Identification and characterization of a novel alpha-amylase from maize endosperm

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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BACKGROUND OF THE INVENTION

Starch is the major storage carbohydrate in higher plants. The biochemical mechanisms of starch biosynthesis and starch utilization are of interest for understanding fundamental aspects of plant physiology and also for their potential utility in manipulating the starch pathway for practical purposes. Not only is starch a critical primary source of dietary carbohydrates, but it is also used extensively for various industrial purposes ranging from formation of packaging materials to ethanol production. Despite its wide availability in nature and its many industrial applications, the mechanisms by which starch is formed and degraded in plant endosperm tissue are not well understood.

Starch consists essentially of a mixture of the homopolysaccharides amylose and amylopectin [11, 24]. Amylose is a linear chain of glucosyl units joined by alpha-1,4 glycosidic bonds and normally constitutes about 25% of the total endosperm starch in maize (Zea mays). Amylopectin comprises many linear chains of glucosyl monomers joined by alpha-1,4 linkages and constitutes approximately 75% of the starch. The chains of amylopectin are joined to each other by alpha-1,6 glycosidic bonds, often referred to as branch linkages. In amylopectin, the organized positioning of branch linkages enables periodic clustering of the linear chains [9, 15]. This permits tight and efficient packaging of glucose units, and confers crystallinity to the granule. The functional properties of starch relate directly to this architectural organization of linear chains and branch linkages in amylopectin [23].

The organization of amylopectin and amylose into higher order structures that lead to granule formation renders starch resistant to degradation. However, starch granules can be completely degraded by a combination of phosphorolysis and hydrolysis when glucose supply is required [2]. As no single enzyme has been shown to completely convert starch to simple sugars, multiple enzymes most likely are involved. Starch debranching enzymes and disproportionating enzymes are potential degradative enzymes, as are the alpha-1,4 linkage-specific hydrolases of the alpha-amylase and beta-amylase classes. Genetic evidence for involvement of an alpha-amylase in starch degradation comes from the sex, 4 mutant of Arabidopsis, which lacks such an enzyme and accumulates abnormally high levels of starch in leaves [34]. Another enzyme that likely participates in starch degradation is phosphorylase, which inserts phosphoryl groups from inorganic pyrophosphate into the alpha-1,4 glucoside bond, releasing glucose-1-phosphate.

Classification of starch degrading enzymes is made according to their behavior: endo- versus exo-mode of attack, inversion versus retention of anomeric configuration of the substrate, preference for length of the glucosyl chain, preference for the nature of the glucosyl bond, and hydrolytic versus glucosyl-transfer activity [31]. Alpha-amylases are endo-acting hydrolytic enzymes that hydrolyze internal alpha-1,4 linkages in alpha D-glucan polymers, such as amylopectin and amylose molecules. Alpha-amylases are widely distributed in nature, and are produced by plants, animals, and microorganisms [28]. Those from different sources are known to have different substrate specificities, acting preferentially on glucan chains of different lengths. This substrate specificity is dependent on the configuration of the active site of the enzyme and results in characteristic products that are formed according to the enzyme source [28]. For example, salivary gland and pancreatic alpha-amylases immediately produce low molecular weight products such as maltose and maltotriose by "multiple attack" on the substrate [27], and barley alpha-amylase primarily produces maltose, maltohexaose, and maltoheptaose without multiple attack [19]. Because alpha-amylases can hydrolyze linkages only so close to a branch point (generated by an alpha-1,6 linkage), activity halts when this physical limitation occurs. When hydrolytic activity of the alpha-amylase reaches this limit, the resulting product is termed a "limit dextrin". To achieve further hydrolysis of the limit dextrin, other enzymes must be employed, such as exo-cleaving beta-amylases or debranching enzymes. All of the alpha-amylase activities that have been described to date hydrolyze alpha D-glucans to maltose, maltotriose, or other small malto-oligosaccharides plus alpha-limit dextrins of various sizes [19, 28, 31].

Hydrolysis of starch with alpha-amylases from bacteria or fungi is routinely used by some starch industries as a first step in the process of the complete degradation of starch to glucose (this step is termed "saccharification"). The hydrolysis of starch to glucose is preliminary to the manufacture of conversion products such as high fructose corn syrup or fuel ethanol [18]. The goal of other starch processing industries is the incomplete hydrolysis of starch by various degradative enzymes, including alpha-amylases, to generate limit dextrins (termed "maltodextrins") in a range of sizes that are used for a variety of industrial purposes. For example, maltodextrins are used in food and pharmaceutical manufacturing as thickening agents, cryoprotectants and binders. They can also be further processed or chemically modified for use as viscosity or hygroscopicity or dissolving agents [13]. Different limit dextrin products are typically produced by varying the combination of enzymes used for the starch digestion, or by varying the digestion conditions. An important industrial goal is the low-energy production of specific starch hydrolysates containing few by-products [18].

In plants, alpha-amylases are believed to be involved in the hydrolysis of transient starch in the leaves, which occurs during the dark cycle of the plant, and in the hydrolysis of storage starch that accumulates in seeds or tubers, which occurs during seed germination or tuber sprouting. The first complete sequence of a plant genome, that of the Arabidopsis genome, reveals that three alpha-amylase genes are present in this plant species [14, 16]. Two are genes that encode predicted polypeptides of approximately 50-60 kilodaltons (kD), and one is a gene that encodes a larger form predicted to have a molecular mass of approximately 100 kD (Genbank Accession No. NM.sub.--105651).

Sequencing of the rice genome reveals the presence of one homolog of the Arabidopsis gene that encodes the large alpha amylase [12]. This rice gene is also predicted to encode a polypeptide of approximately 100 kD (Genbank Accession No. AP003408). In addition, the rice genome contains several genes that code for smaller sized (50-60 kD) alpha-amylases. All of the plant 50-60 kD alpha-amylases are similar in size to those from bacteria, yeast, and mammals that are used commercially. Activities of the 50-60 kD alpha-amylase enzymes from plants also are similar to those of bacterial, fungal, and mammalian alpha-amylase enzymes, in that they result in starch hydrolysis products consisting of maltose, maltotriose, or small oligosaccharides plus alpha limit dextrins. The activities of the 100 kD plant alpha-amylases and the nature of their starch hydrolysis products have not been characterized to date.

Alignment of the amino acid residues of all predicted alpha-amylases (both large and small) reveals they are highly similar in their C-terminal regions, which are believed to contain the catalytic domain of the protein [17]. The two 100 kD alpha-amylases from Arabidopsis and rice also have considerable amino acid sequence similarity, with 37% sequence identity in their N-terminal regions and 59% amino acid identity overall. The N-termini of the Arabidopsis and rice 100 kD alpha-amylases also have two small regions of similarity with another protein from Arabidopsis that has been termed the R1-protein, the product of the sex1 gene [26, 35]. Mutations in the sex1 gene result in excess starch accumulation, suggesting that a functional R1-protein is required for starch degradation. This suggests the larger 100 kD alpha-amylases from plants comprise a distinct isoform class of alpha-amylase enzymes. Further investigation into the role of the large alpha-amylase in starch metabolism, particularly in an agronomically important plant such as maize, is desirable.

BRIEF SUMMARY OF THE INVENTION

The invention is based on the discovery of a novel starch hydrolytic activity (called Starch Hydrolytic Enzyme, or SHE) in developing maize kernels. The specificity of native, purified SHE is similar, in general terms, to previously known alpha-amylases since both activities are able to hydrolyze starch, amylopectin, amylose, and beta-limit dextrin but are not able to hydrolyze the branched polymer pullulan. However, the activity of SHE toward amylopectin results in hydrolysis products that are distinctly different from those of other alpha-amylases. Specifically, they are of the same approximate molecular mass as beta-limit dextrins and do not include maltose or malto-oligosaccharides. This unique activity suggests that the novel maize alpha-amylase is an endo-hydrolytic enzyme that specifically cleaves long amylopectin chains (B.sub.2 or B.sub.3 chains) that extend between unit clusters in the molecule. In contrast, conventional maize amylases, in addition, clip off the smaller side chains of amylopectin. The new enzyme according to the invention, SHE, and its homologous equivalents in other plants such as rice, Arabidopsis, apple and potato, will have value in starch processing for generating different, and perhaps larger sized, alpha-limit dextrins for industrial use, as compared to those generated by previously known alpha-amylases or other starch hydrolytic enzymes. In addition, modification of the expression of this enzyme in transgenic maize plants or in other transgenic organisms (including bacteria, yeast, and other plant species) can be useful for the generation of novel starch forms or altered starch metabolism.

The cDNA encoding the new enzyme according to the invention, SHE, has also been isolated and sequenced. cDNA sequences encoding SHE or portions thereof can be incorporated into replicable expression vectors and the vectors transfected into an appropriate host (e.g., bacterial, yeast, eucaryotic cell culture). Alternatively, genomic DNA fragments encoding SHE can be utilized in situ. The SHE protein, in either naturally occurring or recombinant form, can be used in the starch processing industry or in other industries that employ starch for any purpose. The protein, or fragments thereof, also can be employed as an immunogen in order to raise antibodies against SHE.

Thus, the invention generally features a Starch Hydrolytic Enzyme, SHE, or portions thereof; nucleic acid isolates encoding SHE or portions thereof; methods of producing SHE or portions thereof; cells transformed with a recombinant vector containing a SHE-encoding alpha-amylase3 (Amy3) gene; antibodies to SHE or fragments thereof and methods to produce such antibodies; transgenic plants containing a SHE gene and methods to produce such transgenic plants; and methods of using a protein having SHE hydrolytic activity for starch degradation.

The invention also features a nucleic acid isolate able to hybridize under stringent conditions to the complement of a nucleic acid sequence encoding SHE, and the protein or polypeptide fragment, e.g., immunogenic fragment, thereof encoded by the nucleic acid isolate. The invention, furthermore, features a recombinant expression vector comprising a nucleic acid isolate able to hybridize under stringent conditions to the complement of a sequence encoding SHE, cells transformed with the recombinant expression vector, and methods of expressing the SHE protein or polypeptide fragment encoded within the recombinant expression vector.

Also featured is a method of producing the SHE protein, or polypeptide fragment thereof, comprising transforming a host cell with a nucleic acid able to hybridize under stringent conditions to a nucleic acid sequence encoding the SHE protein and linked to a nucleic acid sequence under the control of an inducible promotor, and inducing the cell to produce a fusion protein comprising the SHE protein, or polypeptide fragment thereof. The invention also features a SHE fusion protein, methods of producing antibodies to a SHE fusion protein and antibodies produced by such method.

As used herein, the terms "isolated" or "purified" refer to a nucleic acid or protein sequence that has been separated or isolated from the environment in which it was prepared or in which it naturally occurs. Such nucleic acid or protein sequences may be in the form of chimeric hybrids or fusions, useful for combining the function of the nucleic acid or protein sequences of the invention with other species and also include recombinant forms. The term "determinant" as used herein includes the site on an antigen at which a given antibody molecule binds. The term "immunogenic fragment" refers to a fragment of SHE protein that reacts with antibodies specific for a determinant of SHE.

