AU783098B2 - Plant like starches and the method of making them in hosts - Google Patents

Description

1

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicants Actual Inventors Address for Service: Invention Title: Exseed Genetics, LLC.

Hanping GUAN and Peter L. KEELING CULLEN CO Patent Trade Mark Attorneys, 239 George Street Brisbane QId 4000 Australian Plant Like Starches and the Method of Making Them in Hosts Details of Associated Parent Application Australian 68828/98 Patent Application No.

The following statement is a full description of this invention, including the best method of performing it, known to us: This invention has been divided from the specification of Australian Patent Application No. 68828/98.

BACKGROUND FIELD OF INVENTION This invention relates to hosts containing constructs with genes from the starch pathway. More specifically the present invention relates to bacterial hosts that form plant like starches. Additionally the present invention relates to plant hosts that have genes from the starch pathway. The invention further relates to the starches produced by said hosts.

T'hs: BACKGROUND DESCRIPTION OF PRIOR ART *"The starch using industry includes diverse industries such as candy makers, makers of adhesives and paints, gravy makers, paper producers, etc. Since the demand for starch, (which is formed of amylose and amylopectin), has been dramatically increasing for specialized food S and industrial uses, efforts have been undertaken to tailor the quantity and quality of starch for specific food and industrial uses.

This industry has overtime looked for a number of different starches having, high viscosity, lower viscosity, higher gelling strength and lower gelling strength, different boiling points etc. Each starch tailored for a number of uses. The industry has utilized mutant starches that have less amylopectin and mutant starches with more amylose for tailored specifications.

For example the increased amylose starch has been used in the gelled candy making area. And the industry has used the increased amylopectin starches formed by mutants such as wx and wx su2 containing little amylose and mostly amylopectin for thicken foods like pudding, pies, gravies, frozen foods and batters, stews, canned foods and baby food. Additionally the mutant starches of different types have usefulness as adhesives and as sizing.

The other method used to address the industry needs for tailored starch is the use of chemical modification of the starch. Chemical derivation of the starch are produced by chemically reacting the starch with the monofunctional reagents to introduce the substituents such as phosphate, acetate, succinate groups to stabilize the starch. Unfortunately, these types of starches can be subjected to government regulation and additionally have less acceptance generally due to the added cost of the starch.

Starch is the major form in which carbohydrates are stored in biological systems. Plant starch in chloroplasts is transitory and storage starch accumulates in storage organs of many plant. Starch can be found in all organs of most higher plants including leaves stems and roots and fruits and embryo and endosperm. In addition to higher plants starch, similar polysaccharide (glycogen) has been found in bacteria. Many bacteria produce a reserve S polysaccharide similar to the glycogen found in animals.

Storage polysaccharide has been classified as being in two groups, group one has storage in the cytsol of the cell and the second group within the plastid. Escherichia coli produces a polysaccharide within the cytsol. Starch producing plants typically store starch in the plastids. Typical starch bearing plants include cassava, potato, corn, peas, rice, wheat, barley. The main starch storing tissue of corn, rice wheat and barley and oats, the cereal grains, is the endosperm.

Starches are also classified by the plant source, for example cereal starches are from cereal grains such as maize, rice, wheat, barley, oats and sorghum; tuber and root starch are S from potatoes and yams and cassava.

The pathway of starch synthesis is not well understood. Generally, as noted above starch from plants, consists of two major components: amylose and amylopectin. These intertwine in the starch granule of the plants. Amylose is a linear polymer of alpha 1 -4 bonded anhydroglucose units while amylopectin is a branched polymer comprised of linear chains alpha 1-4 linked anhydroglucose units with branches resulting from alphal-6 linkages between the linear chains. It has been known for sometime that mutant genes in starch bearing plants can be expressed and that the properties of the starch can be altered. The proportion of the two components and their structures in the mutant primarily determine the physical-chemical properties of the starch.

Thus the lack of a clear understanding of the starch synthesis pathway and the difficulty of employing mutants limited the industry to the use of existing and producible mutant starches (cereals containing mutant starch can show a tremendous yield penalty in field environments) or to the chemical modification that could be made to the starch. In the last decade the industry has been studying the effects of certain starch genes in plants and bacteria in an attempt to more clearly understand starch synthesis.. Since the late 80's it has been possible to transform plants and bacteria to contain isolated genes. In response to this the industry has transformed potatoes with a bacterial gene GS and with starch soluble synthase III gene in the antisense (forming a mutant). As part of these potato starch experiments bacteria has been transformed with certain potato starch genes. For example the SSSIII gene from potato was transformed into E.coli deficient in glgA gene. The effect of glgC and branching enzyme I and II in combination in a mutant E.coli has also been studied and glycogen like product was reported.

The starch industry that is commercial does not have a particular interest in the production of glycogen which is the polysaccharide produced by bacteria and animals the health care industry may have some such interest). The industry has thus not yet been able to generate tailored starches at reasonable prices through plant gene transformation. There remains a need for the industry to find new starches that are useful due to their changed characteristics such as lower viscosity and new starches that are useful because of their higher viscosity and new methods of producing such starches.

Glycogen synthesis in E. coli and starch synthesis in higher plants have similar pathways involving ADPGIc pyrophosphorylase, starch synthase,(SS) or glycogen synthase and branching enzyme It has been suggested that ADPGlc pyrophosphorylase plays a pivotal role in regulating the amount of starch synthesized, while starch synthase and starch branching enzyme primarily determine the starch structure. Multiple forms of SBE and SS have been identified in many plants including maize, rice, pea and potato. In addition to the waxy gene coding for granule bound starch synthase (GBSS), three genes coding for the other forms of SS have been isolated from maize endosperm. Maize is the only cereal crop from which the genes coding for the five forms of SS have been isolated. Clearly a number of these sequences are published and known to those of skill in the art. Genes coding for maize SBE have also been cloned and characterized. Previous reports have demonstrated that maize SBEI 4 has a higher rate of branching amylose than SBEII and preferentially transfers longer chains, while SBEII shows a higher rate of branching amylopectin and preferentially transfers shorter chains. In comparison with SBE, less is known about the specificities and functions of multiple forms of SS. In Waxy maize, which lacks GBSS, only amylopectin is synthesized and amylose is missing. Therefore, it is generally accepted that GBSS, encoded by waxy gene, is primarily responsible for the synthesis of amylose. Study of waxy mutation in Chlamedomonas reinhardtii has suggested that GBSS is also involved in amylopectin synthesis. Although it has been reported that Chlamedomonas reinhardtii SSII controls the synthesis of intermediate size glucans of amylopectin in Chlamedomonas, direct evidence for the functions of SS in higher plants is still missing. Antisense technology has been used to study the functions of SS in potato, however, the results are inconclusive.

9 In an article written by Hanping Guan et al., entitled AMaize Branching Enzyme Catalyzes Synthesis of Glycogen-like Polysaccharide in gig B-deficient Escherichia coli@.

