cc1     Corn (Zea mays L.) callus stage 1**             cc1.mn0002.h1
    Cepe7   Corn (Zea mays L.) epicotyl from 7 day old etiolated
     cepe7.pk0018.g3
            seedling
    cil1c   Corn (Zea mays L.) pooled immature leaf tissue at V4,
     cil1c.pk001.f21
            V6 and V8**
    cr1n    Corn (Zea mays L.) root from 7 day seedlings grown in
     cr1n.pk0143.h10
            light*
    p0002   Corn (Zea mays L.) tassel: premeiotic > early uninucleate
     p0002.cgevb73r
    p0032   Corn (Zea mays L.) regenernerating callus, 10 and 14 days
     p0032.crcba66r
            after auxin removal.
    p0072   Corn (Zea mays L.) 14 days after planting etiolated p0072.comgi92r
            seedling: mesocotyl
    p0097   Corn (Zea mays L.) V9, 7 cm whorl section after p0097.cqran4lr
            application of European Corn Borer
    p0106   Corn (Zea mays L.) 5 days after pollenation whole kernels*
     p0106.cjlpm67r
    p0114   Corn (Zea mays L.) intercalary meristem of expanding p0114.cimm181r
            internodes 5-9 at V10 stage*
    p0128   Corn (Zea mays L.) pooled primary and secondary p0128.cpict38
            immature ear
    Rdslc   Rice (Oryza sativa, YM) developing seeds        rdslc.pk007.n17
    rlr6    Rice (Oryza sativa L.) leaf (15 days after germination)
     rlr6.pk0005.b10
            6 hrs after infection of Magaporthe grisea strain
            4360-R-62 (AVR2-YAMO); Resistant
    r10n    Rice (Oryza sativa L.) 15 day leaf*             r10n.pk102.p24
                                                            r10n.pk107.p2
    rlr12   Rice (Oryza sativa L.) leaf, 15 days after germination,
     rlr12.pk0013.d11
            12 hours after infection of Magaporthe grisea strain
            4360-R-62 (AVR2-YAMO); Resistant
    rls6    Rice (Oryza sativa L.) leaf, 15 days after germination,
     rls6.pk0003.d5
            6 hrs after infection of Magaporthe grisea strain
            4360-R-67 (avr2-yamo); Susceptible
    sdp3c   Soybean (Glycine max L.) developing pods 8-9 mm sdp3c.pk012.i1
    sfl1    Soybean (Glycine max L.) immature flower        sfl1.pk0079.a4
                                                            sfl1.pk0058.h12
    sgs2c   Soybean (Glycine max L.) seeds 14 hrs after germination
     sgs2c.pk004.o17
    sgs4c   Soybean (Glycine max L.) seeds 2 days after germination
     sgs4c.pk005.c9
    srl     Soybean (Glycine max L.) root library           srl.pk0061.g8
    sre     Soybean (Glycine max L.) root elongation        sre.pk0032.h6
    ssl     Soybean (Glycine max L.) seedling 5-10 day      ssl.pk0022.f1
    wlk8    Wheat (Triticum aestivum L.) seedlings 8 hr after wlk8.pk0001.a11
            treatment with fungicide***                     wlk8.pk0001.a12
    wlm1    Wheat (Triticum aestivum L.) seedlings 1 hr after wlm1.pk0012.h1
            inoculation with Erysiphe graminis f. sp tritici
    wlm96   Wheat (Triticum aestivum L.) seedlings 96 hr after wlm96.pk043.e19
            inoculation w/ E. graminis
    wre1n   Wheat (Triticum aestivum L.) root; 7 day old etiolated
     wre1n.pk0006.b4
            seedling*                                       wre1n.pk0062.g6
    *These libraries were normalized essentially as described in U.S. Pat. No.
     5,482,845
    **V4, V6 and V8 refer to stages of corn growth. The descriptions can be
     found in "How a Corn Plant Develops" Special Report No. 48, Iowa State
     University of Science and Technology Cooperative Extension Service Ames,
     Iowa, Reprinted February 1996.
    ***Application of 6-iodo-2-propoxy-3-propyl-4(3H-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.TM. XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). Conversion of the Uni-ZAP.TM. 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 of cDNA Clones

ESTs encoding sugar transport proteins 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 3:266-272 and Altschul, Stephen F., et al. (1997) Nucleic Acids Res. 25:3389-3402) 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.

Example 3

Characterization of cDNA Clones Encoding Arabidopsis thaliana-Like Sugar Transport Proteins

The BLASTX search using the EST sequences from several corn, rice, soybean and wheat clones revealed similarity of the proteins encoded by the cDNAs to a sugar transport protein from Arabidopsis thaliana (NCBI Identifier No. gi 3080420). In the process of comparing the ESTs it was found that many of the clones had overlapping regions of homology. Using this homology it was possible to align the ESTs and assemble several contigs encoding unique corn, rice, soybean and wheat sugar transport proteins. The individual clones and the composition of each assembled contig are shown in Table 3. The BLAST results for each of the contigs and individual ESTs and are also shown in Table 3: 

                             TABLE 3
    BLAST Results for Clones Encoding Polypeptides Homologous
         to Arabidopsis thaliana Sugar Transport Protein
          Clone                         BLAST pLog Score
          Contig composed of clones:         >250.00
          p0032.crcba66r
          p0097.cqran41r
          crln.pk0143.h10
          p0128.cpict38
          p0106.cjlpm67r
          cillc.pk001.f21
          p0072.comgi92r
          p0114.cimm181r
          p0002.cgcvb73r
          Contig composed of clones:              27.70
          rlr12.pk0013.d11
          rdslc.pk007.n17
          rls6.pk0003.d5                      54.00
          Contig composed of clones:         >250.00
          sgs4c.pk005.c9
          sfl1.pk0079.a4
          sdp3c.pk012.i1
          ssl.pk0022.fl                  >250.00
          wlk8.pk0001.a12                     21.30
          Contig composed of clones:             149.00
          wlm96.pk043.el9
          wreln.pk0062.g6
          wreln.pk0006.b4                    117.00


The sequence of the corn contig composed of clones p0032.crcba66r, p0097.cqran41r, cr1n.pk0143.h10, p0128.cpict38, p0106.cjlpm67r, cil1c.pk001.f21, p0072.comgi92p0114 .cimm181r and p0002.cgevb73r is shown in SEQ ID NO:1; the deduced amino acid sequence of this contig, which represents 100% of the protein, is shown in SEQ ID NO:2. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:2 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:2 is 66% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of the rice contig composed of clones rlr12.pk0013.d11 and rds1c.pk007.n17 is shown in SEQ ID NO:3; the deduced amino acid sequence of this contig, which represents 9% of the protein (N-terminal region), is shown in SEQ ID NO:4. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:4 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:2 is 86% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of the entire cDNA insert from clone rls6.pk0003.d5 is shown in SEQ ID NO:5; the deduced amino acid sequence of this cDNA, which represents 18% of the of the protein (C-terminal region), is shown in SEQ ID NO:6. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:6 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:6 is 74% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of the soybean contig composed of clones sgs4c.pk005.c9, sfl1.pk0079.a4 and sdp3c.pk012.i1 is shown in SEQ ID NO:7; the deduced amino acid sequence of this contig, which represents 100% of the protein, is shown in SEQ ID NO:8. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:8 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:8 is 68% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of a portion of the cDNA insert from clone ss1.pk0022.f1 is shown in SEQ ID NO:9; the deduced amino acid sequence of this cDNA, which represents 66% of the of the protein (C-terminal region), is shown in SEQ ID NO:10. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:10 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:10 is 66% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of a portion of the cDNA insert from clone wlk8.pk0001.a12 is shown in SEQ ID NO:11; the deduced amino acid sequence of this cDNA, which represents 7% of the of the protein (N-terminal region), is shown in SEQ ID NO:12. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:12 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:12 is 88% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of the wheat contig composed of clones wlm96.pk043.e19 and wre1n.pk0062.g6 is shown in SEQ ID NO:13; the deduced amino acid sequence of this contig, which represents 45% of the protein (C-terminal region), is shown in SEQ ID NO:14. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:14 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:14 is 65% similar to the Arabidopsis thaliana sugar transport protein.