The SHE protein can be used as an alternative hydrolase, along with bacterial and fungal starch hydrolases and debranching enzymes, for industrial starch processing applications. SHE-encoding cDNA (Amy3), SHE-encoding genomic DNA (Amy3), or portions thereof may be utilized as markers for the identification of specific corn varieties, and for the development of corn varieties with starch properties tailored for specific industrial applications. Amy3 cDNA or genomic DNA fragments can be used to produce these proteins or peptide fragments or as probes to identify nucleic acid molecules encoding related proteins or polypeptides (e.g., homologous polypeptides from related species and heterologous molecules from the same species). Assays for SHE function, production or expression by cells are made possible by the development of antibodies reactive with the SHE protein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings in which:

FIG. 1 is a gel showing two-dimensional separation of starch-metabolizing enzymes from developing maize endosperm;

FIG. 2 is a flow chart showing a purification scheme for purifying the Starch Hydrolytic Enzyme (SHE) according to the invention;

FIG. 3 is a gel analysis of hydrolysis results showing the specificity of SHE toward different polysaccharides;

FIGS. 4A and 4B are graphs showing FACE analysis of hydrolysis products of amylopectin incubated with SHE (FIG. 4A) and a conventional alpha-amylase (FIG. 4B);

FIG. 5 is a graph showing GPG analysis (Sepharose CL-2B column) of the hydrolysis products of amylopectin incubated with purified SHE;

FIG. 6 is a graph showing GPG analysis (Sepharose CL-2B column) of the hydrolysis products of beta-limit dextrin incubated with purified SHE, overlaid on the graph of FIG. 5;

FIG. 7A is a graph showing GPC determination (Superose 6 column) of the molecular weight of SHE under native conditions;

FIG. 7B is a gel showing a molecular weight determination of SHE under denaturing conditions by SDS-PAGE 7% analysis;

FIG. 8 is a chart showing the results of MALDI-TOF analysis of tryptic peptides produced from digestion of the 94 kD form of SHE;

FIGS. 9A and 9B show the nucleotide sequence of ZmAmy3 cDNA (SEQ ID No: 1) aligned with the amino acid sequence (single letter code) of the encoded SHE protein (SEQ ID NO: 2), according to the invention;

FIGS. 10A and 10B show a sequence alignment of the SHE protein according to the invention (SEQ ID NO: 2) with the predicted polypeptide sequences for both the rice (SEQ ID NO: 15) and the Arabidopsis 100 kD alpha-amylases (AMY3) (SEQ ID NO: 16); and

FIG. 11 shows a hypothetical reaction mechanism for SHE activity.

DETAILED DESCRIPTION OF THE INVENTION

Two-dimensional native PAGE/activity gel analysis (i.e., starch zymogram analysis) of proteins from developing maize kernels harvested 20 days after pollination (DAP) was used to identify a novel maize starch hydrolytic activity. As shown in FIG. 1, proteins in specific anion exchange chromatography fractions were separated by electrophoresis through a native polyacrylamide gel and then transferred to another polyacrylamide gel containing starch. Enzymatic activity that altered the starch substrate in the gel was visualized by staining with iodine solution. A distinct white activity band indicating starch hydrolysis, and not correlated with the activity of known starch hydrolytic enzymes, was identified in chromatography fractions 19-23.

This newly identified enzymatic activity was specifically isolated in a series of purification steps, including ammonium sulfate precipitation, anion exchange chromatography, gel permeation chromatography and affinity electrophoresis, as diagramed in FIG. 2. These purification steps resulted in a preparation of the enzyme that was devoid of other starch metabolizing activities. Those fractions containing purified SHE activity were pooled and used to carry out various incubation experiments with different polysaccharide substrates.

The glucan substrate specificity of this novel enzyme, called here Starch Hydrolytic Enzyme (SHE), was determined by zymogram analysis. As shown in FIG. 3, the specificity of SHE is similar, in general terms, to that of previously known alpha-amylases. Both activities are able to hydrolyze starch (Sta), amylopectin (Ap), amylose (Am) and the beta-limit dextrin of amylopectin (.beta.-LD), but they are not able to hydrolyze the branched isomaltotriose polymer called pullulan (Pul).

Differences between SHE activity and that of conventional alpha-amylases were detected, however, after extended incubation of the purified SHE protein with starch, amylopectin, and beta-limit dextrin. Each individual substrate was incubated overnight with SHE or with a lower molecular weight alpha-amylase similarly purified from the same maize endosperm tissue (a "conventional" alpha-amylase). The hydrolysis products were analyzed by fluorophore-assisted capillary electrophoresis (FACE) [7, 25]. As indicated in FIG. 4A, SHE activity does not release small oligosaccharides (i.e., short chains consisting of 1 to 8 units of glucose), in contrast to the activity of the conventional maize alpha-amylase (FIG. 4B).

The hydrolysis products were further characterized by gel permeation chromatography on a Sepharose CL-2B column. FIG. 5 displays the analysis of hydrolysis products that resulted from the incubation of amylopectin with purified SHE. In addition, FIG. 6 compares the analysis of FIG. 5 with that of hydrolysis products resulting from the incubation of beta-limit dextrin with SHE. These experiments demonstrate that purified SHE activity hydrolyzes both branched polysaccharides, as indicated by significant decreases in the molecular mass of each. However, the results also indicate that the hydrolysis products themselves are of high molecular weight, because no short glucosyl chains were detected by the FACE analysis (see FIG. 4).

The apparent molecular weight of SHE was determined by gel permeation chromatography following passage of the purified protein over a Superose-6 column. Comparison of the migration of the activity to that of known molecular weight standards provided the estimate that SHE is approximately 366 kD (FIG. 7A). However, analysis of purified SHE under denaturing conditions by SDS-PAGE, followed by staining with Coomassie-Blue (FIG. 7B), revealed that the molecular weight of the SHE polypeptide monomer is approximately 94 kD. This suggests that the purified SHE activity results from the formation of an enzyme complex that most likely is comprised of four SHE subunits.

The identity of the purified protein shown in FIG. 7B was established by subjecting the 94 kD polypeptide to mass spectrometric analysis (MALDI-TOF) following trypsin digestion. The mass of each tryptic peptide was determined, and these were compared to the masses of tryptic peptides of all known proteins available in the databases. Database analysis determined that the protein identity is closest to that of the 100 kD alpha-amylase that is the predicted product of the rice large alpha-amylase gene (AMY3)(Genbank AP003408) (FIG. 8).

Based on the results of the mass spectrophotometric analysis, the rice Amy3 gene sequence was used to search the Maize Gene Database for similar sequences. This database contains a partial sequence of the maize genome, including "expressed sequence tags" (ESTs) representing partial gene sequences. The database search uncovered a partial maize polypeptide sequence predicted from a 548 nt EST sequence (Genbank PCO139185) that closely matches the predicted polypeptide sequences for both the rice and the Arabidopsis 100 kD alpha-amylases (At1 g69830), as shown in FIG. 10.

The full-length coding sequence for the maize Amy3 (ZmAmy3) cDNA was PCR amplified using gene-specific primers complementary to the 3' end of the maize EST and degenerate primers based on the 5' region of the rice Amy3 gene sequence. The 2640 bp ZmAmy3 cDNA product (FIGS. 9A and 9B, SEQ ID NO.:1) is predicted to code for a polypeptide of approximately 99 kDa (SHE) (FIGS. 9A and 9B, SEQ ID NO.:2). At the amino acid level, the maize and rice sequences are 97% identical over the length of the polypeptide fragment predicted from the maize EST, and at the nucleic acid level the maize and rice sequences are 86% identical for this region. This high degree of sequence identity indicates that the maize EST derives from the maize Amy3 gene and that the purified SHE protein and the predicted rice AMY3 protein are homologous.

Comparisons of the deduced, full-length SHE amino acid sequence with the corresponding AMY3 sequences from rice and Arabidopsis revealed that all three large alpha-amylase polypeptides are closely conserved. Overall, the maize SHE sequence has 79% identity with the rice AMY3 polypeptide and 62% identity with the Arabidopsis AMY3 polypeptide. The rice and Arabidopsis AMY3 amino acid sequences are 59% identical. In the N-terminal regions, the rice and maize AMY3 polypeptides are 60% identical. Because the three known AMY3 polypeptides (including maize SHE) also have high sequence similarity to 50-60 kD AMY1 and AMY2 sequences from rice, Arabidopsis, and maize at their C-termini, they represent divergent plant alpha-amylase isoforms. The enzymatic activity of this class of alpha-amylase isoforms (AMY3) has not been characterized to date.

USE

The starch hydrolytic activity from maize kernels (SHE) described herein exhibits a novel alpha-amylase activity. As indicated above, the activity of this enzyme toward amylopectin results in hydrolysis products that are of the same approximate molecular mass as beta-limit dextrins. However, SHE activity does not result in the production of maltose or malto-oligosaccharides, as would be expected from any known, conventional alpha-amylase. This unique activity suggests that SHE is an endo-hydrolytic enzyme that specifically cleaves long amylopectin chains (B.sub.2 or B.sub.3 chains) that extend between unit clusters in the molecule, thus generating larger sized alpha-limit dextrins.

Referring to FIG. 11, a typical branched glucan substrate such as amylopectin 10, in which branch chains are arranged in distinct clusters 12, 14 connected by single (non-branched) B.sub.2 or B.sub.3 chains 16, would be acted upon differently by SHE and a conventional alpha-amylase. According to this model, SHE, by virtue of its assembly state and size (a 366 kD tetramer 18), would be barred from access (pathway 20) to the glucan chains in the interior regions of amylopectin, clusters 12, 14. Thus, SHE hydrolysis would be limited only to those regions of the amylopectin molecule that are accessible to the enzyme, for example the long linear chains 16 that extend between individual cluster units. No other enzyme is known that cleaves starch specifically in regions external to the unit clusters, producing larger sized alpha-limit dextrins 26. This is in stark contrast to the action (pathway 22) of conventional, smaller sized alpha-amylases, which function as monomers 24 and are likely to penetrate all regions of the amylopectin molecule, producing a mixture of small oligosaccharides and larger products 28.

Use of the ZmAmy3 cDNA that encodes SHE will make possible the isolation of active portions of SHE protein and, thus, the development of highly active, recombinant enzyme preparations for starch processing. In addition, ZmAmy3 cDNA can be used to isolate the cDNA encoding the homologous AMY3 enzymes from other plants with important starch hydrolytic pathways, such as rice, Arabidopsis, apple and potato.

As indicated above, recombinant SHE and/or the native maize enzyme, recombinant AMY3 enzymes from other plant species and active fragments thereof will have value in starch processing for consistently generating different, larger sized alpha-limit dextrins (maltodextrins) for industrial use. In comparison, the previously known alpha-amylases could be used to generate larger sized alpha-limit dextrins only by manipulating enzyme concentrations and/or incubation times, reaction conditions that could not be counted on to consistently produce the same products from batch to batch.

These new maltodextrin products will have value in food production as thickeners, emulsifiers, ice crystal retardants, texturizing agents, and/or fat or oil substitutes [13]. In manufacturing and pharmaceutical industries, the new maltodextrin products of SHE hydrolysis will have value as coating or encapsulation agents (e.g., for tablets or drug delivery), or as adhesive or binding agents.

The new enzyme according to the invention, SHE, and its homologous equivalents also will have value for the production of high MW dextrins that can potentially be used for the manufacture of biopolymers. Native and destructured starches have long been employed as particulate fillers and in commodity plastics [33]. Biopolymer blends containing either chemically modified or native starch forms are continually being examined for their effectiveness as packaging materials and biomedical adhesive agents [8, 22]. Large maltodextrins, such as those produced by SHE, may confer altered tensile properties and reduced water sensitivity to biopolymer blends.

Furthermore, the new enzyme according to the invention, SHE, and its homologous equivalents will have value as unique enzymes that can be added to formulations designed to selectively degrade starch. In addition, modification of the expression of the SHE enzyme and/or its homologous forms in transgenic maize plants or in other transgenic organisms (including bacteria, yeast, and other plant species) can be useful for the generation of novel starch forms or altered starch metabolism.

Experimental Procedures

Maize Stocks and Allele Nomenclature

Wild type maize inbred lines in the W64A or the Oh43 inbred genetic backgrounds are used for analysis. Kernels are harvested 19-21 days after pollination (DAP), quick frozen in liquid nitrogen, and stored at -80.degree. C. Prior to protein extraction, endosperm tissue is separated from embryo and pericarp tissues.

The nomenclature follows the standard maize (Zea mays L.) genetics format [1]. Names and symbols of genetic loci are italicized. Messenger RNAs and cDNAs are designated by italic font with the first letter capitalized, whereas polypeptide symbols are not italicized and are in upper case letters. Species designations for orthologous loci are distinguished by having the first letter of both the genus and the species precede the locus designation (e.g., ZmAmy3 designates the Zea mays Amy3 cDNA).