Published in Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 964-967, February 1995 Plant Biology a specific glycogen like polysaccharide from a transformed E coli was reported. This article taught the transformation of an E coli bacteria with maize BEI and BEII. These enzymes were 9 transformed into two E coli hosts. One of the bacterial hosts was a wild type and the other was a mutant. The mutation to the bacteria was the reduction of the activity of glycogen BE in the AC71(glgB-) so that the mutant was essentially free of BE activity. The paper analyzed the S debranched alpha-glucan isolated from the four different transformants. The first host was E.

coli containing glgB and the second host was the AC71 without any transformed genes then AC71 transformed with maize BEII, and then AC71 with maize BEI, then AC71 with maize BEI and BEII. The resultant polysaccharide products were analyzed by HPLC, by chain length and relative peak area and by mole distribution of chains. The study led to the understanding that BEII could play a role in synthesis of the short chains of amylopectin and BEI could be involved with the longer chains of amylopectin. The paper also noted that the mutant host AC71 produced more chains with chain length of 6 then did the wild type E. Coli. The paper also noted that the maize BE and the GS of the host did not produce amylopectin like polysaccharides. The article suggested that the concerted action of GS with different BE=s could play an important role in determining the final structure of the polysaccharide synthesized. The article by Guan ends by suggesting that his study had established the basis for studying the concerted actions of BE and SS in a bacterial model system.

The expression of E coli GS (glycogen synthases) in potatoes showed a large incidence of highly branched starch. This result was published in an article in Plant Physiol. 104,1159- 1166 by Shewaker et al. This potato does not appear to be of much commercial use at this time.

The industry still needs the option of producing plant like starches in a fermentation process from bacteria and thus without the necessity of breeding and growing environment sensitive plants; and, the option of producing plants that generate the specific tailored starch through a plant host. And the industry needs altered and new starches that are cereal like starches or root and tuber like starches in large quantities and inexpensively thus avoiding having to use chemical modification of starch. The industry needs a host that can be readily transformed to supply different starches tailored to the industry=s need. Specifically the industry needs a host that supplies various different starches including those not capable of Sbeing made in plants or bacteria presently.

OBJECTS AND ADVANTAGES Accordingly, several objects and advantages of the invention are to produce plant like starch through the process of fermentation.

Additional objects and advantages are the production of new starches in plants.

Still further objects and advantages will become apparent from a consideration of the ensuing description and accompanying drawings.

Another object of the present invention is the synthesis of polysaccharides including amylose, amylopectin in E. coli, and/or fungal and yeast by plant starch synthesizing enzymes including SS, SBE, bacterial branching enzyme, glycogen synthase and other enzymes in other living organism.

Yet another object of my invention is using each or combination of these enzymes or 6 modified enzymes studied in this patent to produce or to improve polysaccharides in any living organism including starch synthesis in plants.

SUMMARY OF THE INVENTION The invention provides DNA constructs in a host that include most of the genes in the starch pathway of a plant such that the host produces a plant like polysaccharide. And in one embodiment produces maize starch including slightly different embodiments that make specific maize mutant like starch in a non plant host. This invention encompasses a bacterial host containing a combination of two or more of such genes SSI, SSSIIa, SSIIb, SSSIII, GBSS, BEI and BEII when the combination does not form glycogen like material. This invention encompasses a plant host transformed with any of the following maize genes or a plant host having a combination of two or more of the following maize genes SSI, SSIIa, SSIIb, SSSIII, SGBSS, BEI and BEII in a hybrid or an inbred rice plant.

Additionally the present invention includes new'polysaccharide produced by a transformed host. The host having a wildtype, which does not produce the new polysaccharide, the transformed host expressing at least two exogenous starch synthesis genes, the genes are selected from a group consisting of starch synthesis genes such as SSI SSIIa, SSIIb, SSIII, GBSS and optionally including at least one of the BEI and BEII genes wherein 0 the transformed host is capable of producing such new polysaccharide.

The invention also covers a new polysaccharide wherein the host also expresses the exogenous genes selected from the following group consisting of bacterial glycogen inducing genes are selected from the group glgA, glgB, glgC and any mutants thereof. Or wherein the host also expresses the exogenous genes selected from the following group consisting of plant granule bound enzymes. And the new polysaccharide wherein the starch synthesis genes are selected from the group consisting of BEI and BEII.

The present invention broadly encompasses a host containing a transformed Glg C gene and at least one of the starch branching enzymes genes in a host in combination with at least one other transformed starch gene wherein the host produces a polysaccharide product. And a host containing transformed bacterial gene and at least one of the non starch branching enzymes selected from the group consisting of debranching enzymes and soluble starch synthase A method of producing polysaccharides which are non glycogen like in a host comprising transforming a host capable of being used in a fermentation process, with genes selected from the group which produce starch synthesizing enzymes, or glycogen synthesizing enzymes such that the host produces nonglycogen like starch, and employing the host in a fermentation process that produces polysaccharides. The host is bacteria, or a fungal or a yeast. Additionally the method of this invention includes the use of bacterial genes also such as the glycogen synthesizing genes including the glgC, glgA, glgB genes. A method wherein the genes which produce starch synthesizing enzymes include genes encoding for starch S soluble synthases I, IIa, IIb and SS III (dull). A method wherein the genes which produce starch synthesizing enzymes include genes encoding for starch debranching enzyme and branching enzymes. The invention covers the modified starch synthesizing enzymes including the N-terminally truncated SS.

In other word the invention covers a host transformed to carry a gene active in glycogen production, and at least one nonstarch branching gene active in the production of at least one of the following polysaccharides amylopectin and amylose in its original host. The host can be a monocot or a dicot plant. The host can be a cereal bearing plant. Or the host can be a bacteria.

More specifically the invention includes a host wherein at least one nonstarch genes active in the production of at least one of the following polysaccharides, amylopectin and amylose in its original plant, is selected from the group consisting of starch soluble starch synthase I, lia, IIb, III genes and debranching enzyme gene (sul), GBSS gene, sh2 gene and bt2 gene. A host including at least one of the starch branching enzyme genes such as BEI gene, BEII gene.

The present invention can also be described as a host transformed to carry a gene active in ADPG production, and at least one starch gene active in the production of at least one of the following polysaccharides amylopectin and amylose in its original host wherein the host 8 produces polysaccharides that are plant like starch and not glycogen like.

Additionally the host can be transformed to carry a pyrophosphorylase gene, and glycogen synthase gene.

The scope of the present invention includes a host deficient in alpha 1,4 glucan synthesizing ability and alpha 1,4-1,6 branching enzyme capability transformed to express at least one plant starch soluble synthesis gene. And the host can also include being transformed to express at least one gene encoding for debranching enzyme, and/or a gene encoding for starch soluble synthase I, starch soluble synthase enzyme lia, lib, starch soluble synthase enzyme III.

This host can include being transformed to express at least one gene encoding for starch branching enzyme.

This invention also includes the production of a glycogen like material implants.

Attached hereto are a number of plasmids described by the figures and by table one, that are part of the present invention and are claimed herein. One such example is the plasmid wherein the plasmid is in a carrier host and the plasmid contains the SSIIa gene with the n terminus (SEQ ID SNO:1) GENVMNVIVV and wherein the gene is approximately 1561 base pairs in length. The S invention includes mutant hosts such as mutant plants like waxy rice and potatoes and corn as Sexample and wherein the host is a mutant E. coli, or fungus.