The sequence of a portion of the cDNA insert from clone wre1n.pk0006.b4 is shown in SEQ ID NO:15; the deduced amino acid sequence of this cDNA, which represents 31% of the of the protein (C-terminal region), is shown in SEQ ID NO:16. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:16 and the Arabidopsis thaliana sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:16 is 76% similar to the Arabidopsis thaliana sugar transport protein.

FIG. 1 presents an alignment of the amino acid sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 and 16 with the Arabidopsis thaliana-like sugar transport protein amino acid sequence, SEQ ID NO:29. Alignments were performed using the Clustal algorithm. The percent similarity between the corn, rice, soybean and wheat acid sequences was calculated to range between 16% to 89% using the Clustal algorithm.

BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of sugar transport proteins. These sequences represent the first corn, rice, soybean and wheat sequences encoding Arabidopsis thaliana-like sugar transport proteins.

Example 4

Characterization of cDNA Clones Encoding Beta vulgaris-Like Sugar Transport Proteins

The BLASTX search using the EST sequences from several corn, rice, soybean and wheat clones revealed similarity of the proteins encoded by the cDNAs to a sugar transport protein from Beta vulgaris (NCBI Identifier No. gi 1778093). In the process of comparing the ESTs it was found that several of the rice and soybean clones had overlapping regions of homology. Using this homology it was possible to align the ESTs and assemble two contigs encoding unique rice and soybean B. vulgaris-like sugar transport proteins. The individual clones and the assembled composition of each contig are shown in Table 4. The BLAST results for each of the contigs and individual ESTs and are also shown in Table 4: 

                             TABLE 4
    BLAST Results for Clones Encoding Polypeptides Homologous
             to Beta vulgaris Sugar Transport Protein
          Clone                         BLAST pLog Score
          ccl.mn0002.h1                       53.70
          cepe7.pk0018.g3                    164.00
          Contig composed of clones:         >250.00
          rlr6.pk0005.b10
          rl0n.pk102.p24
          rl0n.pk107.p2
          Contig composed of clones:         >250.00
          srl.pk0061.g8
          sfl1.pk0058.h12
          sgs2c.pk004.o17
          sre.pk0032.h6
          wlk8.pk0001.a11                >250.00
          wlml.pk0012.h1                 >250.00


The sequence of a portion of the cDNA insert from clone cc1.mn0002.h1 is shown in SEQ ID NO:17; the deduced amino acid sequence of this cDNA, which represents 31% of the of the protein (N-terminal region), is shown in SEQ ID NO:18. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:18 and the Beta vulgaris sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:18 is 65% similar to the Beta vulgaris sugar transport protein.

The sequence of the entire cDNA insert from clone cepe7.pk0018.g3 is shown in SEQ ID NO:19; the deduced amino acid sequence of this cDNA, which represents 100% of the of the protein, is shown in SEQ ID NO:20. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:20 and the Beta vulgaris sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:20 is 57% similar to the Beta vulgaris sugar transport protein.

The sequence of the rice contig composed of clones rlr6.pk0005.b10, r10n.pk102.p24 and r10n.pk107.p2 is shown in SEQ ID NO:21; the deduced amino acid sequence of this contig, which represents 100% of the protein, is shown in SEQ ID NO:22. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:22 and the Beta vulgaris sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:22 is 61% similar to the Beta vulgaris sugar transport protein.

The sequence of the soybean contig composed of clones sr1.pk0061.g8, sfl1.pk0058.h12, sgs2c.pk004.o17 and sre.pk0032.h6 is shown in SEQ ID NO:23; the deduced amino acid sequence of this contig, which represents 100% of the protein, is shown in SEQ ID NO :24. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:24 and the Beta vulgaris sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:24 is 66% similar to the Beta vulgaris sugar transport protein.

The sequence of the entire cDNA insert from clone wlk8.pk0001.a11 is shown in SEQ ID NO:25; the deduced amino acid sequence of this cDNA, which represents 100% of the of the protein, is shown in SEQ ID NO:26. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:26 and the Beta vulgaris sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:26 is 61% similar to the Beta vulgaris sugar transport protein.

The sequence of the entire cDNA insert from clone wlm1.pk0012.h1 is shown in SEQ ID NO:27; the deduced amino acid sequence of this cDNA, which represents 100% of the of the protein, is shown in SEQ ID NO:28. A calculation of the percent similarity of the amino acid sequence set forth in SEQ ID NO:28 and the Beta vulgaris sequence (using the Clustal algorithm) revealed that the protein encoded by SEQ ID NO:28 is 56% similar to the Beta vulgaris sugar transport protein.

FIG. 2 presents an alignment of the amino acid sequence set forth in SEQ ID NOS:18, 20, 22, 24, 26 and 28 with the Beta vulgaris-like sugar transport protein amino acid sequence, SEQ ID NO:30. Alignments were performed using the Clustal algorithm. The percent similarity between the corn, rice, soybean and wheat acid sequences was calculated to range between 43% to 81% using the Clustal algorithm.

BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of sugar transport proteins. These sequences represent the first corn, rice, soybean and wheat sequences encoding Beta vulgaris-like sugar transport proteins.

Example 5

Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding sugar transport protein 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 constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML 103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI 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 Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI 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.degree. C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue.TM.M; 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.TM. 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 sugar transport protein, and the 10 kD zein 3' region.

The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn 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.degree. 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 .mu.m in diameter) are coated with DNA using the following technique. Ten .mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5M solution) and spermidine free base (20 .mu.L of a 1.0M 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 .mu.L 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 .mu.L of ethanol. An aliquot (5 .mu.L) of the DNA-coated gold particles can be placed in the center of a Kaptonh.TM. flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), 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 issue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the issue can be transferred to regeneration medium (Fromm et al., (1990) Bio/Technology 8:833-839).

Example 6

Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the .beta. 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 sugar transport proteins 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 Nco 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 a sequence encoding a sugar transport protein. 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.degree. 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.degree. 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) 327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic.TM. 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 Virus (Odell et al. (1985) Nature 313:810-812), 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 sugar transport protein 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 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.l spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfage for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 .mu.L 70% ethanol and resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five .mu.L 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 60.times.15 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 7

Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant sugar transport proteins can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) 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-3 a 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 Nco I site using ligonucleotide-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 protein. This fragment may then be purified on a 1% NuSieve GTGTM low melting agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase.TM. (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 .mu.L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). 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.degree. 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 .mu.g/mL ampicillin. Transformants containing the gene encoding the sugar transport protein 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. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25.degree. C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-.beta.-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25.degree.. Cells are then harvested by centrifugation and re-suspended in 50 .mu.L 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 .mu.g 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. 