Protein Extraction and Activity Gel Analysis

Protein isolation from endosperm is as described [7]. Briefly, frozen kernels (5 g) are ground to a fine powder in liquid nitrogen with a mortar and pestle, and the tissue is suspended in 5 mL of buffer containing 50 mM sodium acetate, pH 6, and 20 mM DTT. All of the lysates are centrifuged at 50,000 g for one hour at 4.degree. C. Protein concentrations are determined according to the method of Bradford [3, 4].

For one-dimensional native PAGE activity gel analysis (i.e., zymogram analysis), total proteins (approximately 100 .mu.g) are separated on a native polyacrylamide gel (16 cm.times.20 cm.times.0.15 cm). The resolving gel contains 7% (w/v) acrylamide (29:1 acrylamide-bisacrylamide [Sigma]) and 375 mM Tris-HCl, pH 8.8. The stacking gel contains 4% (w/v) acrylamide and 63 mM Tris-HCl, pH 6.8. Electrophoresis is conducted at 4.degree. C., 25 V cm.sup.-1 for 4 h using a Protean II cell (Bio-Rad) in an electrode buffer of 25 mM Tris, 192 mM glycine, pH 8.8, and 2 mM DTT. At the end of the run, the gel is electroblotted to a polacrylamide gel of the same size containing 7% acrylamide, 0.3% (w/v) potato starch (Sigma), and 375 mM Tris-HCl, pH 8.8. Alternative substrates to starch in the transfer gel include 0.3% (w/v) amylopectin (Sigma), 0.3% (w/v) amylose (Sigma), 0.3% (w/v) beta limit-dextrin (Megazyme), 0.3% (w/v) oyster glycogen (Sigma), and 0.3% (w/v) azure pullulan (Sigma). The transfer is performed overnight at 20 V in the electrode buffer at room temperature. Starch metabolic activities are observed by staining the gel with I.sub.2/KI solution, and the gel is photographed immediately.

For two-dimensional zymogram analysis, total proteins (40 .mu.g) are extracted as described above and loaded onto an anion exchange chromatography (MonoQ HR 5/5) using AKTA FPLC instrumentation (Amersham-Pharmacia). The MonoQ column is equilibrated with buffer A (50 mM Tris-acetate, pH 7.5; 10 mM DTT). Bound proteins are eluted with a 48 mL-linear gradient of 0 to 500 mM NaCl in buffer A containing 1M NaCl. The flow rate is 0.9 mL/min, and 1 mL fractions are collected. Proteins in each fraction are separated by non-denaturing PAGE. Following electrophoresis, proteins are transferred by electroblotting to a polyacrylamide gel of the same size containing 0.3% (w/v) starch. Starch metabolic activities are observed after staining the gel with I.sub.2/KI solution, as described [7]. Transfer is performed overnight as described for the one-dimensional zymogram.

Protein Purification

The unknown glucan hydrolytic activity (termed "SHE") detected by one- and two-dimensional native PAGE activity gel analysis was purified in a step-wise manner from crude protein extracts isolated from approximately 100 grams mid-development maize kernels. At the conclusion of each purification step, starch zymogram analysis was employed to identify the fraction(s) containing SHE activity. The first step in the purification scheme was fractionation by ammonium sulfate precipitation, in which proteins were precipitated by the slow addition of saturated ammonium sulfate to 40% saturation. After incubation at 0.degree. C. for 30 min, proteins were collected by centrifugation at 20,000 g for 20 min. The protein pellet was dissolved in 5 mL Buffer A and dialyzed twice against 400 mL Buffer A, according to previously described methods [5].

Dialyzed proteins were injected onto a FPLC MonoQ HR 5/5 column using AKTA FPLC instrumentation (Amersham-Pharmacia), preincubated with Buffer A. Bound proteins were eluted with a 48 mL-linear gradient of 0 to 500 mM NaCl in buffer A containing 1M NaCl. The flow rate was 0.9 mL/min, and 1 mL fractions were collected. MonoQ fractions containing SHE activity were pooled and further purified by gel filtration chromatography (GPC) on a Sephacryl S400 column (Amersham-Pharmacia). GPC was performed at 4.degree. C. in buffer A at a flow rate of 0.4 mL/min, and 1 mL fractions were collected. Fractions containing SHE activity were pooled and concentrated using an Amicon centricon microspin column (Millipore) to 500 .mu.L. A second GPC purification was performed by application of 100 .mu.L of the concentrated SHE-containing sample to a Superose 6 column (Amersham-Pharmacia). Superose 6 GPC was at 4.degree. C. in buffer A with a flow rate of 0.2 mL/min, and 0.3 mL fractions were collected. Identification of the Superose 6 fractions containing SHE activity was achieved by starch zymogram analysis, which also provided the final step in the purification process, determination of the affinity of the purified protein for the starch substrate in the gel.

Purification of "conventional" alpha amylase from maize was achieved by the same methods as described for the purification of SHE. In this case, the alpha amylase activity was monitored at each step in the purification process enzymatically, using the Ceralpha kit (Megazyme). Briefly, this method assays the production of p-nitrophenol at 410 nm, which results from the hydrolysis of non-reducing ends that are blocked with p-nitrophenyl maltoheptaoside. Assays for "conventional" alpha amylase activity were conducted at 30.degree. C. for 30 min and were terminated by the addition of 1% (w/v) Trizma base (Sigma).

Characterization of Purified Protein

The approximate molecular mass of SHE was determined by comparison of the elution of SHE from the analytical Superose 6 column to the elution of known MW standards from same column. Standard proteins used to calibrate the column were bovine thyroglobulin (670,000), bovine gamma globulin (158,000), chicken ovalbumin (44,000), horse myoglobin (17,000) and vitamin B-12 (1,350) (Bio-Rad).

Protein in pooled, concentrated Superose 6 fractions containing SHE activity was analyzed by SDS-PAGE on a 7% polyacrylamide gel, followed by staining of the gel with Coomassie brilliant blue and destaining with 50% methanol solution, according to standard procedures [29]. The approximate molecular mass of the strongly stained, abundant polypeptide corresponding to SHE was determined by comparison of the migration distance of the polypeptide with the migration of commercial molecular weight standards (Bio-Rad).

To identify the SHE protein in terms of its amino acid sequence, the band corresponding to SHE was excised from the SDS-polyacrylamide gel and analyzed by time-of-flight mass spectrometry (MALDI-TOF) according to standard methods [21]. At the mass spectrometry facility (Protein Facility, Iowa State University), the polypeptide was digested with the protease trypsin. The peptide fragments were concentrated, fractionated by capillary electrophoresis, and the eluent from the capillary was directly injected into the electrospray mass spectrometer, which separated the individual peptides. Computational algorithms utilized the differences between fragment masses to reveal the amino acid sequence of the original peptide, based on the expectation of fragmentation by cleavage of the peptide bonds. The peptide sequences were then compared to proteins in the public databases, enabling the sequence match of a peptide in a given gel band to a peptide within a protein sequence in the database.

Characterization of Hydrolysis Products

Purified SHE activity was analyzed by incubation of 20 .mu.L of the pooled and concentrated Superose 6 fractions containing SHE activity with 100 .mu.L of a 1% amylopectin (Sigma) or 1% beta limit-dextrin (Megazyme) solution in a total volume of 200 .mu.L. Incubation was at 37.degree. C. for 24 h. Control incubations with both substrates also were conducted using "conventional" maize alpha amylase purified from developing maize endosperm, under the same conditions described for SHE incubation. Equivalent amounts of SHE and the alpha amylase control proteins were determined by quantification of the protein sample according to standard methods [3].

SHE amylopectin hydrolysis products were analyzed using a modified protocol for fluorophore-assisted carbohydrate electrophoresis (FACE) [7, 25]. Briefly, a 10 .mu.L aliquot of the SHE amylopectin hydrolysis products was lyophilized, then resuspended in 30% DMSO and boiled for 10 min. A 10 .mu.L aliquot was diluted to a final volume of 50 .mu.L with 50 mM sodium acetate, pH 4.5. Pseudomonas sp. isoamylase (1 .mu.L, 0.3 units) (Catalog No. E-ISAMY, Megazyme International, Bray, Ireland) was added and the reaction incubated overnight at 42.degree. C. The mixture was heated in boiling water for 5 min and then centrifuged for 2 min at 12,000 g. A 10 .mu.L sample of the reaction was evaporated to dryness in a Speed Vac. The reducing ends of the liberated oligosaccharide chains were derivatized with the fluorescent compound 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) (Catalog No. 09341, Sigma-Aldrich, St. Louis, Mo.) by suspending the dried sample in 2 .mu.L of 1 M sodium cyanoborohydride in tetrahydrafuran (Catalog No. 29,681-3, Sigma-Aldrich) and 2 .mu.L APTS (0.1 mg/.mu.L in 15% acetic acid). The reaction was incubated overnight at 42.degree. C., diluted with 46 .mu.L water, vortexed, and centrifuged briefly in a microfuge. A 5 .mu.L aliquot was added to 195 .mu.L purified water, and this sample was applied to a Beckman P/ACE capillary electrophoresis instrument. The sample injection parameters were 5 s at 0.5 psi. Separation was accomplished at 23.5 kV in an uncoated capillary using Carbohydrate Separation Gel Buffer N (Catalog Nos. 338451 and 477623, respectively, Beckman Coulter, Inc., Fullerton, Calif.).

SHE amylopectin and beta-limit dextrin hydrolysis products also were analyzed by GPC, using a Sephacryl CL-2B column (Amersham-Pharmacia; O=18 cm; H=50 cm) equilibrated with 10 mM NaOH. A 100 .mu.L volume of the hydrolysis product was applied to the CL-2B column, and eluted at a flow rate of 12 mL/h in 1.4 mL fractions. Aliquots (30 .mu.L) of each fraction were incubated with 50 .mu.L amyloglucosidase solution (0.3 U in a 50 mM sodium citrate buffer, pH 4.6; Megazyme). The .mu.g of glucose equivalents in each fraction was determined by the colorimetric glucose oxidase/peroxidase method (Sigma Diagnostics).

PCR Amplification, Cloning of ZmAmy3 cDNA and Nucleotide Sequence Analysis of the ZmAmy3 cDNA

Total RNA was isolated from approximately 10 g Zea mays kernels (Oh43 inbred background) harvested 19 days after pollination, using a modification of the protocol reported by Chomczynski and Sacchi [6]. Briefly, frozen kernels were ground to a powder in liquid nitrogen, and RNA was extracted with Trizol reagent (Invitrogen). Addition of chloroform separated polysaccharides and DNA from the RNA-containing aqueous fraction. The RNA was then precipitated and air-dried, resuspended in water, and treated with DNase. The RNA was further purified using an RNeasy Plant Mini Kit (Qiagen).

Approximately 5 .mu.g total RNA from developing maize kernels was reverse transcribed (RT) using a Superscript III First-Strand Synthesis System for RT-PCR kit (Invitrogen), using the oligo-(dT)18 primer provided. The RT product was used as the template for PCR amplification of a 2451 bp fragment of the ZmAmy3 cDNA. Five ZmArny3 gene-specific primers were designed based on the sequence of an EST fragment in the maize genome (Genbank accession number PCO139185). These primers are designated KS052 (5'-GCC AAG TCT ATG AAG ACG CTT CC-3') (SEQ ID NO: 3), KS055 (5'-GCT GAT GGA GCA GGA AAC TC-3') (SEQ ID NO: 4), KS056 (5'-CTT CAG GCG ACA CAG AAT CA-3') (SEQ ID NO: 5), KS057 (5'-CTA CAA TCA GGA TGC CCA CA-3') (SEQ ID NO: 6), and KS058 (5'-AAC AAA GTT GAC AGC GGC GAT TGG A-3') (SEQ ID NO: 7). Degenerate primers were designed based on predicted orthologous sequences of the Arabidopsis thaliana Amy3 gene (Genbank accession number BT000643) and Oryza sativa Amy3 gene (Genbank accession number NM.sub.13 191752). The degenerate primers are KS047(5'-GGV AAR TGG GTS TTR CAT TGG GG-3') (SEQ ID NO: 8) and KS048 (5'-GGV AAR TGG GTS CTS CAT TGG GG-3') (SEQ ID NO: 9) (V=A, C or G; R=A or G; S=G or C). PCR amplification was conducted according to the protocol specified by the Accuzyme Pfx PCR Amplification kit (Invitrogen), using 500 .eta.g of the template DNA, and equivalent amounts (0.5 .rho.mol, final concentration) of primers KS052, KS047, and KS048. The complete nucleotide sequence of both strands of the amplified fragment was obtained using the three PCR primers as well as primers KS055, KS056, and KS057.