More specifically, the invention provides in a first embodiment plant transformed with a nucleic acid encoding a mutant E. coli ADP glucose pyrophosphorylase having an amino acid S sequence as set forth in SEQ ID NO: 76.

In a second embodiment, there is provided a nucleic acid encoding a mutant E. coli ADP glucose pyrophosphorylase having an amino acid sequence as set forth in SEQ ID NO: 76.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a graph which gives the relative peak area in percent and the chain length of glycogen and starch soluble synthase I (SSI), starch soluble synthase II (SSIIa), starch soluble synthase IIb (SSIIb). Thus this shows the specificities of Maize SS=s in chain elongation.

FIG. 2 shows plasmid pEXSC-MBEI with 7661 base pairs and promoter T7 and a Kanamycin gene and glgC and the maize starch branching enzyme I (MBEI).

FIG. 3 shows plasmid pEXSC3C with 7461 base pairs and promoter T7 and ampicillin gene and the maize starch soluble synthase gene lia. pEXS3c is the 1082 bp Nde I-EcoRI fragment containing the N-terminus of MSSSIIa (from MSSIIa in pBSK) subcloned into the Nde I-EcoRI sites of pEXS3a, replacing the N-terminus of IIA-2 with the longer IIa Nterminus. MSSIIa is the mature maize SSIIa and is 2090 bp long. The following sites are not contained in the MSSIIc insert: Apa I, BglII, Eco V, Not, Spe I, and Xba I. The N-terminus of this plasmid is (SEQ ID NO:2) AEAEAGGKD.

FIG. 4 shows plasmid pEXSC-MBEI-MBEII with 9971 base pairs and promoter T7 and a Kanamycin gene and glgC and the maize starch branching enzyme I (MBEI) and the maize starch branching enzyme II (MBEII).

FIG. 5 shows plasmid pEXSC-MBEII with 7521 base pairs and promoter T7 and a Kanamycin gene and glgC and the maize starch branching enzyme II (MBEII).

FIG. 6 shows plasmid pEXSC-3a with 7990 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize N-terminally truncated starch synthase gene IIa (MSSIIa-2). The N-terminal sequence is (SEQ ID NO:61) GENVMNVI.

FIG. 7 shows plasmid pEXSC-8 with 7079 base pairs and promoter T7 and a oo Kanamycin gene and the glgC gene and the maize starch soluble synthase gene I and version I- 2.(MSSI-2), An N-terminally truncated SSI.

FIG. 8 shows plasmid pEXSC-9 with 7551 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize starch soluble synthase gene IIb (SSIIb).

The N-terminal sequence is (SEQ ID NO:10) AAAPAGEE.

FIG. 9 shows plasmid pEXSC-10 with 7211 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the maize starch soluble synthase gene I, the full length SSI. The N-terminal sequence is (SEQ ID NO: 11) CVAELSREGPA FIG. 10 shows plasmid pEXSCA with 6738 base pairs and promoter T7 and a Kanamycin gene and the glgC gene and the glgA gene.

FIG. 11 shows plasmid pEXSC9a with 7240 base pairs and promoter T7 and ampicillin gene and the maize starch soluble synthase gene IIb-2 (Maize SS IIb-2), an N-terminallytruncated SSIIb. The N-terminal sequence is (SEQ ID NO:12) MNVVVVASECAP.

FIG. 12 shows plasmid pEXSWX with 6968 base pairs and promoter T7 and an ampicillin gene and the N-terminally-truncated maize WX (maize granular bound starch synthase). The N-terminal sequence for wx is (SEQ ID NO: 13) ASAGMNVFVGAEMA.

FIG. 13 shows plasmid pEXSWX2 with 6980 base pairs and promoter T7 and an ampicillin gene and the N-terminally-truncated maize WX termed as wx2. The N-terminus of wx2 is (SEQ ID NO:14) MNVVFVGAEMA.

FIG. 14 shows plasmid pEXSC9 with 7780 base pairs and promoter T7 and ampicillin gene and E. coli glgC gene and the maize starch soluble synthase genellb (Maize SS IIb).

FIG. 15 shows plasmid pEXSC1.Od with 7112 base pairs and promoter T7 and ampicillin gene, E. coli glgC gene and the N-terminally-truncated maize starch soluble synthase gene I termed as Maize SSI-3). The N-terminus of maize SSI-3 is (SEQ ID NO: 15) MSIVFTGEASPYA.

FIG. 16 shows plasmid pEXS10 with 5300 base pairs and promoter T7 and ampicillin gene and the full length maize starch soluble synthase gene I termed as Maize SS I.

FIG. 17 shows plasmid pEXS8 with 7259 base pairs and promoter T7 and ampicillin gene and the N-terminally-truncated maize starch synthase gene I termed as SSI-2. The Nterminal sequence is (SEQ ID NO:16) CVAELSRDLGLEPEG.

FIG. 18 shows plasmid pEXSCA1 with 5128 base pairs and promoter T7 and ampicillin gene and the glgA. pESCA1 is a 1551 bp SpeI-Sac I fragment containing glgA (from glgA in pBSK) subcloned into the Xba I- Sac I sites of p ET-23d which is commercially available from Novagen in Madision Wisconsin under catalog number 69748-1 and called ET- 23d(+) DNA.

FIG. 19 shows the spectrum of the iodine glucan complex of the product produced by the host containing the glgC and glgA, and the pEXSC9, pEXSC3, pEXSC8, pEXSCwx the X-axis is listing nm and the Y axis is reading absorbance.

FIG. 20 shows the spectrum of the iodine glucan complex of the product produced by the host transformed with plasmids containing the glgC, the BEI, the BEII genes and glgA; glgC, the BEI, the BEII genes and maize SSI, SSI-2 and glgC, the BEI, the BEII genes and maize SSIIb, and glgC, the BEI, the BEII genes and maize SSIIa-2, and glgC, the BEI, the BEII genes, the X-axis is listing rim and the Y axis is reading absorbance.

FIG. 21 shows the product produced by the host in small bottles including the product from the host containing glgC, the BEI, the BEII genes and maize SS=s genes. Encoded as (C- I-II+8), glgC, the BEI, the BEII and maize SSI-2 genes and pEXSC10 encoded as glgC, the BEI, the BEII and maize SSI genes and pEXSC9 encoded as (C-I-II+9),glgC, the BEI, the BEII and maize SSIIb genes and pEXSC3a encoded as (C-I-II+3a), glgC, the BEI, the BEII and maize SSIIa-2 genes and pEXSCWX encoded as (C-I-II+WX),glgC, the BEI, the BEII and maize waxy genes and pEXSCAl encoded as (C-I-II+AI), containing maize BEI, BEII and E.coli glgA genes, potato dextrin, waxy maize starch, corn amylopectin, rice starch, corn starch, pEXSC8.