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    <400> SEQUENCE:  1
    cccacccccc tccactccac taccacggng gcacggcctg cctctgcagc tctgccctgc     60
    tccgcacccc tcgctctcca accccaacgc gcggcgttgc taaaattcac ctcagcgcgt    120
    actccagttt ggccacctca ccacccgccg ccgctgttta agaaggcccc gcgcccgatc    180
    ggggatcacg aaccttggcc gccgctgccg gagtgggggc gtagatttcc ggcggccatg    240
    gggggcgccg tgatggtcgc catcgcggcc tctatcggca acttgctgca gggctgggac    300
    aatgcgacaa ttgctggagc cgtcctgtac ataaagaagg aattcaacct gcagagcgag    360
    cctctgatcg aaggcctcat cgtcgccatg ttcctcattg gggcaacagt catcacaaca    420
    tctccggggc caagggctga ctgcgttggt aggaggccca tgctggtcgc ctcggctgtc    480
    ctctacttcg tcagtgggct ggtgatgctt tgggcgccaa ttgtgtacat cttgctcctc    540
    gcaaggctca ttgatgggtt cggtatcggt ttggcggtca cacttgttcc tctctacatc    600
    tccgaaactg caccgcacag anattcttgg ggctgntnga acacgttgcc gcagttcatt    660
    ggggtcagng gagggatgtt cctctcctac tgcatggtgt ttgggatgtc cctcatgccc    720
    aaacctgatt ggaggctcat gcttggagtt ctgtcgatcc cgtcacttat ntactttgga    780
    ctgactgtct tctacttgcc tgaatcacca aggtggcttg tnagcaaagg aaggatggcg    840
    gaggcgaaga gagtgntgca aaggctgcgg ggaagagaag atgtctcang ggaganggct    900
    cttctagttg aaggtttggg ggtcggtaaa gatacacgta tttnagagta catcattgga    960
    cctgccaccg aggcagccga tgatcttgta actgacggtg ataaggaaca aatcacactt   1020
    tatgggcctg aagaaggcca gtcatggatt gctcgacctt ctaagggacc catcatgctt   1080
    ggaagtgtgc tttctcttgc atctcgtcat gggagcatgg tgaaccagag tgtacccctt   1140
    atggatccga ttgtgacact ttttggtagt gtccatgaga atatgcctca agctggagga   1200
    agtatgagga gcacattgtt tccaaacttt ggaagtatgt tcagtgtcac agatcagcat   1260
    gccaaaaatg agcagtggga tgaagagaat cttcataggg atgacgagga gtacgcatct   1320
    gatggtgcag gaggtgacta tgaggacaat ctccatagcc cattgctgtc caggcaggca   1380
    acaggtgcgg aagggaagga cattgtgcac catggtcacc gtggaagtgc tttgagcatg   1440
    agaaggcaaa gcctcttagg ggagggtgga gatggtgtga gcagcactga tatcggtggg   1500
    ggatggcagc ttgcttggaa atggtcagag aaggaaggtg agaatggtag aaaggaaggt   1560
    ggtttcaaaa gagtctactt gcaccaagag ggagttcctg gctcaagaag gggctcaatt   1620
    gtttcacttc ccggtggtgg cgatgttctt gagggtagtg agtttgtaca tgctgctgct   1680
    ttagtaagtc agtcagcact tttctcaaag ggtcttgctg aaccacgcat gtcagatgct   1740
    gccatggttc acccatctga ggtagctgcc aaaggttcac gttggaaaga tttgtttgaa   1800
    cctggagtga ggcgtgccct gttagtcggt gttggaattc agatccttca acagtttgct   1860
    ggaataaacg gtgttctgta ctatacccca caaattcttg agcaagctgg tgtggcagtt   1920
    attctttcca aatttggtct cagctcggca tcagcatcca tcttgatcag ttctctcact   1980
    accttactaa tgcttccttg cattggcttt gccatgctgc ttatggatct ttccggaaga   2040
    aggtttttgc tgctaggcac aattccaatc ttgatagcat ctctagttat cctggttgtg   2100
    tccaatctaa ttgatttggg tacactagcc catgctttgc tctccaccat cagtgttatc   2160
    gtctacttct gctgcttcgt tatgggattt ggtcccatcc ccaacatttt atgtgcagag   2220
    atctttccaa ccagggttcg tggcctctgt attgccattt gtgcctttac attctggatc   2280
    ggagatatca tcgtcaccta cagccttcct gtgatgctga atgctattgg actggcgggt   2340
    gttttcagca tatatgcagt cgtatgcttg atttcctttg tgttcgtctt ccttaaggtc   2400
    cctgagacaa aggggatgcc ccttgaggtt attaccgaat tctttgcagt tggtgcgaag   2460
    caagcggctg caaaagccta atttctttgg tacctttgtg tgcaactatt gcactgtaag   2520
    ttagaaactt gaaggggttt caccaagaag ctcggagaat tactttggat ttgtgtaaat   2580
    gttaagggaa cgaacatctg ctcatgctcc tcaaacggta aaaaagagtc cctcaatggc   2640
    aaataggagt cgttaagttg tcaatgtcat ttaccatatg ttttacctat ttgtactgta   2700
    ttataagtca agctattcaa cgctggttgt tgctagaaat ctttagaaca aagatgataa   2760
    tgatctgatc tgatgttata atattcaaat ctcaaataaa gaaaatatcg tttctcaaaa   2820
    aaaa                                                                2824
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    Met Gly Gly Ala Val Met Val Ala Ile Ala Ala Ser Ile Gly Asn Leu
      1               5                  10                  15
    Leu Gln Gly Trp Asp Asn Ala Thr Ile Ala Gly Ala Val Leu Tyr Ile
                 20                  25                  30
    Lys Lys Glu Phe Asn Leu Gln Ser Glu Pro Leu Ile Glu Gly Leu Ile
             35                  40                  45
    Val Ala Met Phe Leu Ile Gly Ala Thr Val Ile Thr Thr Ser Pro Gly
         50                  55                  60
    Pro Arg Ala Asp Cys Val Gly Arg Arg Pro Met Leu Val Ala Ser Ala
     65                  70                  75                  80
    Val Leu Tyr Phe Val Ser Gly Leu Val Met Leu Trp Ala Pro Ile Val
                     85                  90                  95
    Tyr Ile Leu Leu Leu Ala Arg Leu Ile Asp Gly Phe Gly Ile Gly Leu
                100                 105                 110
    Ala Val Thr Leu Val Pro Leu Tyr Ile Ser Glu Thr Ala Pro His Arg
            115                 120                 125
    Xaa Ser Trp Gly Xaa Xaa Asn Thr Leu Pro Gln Phe Ile Gly Val Xaa
        130                 135                 140
    Gly Gly Met Phe Leu Ser Tyr Cys Met Val Phe Gly Met Ser Leu Met
    145                 150                 155                 160
    Pro Lys Pro Asp Trp Arg Leu Met Leu Gly Val Leu Ser Ile Pro Ser
                    165                 170                 175
    Leu Xaa Tyr Phe Gly Leu Thr Val Phe Tyr Leu Pro Glu Ser Pro Arg
                180                 185                 190