To obtain the 5' end of the ZmAmy3 cDNA, the rapid amplification of cDNA ends (RACE) [10] protocol was employed, using the GeneRacer kit (Invitrogen), according to instructions provided with the kit. Briefly, 5 .mu.g RNA from maize kernels was reverse transcribed using the ZmAmy3-specific primer KS052 (0.5 .rho.mol, final concentration). PCR amplification of the 5' end was performed with Accuzyme Pfx polymerase, according to the kit protocol, using primer KS052 and a primer provided by the GeneRacer kit (5'-CGA CTG GAG CAC GAG GAC ACT GA-3') (SEQ ID NO: 10). The amplified DNA fragments were gel purified using the Qiaquick Gel Extraction kit (Qiagen). The purified PCR product was used as the template for a second-round PCR reaction using a nested ZmAmy3-specific primer KS059 (5'-GGG CTG TCC TTC TGA ATT GGG CAA A-3') (SEQ ID NO: 11) and a nested GeneRacer primer (5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3') (SEQ ID NO: 12). The amplified products were gel purified and sequenced using the same primers that were used for the amplification.

The PCR fragment containing the amplified ZmArny3 cDNA was re-amplified for the purpose of cloning the fragment into a plasmid vector. Following a protocol based on the recombination-mediated cloning strategy of the Gateway Technology system (Invitrogen), two new PCR primers were used for the amplification: KS062 (5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTG GGA AGT GGG TAC TGC ACT GGG G-3') (SEQ ID NO: 13), and KS063 (5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG CCA AGT CTA TGA AGA CGC TTC C-3') (SEQ ID NO: 14). The PCR product was gel purified and cloned into the Gateway cloning vector pDONR221 using the Invitrogen BP Clonase Enzyme Mix, according to the recommended protocol, generating plasmid pKS024. E. coil cells (DH-5.alpha.) were transformed with the plasmid DNA and screened for successful transformation events on LB media containing kanamycin. Plasmid DNA was isolated from successful transformants and the identity of pKS024 is confirmed by restriction enzyme analysis. Plasmid DNA was digested with both ApaI and HindIII, which produces fragments of 3738 and 1114 bp; BamHI, which produces fragments of 4449 and 553 bp; and with EcoRV and produces fragments of 2903 and 2100 bp.

The nucleotide sequence of plasmid pKS024 was determined by the chain termination method [30] using Sequenase Version 2.0 (U.S. Biochemical Corp.). The plasmid has been deposited with the American Type Culture Collection.

DEPOSITS

Plasmid pKS024 was deposited on Sep. 24, 2004, with the American Type Culture Collection (ATCC), P.O. Box 1549, Nanassas, Va. 20108 USA, as ATCC No. PTA-6235.

Applicants' assignee, Iowa State University Research Foundation, represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited microorganism, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicants' assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.

REFERENCES

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P., Reis, R. L. and Hunt, J. A. 2002. The biocompatibility of novel starch-based polymers and composites: in vitro studies. Biomaterials 23: 1471-1478. 23. Martin, C., Smith A. M. 1995. Starch biosynthesis. Plant Cell 7: 971-985. 24. Myers, A. M., Morell, M. K., James, M. G. and Ball, S. G. 2000. Recent progress toward understanding the amylopectin crystal. Plant Physiol 122. 25. O'Shea, M. G., Samuel, M. S., Konik, C. M. and Morell, M. K. 1998. Fluorophore-assisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labelling and high-resolution separation. Carbohydr Res 307: 1-12. 26. Ritte, G., Lorberth, R. and Steup, M. 2000. Reversible binding of the starch-related R1 protein to the surface of transitory starch granules [In Process Citation]. Plant J 21: 387-391. 27. Robyt, J. F.: Enzymes in the hydrolysis and synthesis of starch. In: Whistler, R. L., BeMiller, J. M. and Paschall, E. F. (eds) Starch: Chemistry and technology, pp. 87-123. Academic Press, Orlando (1984). 28. Robyt, J. F.: Essentials of Carbohydrate Chemistry. Springer, N.Y. (1998). 29. Sambrook, J., Fritsch, E. F. and Maniatis, T.: Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). 30. Sanger, F., Nicklen, S. and Coulson, A. R. 1977. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences 74: 5463-5467. 31. Sogaard, M., Abe, J., Martin-Eauclaire, F. and Svensson, B. 1993. .alpha.-Amylases: structure and function. Carbohydrate Polymers 21: 137-146. 32. Svensson, B. 1994. Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Mol Biol 25: 141-157. 33. van Soest, J. J. and Vliegenthart, J. F. 1997. Crystallinity in starch plastics: consequences for material properties. Trends Biotechnol 15: 208-213. 34. Zeeman, S. C., Northrop, F., Smith, A. M. and Rees, T. 1998. A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme. Plant J 15: 357-365. 35. Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A.M. and Chen, J. 1998. A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and phytoglycogen. Plant Cell 10: 1699-1712.