FIG. 22 shows pExs-trc has 4178 base pairs with the trc promoter and the ampicillin gene. PEXS trc is pTrc99A-Ndel which has been mutagenixed. (Nco I site in multiple cloning site ofp Trc99A-Ndel is mutagenixed to Nde I using primers EXS63 AND EXS64.) pEXS-trc contains only one Nde I site and no Nco I sites. The following sites are not contained in pEXS-trc; Bgl II, Cla I, Nco I, Not I, Sac II, SnaB I, Spe I, and Xho I.

FIG. 23 shows pEXS-trc3 has 4129 base pairs with the trc promoter and the ampicillin gene in partial and the Kanamycin gene. The pEXS-trc3 is pEXS-trcl cut with BglI (filled in)- Sca I and religated, deleting most of the Amp gene (304 nt from the 5" end remain). The following sites are Not contained in p EXS-trc3: Apa I, BgI II, Eco V, Nco I, Not I, SnaB I, and Spe I.

12 FIG. 24 shows the plasmid pEXS 102 having 7190 base pairs, adapted for plant transformation containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the Waxy 2 gene and the nos terminator and the ampicillin gene.

FIG. 25 shows the plasmid pEXS 103 having 6607 base pairs, adapted for plant use containing the maize 10KD zein promoter,the gene coding for the maize starch synthase I transit peptide and the Waxy 2 gene and the nos terminator and the ampicillin gene.

FIG. 26 shows the plasmid pEXS 101 having 6979 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide and the gig B gene and the nos terminator and the ampicillin gene.

FIG. 27 shows the plasmid pEXS 100 having 7557 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the gig B gene and the nos terminator and the ampicillin gene.

FIG. 28 shows the plasmid pEXS 101 having 6273 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the gig A gene and the nos terminator and the ampicillin gene.

FIG. 29 shows the plasmid pEXS 66 having 6001 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the gig C 3 gene and the nos terminator and the ampicillin gene.

FIG. 30 shows the plasmid pEXS 65 having 6373 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the maize waxy gene and the nos terminator and the ampicillin gene.

FIG. 31 shows the plasmid pEXS 64 having 7073 base pairs, adapted for plant use containing the maize 10KD zein promoter,the gene coding for the maize starch synthase I 13 transit peptide, and the maize soluble starch synthase IIa gene and the nos terminator and the ampicillin gene.

FIG. 32 shows the plasmid pEXS 63 having 6473 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase IIa gene and the nos terminator and the ampicillin gene.

FIG. 33 shows the plasmid pEXS 62 having 6773 base pairs, adapted for plant use containing the maize 10KD zein promoter,the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase I-2 gene and the nos terminator and the S* ampicillin gene FIG. 34 shows the plasmid pEXS 61 having 7013 base pairs, adapted for plant use containing the maize 10KD zein promoter, the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase IIb gene and the nos terminator and the ampicillin gene.

FIG. 35 shows the plasmid pEXS 59 having 6858 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the E.coli glgA gene and the nos terminator and the ampicillin gene FIG. 36 shows the plasmid pEXS 58 having 7658 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the maize soluble starch synthase IIa gene and the nos terminator and the ampicillin gene.

FIG. 37 shows the plasmid pEXS 56 having 6586 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the glg C, gene and the nos terminator and the ampicillin gene.

14 FIG. 38 shows the plasmid pEXS 54 having 7658 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the Maize SS IIa gene and the nos terminator and the ampicillin gene.

FIG. 39 shows the plasmid pEXS 53 having 7058 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and the maize starch soluble synthase IIa-2 gene and the nos terminator and the ampicillin gene.

FIG. 40 shows the plasmid pEXS 52 having 7358 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and maize starch soluble synthase I-2 gene and the nos terminator and the ampicillin gene.

FIG. 41 shows the plasmid pEXS 51 having 7398 base pairs, adapted for plant use containing the maize 10KD zein promoter, and maize adh I intron, the gene coding for the maize starch synthase I transit peptide, and maize starch soluble synthase IIb gene and the nos terminator and the ampicillin gene.

.FIG. 42 shows photograph of eleven products of altered starch produced with the present invention. The titled are encoded C-I-II=the glgC gene and the BEI and the BEII genes the following number or alternatively designation means pEXS-and the number. Thus C-I- II=the glgC gene and the BEI and the BEII and EXS-10 plasmid that contains the gene SSI, having the N-terminus shown in Table 1.

FIG. 43 shows the DNA sequence (SEQ ID NO:33) and the protein sequence (SEQ ID NO:34) for glgA having 1488 base pairs.

FIG. 44 shows the DNA sequence (SEQ ID NO:35) and the protein sequence (SEQ ID NO:36) for glgB having 2361 base pairs.

FIG. 45a shows the DNA and amino acid sequences (SEQ ID Nos: 38 37 respectively) for Zea mays 10-kDa zein gene having 2562 base pairs.

FIG. 45b shows the DNA and amino acid sequences (SEQ ID Nos. 39 37 respectively) for Zea mays 10-kDa zein portion of the gene used as the promoter in a number of the plasmids discussed herein.

FIG. 46 shows the DNA sequence (SEQ ID NO:40) and the protein sequence (SEQ ID Nos:41, 76 77) for glgC3 (glgC 3 having 1328 base pairs containing two mutations P295D, K296E. This is a mutant of the wild type glgC gene.

FIG. 47 shows the DNA sequence (SEQ ID NO:42) and the protein sequence (SEQ ID NO: 43) for glgC (glgC) having 1328 base pairs.

FIG. 48 shows the DNA sequence (SEQ ID NO:44) and the protein sequence (SEQ ID NO:45) for glgCwt (glgCwt) having 1328 base pairs. This is the glgC gene that is found in nature.

FIG. 49 shows the DNA sequence (SEQ ID NO:46) and the protein sequence (SEQ SID NO:47) for the maize waxy gene denoted wx herein.

FIG. 50 shows the DNA sequence (SEQ ID NO:48) and the protein sequence (SEQ ID NO:49) for the maize starch soluble synthase lib encoding gene having 2423 base pairs.

FIG. 51 shows the DNA sequence (SEQ ID NO:50) and the protein sequence

(SEQ

ID NO:51) for the maize starch soluble synthase IIa.

FIG. 52 shows the DNA sequence (SEQ ID NO:52) and the protein sequence (SEQ ID NO:53) for the maize starch soluble synthase I-2 having 1749 base pairs.

FIG. 53 shows the DNA sequence (SEQ ID NO:55) and the protein sequence (SEQ ID NOs:54 and 70-75) for the maize branching enzyme II.

FIG. 54 shows the DNA sequence (SEQ ID NO:57) and the protein sequence (SEQ ID NO:56) for the maize branching enzyme I.

FIG. 55 shows the DNA sequence (SEQ ID NO:58) and the protein sequence (SEQ ID NO:59) (153) for the transit peptide portion of the maize starch soluble synthase I.

FIG. 56, PCR analysis of transgenic rice plants. The genomic DNA isolated from rice plants were PCR amplified using specific primers for the inserted gene. The specific bands were identified on 1% agarose gel compared with non-transgenic rice plant.