    Trp Leu Val Ser Lys Gly Arg Met Ala Glu Ala Lys Arg Val Xaa Gln
            195                 200                 205
    Arg Leu Arg Gly Arg Glu Asp Val Ser Xaa Glu Xaa Ala Leu Leu Val
        210                 215                 220
    Glu Gly Leu Gly Val Gly Lys Asp Thr Arg Ile Xaa Glu Tyr Ile Ile
    225                 230                 235                 240
    Gly Pro Ala Thr Glu Ala Ala Asp Asp Leu Val Thr Asp Gly Asp Lys
                    245                 250                 255
    Glu Gln Ile Thr Leu Tyr Gly Pro Glu Glu Gly Gln Ser Trp Ile Ala
                260                 265                 270
    Arg Pro Ser Lys Gly Pro Ile Met Leu Gly Ser Val Leu Ser Leu Ala
            275                 280                 285
    Ser Arg His Gly Ser Met Val Asn Gln Ser Val Pro Leu Met Asp Pro
        290                 295                 300
    Ile Val Thr Leu Phe Gly Ser Val His Glu Asn Met Pro Gln Ala Gly
    305                 310                 315                 320
    Gly Ser Met Arg Ser Thr Leu Phe Pro Asn Phe Gly Ser Met Phe Ser
                    325                 330                 335
    Val Thr Asp Gln His Ala Lys Asn Glu Gln Trp Asp Glu Glu Asn Leu
                340                 345                 350
    His Arg Asp Asp Glu Glu Tyr Ala Ser Asp Gly Ala Gly Gly Asp Tyr
            355                 360                 365
    Glu Asp Asn Leu His Ser Pro Leu Leu Ser Arg Gln Ala Thr Gly Ala
        370                 375                 380
    Glu Gly Lys Asp Ile Val His His Gly His Arg Gly Ser Ala Leu Ser
    385                 390                 395                 400
    Met Arg Arg Gln Ser Leu Leu Gly Glu Gly Gly Asp Gly Val Ser Ser
                    405                 410                 415
    Thr Asp Ile Gly Gly Gly Trp Gln Leu Ala Trp Lys Trp Ser Glu Lys
                420                 425                 430
    Glu Gly Glu Asn Gly Arg Lys Glu Gly Gly Phe Lys Arg Val Tyr Leu
            435                 440                 445
    His Gln Glu Gly Val Pro Gly Ser Arg Arg Gly Ser Ile Val Ser Leu
        450                 455                 460
    Pro Gly Gly Gly Asp Val Leu Glu Gly Ser Glu Phe Val His Ala Ala
    465                 470                 475                 480
    Ala Leu Val Ser Gln Ser Ala Leu Phe Ser Lys Gly Leu Ala Glu Pro
                    485                 490                 495
    Arg Met Ser Asp Ala Ala Met Val His Pro Ser Glu Val Ala Ala Lys
                500                 505                 510
    Gly Ser Arg Trp Lys Asp Leu Phe Glu Pro Gly Val Arg Arg Ala Leu
            515                 520                 525
    Leu Val Gly Val Gly Ile Gln Ile Leu Gln Gln Phe Ala Gly Ile Asn
        530                 535                 540
    Gly Val Leu Tyr Tyr Thr Pro Gln Ile Leu Glu Gln Ala Gly Val Ala
    545                 550                 555                 560
    Val Ile Leu Ser Lys Phe Gly Leu Ser Ser Ala Ser Ala Ser Ile Leu
                    565                 570                 575
    Ile Ser Ser Leu Thr Thr Leu Leu Met Leu Pro Cys Ile Gly Phe Ala
                580                 585                 590
    Met Leu Leu Met Asp Leu Ser Gly Arg Arg Phe Leu Leu Leu Gly Thr
            595                 600                 605
    Ile Pro Ile Leu Ile Ala Ser Leu Val Ile Leu Val Val Ser Asn Leu
        610                 615                 620
    Ile Asp Leu Gly Thr Leu Ala His Ala Leu Leu Ser Thr Ile Ser Val
    625                 630                 635                 640
    Ile Val Tyr Phe Cys Cys Phe Val Met Gly Phe Gly Pro Ile Pro Asn
                    645                 650                 655
    Ile Leu Cys Ala Glu Ile Phe Pro Thr Arg Val Arg Gly Leu Cys Ile
                660                 665                 670
    Ala Ile Cys Ala Phe Thr Phe Trp Ile Gly Asp Ile Ile Val Thr Tyr
            675                 680                 685
    Ser Leu Pro Val Met Leu Asn Ala Ile Gly Leu Ala Gly Val Phe Ser
        690                 695                 700
    Ile Tyr Ala Val Val Cys Leu Ile Ser Phe Val Phe Val Phe Leu Lys
    705                 710                 715                 720
    Val Pro Glu Thr Lys Gly Met Pro Leu Glu Val Ile Thr Glu Phe Phe
                    725                 730                 735
    Ala Val Gly Ala Lys Gln Ala Ala Ala Lys Ala
                740                 745
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    gaagagctca cccccccccc ctcggccctg gactccctcc tccaaatctc ccctaaaagc     60
    ttcccaattt ggcgagaatt ccccatatat ttgccccatc tcggcgtccc aacgagccct    120
    tccagattcc cagccgcctc tcttcttgtt aggggatccg aaatctcggt ggacgagaga    180
    cttggtggta atnattcgcc ggccatggcg ggcgccgtgc tggtcgccat cgcggcctcc    240
    atcggcaact tgctgcaggg ctgggataat gcaaccattg caggtgcggt actgtacatc    300
    aagaaggaat tcaacttgca tagcgacccc cttatcgaag gtctgatcgt ggccatgtcg    360
    ctcattgggg ccaccatcat cacgacgntc tctgcgagca ggtggctgac tcttttggta    420
    tggcggccca tgctnatcnc ttc                                            443
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    <400> SEQUENCE:  4
    Glu Glu Leu Thr Pro Pro Pro Ser Ala Leu Asp Ser Leu Leu Gln Ile
      1               5                  10                  15