While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

SEQUENCE LISTINGS

1

16 1 2980 DNA Zea maize CDS (1)...(2640) misc_feature 4, 6, 45, 81, 86, 87, 88 n = A,T,C or G 1 atg nan aaa tct tct ctc tct ctc tcc cgc tct att ttt ctc atn cgc 48 Met Xaa Lys Ser Ser Leu Ser Leu Ser Arg Ser Ile Phe Leu Xaa Arg 1 5 10 15 ttc ggc tcc ctc aca cac tca cac gca cac acn aga gnn ncg cac aca 96 Phe Gly Ser Leu Thr His Ser His Ala His Thr Arg Xaa Xaa His Thr 20 25 30 ccc gct ctc tca gag aga gag aga gag aga gct agc cat gtc ggt ggg 144 Pro Ala Leu Ser Glu Arg Glu Arg Glu Arg Ala Ser His Val Gly Gly 35 40 45 gag ttg ttg cat tcg tgc tat ccc ggg gcc gcg gag agc gcg gtg gct 192 Glu Leu Leu His Ser Cys Tyr Pro Gly Ala Ala Glu Ser Ala Val Ala 50 55 60 ggc act gac gag gac gct gga gcg gcg ttc tcc gag acg ttc ccc ctg 240 Gly Thr Asp Glu Asp Ala Gly Ala Ala Phe Ser Glu Thr Phe Pro Leu 65 70 75 80 cgc cga tgc caa gct gtg gaa ggg aag gcg tgg gtg agg gtg gac gca 288 Arg Arg Cys Gln Ala Val Glu Gly Lys Ala Trp Val Arg Val Asp Ala 85 90 95 gag ccg gac tcc gaa ggc aag tgc aag gtc gtt gtt ggg tgt aat gtg 336 Glu Pro Asp Ser Glu Gly Lys Cys Lys Val Val Val Gly Cys Asn Val 100 105 110 gcg ggg aag tgg gta ctg cac tgg ggt gtc tcg tac gat gat gaa cat 384 Ala Gly Lys Trp Val Leu His Trp Gly Val Ser Tyr Asp Asp Glu His 115 120 125 gga aga gaa tgg gat cag cct cct tca gaa atg aga cca cct ggt tca 432 Gly Arg Glu Trp Asp Gln Pro Pro Ser Glu Met Arg Pro Pro Gly Ser 130 135 140 gtt gca atc aag gac tat gca att gaa aca cca ttg gag att ttg ccc 480 Val Ala Ile Lys Asp Tyr Ala Ile Glu Thr Pro Leu Glu Ile Leu Pro 145 150 155 160 aat tca gaa gga cag ccc ctt tat gaa atg caa atc aaa ttt gat aaa 528 Asn Ser Glu Gly Gln Pro Leu Tyr Glu Met Gln Ile Lys Phe Asp Lys 165 170 175 gac att cca atc gcc gct gtc aac ttt gtt cta aag gaa gag gaa aca 576 Asp Ile Pro Ile Ala Ala Val Asn Phe Val Leu Lys Glu Glu Glu Thr 180 185 190 ggt gca tgg ttt cag cat aag ggc agg gat ttc aga ata ccc tta aat 624 Gly Ala Trp Phe Gln His Lys Gly Arg Asp Phe Arg Ile Pro Leu Asn 195 200 205 gga tcc ttc aat gat ggc gga aaa caa gat att gat atc tgg cca gga 672 Gly Ser Phe Asn Asp Gly Gly Lys Gln Asp Ile Asp Ile Trp Pro Gly 210 215 220 gat ttg ggg cat gta ttg aag aaa tct gaa ggc tct agt tct cag cca 720 Asp Leu Gly His Val Leu Lys Lys Ser Glu Gly Ser Ser Ser Gln Pro 225 230 235 240 caa aac act tca cct gag gat aca ggt ttg agt ggc aaa cat ata tca 768 Gln Asn Thr Ser Pro Glu Asp Thr Gly Leu Ser Gly Lys His Ile Ser 245 250 255 ggg ttc tat gag gaa tac ccc atc ctt aaa tca gag tat gtt cag aat 816 Gly Phe Tyr Glu Glu Tyr Pro Ile Leu Lys Ser Glu Tyr Val Gln Asn 260 265 270 ctt gtt act gtc act gtg agg aga gac att gaa gcg cat aaa aga ctt 864 Leu Val Thr Val Thr Val Arg Arg Asp Ile Glu Ala His Lys Arg Leu 275 280 285 gtg gaa ttt gac act gat att cct gga gaa gtt atc att cat tgg gga 912 Val Glu Phe Asp Thr Asp Ile Pro Gly Glu Val Ile Ile His Trp Gly 290 295 300 gtt tgc aga gac aat act atg aca tgg gag atc cca cca gaa cca cat 960 Val Cys Arg Asp Asn Thr Met Thr Trp Glu Ile Pro Pro Glu Pro His 305 310 315 320 cca cca aaa acg aaa ata ttc cga cac aaa gct ctt caa act ttg ctg 1008 Pro Pro Lys Thr Lys Ile Phe Arg His Lys Ala Leu Gln Thr Leu Leu 325 330 335 cag caa aaa gct gat gga gca gga aac tca att tca ttc tca ctt gat 1056 Gln Gln Lys Ala Asp Gly Ala Gly Asn Ser Ile Ser Phe Ser Leu Asp 340 345 350 gca gag tat tct tgt ctg ttt ttt gtg ctc aaa ctt gac gag tat act 1104 Ala Glu Tyr Ser Cys Leu Phe Phe Val Leu Lys Leu Asp Glu Tyr Thr 355 360 365 tgg ttg aga aat ctt gag aat gga tct gat ttc tat gtg cca ctt aca 1152 Trp Leu Arg Asn Leu Glu Asn Gly Ser Asp Phe Tyr Val Pro Leu Thr 370 375 380 aga gtg ggg cag tat ggc agc act cag gat cct gac aag gct gag gca 1200 Arg Val Gly Gln Tyr Gly Ser Thr Gln Asp Pro Asp Lys Ala Glu Ala 385 390 395 400 cag aaa ata gag gat aag tct tca cag gct gat ggc tta atc agt gat 1248 Gln Lys Ile Glu Asp Lys Ser Ser Gln Ala Asp Gly Leu Ile Ser Asp 405 410 415 ata aga aat ctg gtg gtt ggc cta tcg tct aga aga ggt cag aaa gct 1296 Ile Arg Asn Leu Val Val Gly Leu Ser Ser Arg Arg Gly Gln Lys Ala 420 425 430 aag aat aaa gtt ctt caa gag gac atc cta caa gaa atc gaa aga ctt 1344 Lys Asn Lys Val Leu Gln Glu Asp Ile Leu Gln Glu Ile Glu Arg Leu 435 440 445 gca gca gaa gct tat agc att ttc agg agc ccc act att gat tcc gta 1392 Ala Ala Glu Ala Tyr Ser Ile Phe Arg Ser Pro Thr Ile Asp Ser Val 450 455 460 gat gaa tct gta cag ctc gat gac aca tta agc gca aag cca gca tgt 1440 Asp Glu Ser Val Gln Leu Asp Asp Thr Leu Ser Ala Lys Pro Ala Cys 465 470 475 480 tct ggc act gga tct ggt ttt gag ata ttg tgc caa gga ttt aac tgg 1488 Ser Gly Thr Gly Ser Gly Phe Glu Ile Leu Cys Gln Gly Phe Asn Trp 485 490 495 gaa tct cat aaa tca ggg aaa tgg tat gtt gag ctt ggc aca aag gcc 1536 Glu Ser His Lys Ser Gly Lys Trp Tyr Val Glu Leu Gly Thr Lys Ala 500 505 510 aag gag ttg tcg tcc ttg ggt ttc acc att gtc tgg tca cca cca cca 1584 Lys Glu Leu Ser Ser Leu Gly Phe Thr Ile Val Trp Ser Pro Pro Pro 515 520 525 act gat tct gtg tca cct gaa gga tac atg cca agg gat cta tat aat 1632 Thr Asp Ser Val Ser Pro Glu Gly Tyr Met Pro Arg Asp Leu Tyr Asn 530 535 540 cta aac tca cga tat ggg tcc atg gat gag ctg aag gaa ctt gtg aag 1680 Leu Asn Ser Arg Tyr Gly Ser Met Asp Glu Leu Lys Glu Leu Val Lys 545 550 555 560 att ttc cat gaa gct ggt atc aag gtt ctt ggc gac gct gtt cta aat 1728 Ile Phe His Glu Ala Gly Ile Lys Val Leu Gly Asp Ala Val Leu Asn 565 570 575 cat agg tgt gct cag ttt cag aac aac aat ggt gtc tgg aat ata ttt 1776 His Arg Cys Ala Gln Phe Gln Asn Asn Asn Gly Val Trp Asn Ile Phe 580 585 590 ggt ggt cgt atg aac tgg gat gat cga gca gtt gtt gct gat gat cca 1824 Gly Gly Arg Met Asn Trp Asp Asp Arg Ala Val Val Ala Asp Asp Pro 595 600 605 cat ttc cag gga aga gga aac aag agc agt gga gat aat ttc cat gca 1872 His Phe Gln Gly Arg Gly Asn Lys Ser Ser Gly Asp Asn Phe His Ala 610 615 620 gca cca aac att gat cac tcc caa gag ttt gtg agg aat gat ctt aaa 1920 Ala Pro Asn Ile Asp His Ser Gln Glu Phe Val Arg Asn Asp Leu Lys 625 630 635 640 gaa tgg ctt tgc tgg atg aga aag gaa gtc ggc tac gat gga tgg aga 1968 Glu Trp Leu Cys Trp Met Arg Lys Glu Val Gly Tyr Asp Gly Trp Arg 645 650 655 ctt gac ttt gtt cgt ggt ttc tgg ggt gga tat gtc aag gac tat ttg 2016 Leu Asp Phe Val Arg Gly Phe Trp Gly Gly Tyr Val Lys Asp Tyr Leu 660 665 670 gaa gca agt gaa cca tac ttt gca gta gga gag tac tgg gac tcc ctc 2064 Glu Ala Ser Glu Pro Tyr Phe Ala Val Gly Glu Tyr Trp Asp Ser Leu 675 680 685 agt tat act tat ggt gaa atg gac tac aat cag gat gcc cac agg cag 2112 Ser Tyr Thr Tyr Gly Glu Met Asp Tyr Asn Gln Asp Ala His Arg Gln 690 695 700 aga att gtt gat tgg ata aat gct aca