FIG. 57. Activity staining of starch synthase on renaturing SDS-PAGE gel with Iodine solution: The positive staining of maize SSI-2 indicated the expression of maize SSI-2 in transgenic rice plants.

FIG. 58 SSI-1, SSI-2 and SSI-3 construct diagram. Three forms of SSI were constructed in the pET expression system (see Methods). pExsl0a encodes SSI-1 (SEQ ID NO:62), the full length maize SSI (583 amino acids). pExs8 encodes a truncated SSI, SSI-2 (SEQ ID NO:60), with amino acids #8-52 deleted from the N-terminus of SSI-1. pExsld encodes the most truncated form of SSI, SSI-3 (SEQ ID NO:63), with the first 93 amino acids deleted from SSI-1. A depiction of the waxy gene, encoding GBSS, is also included for comparison (SEQ ID NO:64). The amino acid motif (SEQ ID NO:17) KS/TGGL, the putative binding site for ADPGlc, is indicated by the triangles. The KS/TGGL motif (SEQ ID NO:17) is located 18 amino acids from the N-terminus in GBSS, while the motif is 106 amino acids from the N-terminus in maize SSI. Drawing not to scale.

FIG. 59. SSIIa-1 and SSIIa-2 construct diagram. Two forms of SSIIa were constructed in the pET expression system. pExs3c encodes SSIIb-1 (SEQ ID NO:65), the putative full length maize SSIIb. N-terminal sequencing of SSIIa-1 revelaed that the polypeptide chain started at amino acid so the length of SSIIa-1 is 669 amino acids.

pExs3a encodes a truncated form of SSIIa, SSIIa-2 (SEQ ID NO:66), with the first 176 Nterminal amino acids deleted from SSIIa (493 amino acids total). A depiction of the waxy gene, encoding GBSS, is also included for comparison (SEQ ID NO:64). The amino acid motif (SEQ ID NO:17) KTGGL, the putative binding site for ADPGlc, is indicated by the triangles. The KTGGL motif (SEQ ID NO:17) is located 18 amino acids from the Nterminus in GBSS, while the motif is 194 amino acids from the N-terminus in maize SSIIa.

FIG. 60. SSIIb-1 and SSIIb-2 construct diagram. Two forms of SSIIb were constructed in the pET expression system (see Methods). pExs9 encodes SSIIb-1 (SEQ ID NOs:67 68 respectively), the putative full length maize SSIIb. N-terminal sequencing of SSIIb-1 revealed that the polypeptide chain started at amino acid so the length of SSIIb-1 is 637 amino acids. pExs9a encodes a truncated form of SSIIb, SSIIb-2 (SEQ ID NO:69), with the first 144 N-terminal amino acids deleted from SSIIb (492 amino acids total). A depiction of the waxy gene, encoding GBSS, is also included for comparison (SEQ ID NO:64). The amino acid motif (SEQ ID NO:17) KTGGL, the putative binding site for ADPGlc, is indicated by the triangles. The KTGGL motif (SEQ ID NO: 17) is located 18 amino acids from the N-terminus in GBSS, while the motif is 158 amino acids from the Nterminus in maize SSIIb.

0 FIG. 61. Temperature Curvesfor SSI enzymes. All assay components, except enzyme and 4 C]-ADPGlc, were mixed and then preincubated at each temperature .for 3 minutes before addition of enzyme and ADPGlc. For all assays, the final concentration of 4

C]-

S ADPGlc was 3 mM, while amylopectin was 6 mg/ml. Each point is an average of three separate determinations.

FIG.62. Temperature Optima of SSIIa-1 and SSIIa-2. All assay components, except enzyme and [U- 14 C]-ADPGlc, were mixed and then preincubated at each temperature for 3 minutes before addition of enzyme and ADPGlc. For assays in the presence of 0.5 M citrate, mg/ml amylopectin was used as primer. For assays without citrate, 10 mg/ml amylopectin was used. For all assays, the concentration of 4 C]-ADPGlc was 3 mM. Each point is an average of three separate determinations.

FIG. 63. Temperature Optima of SSIIb-1 and SSIIb-2. All assay components, except enzyme and [U-'4C]ADPGlc, were mixed and then preincubated at each temperature for 3 minutes before addition of enzyme and ADPGlc. For all assays, the concentration of [U- 4 C]ADPGlc was 3 mM and the concentration of glycogen was 40 mg/ml. Each point is an average of three separate determinations.

PREFERRED EMBODIMENT

DESCRIPTION

Gene shall mean the entire gene sequence or any mutations or varieties of the codon that produce the desired activity in the host or alternatively the section or sections of the gene sequence necessary to produce the desired activity in the host. For example glgC gene shall mean glgCI 6 glgC 3 and other mutants that produce the desired activity in the host. Starch synthase gene shall mean full length SS, N-terminally-truncated SS or mutated SS with starch synthase activity.

Glycogen like-shall mean polysaccharide material such as those produced as the main starch product by E.coli in its native state and by the hosts as taught in the above described paper by Hanping Guan.

Non Glycogen like- shall mean polysaccharide material which is plant like and is not .produced as the main starch product by E.coli in its native state and by the hosts as taught in the above described paper by Hanping Guan.

Plant like starch- is non glycogen like.

S* Transformed gene-shall mean a gene that was somewhere in the lineage of the plant or bacteria introduced into the plant by means other than nature. Thus the progeny of a transformed host would continue to contain a transformed gene.

Transformed host- shall mean any organism containing one or more of the novel plasmids and/or a novel combination of starch synthetic genes discussed herein.

Within this application a number of different protocols have been employed to designate the same gene or synthase. MSS#=maize soluble starch synthase, SS#will likewise mean starch synthase though not necessarily maize. STS#will also designate soluble starch synthase.

GBSS=granule bound starch synthase. SBE#=starch branching enzyme, MBE= maize starch branching enzyme, MSBE#=maize starch branching enzyme, and BE#=starch branching enzyme.

The present invention broadly encompasses transforming hosts such as bacteria or plants with plant starch synthetic genes that produce a non glycogen like material (a bacteria containing BEI and BEII from maize produces a glycogen like material). Starch bearing plants and organisms hereinafter are referred to as the host. One of the primary aspects of this invention is the generation of plant like starch from a bacterial host and the production of altered starch in a plant host. The present invention has been exemplified in both bacteria and in transformed rice plants. The host can contain though it is not a limitation, an unlimited supply of ADPG from the addition of the glgC gene (the bacterial gene) to the plant.

Additionally the present invention encompasses plasmids that contain the maize genes and/or "i the bacterial genes in a construct adapted for use in a bacteria and constructs adapted for use in a plant. The plasmids in the plant construct preferably containing an active promoter recognized by the plant, a transit peptide, and the cleavage site that permits the protein to cleave from the transit peptide when crossing into the amyloplast in the plant. The plasmids used in the rice transformation specifically encompassed the maize 10 kd zein promoter, and the transit peptide from the maize SSI gene in the constructs adapted for plant use. The present invention also encompasses the plant producing the altered starch in the starch storage section of the plant or within the host cell and the altered starch itself. Additionally the present invention encompasses the combination of a number of starch genes in combination being active in a host such that the host produces differing non glycogen polysaccharides. Still further the present invention encompasses a method of making plant like starch in a bacterial host and the method of making altered plant like starch (altered in relationship to the type or amount of starch that the host makes without the constructs containing the genes), in a plant.