    Ser Pro Lys Ser Phe Pro Ile Trp Arg Glu Phe Pro Ile Tyr Leu Pro
                 20                  25                  30
    His Leu Gly Val Pro Thr Ser Pro Ser Arg Phe Pro Ala Ala Ser Leu
             35                  40                  45
    Leu Val Arg Gly Ser Glu Ile Ser Val Asp Glu Arg Leu Gly Gly Asn
         50                  55                  60
    Xaa Ser Pro Ala Met Ala Gly Ala Val Leu Val Ala Ile Ala Ala Ser
     65                  70                  75                  80
    Ile Gly Asn Leu Leu Gln Gly Trp Asp Asn Ala Thr Ile Ala Gly Ala
                     85                  90                  95
    Val Leu Tyr Ile Lys Lys Glu Phe Asn Leu His Ser Asp Pro Leu Ile
                100                 105                 110
    Glu Gly Leu Ile Val Ala Met Ser Leu Ile Gly Ala Thr Ile Ile Thr
            115                 120                 125
    Thr Xaa Ser
        130
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 5
    <211> LENGTH: 870
    <212> TYPE: DNA
    <213> ORGANISM: Oryza sativa
    <400> SEQUENCE:  5
    gcacgaggtt ctaaccttga ttctggtcaa tattctggat gtggggacca tggttcatgc     60
    ctcactgtcc acagtcagtg tcatactcta cttctgcttc tttgtcatgg ggttcgggcc    120
    tattccaaac attctctgtg cagagatttt cccgaccacc gttcgtggca tctgcatagc    180
    catctgtgcc ctaacattct ggatcggtga tatcattgtg acatacaccc tccccgtgat    240
    gctcaacgcc attggactcg ctggagtgtt tggaatctac gcagtggtct gcatactggc    300
    tttcctgttt gtcttcatga aggtgccgga gacaaagggc atgcctcttg aagtcatcac    360
    cgagttcttc tctgtcggag caaagcaggc caaggaggac tagttgctcg gatcaagtga    420
    tcaatcagat tgctggtggt aattttgttg cttccaaatc gcgctgcggg ttaaacctgt    480
    gatggatgct ttgttaaaga atcttggaag agatcaaaat gcagtgagcc taaagagatg    540
    atttggctgt acatcatgag gctgaatcct gtcgtagact ggattttgga gcttaggata    600
    tgtagatcat ctgttccttt tggtttggtc attttccatt tgtgtttctt tggaattctt    660
    ctccctgtaa ctagtggtct atcacagttg tgttactggt tttgccttac tcttgagttt    720
    gttttcttct ctcggttgtg agttctgaat attagcatag ccgagtacta gttctgaatt    780
    ggtttcctct ctgctgaaca tctttcattg atgcttggat ttcatcaaaa aaaaaaaaaa    840
    aaaactcgag ggggagcccg gtacacatct                                     870
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 6
    <211> LENGTH: 131
    <212> TYPE: PRT
    <213> ORGANISM: Oryza sativa
    <400> SEQUENCE:  6
    Val Leu Thr Leu Ile Leu Val Asn Ile Leu Asp Val Gly Thr Met Val
      1               5                  10                  15
    His Ala Ser Leu Ser Thr Val Ser Val Ile Leu Tyr Phe Cys Phe Phe
                 20                  25                  30
    Val Met Gly Phe Gly Pro Ile Pro Asn Ile Leu Cys Ala Glu Ile Phe
             35                  40                  45
    Pro Thr Thr Val Arg Gly Ile Cys Ile Ala Ile Cys Ala Leu Thr Phe
         50                  55                  60
    Trp Ile Gly Asp Ile Ile Val Thr Tyr Thr Leu Pro Val Met Leu Asn
     65                  70                  75                  80
    Ala Ile Gly Leu Ala Gly Val Phe Gly Ile Tyr Ala Val Val Cys Ile
                     85                  90                  95
    Leu Ala Phe Leu Phe Val Phe Met Lys Val Pro Glu Thr Lys Gly Met
                100                 105                 110
    Pro Leu Glu Val Ile Thr Glu Phe Phe Ser Val Gly Ala Lys Gln Ala
            115                 120                 125
    Lys Glu Asp
        130
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 7
    <211> LENGTH: 2601
    <212> TYPE: DNA
    <213> ORGANISM: Glycine max
    <400> SEQUENCE:  7
    gttgcttaac ccttgttgag tgaagtgagc aaggggaatg gcgatctgaa attcggatac     60
    tttaattgct tctcgctttc accgaccgaa ctcaatttat agatactccg tcaacctcaa    120
    tcccaactaa ctagcagttc cttgctgctg ctccttcttc accatatcgc agtaatgaaa    180
    ggtgccgtcc ttgttgctat tgccgcttcc attggtaatt tcctccaagg atgggataat    240
    gctaccatcg ccggggctaa tggttacatt aagaaagacc ttgctttggg aacaactatg    300
    gaaaggcttg tggtgggcat gtccctgatt ggagcaacgg taatcaccac atgctctggt    360
    cctatagcgg attggctcgg tcggcgaccc atgatgataa tctcatctgt gctctatttc    420
    ttgggtggtt tggtgatgct gtggtcccca aatgtgtatg tgttgtgctt ggcgaggcta    480
    cttgatggat ttgggattgg ccttgctgtg actcttgtcc cggtctatat atctgaaacg    540
    gcgccgtctg aaataagggg gtcgttgaat acgcttcctc agttcagtgg ctctggagga    600
    atgtttttgt cgtactgtat ggtttttggc atgtcattga gtcccgcgcc tagctggagg    660
    ctcatgcttg gggttctgtc tattccttct ctcttgtatt ttgcattgac catttttttc    720
    ttgcccgagt ctcctcggtg gctggtcagc aaaggaagga tgctcgaggc taagaaggtg    780
    ctccaaagat tgcgcggaag ggaggatgtg tcaggcgaga tggcattgct ggttgaaggt    840
    ctcgggattg ggggtgatac atctatcgaa gagtacataa ttggccctgc tgacgatgtg    900
    gctgatggtc atgaacatgc aacagagaaa gataaaattc gattatatgg atcccaagca    960
    ggcctttctt ggttatcaaa acctgtcact ggacagagtt ctattggcct tgcgtcacac   1020
    catggaagca tcatcaacca aagcatgccc ctcatggatc ctctggtgac actgtttggt   1080
    agcattcatg agaagctccc cgagacagga gcaagaggaa gcatgcgaag cactctgttt   1140
    ccaaattttg gaagcatgtt cagcactgct gagccgcatg ctaaaattga acaatgggat   1200
    gaagaaagct tacaaaggga acgtgaggac tacatgtcag atgcaacccg tggggactcc   1260
    gatgataatt tgcacagtcc tttaatctca cgccaaacaa caagccttga aaaagactta   1320
    cctcctcctc cttcccatgg cagtatcctt ggcagcatga ggcgtcacag tagtctcatg   1380
    caagggtcag gtgagcaagg tggtagtaca ggtattggtg gtggctggca actggcatgg   1440
    aaatggactg ataaaggtga ggatggaaaa caacaaggag ggtttaaaag gatttattta   1500
    catgaggagg gagtttctgc atctcgtcgt ggatccattg tatcgattcc cggtgaaggc   1560
    gaatttgtcc aggctgctgc cttggtaagc caacccgctc tttactccaa ggagcttatt   1620
    gatggacacc cagttgggcc tgcaatggtt cacccatctg agacagcttc aaaggggcca   1680
    agttggaaag ctcttcttga accaggggtt aagcatgcat tggttgttgg agttggaata   1740
    caaatacttc agcagttttc agggataaat ggggttctat attacacacc tcaaatcctt   1800
    gaagaggccg gtgttgaagt tcttctttca gatataggca ttggctcaga gtcggcatca   1860
    ttccttatca gtgctttcac aaccttcttg atgcttccct gtataggcgt agccatgaag   1920
    ctcatggatg tttcaggcag aaggcagttg ctacttacta caatccccgt gctgattgtg   1980
    tcactcatta ttttggtcat tggaagcctg gtaaattttg gcaatgtcgc ccatgcagca   2040
    atctcaacag tatgcgttgt ggtttatttc tgctgctttg tgatgggtta tggaccaatt   2100
    ccaaacatcc tttgctcaga gattttcccc actagggtgc gtggcctctg cattgctatc   2160
    tgtgcattag tgttctggat tggagacatc atcatcacat actcgctgcc tgtgatgctc   2220
    ggctctttag gacttggtgg tgtattcgcc atttacgcag ttgtttgttt catctcgtgg   2280
    atatttgtgt ttttgaaggt tccagaaaca aagggcatgc cccttgaagt catctctgaa   2340
    ttcttttctg ttggagcaaa gcaggctgct tctgccaaga atgagtgaca caacacaagt   2400
    ccgttatata ctctgtaact ttagttgtta aagccatcat ctctcgtctt tacagatttt   2460
    gcttttcata agtttatttg gaggaagata ttttgaaaca tatgggtttt tttttctttc   2520
    ataaaaataa aacccttccc tttttgggtg gggaaaagaa aaaaaaaaaa aaaaaaaaaa   2580
    aaaaaaaaaa aaaaaaaaaa a                                             2601
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 8
    <211> LENGTH: 737
    <212> TYPE: PRT
    <213> ORGANISM: Glycine max
    <400> SEQUENCE:  8
    Met Lys Gly Ala Val Leu Val Ala Ile Ala Ala Ser Ile Gly Asn Phe
      1               5                  10                  15
    Leu Gln Gly Trp Asp Asn Ala Thr Ile Ala Gly Ala Asn Gly Tyr Ile
                 20                  25                  30
    Lys Lys Asp Leu Ala Leu Gly Thr Thr Met Glu Arg Leu Val Val Gly
             35                  40                  45
    Met Ser Leu Ile Gly Ala Thr Val Ile Thr Thr Cys Ser Gly Pro Ile
         50                  55                  60
    Ala Asp Trp Leu Gly Arg Arg Pro Met Met Ile Ile Ser Ser Val Leu
     65                  70                  75                  80
    Tyr Phe Leu Gly Gly Leu Val Met Leu Trp Ser Pro Asn Val Tyr Val
                     85                  90                  95