aat gga act gct ggc gca ttt 2160 Arg Ile Val Asp Trp Ile Asn Ala Thr Asn Gly Thr Ala Gly Ala Phe 705 710 715 720 gat gtt acc act aaa gga ata ctt cat gcg gcg ctt gaa aga tca gag 2208 Asp Val Thr Thr Lys Gly Ile Leu His Ala Ala Leu Glu Arg Ser Glu 725 730 735 tat tgg cgc ctg tcc gat gaa aaa ggg aaa ccc cct gga gta ttg ggt 2256 Tyr Trp Arg Leu Ser Asp Glu Lys Gly Lys Pro Pro Gly Val Leu Gly 740 745 750 tgg tgg cct tca aga gca gtc aca ttt ata gag aat cat gat act ggt 2304 Trp Trp Pro Ser Arg Ala Val Thr Phe Ile Glu Asn His Asp Thr Gly 755 760 765 tct act cag ggc cat tgg agg ttc ccc tat ggt atg gaa ctg caa gga 2352 Ser Thr Gln Gly His Trp Arg Phe Pro Tyr Gly Met Glu Leu Gln Gly 770 775 780 tac gcc tac atc ctg aca cac cct ggc act ccc gca gtc ttc tat gac 2400 Tyr Ala Tyr Ile Leu Thr His Pro Gly Thr Pro Ala Val Phe Tyr Asp 785 790 795 800 cac ata ttt tca cac tta caa cca gag atc gct aaa ttt att tcc att 2448 His Ile Phe Ser His Leu Gln Pro Glu Ile Ala Lys Phe Ile Ser Ile 805 810 815 cga cac cgt caa aag att cat tgc cgc agc aag atc aag ata cta aag 2496 Arg His Arg Gln Lys Ile His Cys Arg Ser Lys Ile Lys Ile Leu Lys 820 825 830 gca gag agg agt tta tat gcg gct gaa att gat gag aag gta aca atg 2544 Ala Glu Arg Ser Leu Tyr Ala Ala Glu Ile Asp Glu Lys Val Thr Met 835 840 845 aaa atc gga tca gaa cat ttt gag cca agc ggt ccc cag aac tgg att 2592 Lys Ile Gly Ser Glu His Phe Glu Pro Ser Gly Pro Gln Asn Trp Ile 850 855 860 gtt gct gct gag ggt caa gat tac aaa atc tgg gaa gcg tct tca tag 2640 Val Ala Ala Glu Gly Gln Asp Tyr Lys Ile Trp Glu Ala Ser Ser * 865 870 875 acttggcggg ctgcgagtgc catataactg ctcaaagact aaaaaggaag ctacaagaaa 2700 gcataatgct aggagatggc gcctcagaat ggtcagctgg cgggagctgc tgccgctgcc 2760 gccatggagc agcaaaattc aacaacataa gctatggaag cacttgccac ggcgatagga 2820 ttatctaata ggactactgt tgaacattcg atatatccag aaccacccat catttgcagg 2880 cacattggcc attaaaattt agcttcgtcc tctattttgg agctatgtaa aagcatgcac 2940 aactctattt atgtgtgaaa taaaatttga aaacgcgtgg 2980 2 879 PRT Zea maize VARIANT 2, 15, 29, 30 Xaa = Any Amino Acid 2 Met Xaa Lys Ser Ser Leu Ser Leu Ser Arg Ser Ile Phe Leu Xaa Arg 1 5 10 15 Phe Gly Ser Leu Thr His Ser His Ala His Thr Arg Xaa Xaa His Thr 20 25 30 Pro Ala Leu Ser Glu Arg Glu Arg Glu Arg Ala Ser His Val Gly Gly 35 40 45 Glu Leu Leu His Ser Cys Tyr Pro Gly Ala Ala Glu Ser Ala Val Ala 50 55 60 Gly Thr Asp Glu Asp Ala Gly Ala Ala Phe Ser Glu Thr Phe Pro Leu 65 70 75 80 Arg Arg Cys Gln Ala Val Glu Gly Lys Ala Trp Val Arg Val Asp Ala 85 90 95 Glu Pro Asp Ser Glu Gly Lys Cys Lys Val Val Val Gly Cys Asn Val 100 105 110 Ala Gly Lys Trp Val Leu His Trp Gly Val Ser Tyr Asp Asp Glu His 115 120 125 Gly Arg Glu Trp Asp Gln Pro Pro Ser Glu Met Arg Pro Pro Gly Ser 130 135 140 Val Ala Ile Lys Asp Tyr Ala Ile Glu Thr Pro Leu Glu Ile Leu Pro 145 150 155 160 Asn Ser Glu Gly Gln Pro Leu Tyr Glu Met Gln Ile Lys Phe Asp Lys 165 170 175 Asp Ile Pro Ile Ala Ala Val Asn Phe Val Leu Lys Glu Glu Glu Thr 180 185 190 Gly Ala Trp Phe Gln His Lys Gly Arg Asp Phe Arg Ile Pro Leu Asn 195 200 205 Gly Ser Phe Asn Asp Gly Gly Lys Gln Asp Ile Asp Ile Trp Pro Gly 210 215 220 Asp Leu Gly His Val Leu Lys Lys Ser Glu Gly Ser Ser Ser Gln Pro 225 230 235 240 Gln Asn Thr Ser Pro Glu Asp Thr Gly Leu Ser Gly Lys His Ile Ser 245 250 255 Gly Phe Tyr Glu Glu Tyr Pro Ile Leu Lys Ser Glu Tyr Val Gln Asn 260 265 270 Leu Val Thr Val Thr Val Arg Arg Asp Ile Glu Ala His Lys Arg Leu 275 280 285 Val Glu Phe Asp Thr Asp Ile Pro Gly Glu Val Ile Ile His Trp Gly 290 295 300 Val Cys Arg Asp Asn Thr Met Thr Trp Glu Ile Pro Pro Glu Pro His 305 310 315 320 Pro Pro Lys Thr Lys Ile Phe Arg His Lys Ala Leu Gln Thr Leu Leu 325 330 335 Gln Gln Lys Ala Asp Gly Ala Gly Asn Ser Ile Ser Phe Ser Leu Asp 340 345 350 Ala Glu Tyr Ser Cys Leu Phe Phe Val Leu Lys Leu Asp Glu Tyr Thr 355 360 365 Trp Leu Arg Asn Leu Glu Asn Gly Ser Asp Phe Tyr Val Pro Leu Thr 370 375 380 Arg Val Gly Gln Tyr Gly Ser Thr Gln Asp Pro Asp Lys Ala Glu Ala 385 390 395 400 Gln Lys Ile Glu Asp Lys Ser Ser Gln Ala Asp Gly Leu Ile Ser Asp 405 410 415 Ile Arg Asn Leu Val Val Gly Leu Ser Ser Arg Arg Gly Gln Lys Ala 420 425 430 Lys Asn Lys Val Leu Gln Glu Asp Ile Leu Gln Glu Ile Glu Arg Leu 435 440 445 Ala Ala Glu Ala Tyr Ser Ile Phe Arg Ser Pro Thr Ile Asp Ser Val 450 455 460 Asp Glu Ser Val Gln Leu Asp Asp Thr Leu Ser Ala Lys Pro Ala Cys 465 470 475 480 Ser Gly Thr Gly Ser Gly Phe Glu Ile Leu Cys Gln Gly Phe Asn Trp 485 490 495 Glu Ser His Lys Ser Gly Lys Trp Tyr Val Glu Leu Gly Thr Lys Ala 500 505 510 Lys Glu Leu Ser Ser Leu Gly Phe Thr Ile Val Trp Ser Pro Pro Pro 515 520 525 Thr Asp Ser Val Ser Pro Glu Gly Tyr Met Pro Arg Asp Leu Tyr Asn 530 535 540 Leu Asn Ser Arg Tyr Gly Ser Met Asp Glu Leu Lys Glu Leu Val Lys 545 550 555 560 Ile Phe His Glu Ala Gly Ile Lys Val Leu Gly Asp Ala Val Leu Asn 565 570 575 His Arg Cys Ala Gln Phe Gln Asn Asn Asn Gly Val Trp Asn Ile Phe 580 585 590 Gly Gly Arg Met Asn Trp Asp Asp Arg Ala Val Val Ala Asp Asp Pro 595 600 605 His Phe Gln Gly Arg Gly Asn Lys Ser Ser Gly Asp Asn Phe His Ala 610 615 620 Ala Pro Asn Ile Asp His Ser Gln Glu Phe Val Arg Asn Asp Leu Lys 625 630 635 640 Glu Trp Leu Cys Trp Met Arg Lys Glu Val Gly Tyr Asp Gly Trp Arg 645 650 655 Leu Asp Phe Val Arg Gly Phe Trp Gly Gly Tyr Val Lys Asp Tyr Leu 660 665 670 Glu Ala Ser Glu Pro Tyr Phe Ala Val Gly Glu Tyr Trp Asp Ser Leu 675 680 685 Ser Tyr Thr Tyr Gly Glu Met Asp Tyr Asn Gln Asp Ala His Arg Gln 690 695 700 Arg Ile Val Asp Trp Ile Asn Ala Thr Asn Gly Thr Ala Gly Ala Phe 705 710 715 720 Asp Val Thr Thr Lys Gly Ile Leu His Ala Ala Leu Glu Arg Ser Glu 725 730 735 Tyr Trp Arg Leu Ser Asp Glu Lys Gly Lys Pro Pro Gly Val Leu Gly 740 745 750 Trp Trp Pro Ser Arg Ala Val Thr Phe Ile Glu Asn His Asp Thr Gly 755 760 765 Ser Thr Gln Gly His Trp Arg Phe Pro Tyr Gly Met Glu Leu Gln Gly 770 775 780 Tyr Ala Tyr Ile Leu Thr His Pro Gly Thr Pro Ala Val Phe Tyr Asp 785 790 795 800 His Ile Phe Ser His Leu Gln Pro Glu Ile Ala Lys Phe Ile Ser Ile 805 810 815 Arg His Arg Gln Lys Ile His Cys Arg Ser Lys Ile Lys Ile Leu Lys 820 825 830 Ala Glu Arg Ser Leu Tyr Ala Ala Glu Ile Asp Glu Lys Val Thr Met 835 840 845 Lys Ile Gly Ser Glu His Phe Glu Pro Ser Gly Pro Gln Asn Trp Ile 850 855 860 Val Ala Ala Glu Gly Gln Asp Tyr Lys Ile Trp Glu Ala Ser Ser 865 870 875 3 23 DNA Artificial Sequence PCR Primer 3 gccaagtcta tgaagacgct tcc 23 4 20 DNA Artificial Sequence PCR Primer 4 gctgatggag caggaaactc 20 5 20 DNA Artificial Sequence PCR Primer 5 cttcaggcga cacagaatca 20 6 20 DNA Artificial Sequence PCR Primer 6 ctacaatcag gatgcccaca 20 7 25 DNA Artificial Sequence PCR Primer 7 aacaaagttg acagcggcga ttgga 25 8 23 DNA