Yet another object of the present invention is the addition of a gene that encodes for the substrate ADPG used to form starch.

The present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a plant and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I or its mutant form. The present invention also encompasses the combination of a promoter adapted for use in a plant and optionally a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I or its mutant form, and at least one gene encoding for branching enzyme transformed into a plant host.

The present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a plant and a gene encoding for ADPGlc pyrophosphorylase, preferably a bacterial gene, and a gene encoding for starch synthase Iia or its mutant form. The present invention also encompasses the combination of a promoter adapted for use in a plant and optionally a gene encoding for ADPGlc pyrophosphorylase, and a gene encoding for starch synthase lia or its mutant form, and at least one gene encoding for branching enzyme transformed in to a plant host.

The present invention encompasses a plasmid having a promoter adapted for use in a plant and a gene encoding for ADPGlc pyrophosphorylase, preferably a bacterial gene. and a gene encoding for starch synthase IIb and its mutant form. The present invention also encompasses the combination for a promoter adapted of use in a plant and an optional gene encoding for ADPGlc pyrophosphorylase, and a gene encoding for starch synthase IIb or its mutant form and at least one gene encoding for branching enzyme transformed in to a plant host.

The present invention encompasses a plasmid having a promoter adapted for use in a plant and a gene encoding for Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa, starch synthase IIb, DU1. The present invention also encompasses the combination of a promoter adapted for use in a plant and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa, starch synthase lib and DU1,, and at least one gene encoding for branching enzyme transformed in to a plant host.

The present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a plant and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa, IIb and starch synthase III (DU1). The present invention also encompasses the combination of a promoter adapted for use in a plant and an optional gene encoding for ADPGlc pyrophosphorylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa, IIb starch synthase III(DUI), and at least one gene encoding for branching enzyme, and at least one gene encoding for the debranching enzyme transformed in to a plant host.

The present invention encompasses a plasmid or combination of plasmids in the host having a promoter adapted for use in a bacteria or yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I.

The present invention also encompasses the combination of a promoter adapted for use in a bacteria or yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase I, and at least one gene encoding for branching enzyme transformed in to a bacteria or yeast host.

The present invention encompasses a plasmid or combination of plasmids in the host having a promoter adapted for use in a bacteria or yeast and a gene encoding for Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase IIa.

The present invention also encompasses the combination of a promoter adapted for use in a bacteria or yeast and optionally a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase IIa, and at least one gene encoding for branching enzyme transformed in to a bacteria or yeast host.

The present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in a bacteria or yeast, and a maize gene encoding for starch synthase III(DUl). The present invention also encompasses the combination of a promoter adapted for use in a bacteria or yeast and an optional gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and a gene encoding for starch synthase III, and at least one gene encoding for branching enzyme transformed in to a bacteria or yeast host.

The present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in bacteria or in yeast and a gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III(DUl). The present invention also encompasses the combination of a promoter adapted for 22 use in bacteria or in yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIaIIb, starch synthase III, and at least one gene encoding for branching enzyme transformed in to bacteria or into yeast hosts.

The present invention encompasses a plasmid or combination of plasmids in the same host having a promoter adapted for use in bacteria or in yeast and a gene encoding for ADPGlc Pyrophosphoroylase, preferably a bacterial gene, and genes encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III The present invention also encompasses the combination of a promoter adapted for use in bacteria or in yeast and a gene encoding for Pyrophosphoroylase, preferably a bacterial gene, and genes o: encoding for at least one of the following genes starch synthase I, starch synthase IIa,IIb, starch synthase III(DU1), and at least one gene encoding for branching enzyme, and at least one gene encoding for the debranching enzyme transformed in to a bacteria or into a yeast host.

The present invention encompasses the truncated versions of the SSI and the SSII and the SSIII genes that still provide protein that is sufficient to make the polysaccharide.

SBy transforming different combinations ofSS and SBE into E. coli HPG204(DE3) or G6MD3 defective in GS and GBE, we obtained the first evidence that maize SSI, SSII and SSIII have different specificities in the size of glucans synthesized see fig one.. Herein, we present the .i model system to produce differing polysaccharides from hosts with SS and SBE in E.coli by metabolic engineering. We also demonstrated that the truncated forms of SS had different Vmax, temperature stability and kinetic properties (Table, Fig).

We also demonstrated that transformation of starch synthase and/or branching enzyme in E .coli resulted in production of polysaccharides differing in size and structure. These polysaccharides can be used in food and nonfood industries to replace and/or complement starch functionalities. A large amount of these polysaccharides can be produced with fermentation technology.

Starch.biosynthesis in higher plants and glycogen biosynthesis in E. coli have similar reactions which use adenosine diphosphate glucose (ADPGlc) as a substrate. This similarity 23 allows us to use plant starch synthase (SS) and starch branching enzyme (SBE) to complement the functions of glycogen synthase (GS) and glycogen branching enzyme (GBE) in E. coli G6MD3, which is deficient in GS and GBE. Transformation of E.coli glgC gene and maize starch synthase gene in E.coli G6MD3 produced linear a 1,4 glucan similar to amylose.

coexpression of the glgC, maize starch synthase and maize branching enzyme produced branched polysaccharides. However, distinct properties of plant starch branching enzyme and starch synthase make it possible to synthesize different polysaccharides in E.coli. While maize SSI preferentially synthesis short chains (dp 6 15), SSII and SSIII preferentially transferred long chains (dp 24) and intermediate chains (dp 16 24) respectively. Transformation of different maize starch synthases, E. coli glycogen synthase (glgA) and/or maize branching enzymes into E.coli HPG96 or E. coli G6MD3 resulted in the synthesis of different sizes of polysaccharide with DP 500-4000.These polysaccharides synthesized in E .coli by maize SS have different physical-chemical properties than polysaccharides synthesized in natural organisms including starch from plant sources and glycogen from animals. The polysaccharide can be used in food and nonfood industries to replace and/ or complement starch, functionalities. A large amount of these polysaccharide can be produced by fermentation technology. The following materials were employed in the construction of the present invention some of the starting material are commercially available from Novagen in Madision t Wisconsin ET-23d(+) DNA under catalog number 69748-1 and BL21(DE3) under catalog number 69387-1; ET-21a(+) DNA under catalog number 697401.

Plant Hosts The following plasmids have been transformed into rice plants Transgenic 1, MSTSIA(pExs52) and glgC 3 (pExs66), MSTSIIa and glgC 3 (pExs53 and pExs56). The second group of rice transformatns contain MSTSIIc and glgC 3 (pExs54 and pExs56).The third group of transformation: transgenic 5 MSTSIII and glgC 3 pExs 61 and pExs 66); transgenic 6 Mwx glgC 3 p-Exs65 and pExs66). Generally see figures 25-41 for plasmid maps and figure 43-55 for sequences used in the plasmid. Additionally, glgA and glgB and glgC were combined and transformed into rice. This is combining the rice plants starch pathway with the gene encoding for ADPG and the genes encoding for at least one of the following enzymes, SSI, SSII, SSIII, Debranching enzymes, BEI, BEII, GBSS (wx).