    Leu Cys Leu Ala Arg Leu Leu Asp Gly Phe Gly Ile Gly Leu Ala Val
                100                 105                 110
    Thr Leu Val Pro Val Tyr Ile Ser Glu Thr Ala Pro Ser Glu Ile Arg
            115                 120                 125
    Gly Ser Leu Asn Thr Leu Pro Gln Phe Ser Gly Ser Gly Gly Met Phe
        130                 135                 140
    Leu Ser Tyr Cys Met Val Phe Gly Met Ser Leu Ser Pro Ala Pro Ser
    145                 150                 155                 160
    Trp Arg Leu Met Leu Gly Val Leu Ser Ile Pro Ser Leu Leu Tyr Phe
                    165                 170                 175
    Ala Leu Thr Ile Phe Phe Leu Pro Glu Ser Pro Arg Trp Leu Val Ser
                180                 185                 190
    Lys Gly Arg Met Leu Glu Ala Lys Lys Val Leu Gln Arg Leu Arg Gly
            195                 200                 205
    Arg Glu Asp Val Ser Gly Glu Met Ala Leu Leu Val Glu Gly Leu Gly
        210                 215                 220
    Ile Gly Gly Asp Thr Ser Ile Glu Glu Tyr Ile Ile Gly Pro Ala Asp
    225                 230                 235                 240
    Asp Val Ala Asp Gly His Glu His Ala Thr Glu Lys Asp Lys Ile Arg
                    245                 250                 255
    Leu Tyr Gly Ser Gln Ala Gly Leu Ser Trp Leu Ser Lys Pro Val Thr
                260                 265                 270
    Gly Gln Ser Ser Ile Gly Leu Ala Ser His His Gly Ser Ile Ile Asn
            275                 280                 285
    Gln Ser Met Pro Leu Met Asp Pro Leu Val Thr Leu Phe Gly Ser Ile
        290                 295                 300
    His Glu Lys Leu Pro Glu Thr Gly Ala Arg Gly Ser Met Arg Ser Thr
    305                 310                 315                 320
    Leu Phe Pro Asn Phe Gly Ser Met Phe Ser Thr Ala Glu Pro His Ala
                    325                 330                 335
    Lys Ile Glu Gln Trp Asp Glu Glu Ser Leu Gln Arg Glu Arg Glu Asp
                340                 345                 350
    Tyr Met Ser Asp Ala Thr Arg Gly Asp Ser Asp Asp Asn Leu His Ser
            355                 360                 365
    Pro Leu Ile Ser Arg Gln Thr Thr Ser Leu Glu Lys Asp Leu Pro Pro
        370                 375                 380
    Pro Pro Ser His Gly Ser Ile Leu Gly Ser Met Arg Arg His Ser Ser
    385                 390                 395                 400
    Leu Met Gln Gly Ser Gly Glu Gln Gly Gly Ser Thr Gly Ile Gly Gly
                    405                 410                 415
    Gly Trp Gln Leu Ala Trp Lys Trp Thr Asp Lys Gly Glu Asp Gly Lys
                420                 425                 430
    Gln Gln Gly Gly Phe Lys Arg Ile Tyr Leu His Glu Glu Gly Val Ser
            435                 440                 445
    Ala Ser Arg Arg Gly Ser Ile Val Ser Ile Pro Gly Glu Gly Glu Phe
        450                 455                 460
    Val Gln Ala Ala Ala Leu Val Ser Gln Pro Ala Leu Tyr Ser Lys Glu
    465                 470                 475                 480
    Leu Ile Asp Gly His Pro Val Gly Pro Ala Met Val His Pro Ser Glu
                    485                 490                 495
    Thr Ala Ser Lys Gly Pro Ser Trp Lys Ala Leu Leu Glu Pro Gly Val
                500                 505                 510
    Lys His Ala Leu Val Val Gly Val Gly Ile Gln Ile Leu Gln Gln Phe
            515                 520                 525
    Ser Gly Ile Asn Gly Val Leu Tyr Tyr Thr Pro Gln Ile Leu Glu Glu
        530                 535                 540
    Ala Gly Val Glu Val Leu Leu Ser Asp Ile Gly Ile Gly Ser Glu Ser
    545                 550                 555                 560
    Ala Ser Phe Leu Ile Ser Ala Phe Thr Thr Phe Leu Met Leu Pro Cys
                    565                 570                 575
    Ile Gly Val Ala Met Lys Leu Met Asp Val Ser Gly Arg Arg Gln Leu
                580                 585                 590
    Leu Leu Thr Thr Ile Pro Val Leu Ile Val Ser Leu Ile Ile Leu Val
            595                 600                 605
    Ile Gly Ser Leu Val Asn Phe Gly Asn Val Ala His Ala Ala Ile Ser
        610                 615                 620
    Thr Val Cys Val Val Val Tyr Phe Cys Cys Phe Val Met Gly Tyr Gly
    625                 630                 635                 640
    Pro Ile Pro Asn Ile Leu Cys Ser Glu Ile Phe Pro Thr Arg Val Arg
                    645                 650                 655
    Gly Leu Cys Ile Ala Ile Cys Ala Leu Val Phe Trp Ile Gly Asp Ile
                660                 665                 670
    Ile Ile Thr Tyr Ser Leu Pro Val Met Leu Gly Ser Leu Gly Leu Gly
            675                 680                 685
    Gly Val Phe Ala Ile Tyr Ala Val Val Cys Phe Ile Ser Trp Ile Phe
        690                 695                 700
    Val Phe Leu Lys Val Pro Glu Thr Lys Gly Met Pro Leu Glu Val Ile
    705                 710                 715                 720
    Ser Glu Phe Phe Ser Val Gly Ala Lys Gln Ala Ala Ser Ala Lys Asn
                    725                 730                 735
    Glu
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 9
    <211> LENGTH: 1692
    <212> TYPE: DNA
    <213> ORGANISM: Glycine max
    <400> SEQUENCE:  9
    gcacgaggga tccgtccaga gaaaaagatc aaattaagtt gtatggacca gaacaaggcc     60
    agtcctgggt tgctagacct gttgctggac caaattctgt tggccttgta tctaggaaag    120
    gaagcatggc aaatccaagc agtctagtgg accctctagt gaccctcttt ggtagtgtac    180
    atgagaagct cccagaaaca ggaagcaccc tttttccaca ctttgggagt atgttcagtg    240
    ttgggggaaa tcagccaagg aatgaagatt gggatgagga aagcctagcc agagagggtg    300
    atgattatgt ctctgatgct ggtgattctg atgacaattt gcagagtcca ttgatctcac    360
    gtcaaacaac gagtctggat aaggacatac ctcctcatgc ccatagtaac cttgcaagca    420
    tgaggcaagg tagtctttta catggaaatt caggagaacc cactggtagt actgggattg    480
    gtggtggttg gcagctagca tggaaatggt ctgaaagaga gggcccagat ggaaagaagg    540
    aaggtggctt caagagaata tatttacacc aagatggtgg ttctggatct agacgtgggt    600
    ctgtggtttc actccctggc ggtgatttac caactgacag tgaggttgta caggctgctg    660
    ctctggtgag tcagcctgcc ctttataatg aggaccttat gcgtcaacgg ccagttggac    720
    cagctatgat tcatccctct gaaacaattg caaaagggcc aagttggagt gatctttttg    780
    aacctggggt gaagcatgca ttgattgtgg gggtgggaat gcaaattctt cagcagttct    840
    ctggtataaa tggggtcctc tactatacgc ctcaaattct tgagcaggca ggtgttggtt    900
    atcttctttc aagcctaggc cttggttcta cttcttcatc ctttcttatt agtgcggtga    960
    caaccttgtt gatgcttcct tgtatagcca ttgccatgag gctcatggat atttcaggca   1020
    gaaggacttt gctgctcagt acaatccccg tcctaatagc agctcttctc atattagtcc   1080
    tgggaagtct tgtggatttg ggatccactg caaatgcatc aatctcaacc attagtgtta   1140
    ttgtctattt ctgtttcttt gtcatgggat ttggaccaat tcctaatata ctttgtgcag   1200
    agatcttccc cactcgagtt cgtggtctct gcattgctat ttgtgccctt accttttgga   1260
    tctgtgatat cattgtcacc tacacactcc cagttatgct caattctgta ggcctcgctg   1320
    gtgtttttgg tatttatgct gtcgtgtgct tcatagcatg ggtgtttgtc tttttgaaag   1380
    ttccagaaac caagggcatg ccactggaag tgatcattga gttcttctct gtcggagcaa   1440
    aacagtttga cgatgccaag cacaactgac ccaaggacat gataaattca aagttttgac   1500
    ggtaccttct aattattttc aatctacggc tgtttgaaat tttcccctct tttaaaattt   1560
    tattttctat ttattctctc ttttccgtgg gttgagattg agaaacaaga aactttgttt   1620
    ctgtaaagaa aaatgttcat tttctggttc atttatggaa ctttatatac ttcctaaaaa   1680
    aaaaaaaaaa aa                                                       1692
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 10
    <211> LENGTH: 486
    <212> TYPE: PRT
    <213> ORGANISM: Glycine max
    <400> SEQUENCE:  10
    Asp Pro Ser Arg Glu Lys Asp Gln Ile Lys Leu Tyr Gly Pro Glu Gln
      1               5                  10                  15
    Gly Gln Ser Trp Val Ala Arg Pro Val Ala Gly Pro Asn Ser Val Gly
                 20                  25                  30