Artificial Sequence PCR Primer 8 ggvaartggg tsttrcattg ggg 23 9 23 DNA Artificial Sequence PCR Primer 9 ggvaartggg tsctscattg ggg 23 10 23 DNA Artificial Sequence PCR Primer 10 cgactggagc acgaggacac tga 23 11 25 DNA Artificial Sequence PCR Primer 11 gggctgtcct tctgaattgg gcaaa 25 12 26 DNA Artificial Sequence PCR Primer 12 ggacactgac atggactgaa ggagta 26 13 52 DNA Artificial Sequence PCR Primer 13 ggggacaagt ttgtacaaaa aagcaggctg ggaagtgggt actgcactgg gg 52 14 51 DNA Artificial Sequence PCR Primer 14 ggggaccact ttgtacaaga aagctgggtg ccaagtctat gaagacgctt c 51 15 906 PRT Oryza sativa 15 Met Ala Val Ala Ser Trp Ser Ile Pro Ala Ile Pro Arg Ala Gly Pro 1 5 10 15 Thr Ala Arg Gly Val Leu Leu Gly Gly Ala Phe Val Thr Ala Ala Arg 20 25 30 Pro Pro Val Ala Trp Arg Cys Arg Ala Thr Leu Pro Arg Arg Val Arg 35 40 45 Leu Gly Gly Val Val Ala Arg Ala Gly Ala Ala Glu Thr Pro Val Ala 50 55 60 Gly Ser Gly Glu Ala Gly Leu Leu Phe Ser Glu Lys Phe Pro Leu Arg 65 70 75 80 Arg Ser Arg Thr Val Glu Gly Lys Ala Trp Val Arg Val Asp Ala Glu 85 90 95 Pro Asp Gly Glu Gly Lys Cys Lys Val Val Ile Gly Cys Asp Val Glu 100 105 110 Gly Lys Trp Val Leu His Trp Gly Val Ser Tyr Asp Gly Glu Gln Gly 115 120 125 Arg Glu Trp Asp Gln Pro Pro Ser Asp Met Arg Pro Pro Gly Ser Val 130 135 140 Pro Ile Lys Asp Tyr Ala Ile Glu Thr Ser Leu Asp Thr Pro His Asn 145 150 155 160 Ser Glu Gly Lys Thr Ile His Glu Val Gln Ile Lys Ile Asp Lys Gly 165 170 175 Thr Ser Ile Ala Ala Ile Asn Phe Val Leu Lys Val Gln Ile Leu Arg 180 185 190 Cys Cys Ile Leu Tyr His Val Ser Lys Gly Leu Glu Val Tyr Asp Trp 195 200 205 Pro Ile Arg Phe Val Lys Leu Leu Lys Val Pro Lys Glu Glu Glu Thr 210 215 220 Gly Ala Trp Phe Gln His Lys Gly Gln Asp Phe Arg Ile Pro Leu Ser 225 230 235 240 Gly Ser Phe Gly Gly Asp Leu Leu Gly Thr Glu Gln Asp Ile Asp Val 245 250 255 Arg Pro Gly His Leu Ser Asn Val Leu Gln Lys Pro Glu Gly Pro Ile 260 265 270 Ala Glu Pro His Lys Thr Val Pro Asp Asp Lys Gly Ser Arg Thr Lys 275 280 285 His Ile Ser Gly Phe Tyr Glu Glu Tyr Pro Ile Leu Lys Thr Val Tyr 290 295 300 Val Gln Asn Phe Ile Thr Val Asn Val Arg Glu Asn Asn Gly Thr Thr 305 310 315 320 Lys His Ala Val Glu Phe Asp Thr Asp Ile Pro Gly Glu Val Ile Ile 325 330 335 His Trp Gly Val Cys Lys Asp Asn Thr Met Thr Trp Glu Ile Pro Pro 340 345 350 Glu Pro His Pro Pro Ala Thr Lys Ile Phe Arg Gln Lys Ala Leu Gln 355 360 365 Thr Met Leu Gln Gln Lys Ala Asp Gly Thr Gly Asn Ser Leu Ser Phe 370 375 380 Leu Leu Asp Gly Glu Tyr Ser Gly Leu Ile Phe Val Val Lys Leu Asp 385 390 395 400 Glu Tyr Thr Trp Leu Arg Asn Val Glu Asn Gly Phe Asp Phe Tyr Ile 405 410 415 Pro Leu Thr Arg Ala Asp Ala Glu Ala Asp Lys Gln Lys Ala Asp Asp 420 425 430 Lys Ser Ser Gln Asp Asp Gly Leu Ile Ser Asp Ile Arg Asn Leu Val 435 440 445 Val Gly Leu Ser Ser Arg Arg Gly Gln Arg Ala Lys Asn Lys Val Leu 450 455 460 Gln Glu Asp Ile Leu Gln Glu Ile Glu Arg Leu Ala Ala Glu Ala Tyr 465 470 475 480 Ser Ile Phe Arg Ser Pro Thr Ile Asp Thr Val Glu Glu Ser Val Tyr 485 490 495 Ile Asp Asp Ser Ser Ile Val Lys Pro Ala Cys Ser Gly Thr Gly Ser 500 505 510 Gly Phe Glu Ile Leu Cys Gln Gly Phe Asn Trp Glu Ser His Lys Ser 515 520 525 Gly Lys Trp Tyr Val Glu Leu Gly Ser Lys Ala Lys Glu Leu Ser Ser 530 535 540 Met Gly Phe Thr Ile Val Trp Ser Pro Pro Pro Thr Asp Ser Val Ser 545 550 555 560 Pro Glu Gly Tyr Met Pro Arg Asp Leu Tyr Asn Leu Asn Ser Arg Tyr 565 570 575 Gly Thr Met Glu Glu Leu Lys Glu Ala Val Lys Arg Phe His Glu Ala 580 585 590 Gly Met Lys Val Leu Gly Asp Ala Val Leu Asn His Arg Cys Ala Gln 595 600 605 Phe Gln Asn Gln Asn Gly Val Trp Asn Ile Phe Gly Gly Arg Leu Asn 610 615 620 Trp Asp Asp Arg Ala Val Val Ala Asp Asp Pro His Phe Gln Gly Arg 625 630 635 640 Gly Asn Lys Ser Ser Gly Asp Asn Phe His Ala Ala Pro Asn Ile Asp 645 650 655 His Ser Gln Glu Phe Val Arg Ser Asp Leu Lys Glu Trp Leu Cys Trp 660 665 670 Met Arg Lys Glu Val Gly Tyr Asp Gly Trp Arg Leu Asp Phe Val Arg 675 680 685 Gly Phe Trp Gly Gly Tyr Val His Asp Tyr Leu Glu Ala Ser Glu Pro 690 695 700 Tyr Phe Ala Val Gly Glu Tyr Trp Asp Ser Leu Ser Tyr Thr Tyr Gly 705 710 715 720 Glu Met Asp Tyr Asn Gln Asp Ala His Arg Gln Arg Ile Val Asp Trp 725 730 735 Ile Asn Ala Thr Asn Gly Thr Ala Gly Ala Phe Asp Val Thr Thr Lys 740 745 750 Gly Ile Leu His Ser Ala Leu Glu Arg Ser Glu Tyr Trp Arg Leu Ser 755 760 765 Asp Glu Lys Gly Lys Pro Pro Gly Val Leu Gly Trp Trp Pro Ser Arg 770 775 780 Ala Val Thr Phe Ile Glu Asn His Asp Thr Gly Ser Thr Gln Gly His 785 790 795 800 Trp Arg Phe Pro Phe Gly Met Glu Leu Gln Gly Tyr Val Tyr Ile Leu 805 810 815 Thr His Pro Gly Thr Pro Ala Ile Phe Tyr Asp His Ile Phe Ser His 820 825 830 Leu Gln Pro Glu Ile Ala Lys Leu Ile Ser Ile Arg Asn Arg Gln Lys 835 840 845 Ile His Cys Arg Ser Lys Ile Lys Ile Leu Lys Ala Glu Gly Asn Leu 850 855 860 Tyr Ala Ala Glu Ile Asp Glu Arg Val Thr Met Lys Ile Gly Ala Gly 865 870 875 880 His Phe Glu Pro Ser Gly Pro Thr Asn Trp Val Val Ala Ala Glu Gly 885 890 895 Gln Asp Tyr Lys Val Trp Glu Val Ser Ser 900 905 16 2980 PRT Zea miaze 16 Ala Thr Gly Asn Ala Asn Ala Ala Ala Thr Cys Thr Thr Cys Thr Cys 1 5 10 15 Thr Cys Thr Cys Thr Cys Thr Cys Thr Cys Cys Cys Gly Cys Thr Cys 20 25 30 Thr Ala Thr Thr Thr Thr Thr Cys Thr Cys Ala Thr Asn Cys Gly Cys 35 40 45 Thr Thr Cys Gly Gly Cys Thr Cys Cys Cys Thr Cys Ala Cys Ala Cys 50 55 60 Ala Cys Thr Cys Ala Cys Ala Cys Gly Cys Ala Cys Ala Cys Ala Cys 65 70 75 80 Asn Ala Gly Ala Gly Asn Asn Asn Cys Gly Cys Ala Cys Ala Cys Ala 85 90 95 Cys Cys Cys Gly Cys Thr Cys Thr Cys Thr Cys Ala Gly Ala Gly Ala 100 105 110 Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Cys 115 120 125 Thr Ala Gly Cys Cys Ala Thr Gly Thr Cys Gly Gly Thr Gly Gly Gly 130 135 140 Gly Ala Gly Thr Thr Gly Thr Thr Gly Cys Ala Thr Thr Cys Gly Thr 145 150 155 160 Gly Cys Thr Ala Thr Cys Cys Cys Gly Gly Gly Gly Cys Cys Gly Cys 165 170 175 Gly Gly Ala Gly Ala Gly Cys Gly Cys Gly Gly Thr Gly Gly Cys Thr 180 185 190 Gly Gly Cys Ala Cys Thr Gly Ala Cys Gly Ala Gly Gly Ala Cys Gly 195 200 205 Cys Thr Gly Gly Ala Gly Cys Gly Gly Cys Gly Thr Thr Cys Thr Cys 210 215 220 Cys Gly Ala Gly Ala Cys Gly Thr Thr Cys Cys Cys Cys Cys Thr Gly 225 230 235 240 Cys Gly Cys Cys Gly Ala Thr Gly Cys Cys Ala Ala Gly Cys Thr Gly 245 250 255 Thr Gly Gly Ala Ala Gly Gly Gly Ala Ala Gly Gly Cys Gly Thr Gly 260 265 270 Gly Gly Thr Gly Ala Gly Gly Gly Thr Gly Gly Ala Cys Gly Cys Ala 275 280 285 Gly Ala Gly Cys Cys Gly Gly Ala Cys Thr Cys Cys Gly Ala Ala Gly 290 295 300 Gly Cys Ala Ala Gly Thr Gly Cys Ala Ala Gly Gly Thr Cys Gly Thr 305 310 315 320 Thr Gly Thr Thr Gly Gly Gly Thr Gly Thr Ala Ala Thr Gly Thr Gly 325 330 335 Gly Cys Gly Gly Gly Gly Ala Ala Gly Thr Gly Gly Gly Thr Ala Cys 340 345 350 Thr Gly Cys Ala Cys Thr Gly Gly Gly Gly Thr Gly Thr Cys Thr Cys 355 360 365 Gly Thr Ala Cys Gly Ala Thr Gly Ala Thr Gly Ala Ala Cys Ala Thr 370 375 380 Gly Gly Ala Ala Gly Ala Gly Ala Ala Thr Gly Gly Gly Ala Thr Cys 385 390 395 400 Ala Gly Cys Cys Thr Cys Cys Thr Thr Cys Ala Gly Ala Ala Ala Thr 405 410 415 Gly Ala Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly Thr Thr Cys Ala 420 425 430 Gly Thr Thr Gly Cys Ala Ala Thr Cys Ala Ala Gly Gly Ala Cys Thr 435 440 445 Ala Thr Gly Cys Ala Ala Thr Thr Gly Ala Ala Ala Cys Ala Cys Cys 450 455 460 Ala Thr Thr Gly Gly Ala Gly Ala Thr Thr Thr Thr Gly Cys Cys Cys 465 470 475 480 Ala Ala Thr Thr Cys Ala Gly Ala Ala Gly Gly Ala Cys Ala Gly Cys 485 490 495 Cys Cys Cys Thr Thr Thr Ala Thr Gly Ala Ala Ala Thr Gly Cys Ala 500 505 510 Ala Ala Thr Cys Ala Ala Ala Thr Thr Thr Gly Ala Thr Ala Ala Ala 515 520 525 Gly Ala Cys Ala Thr Thr Cys Cys Ala Ala Thr Cys Gly Cys Cys Gly 530 535 540 Cys Thr Gly Thr Cys Ala Ala Cys Thr Thr Thr Gly Thr Thr Cys Thr 545 550 555 560 Ala Ala Ala Gly Gly Ala Ala Gly Ala Gly Gly Ala Ala Ala Cys Ala 565 570 575 Gly Gly Thr Gly Cys Ala Thr Gly Gly Thr Thr Thr Cys Ala Gly Cys 580 585 590 Ala Thr Ala Ala Gly Gly Gly Cys Ala Gly Gly Gly Ala Thr Thr Thr 595 600 605 Cys Ala Gly Ala Ala Thr Ala Cys Cys Cys Thr Thr Ala Ala Ala Thr 610 615 620 Gly Gly Ala Thr Cys Cys Thr Thr Cys Ala Ala Thr Gly Ala Thr Gly 625 630 635 640 Gly Cys Gly Gly Ala Ala Ala Ala Cys Ala Ala Gly Ala Thr Ala Thr 645 650 655 Thr Gly Ala Thr Ala Thr Cys Thr Gly Gly Cys Cys Ala Gly Gly Ala 660 665 670 Gly Ala Thr Thr Thr Gly Gly Gly Gly Cys Ala Thr Gly Thr Ala Thr 675 680 685 Thr Gly Ala Ala Gly Ala Ala Ala Thr Cys Thr Gly Ala Ala Gly Gly 690 695 700 Cys Thr Cys Thr Ala Gly Thr Thr Cys Thr Cys Ala Gly Cys Cys Ala 705 710 715 720 Cys Ala Ala Ala Ala Cys Ala Cys Thr Thr Cys Ala Cys Cys Thr Gly 725 730 735 Ala Gly Gly Ala Thr Ala Cys Ala Gly Gly Thr Thr Thr Gly Ala Gly 740 745 750 Thr Gly Gly Cys Ala Ala Ala Cys Ala Thr Ala Thr Ala Thr Cys Ala 755 760 765 Gly Gly Gly Thr Thr Cys Thr Ala Thr Gly Ala Gly Gly Ala Ala Thr 770 775 780 Ala Cys Cys Cys Cys Ala Thr Cys Cys Thr Thr Ala Ala Ala Thr Cys 785 790 795 800 Ala Gly Ala Gly Thr Ala Thr Gly Thr Thr Cys Ala Gly Ala Ala Thr 805 810 815 Cys Thr Thr Gly Thr Thr Ala Cys Thr Gly Thr Cys Ala Cys Thr Gly 820 825 830 Thr Gly Ala Gly Gly Ala Gly Ala Gly Ala Cys Ala Thr Thr Gly Ala 835 840 845 Ala Gly Cys Gly Cys Ala Thr Ala Ala Ala Ala Gly Ala Cys Thr Thr 850 855 860 Gly Thr Gly Gly Ala Ala Thr Thr Thr Gly Ala Cys Ala Cys Thr Gly 865 870 875 880 Ala Thr Ala Thr Thr Cys Cys Thr Gly Gly Ala Gly Ala Ala Gly Thr 885 890 895 Thr Ala Thr Cys Ala Thr Thr Cys Ala Thr Thr Gly Gly Gly Gly Ala 900 905 910 Gly Thr Thr Thr Gly Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Ala 915 920 925 Cys Thr Ala Thr Gly Ala Cys Ala Thr Gly Gly Gly Ala Gly Ala Thr 930 935 940 Cys Cys Cys Ala Cys Cys Ala Gly Ala Ala Cys Cys Ala Cys Ala Thr 945 950 955 960 Cys Cys Ala Cys Cys Ala Ala Ala Ala Ala Cys Gly Ala Ala Ala Ala 965 970 975 Thr Ala Thr Thr Cys Cys Gly Ala Cys Ala Cys Ala Ala Ala Gly Cys 980 985 990 Thr Cys Thr Thr Cys Ala Ala Ala Cys Thr Thr Thr Gly Cys Thr Gly 995 1000 1005 Cys Ala Gly Cys Ala Ala Ala Ala Ala Gly Cys Thr Gly Ala Thr Gly 1010 1015 1020 Gly Ala Gly Cys Ala Gly Gly Ala Ala Ala Cys Thr Cys Ala Ala Thr 1025 1030 1035 1040 Thr Thr Cys Ala Thr Thr Cys Thr Cys Ala Cys Thr Thr Gly Ala Thr 1045 1050 1055 Gly Cys Ala Gly Ala Gly Thr Ala Thr Thr Cys Thr Thr Gly Thr Cys 1060 1065 1070 Thr Gly Thr Thr Thr Thr Thr Thr Gly Thr Gly Cys Thr Cys Ala Ala 1075 1080 1085 Ala Cys Thr Thr Gly Ala Cys Gly Ala Gly Thr Ala Thr Ala Cys Thr 1090 1095 1100 Thr Gly Gly Thr Thr Gly Ala Gly Ala Ala Ala Thr Cys Thr Thr Gly 1105 1110 1115 1120 Ala Gly Ala Ala Thr Gly Gly Ala Thr Cys Thr Gly Ala Thr Thr Thr 1125 1130 1135 Cys Thr Ala Thr Gly Thr Gly Cys Cys Ala Cys Thr Thr Ala Cys Ala 1140 1145 1150 Ala Gly Ala Gly Thr Gly Gly Gly Gly Cys Ala Gly Thr Ala Thr Gly 1155 1160 1165 Gly Cys Ala Gly Cys Ala Cys Thr Cys Ala Gly Gly Ala Thr Cys Cys 1170 1175 1180 Thr Gly Ala Cys Ala Ala Gly Gly Cys Thr Gly Ala Gly Gly Cys Ala 1185 1190 1195 1200 Cys Ala Gly Ala Ala Ala Ala Thr Ala Gly Ala Gly Gly Ala Thr Ala 1205 1210 1215 Ala Gly Thr Cys Thr Thr Cys Ala Cys Ala Gly Gly Cys Thr Gly Ala 1220 1225 1230 Thr Gly Gly Cys Thr Thr Ala Ala Thr Cys Ala Gly Thr Gly Ala Thr 1235 1240 1245 Ala Thr Ala Ala Gly Ala Ala Ala Thr Cys Thr Gly Gly Thr Gly Gly 1250 1255 1260 Thr Thr Gly Gly Cys Cys Thr Ala Thr Cys Gly Thr Cys Thr Ala Gly 1265 1270 1275 1280 Ala Ala Gly Ala Gly Gly Thr Cys Ala Gly Ala Ala Ala Gly Cys Thr 1285 1290 1295 Ala Ala Gly Ala Ala Thr Ala Ala Ala Gly Thr Thr Cys Thr Thr Cys 1300 1305 1310 Ala Ala Gly Ala Gly Gly Ala Cys Ala Thr Cys Cys Thr Ala Cys Ala 1315 1320 1325 Ala Gly Ala Ala Ala Thr Cys Gly Ala Ala Ala Gly Ala Cys Thr Thr 1330 1335 1340 Gly Cys Ala Gly Cys Ala Gly Ala Ala Gly Cys Thr Thr Ala Thr Ala 1345 1350 1355 1360 Gly Cys Ala Thr Thr Thr Thr Cys Ala Gly Gly Ala Gly Cys Cys Cys 1365 1370 1375 Cys Ala Cys Thr Ala Thr Thr Gly Ala Thr Thr Cys Cys Gly Thr Ala 1380 1385 1390 Gly Ala Thr Gly Ala Ala Thr Cys Thr Gly Thr Ala Cys Ala Gly Cys 1395 1400 1405 Thr Cys Gly Ala Thr Gly Ala Cys Ala Cys Ala Thr Thr Ala Ala Gly 1410 1415