24 These plasmids could have been transformed into other cereals such as corn, wheat, barley, oats, sorghum, milo in substantially the plasmid that is shown in the figures for the plant host. The promoter could be the waxy gene which is published, other additional zein promoters are known and could be used as the promoter. The promoter used herein is described in Figures 45aand Additionally these plasmid with little additional work could be transformed into dicots such as potatoes, sweet potato, taro, yam, lotus cassava, peanuts, peas, soybean, beans, chickpeas. The promoter could be selected to target the starch storage area of the particular dicots (some are roots some are tubers). Various method of transforming monocots and dicots are known in the industry and the method of transforming the genes is not critical to the present invention. The plasmid can be introduced into Agrobacterium tumefaciens by the freeze-thaw o methodof An et al.(1988) Binary vectors. In Plant Molecular Biology Manual A3, S.B. Gelvin and R.A. Schilperoot,eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 1- 19. Preparation of Agrobacteruim inoculum carrying the construct and inoculation of plant material regeneration of shoots, and rooting of shoots are described in Edwards et al. (1995).

Biochemical and molecular characterization of a novel starch synthase from potatoes. Plant J.

S8, 283-294. Additionally promoters for different dicots are known particularly 35sCaMV and Monsanto has also published a promoter that is useful in potatoes called a patatin promoter.

A number of monocots are also starch bearing plants but until about a decade ago monocots were difficult to develop transformants. The most prominent methods of transformation presently used in monocots is the gunning of micro projectiles into the plants or using Agrobacterium and subsequent regeneration of the plants from the transformed materials.

Various tissues and cells can now be transformed with plasmids into monocot hosts. In fact there are teaching from at least five ago on methods of transforming not only callus but also cotyledons. The methods of transforming plants and selecting for the transformants with either selectable or screen able markers are also well known. The use of the marker in the same plasmid and the use of the markers in a separate plasmid that is co transformed into the host are well known in the art by those of ordinary skill in the art. The biotechnology methods of forming plasmids and transforming plants are listed in the book entitled AShort Protocols In Molecular Biology@, 3rd ed. Published in 1995 by JOHN WILEY& Sons, Inc. Additionally, methods of transforming with the gun and with protoplasts are taught in a number of issued patents to Dekalb and Agracetus and Ciba.

PREFERRED EMBODIMENT OPERATION EXAMPLE 1 Construction of the E. coli expression vector.

The expression vector pExs2 was derived from pET-23d (Novagen) and pGP1-2 .The expression vectors pExs-trc and pExs-trc3 were derived from pTrc99a (Pharmacia) and pGP1-2. The BglII/PstI fragment (2192 bp) containing the pBR322 origin of replication was S: deleted from pET-23d and replaced with the BamHI/PstI fragment (3 kb) containing origin pl5A and kanamycin resistance gene from pGP1-2. This process generated plasmid pEXS1 containing both ampicillin and kanamycin resistance genes. The ampicillin resistance gene was inactivated by deletion of the Scal BglI fragment (360 bp, BglI end was filled in and bluntgo end ligated with Scal end). Inactivation of the ampicillin resistance gene in pEXS1 generated the expression plasmid pEXS2, containing the T7 promoter, T7 terminator, kanamycin Sresistance gene and pl 5A origin of replication. Plasmid pTrc99a was digested with Nde 1, filled in with kelnow fragment and blunt-end ligated to remove NdeI site. A Ndel site was introduced at the NcoI site by mutagenesis to generate plasmid pExs-trc. The BglI and PvuII fragment(2.48 kb) in pExs-trc containing the pBR322 origin of replication was replaced by BglI/BamHI (filled in with Klenow fragment) fragment (3 kb) containing origin pl5A and kanamycin resistance gene from pGP1-2 to generate pExs-trc2. The ampicillin resistance gene was inactivated by deletion of the Scal BglI fragment (360 bp, BglI end was filled in and blunt-end ligated with ScaI end). Inactivation of the ampicillin resistance gene in pExs-trc2 generated the expression plasmid pExs-trc3.

Construction of expression plasmids for maize SS. For expression of maize SS in E.

coli, The PCR method was used to modify the N-terminus of maize SS using the following nucleotides (SEQ ID NOs: 18-30 respectively, in order of appearance) primer Exs4 (5'CAAGAATGCTGCGGGAGTC-3'), primer Exs23 AAGTCGACATATGTGCGTCGCGGAGCTGAGCAG-3'), primer Exs 57 GGGCCCCATATGAGCATTGTCTTTGTAACCGG-3'), primer Exsl 26 CTCGGGCCCATATGGGGGAGAATGTTATGAA..3'), primer Exs;2 GAGGCATCAATGAACACAAAGTCG-3'), Primer Exs33 GAAGGGCCCCATATGGCTGAGGCTGAGGCCGGGGGCAAG.3), primer Exsl 6 TTGGATCCATATGGGAGCTGCGGTTrGCATTGGG.3') and primer Exsl17 CCTGCGGGCTCTGGCTTCACC), primer Exs 55 TTGGATCCATATGAACGTCGTCGTGGTGiCTrrC.3'), primer 56 GCATACCATGGAACCTCAACAGC-3'), primer 53 GGTACCATATGAACGTCGTCTrCGGCG..3'), primer Exs 54 GACAGGCCCGTAGATCTTCTCC-3'), primer Exs -wx TTGGTACCATATGGCCAGCGCCGCCGGCATGAAJCG.3'). Primer Exs 4* paired respectively with primer Exs23 and Exs; 57 was to modify the N-terminus of maize S SSI gene to generate pExs-lO and pExs-Id. Primer Exs2 paired individually with primer Exs33 and Exsl was to ffiodify the N-terminus of maize SSSII to generate pExs3c and pExs3a. Primer Exsl 7 paired individually with primer Exsl6 and Exs55 was to modify the N-terminus of maize- SSSIII to generate pExs-9 and pExs-9a. Primer Exs54 paired individually with primer Exs-wx and Exs53 was used to modify-the N-terminus of maize GBSS to generate pExs-wx and pExswx2. The modified N-terminus was recombined with the rest of the SS gene in pBluescript SK plasmid. The reconstructed of maize SS was subcloned from pBluescript Sk to the NdeI/NotI sites of the expression vector pET-21a (Novagen) pExs-trc, pExs-trc3 (maps are attached, *.*Table I shows the N-terminal sequence of SSS).

EXAMPLE 2 Construction of expression plasmids for E. coli ADPGlc pyrophosphorylase,

BE

and maize SBE.