    Leu Val Ser Arg Lys Gly Ser Met Ala Asn Pro Ser Ser Leu Val Asp
             35                  40                  45
    Pro Leu Val Thr Leu Phe Gly Ser Val His Glu Lys Leu Pro Glu Thr
         50                  55                  60
    Gly Ser Thr Leu Phe Pro His Phe Gly Ser Met Phe Ser Val Gly Gly
     65                  70                  75                  80
    Asn Gln Pro Arg Asn Glu Asp Trp Asp Glu Glu Ser Leu Ala Arg Glu
                     85                  90                  95
    Gly Asp Asp Tyr Val Ser Asp Ala Gly Asp Ser Asp Asp Asn Leu Gln
                100                 105                 110
    Ser Pro Leu Ile Ser Arg Gln Thr Thr Ser Leu Asp Lys Asp Ile Pro
            115                 120                 125
    Pro His Ala His Ser Asn Leu Ala Ser Met Arg Gln Gly Ser Leu Leu
        130                 135                 140
    His Gly Asn Ser Gly Glu Pro Thr Gly Ser Thr Gly Ile Gly Gly Gly
    145                 150                 155                 160
    Trp Gln Leu Ala Trp Lys Trp Ser Glu Arg Glu Gly Pro Asp Gly Lys
                    165                 170                 175
    Lys Glu Gly Gly Phe Lys Arg Ile Tyr Leu His Gln Asp Gly Gly Ser
                180                 185                 190
    Gly Ser Arg Arg Gly Ser Val Val Ser Leu Pro Gly Gly Asp Leu Pro
            195                 200                 205
    Thr Asp Ser Glu Val Val Gln Ala Ala Ala Leu Val Ser Gln Pro Ala
        210                 215                 220
    Leu Tyr Asn Glu Asp Leu Met Arg Gln Arg Pro Val Gly Pro Ala Met
    225                 230                 235                 240
    Ile His Pro Ser Glu Thr Ile Ala Lys Gly Pro Ser Trp Ser Asp Leu
                    245                 250                 255
    Phe Glu Pro Gly Val Lys His Ala Leu Ile Val Gly Val Gly Met Gln
                260                 265                 270
    Ile Leu Gln Gln Phe Ser Gly Ile Asn Gly Val Leu Tyr Tyr Thr Pro
            275                 280                 285
    Gln Ile Leu Glu Gln Ala Gly Val Gly Tyr Leu Leu Ser Ser Leu Gly
        290                 295                 300
    Leu Gly Ser Thr Ser Ser Ser Phe Leu Ile Ser Ala Val Thr Thr Leu
    305                 310                 315                 320
    Leu Met Leu Pro Cys Ile Ala Ile Ala Met Arg Leu Met Asp Ile Ser
                    325                 330                 335
    Gly Arg Arg Thr Leu Leu Leu Ser Thr Ile Pro Val Leu Ile Ala Ala
                340                 345                 350
    Leu Leu Ile Leu Val Leu Gly Ser Leu Val Asp Leu Gly Ser Thr Ala
            355                 360                 365
    Asn Ala Ser Ile Ser Thr Ile Ser Val Ile Val Tyr Phe Cys Phe Phe
        370                 375                 380
    Val Met Gly Phe Gly Pro Ile Pro Asn Ile Leu Cys Ala Glu Ile Phe
    385                 390                 395                 400
    Pro Thr Arg Val Arg Gly Leu Cys Ile Ala Ile Cys Ala Leu Thr Phe
                    405                 410                 415
    Trp Ile Cys Asp Ile Ile Val Thr Tyr Thr Leu Pro Val Met Leu Asn
                420                 425                 430
    Ser Val Gly Leu Ala Gly Val Phe Gly Ile Tyr Ala Val Val Cys Phe
            435                 440                 445
    Ile Ala Trp Val Phe Val Phe Leu Lys Val Pro Glu Thr Lys Gly Met
        450                 455                 460
    Pro Leu Glu Val Ile Ile Glu Phe Phe Ser Val Gly Ala Lys Gln Phe
    465                 470                 475                 480
    Asp Asp Ala Lys His Asn
                    485
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 11
    <211> LENGTH: 510
    <212> TYPE: DNA
    <213> ORGANISM: Triticum aestivum
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (421)
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (434)
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (441)
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (458)
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (483)
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (493)
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (498)
    <400> SEQUENCE:  11
    cggtggcagc cggggcagtg aaggaggggt agctcttggc tcctatttga ggcggcttcg     60
    ctcggttctg atctaccgca ccacaccacc acaccacacc aggggcctgc cgcttcttgg    120
    gcttctccat ctcatctcct tggttggttc tctactagag aggcgcagct gcagggatcc    180
    ttggtggaga ggagggaaga agatgtcggg tgctgcactg gtcgcgattg cggcttccat    240
    tggcaatctg ctgcaggggt gggacaatgc caccatcgct ggtgctgttc tgtacatcaa    300
    gaaggaattc cagctcgaaa ataatccgac tgtggagggg ctcatcgtgg catgtcctca    360
    tcgggtgcaa catcatcaca cattctccgg gccagtatca aactgggttg ccgggcccta    420
    ngccatctcc ttgntttcaa ntcccaaggg ctaatcanct aggcaccaat gtcaatgtgc    480
    gcnccggaac ctntcaangg ttggaacgtt                                     510
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 12
    <211> LENGTH: 117
    <212> TYPE: PRT
    <213> ORGANISM: Triticum aestivum
    <400> SEQUENCE:  12
    Gly Gly Ser Arg Gly Ser Glu Gly Gly Val Ala Leu Gly Ser Tyr Leu
      1               5                  10                  15
    Arg Arg Leu Arg Ser Val Leu Ile Tyr Arg Thr Thr Pro Pro His His
                 20                  25                  30
    Thr Arg Gly Leu Pro Leu Leu Gly Leu Leu His Leu Ile Ser Leu Val
             35                  40                  45
    Gly Ser Leu Leu Glu Arg Arg Ser Cys Arg Asp Pro Trp Trp Arg Gly
         50                  55                  60
    Gly Lys Lys Met Ser Gly Ala Ala Leu Val Ala Ile Ala Ala Ser Ile
     65                  70                  75                  80
    Gly Asn Leu Leu Gln Gly Trp Asp Asn Ala Thr Ile Ala Gly Ala Val
                     85                  90                  95
    Leu Tyr Ile Lys Lys Glu Phe Gln Leu Glu Asn Asn Pro Thr Val Glu
                100                 105                 110
    Gly Leu Ile Val Ala
            115
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 13
    <211> LENGTH: 1487
    <212> TYPE: DNA
    <213> ORGANISM: Triticum aestivum
    <400> SEQUENCE:  13
    tctcttggaa agagggtggg gaggcagtca gcagcactgg tattggtggg gggtggcaac     60
    tcgcatggaa atggtcggag cgacaaggcg aggatggcaa gaaggaagga ggcttcaaaa    120
    gaatctactt gcaccaagag ggggtggccg actcaagaag gggctctgtt gtttcacttc    180
    ctggtggggg tgatgccacg caagggggca gtgggtttat acatgctgct gctttggtaa    240
    gccactcggc tctttactcc aaggatctta tggaagagcg tatggcggcc ggtccagcca    300
    tgattcatcc attggaggca gctcccaaag gttcaatctg gaaagatctg tttgaacctg    360
    gtgtgaggcg tgcattgttc gtcggtgttg gaattcagat gcttcagcag tttgctggaa    420
    taaatggagt tctctactat actcctcaaa ttctggagca agctggtgtg gctgttcttc    480
    tttccaatct tggcctcagt tcagcatcag catccatctt gatcagttct ctcaccacct    540
    tactcatgct cccaagcatt ggtgtagcca tgagacttat ggatatatct ggaagaaggt    600
    ttctgctact gggcacaatt cccatcttga tagcatccct aattgttttg ggtgtggtca    660
    atgttatcaa cttgagtacg gtgccccacg ctgtgctctc cacagttagc gtcattgtct    720
    acttctgctg ctttgtcatg ggctttggcc cgatccccaa cattctatgt gcagagattt    780