1420 Cys Gly Cys Ala Ala Ala Gly Cys Cys Ala Gly Cys Ala Thr Gly Thr 1425 1430 1435 1440 Thr Cys Thr Gly Gly Cys Ala Cys Thr Gly Gly Ala Thr Cys Thr Gly 1445 1450 1455 Gly Thr Thr Thr Thr Gly Ala Gly Ala Thr Ala Thr Thr Gly Thr Gly 1460 1465 1470 Cys Cys Ala Ala Gly Gly Ala Thr Thr Thr Ala Ala Cys Thr Gly Gly 1475 1480 1485 Gly Ala Ala Thr Cys Thr Cys Ala Thr Ala Ala Ala Thr Cys Ala Gly 1490 1495 1500 Gly Gly Ala Ala Ala Thr Gly Gly Thr Ala Thr Gly Thr Thr Gly Ala 1505 1510 1515 1520 Gly Cys Thr Thr Gly Gly Cys Ala Cys Ala Ala Ala Gly Gly Cys Cys 1525 1530 1535 Ala Ala Gly Gly Ala Gly Thr Thr Gly Thr Cys Gly Thr Cys Cys Thr 1540 1545 1550 Thr Gly Gly Gly Thr Thr Thr Cys Ala Cys Cys Ala Thr Thr Gly Thr 1555 1560 1565 Cys Thr Gly Gly Thr Cys Ala Cys Cys Ala Cys Cys Ala Cys Cys Ala 1570 1575 1580 Ala Cys Thr Gly Ala Thr Thr Cys Thr Gly Thr Gly Thr Cys Ala Cys 1585 1590 1595 1600 Cys Thr Gly Ala Ala Gly Gly Ala Thr Ala Cys Ala Thr Gly Cys Cys 1605 1610 1615 Ala Ala Gly Gly Gly Ala Thr Cys Thr Ala Thr Ala Thr Ala Ala Thr 1620 1625 1630 Cys Thr Ala Ala Ala Cys Thr Cys Ala Cys Gly Ala Thr Ala Thr Gly 1635 1640 1645 Gly Gly Thr Cys Cys Ala Thr Gly Gly Ala Thr Gly Ala Gly Cys Thr 1650 1655 1660 Gly Ala Ala Gly Gly Ala Ala Cys Thr Thr Gly Thr Gly Ala Ala Gly 1665 1670 1675 1680 Ala Thr Thr Thr Thr Cys Cys Ala Thr Gly Ala Ala Gly Cys Thr Gly 1685 1690 1695 Gly Thr Ala Thr Cys Ala Ala Gly Gly Thr Thr Cys Thr Thr Gly Gly 1700 1705 1710 Cys Gly Ala Cys Gly Cys Thr Gly Thr Thr Cys Thr Ala Ala Ala Thr 1715 1720 1725 Cys Ala Thr Ala Gly Gly Thr Gly Thr Gly Cys Thr Cys Ala Gly Thr 1730 1735 1740 Thr Thr Cys Ala Gly Ala Ala Cys Ala Ala Cys Ala Ala Thr Gly Gly 1745 1750 1755 1760 Thr Gly Thr Cys Thr Gly Gly Ala Ala Thr Ala Thr Ala Thr Thr Thr 1765 1770 1775 Gly Gly Thr Gly Gly Thr Cys Gly Thr Ala Thr Gly Ala Ala Cys Thr 1780 1785 1790 Gly Gly Gly Ala Thr Gly Ala Thr Cys Gly Ala Gly Cys Ala Gly Thr 1795 1800 1805 Thr Gly Thr Thr Gly Cys Thr Gly Ala Thr Gly Ala Thr Cys Cys Ala 1810 1815 1820 Cys Ala Thr Thr Thr Cys Cys Ala Gly Gly Gly Ala Ala Gly Ala Gly 1825 1830 1835 1840 Gly Ala Ala Ala Cys Ala Ala Gly Ala Gly Cys Ala Gly Thr Gly Gly 1845 1850 1855 Ala Gly Ala Thr Ala Ala Thr Thr Thr Cys Cys Ala Thr Gly Cys Ala 1860 1865 1870 Gly Cys Ala Cys Cys Ala Ala Ala Cys Ala Thr Thr Gly Ala Thr Cys 1875 1880 1885 Ala Cys Thr Cys Cys Cys Ala Ala Gly Ala Gly Thr Thr Thr Gly Thr 1890 1895 1900 Gly Ala Gly Gly Ala Ala Thr Gly Ala Thr Cys Thr Thr Ala Ala Ala 1905 1910 1915 1920 Gly Ala Ala Thr Gly Gly Cys Thr Thr Thr Gly Cys Thr Gly Gly Ala 1925 1930 1935 Thr Gly Ala Gly Ala Ala Ala Gly Gly Ala Ala Gly Thr Cys Gly Gly 1940 1945 1950 Cys Thr Ala Cys Gly Ala Thr Gly Gly Ala Thr Gly Gly Ala Gly Ala 1955 1960 1965 Cys Thr Thr Gly Ala Cys Thr Thr Thr Gly Thr Thr Cys Gly Thr Gly 1970 1975 1980 Gly Thr Thr Thr Cys Thr Gly Gly Gly Gly Thr Gly Gly Ala Thr Ala 1985 1990 1995 2000 Thr Gly Thr Cys Ala Ala Gly Gly Ala Cys Thr Ala Thr Thr Thr Gly 2005 2010 2015 Gly Ala Ala Gly Cys Ala Ala Gly Thr Gly Ala Ala Cys Cys Ala Thr 2020 2025 2030 Ala Cys Thr Thr Thr Gly Cys Ala Gly Thr Ala Gly Gly Ala Gly Ala 2035 2040 2045 Gly Thr Ala Cys Thr Gly Gly Gly Ala Cys Thr Cys Cys Cys Thr Cys 2050 2055 2060 Ala Gly Thr Thr Ala Thr Ala Cys Thr Thr Ala Thr Gly Gly Thr Gly 2065 2070 2075 2080 Ala Ala Ala Thr Gly Gly Ala Cys Thr Ala Cys Ala Ala Thr Cys Ala 2085 2090 2095 Gly Gly Ala Thr Gly Cys Cys Cys Ala Cys Ala Gly Gly Cys Ala Gly 2100 2105 2110 Ala Gly Ala Ala Thr Thr Gly Thr Thr Gly Ala Thr Thr Gly Gly Ala 2115 2120 2125 Thr Ala Ala Ala Thr Gly Cys Thr Ala Cys Ala Ala Ala Thr Gly Gly 2130 2135 2140 Ala Ala Cys Thr Gly Cys Thr Gly Gly Cys Gly Cys Ala Thr Thr Thr 2145 2150 2155 2160 Gly Ala Thr Gly Thr Thr Ala Cys Cys Ala Cys Thr Ala Ala Ala Gly 2165 2170 2175 Gly Ala Ala Thr Ala Cys Thr Thr Cys Ala Thr Gly Cys Gly Gly Cys 2180 2185 2190 Gly Cys Thr Thr Gly Ala Ala Ala Gly Ala Thr Cys Ala Gly Ala Gly 2195 2200 2205 Thr Ala Thr Thr Gly Gly Cys Gly Cys Cys Thr Gly Thr Cys Cys Gly 2210 2215 2220 Ala Thr Gly Ala Ala Ala Ala Ala Gly Gly Gly Ala Ala Ala Cys Cys 2225 2230 2235 2240 Cys Cys Cys Thr Gly Gly Ala Gly Thr Ala Thr Thr Gly Gly Gly Thr 2245 2250 2255 Thr Gly Gly Thr Gly Gly Cys Cys Thr Thr Cys Ala Ala Gly Ala Gly 2260 2265 2270 Cys Ala Gly Thr Cys Ala Cys Ala Thr Thr Thr Ala Thr Ala Gly Ala 2275 2280 2285 Gly Ala Ala Thr Cys Ala Thr Gly Ala Thr Ala Cys Thr Gly Gly Thr 2290 2295 2300 Thr Cys Thr Ala Cys Thr Cys Ala Gly Gly Gly Cys Cys Ala Thr Thr 2305 2310 2315 2320 Gly Gly Ala Gly Gly Thr Thr Cys Cys Cys Cys Thr Ala Thr Gly Gly 2325 2330 2335 Thr Ala Thr Gly Gly Ala Ala Cys Thr Gly Cys Ala Ala Gly Gly Ala 2340 2345 2350 Thr Ala Cys Gly Cys Cys Thr Ala Cys Ala Thr Cys Cys Thr Gly Ala 2355 2360 2365 Cys Ala Cys Ala Cys Cys Cys Thr Gly Gly Cys Ala Cys Thr Cys Cys 2370 2375 2380 Cys Gly Cys Ala Gly Thr Cys Thr Thr Cys Thr Ala Thr Gly Ala Cys 2385 2390 2395 2400 Cys Ala Cys Ala Thr Ala Thr Thr Thr Thr Cys Ala Cys Ala Cys Thr 2405 2410 2415 Thr Ala Cys Ala Ala Cys Cys Ala 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2620 Gly Gly Ala Ala Gly Cys Gly Thr Cys Thr Thr Cys Ala Thr Ala Gly 2625 2630 2635 2640 Ala Cys Thr Thr Gly Gly Cys Gly Gly Gly Cys Thr Gly Cys Gly Ala 2645 2650 2655 Gly Thr Gly Cys Cys Ala Thr Ala Thr Ala Ala Cys Thr Gly Cys Thr 2660 2665 2670 Cys Ala Ala Ala Gly Ala Cys Thr Ala Ala Ala Ala Ala Gly Gly Ala 2675 2680 2685 Ala Gly Cys Thr Ala Cys Ala Ala Gly Ala Ala Ala Gly Cys Ala Thr 2690 2695 2700 Ala Ala Thr Gly Cys Thr Ala Gly Gly Ala Gly Ala Thr Gly Gly Cys 2705 2710 2715 2720 Gly Cys Cys Thr Cys Ala Gly Ala Ala Thr Gly Gly Thr Cys Ala Gly 2725 2730 2735 Cys Thr Gly Gly Cys Gly Gly Gly Ala Gly Cys Thr Gly Cys Thr Gly 2740 2745 2750 Cys Cys Gly Cys Thr Gly Cys Cys Gly Cys Cys Ala Thr Gly Gly Ala 2755 2760 2765 Gly Cys Ala Gly Cys Ala Ala Ala Ala Thr Thr Cys Ala Ala Cys Ala 2770 2775 2780 Ala Cys Ala Thr Ala Ala Gly Cys Thr Ala Thr Gly Gly Ala Ala Gly 2785 2790 2795 2800 Cys Ala Cys Thr Thr Gly Cys Cys Ala Cys Gly Gly Cys Gly Ala Thr 2805 2810 2815 Ala Gly Gly Ala Thr Thr Ala Thr Cys Thr Ala Ala Thr Ala Gly Gly 2820 2825 2830 Ala Cys Thr Ala Cys Thr Gly Thr Thr Gly Ala Ala Cys Ala Thr Thr 2835 2840 2845 Cys Gly Ala Thr Ala Thr Ala Thr Cys Cys Ala Gly Ala Ala Cys Cys 2850 2855 2860 Ala Cys Cys Cys Ala Thr Cys Ala Thr Thr Thr Gly Cys Ala Gly Gly 2865 2870 2875 2880 Cys Ala Cys Ala Thr Thr Gly Gly Cys Cys Ala Thr Thr Ala Ala Ala 2885 2890 2895 Ala Thr Thr Thr Ala Gly Cys Thr Thr Cys Gly Thr Cys Cys Thr Cys 2900 2905 2910 Thr Ala Thr Thr Thr Thr Gly Gly Ala Gly Cys Thr Ala Thr Gly Thr 2915 2920 2925 Ala Ala Ala Ala Gly Cys Ala Thr Gly Cys Ala Cys Ala Ala Cys Thr 2930 2935 2940 Cys Thr Ala Thr Thr Thr Ala Thr Gly Thr Gly Thr Gly Ala Ala Ala 2945 2950 2955 2960 Thr Ala Ala Ala Ala Thr Thr Thr Gly Ala Ala Ala Ala Cys Gly Cys 2965 2970 2975 Gly Thr Gly Gly 2980