    tccccaccag agtccgtggt gtctgcatcg ctatttgcgc cctcacattc tggatttgtg    840
    acattattgt tacctacagc ctgcctgtga tgctgaatgc tattggtcta gcgggtgtct    900
    ttggtatata tgcagtcgtt tgctgcattg cctttgtgtt cgtctaccta aaggtcccag    960
    agacaaaggg catgcccctc gaggtcatca ccgagttctt tgcggttggg gcgaagcaag   1020
    cgcaggccac cattgcctga ttcatcatgg agctttgttt tcagtttgca cactgcggtc   1080
    tgcgctgaaa attgcaaatt ggacgggtcc tcgtgaggaa cggaaaaact tttgagttgt   1140
    aaatgagaca gctacccaaa gagctcatca cgaggaacgg gaagctgtaa aagtaggagg   1200
    atctcatgcc cccatttcat cgtctattat tgcttattag tactgtactg taatcgtcat   1260
    tagttgctgt agggttgttc aacttgctaa tctgattctg aactaccatg ctgatgtccg   1320
    aaataaagaa aaagcatgtt tttttttgtg tcaacttgca aactttcttt taaacattgt   1380
    gcaatgtatt gtaaatttct ttatcaactt ccctcgattc agagagaagc acttgtttgt   1440
    aagtcatgaa agatttttct cgacaaaaaa aaaaaaaaaa aaaaaaa                 1487
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 14
    <211> LENGTH: 345
    <212> TYPE: PRT
    <213> ORGANISM: Triticum aestivum
    <400> SEQUENCE:  14
    Ser Trp Lys Glu Gly Gly Glu Ala Val Ser Ser Thr Gly Ile Gly Gly
      1               5                  10                  15
    Gly Trp Gln Leu Ala Trp Lys Trp Ser Glu Arg Gln Gly Glu Asp Gly
                 20                  25                  30
    Lys Lys Glu Gly Gly Phe Lys Arg Ile Tyr Leu His Gln Glu Gly Val
             35                  40                  45
    Ala Asp Ser Arg Arg Gly Ser Val Val Ser Leu Pro Gly Gly Gly Asp
         50                  55                  60
    Ala Thr Gln Gly Gly Ser Gly Phe Ile His Ala Ala Ala Leu Val Ser
     65                  70                  75                  80
    His Ser Ala Leu Tyr Ser Lys Asp Leu Met Glu Glu Arg Met Ala Ala
                     85                  90                  95
    Gly Pro Ala Met Ile His Pro Leu Glu Ala Ala Pro Lys Gly Ser Ile
                100                 105                 110
    Trp Lys Asp Leu Phe Glu Pro Gly Val Arg Arg Ala Leu Phe Val Gly
            115                 120                 125
    Val Gly Ile Gln Met Leu Gln Gln Phe Ala Gly Ile Asn Gly Val Leu
        130                 135                 140
    Tyr Tyr Thr Pro Gln Ile Leu Glu Gln Ala Gly Val Ala Val Leu Leu
    145                 150                 155                 160
    Ser Asn Leu Gly Leu Ser Ser Ala Ser Ala Ser Ile Leu Ile Ser Ser
                    165                 170                 175
    Leu Thr Thr Leu Leu Met Leu Pro Ser Ile Gly Val Ala Met Arg Leu
                180                 185                 190
    Met Asp Ile Ser Gly Arg Arg Phe Leu Leu Leu Gly Thr Ile Pro Ile
            195                 200                 205
    Leu Ile Ala Ser Leu Ile Val Leu Gly Val Val Asn Val Ile Asn Leu
        210                 215                 220
    Ser Thr Val Pro His Ala Val Leu Ser Thr Val Ser Val Ile Val Tyr
    225                 230                 235                 240
    Phe Cys Cys Phe Val Met Gly Phe Gly Pro Ile Pro Asn Ile Leu Cys
                    245                 250                 255
    Ala Glu Ile Phe Pro Thr Arg Val Arg Gly Val Cys Ile Ala Ile Cys
                260                 265                 270
    Ala Leu Thr Phe Trp Ile Cys Asp Ile Ile Val Thr Tyr Ser Leu Pro
            275                 280                 285
    Val Met Leu Asn Ala Ile Gly Leu Ala Gly Val Phe Gly Ile Tyr Ala
        290                 295                 300
    Val Val Cys Cys Ile Ala Phe Val Phe Val Tyr Leu Lys Val Pro Glu
    305                 310                 315                 320
    Thr Lys Gly Met Pro Leu Glu Val Ile Thr Glu Phe Phe Ala Val Gly
                    325                 330                 335
    Ala Lys Gln Ala Gln Ala Thr Ile Ala
                340                 345
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 15
    <211> LENGTH: 1009
    <212> TYPE: DNA
    <213> ORGANISM: Triticum aestivum
    <400> SEQUENCE:  15
    tgaacctgga gtgaagcatg cactgttcgt tggcatagga ttacagatcc tgcagcagtt     60
    tgcgggtatc aatggagtcc tctactacac acctcagata cttgagcaag caggtgtcgg    120
    ggttcttcta tcaaacattg gactaagctc ttcctcagca tctattctta ttagtgcctt    180
    gacaaccttg ctgatgcttc ccagcattgg catcgccatg agactcatgg atatgtcagg    240
    aagaaggttt cttctccttt caacaatccc tgtcttgata gtagcgctag ctgtcttggt    300
    tttagtgaat gttctggatg tcggaaccat ggtgcacgct gcgctctcaa cgatcagcgt    360
    catcgtctat ttctgcttct tcgtcatggg gtttgggcct atcccaaata ttctctgcgc    420
    ggagattttc cccacctctg tccgtggcat ctgcatagcc atctgcgcgc taaccttctg    480
    gatcggcgac atcatcgtga catacactct ccccgtgatg ctcaatgcca ttggtctcgc    540
    tggagtcttc ggcatatatg ccatcgtttg tgtactagcc tttgtattcg tctacatgaa    600
    ggtccctgag acaaagggca tgcccctgga ggtcatcacc gagttcttct ctgtcggggc    660
    aaagcagggc aaggaagcca cggactagtt gctctgatcc ggtgatccgc gtcgctggtg    720
    gtaattttgt ggtgtcataa ctactactac actggttaac ctgcgatgct ttggtgaaga    780
    aacttcaaag agagcagata cggaagactt tacatcgtga ggctgaattg tgtcgtcgta    840
    ggccggcttt tggaagtagg atatgtactt agatcatctg ctcttttcgc tttggaactt    900
    tctatttgtg ttattcagaa tttcttgccc atgtaactag tgctgttatc acaatttatg    960
    tcgattatgt gtttgcctaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa               1009
    <200> SEQUENCE CHARACTERISTICS:
    <210> SEQ ID NO 16
    <211> LENGTH: 228
    <212> TYPE: PRT
    <213> ORGANISM: Triticum aestivum
    <400> SEQUENCE:  16
    Glu Pro Gly Val Lys His Ala Leu Phe Val Gly Ile Gly Leu Gln Ile
      1               5                  10                  15
    Leu Gln Gln Phe Ala Gly Ile Asn Gly Val Leu Tyr Tyr Thr Pro Gln
                 20                  25                  30
    Ile Leu Glu Gln Ala Gly Val Gly Val Leu Leu Ser Asn Ile Gly Leu
             35                  40                  45
    Ser Ser Ser Ser Ala Ser Ile Leu Ile Ser Ala Leu Thr Thr Leu Leu
         50                  55                  60
    Met Leu Pro Ser Ile Gly Ile Ala Met Arg Leu Met Asp Met Ser Gly
     65                  70                  75                  80
    Arg Arg Phe Leu Leu Leu Ser Thr Ile Pro Val Leu Ile Val Ala Leu
                     85                  90                  95
    Ala Val Leu Val Leu Val Asn Val Leu Asp Val Gly Thr Met Val His
                100                 105                 110
    Ala Ala Leu Ser Thr Ile Ser Val Ile Val Tyr Phe Cys Phe Phe Val
            115                 120                 125
    Met Gly Phe Gly Pro Ile Pro Asn Ile Leu Cys Ala Glu Ile Phe Pro
        130                 135                 140
    Thr Ser Val Arg Gly Ile Cys Ile Ala Ile Cys Ala Leu Thr Phe Trp
    145                 150                 155                 160
    Ile Gly Asp Ile Ile Val Thr Tyr Thr Leu Pro Val Met Leu Asn Ala
                    165                 170                 175
    Ile Gly Leu Ala Gly Val Phe Gly Ile Tyr Ala Ile Val Cys Val Leu
                180                 185                 190
    Ala Phe Val Phe Val Tyr Met Lys Val Pro Glu Thr Lys Gly Met Pro
            195