E. coli glgB gene was excised from plasmid pOP 12 The BstXlI (filled in) Hindul fragment containing the glgB ribosome binding site and the full length glgB gene was cloned at the SinaI site of pBluescriptSK- (Stratagene). The glgB gene in pBluscriptSK- was subsequently cloned into pEXS2 at the XbaI Sall sites to generate plasmid pEXSB. Primer G (SEQ ID NO:31) (5'-GAAGATCTGGCAGGGACCTGCACAC..3') and primer H (SEQ ID NO:32) (5'-GGACTAGTGCATTATCGCTCCTG=r'AT..3') were used to PCT the E. Coli glgC gene coding for ADPGlc pyrophosphorylase from plasmid pOP 12. A BglII site and a SpeI site introduced by PCT to the N-terminal and C-terminal site respectively, were used to clone the 27 PCR product into pBluscript SK- at the BamHI and Spel sites. The glgC gene including its own ribosome binding site was subcloned into expression plasmid pEXS2 at the XbaI (filled in with Klenow fragment) and NotI site to generate plasmid pEXSc. The genes coding for mature maize SBEI and SBEII along with a ribosome binding site were subcloned from plasmids pET- 23d-SBEI and pET-23d-SBEII into the plasmid pEXSc at the Spel site to form the plasmids pEXSc-SBEI and pEXSc-SBEII. The gene coding for mature maize SBEII including a ribosome binding site was cloned into pEXSc-SBEI at the Xbal/NotI sites to form plasmid pEXSc-SBEI-SBEII. E.coli glgc gene and genes encoding maize SBEI and SBEII were also cloned in plasmid pExs-trc and pExs-trc3 respectively and together as described for pExs2.

EXAMPLE 3 i Isolation of E. coli HPG204 deficient in GBE and GS activities Homologous recombination was used for the strain construction. This was done according to the method described by Hamilton et al Journal of Bacteriology, 1989, S171:4617-4622.) A temperature-sensitive pSC101 replicon was used to facilitate the selection.

The gene coding for spectinomycin adenyltransferase was inserted at PvuII sites in plasmid pOP12 to form plasmid HPG9 which has spectinomycin resistance and has C-terminus of glgB gene and N-terminus of glgA gene deleted. The DNA fragment B=SA= with Spectinomycin resistant gene inserted between partial truncated glgB and glgA was subcloned into plasmid pMAK705 at XbaI site containing temperature sensitive replicon (Hamilton et al. Journal of Bacteriology, 1989, 171:4617-4622.) to form Plasmid pMak705B=SA=. Plasmid pMak705B=SA= was transformed into TG1 cell. After the transformed cell was cultured in 3 mL LB with 100 mg/mL Spectinomycin at room temperature overnight, the cells were plated on LB agar plate containing 100 mg/mL spectinomycin and incubated at 44 0 C overnight.

Single colonies were inoculated on LB agar plate containing 100 mg/mL spectinomycin and 0.2% glucose and incubated at 44 0 C and at 37 0 C overnight. The colonies at 37 0 C were stained with iodine. The colony with negative staining was selected and grown in 100 mL LB at 37 0

C

overnight. The cells were harvested and homogenized in an extraction buffer for assaying glycogen synthase and branching enzyme activities. The cell lacking glgA and glgB activities was named as HPG204 [F=traD36 LacI q D(glgBXCA) D(LacZ)M15proA'B*/SupED (hsdMmk, McrB)thiD(lac-proAB),SpectinomycinR, ChloramphenicolR .The IDE3 28 lysogenization kit fromNovagen was used for site specific integration of 1DE3 prophage into E. coli HPG204 to form E. coli HPG204(DE3)[ was DThe lysate was prepared with Plvir and its transduction into E. coli BL21 (DE3) [F=traD36 LacI D(glgBXCA) (hsdM-mcrB)5(r; mn; McrB)thiD(lac-proAB),SpectinomycinR, Chloramphenicol R EXAMPLE 4 Expression of maize SS and SBE in E. coli.

Plasmid pExs-2 and pExs-trc3 has kanamycin resistance and pl5A origin of replication.

It is compatible with plasmid pET21a, pExs-trc, pTrc99A containing pBR322 origin.

Expression plasmids pExs-2 and pET-21a were used to express SS and SBE in E. coli SHPG204(DE3). Expression plasmids pExs-trc and pExs-trc3 were used for expression in E coli G6MD3. This made it possible to transform different combinations of maize SS and SBE in E. coli HPG204(DE3), or G6MD3 which is deficient in GS and GBE activity. An overnight culture of cells transformed with maize SS and SBE was diluted 1:20 in fresh LB containing 0.2 glucose, 100 mg/mL ampicillin and 50 mg/mL kanamycin. The cells were grown at 37 0 C for about 2 h to A600nm =0.6 before the expression of maize SBE and/or SS was induced by adding isopropyl b-D-thiogalactoside to 0.5 mM. Following growth at 25 0 C for 4 h, the cells were harvested in a refrigerated centrifuge.

Isolation of highly branched a-glucan from E. coli.

Cell pellet (30 g) was resuspended and lysed by sonication in 150 mL 50 mM trisacetate buffer (pH 7.5) containing 10 mM EDTA and 5 mM DTT. After a fraction of the homogenate was saved for assaying the STS and SBE activities, the homogenate was centrifuged at 20, 000g for 50 min at 4 0 C. After collecting the supernatant, the pellet was resuspended in 150 mL water and boiled for 15 min with occasional stirring. The resuspension was centrifuged at 20, 000g for 30 at room temperature. After collecting the supernatant, the pellet was washed again with 100 mL water as above. 0.1 volumes of 50% Trichloric acid (TCA) were added to the pooled fractions. After storing on ice for 30 min, the precipitate was spun down at 15,000g for 20 min, then washed with 30 mL 5% TCA and centrifuged as above.

The supematant and wash were pooled and one volume of absolute ethanol was added. After storing on ice for 30 min, the polysaccharide was collected by centrifuging at 15, 000g for min. The polysaccharide was redissolved in water and precipitated with ethanol. This step was repeated twice. The pellet was washed with methanol twice, acetone twice and dried over silica gel at room temperature.

EXAMPLE 6 Isolation of linear a 1,4 polysaccharide from E. coli Resuspend 50 grams of cell pellet in 250 mL of 50 mM Tris acetate buffer, pH containing 10 mM EDTA and 5 mM DTT. Sonicate for 3 minutes (45 seconds/time, output S8, repeat 4 times with 30 seconds interval). The homogenate is centrifuged at 12,000 rpm S(SA1500) for 50 minutes. The supernatant is checked with iodine staining and discarded.

(Same 1 mL homogenate and 1 mL supernatant for enzyme assay. The pellet is resuspended extracted in 100 mL DMSO. Extract the polysaccharide by heating and stirring in boiling water bath for 15 min. Let it cool down to below 40 OC and centrifuge at 12,000 rpm for 30 min at room temperature. The supernatant is pooled. The pellet is extracted two more times with 100 mL DMSO. Equal volume of absolute ethanol is added into the pooled supernatant, mixed and stored on ice for 30 minutes. Centrifuge at 12,000 rpm for 30 min at 4 OC The pellet is redissolved in 20 mL DMSO by heating in boiling water bath. 80 mL water is added and mixed Swell. After adding 10 mL butanol to the solution, the solution is mixed and stored at 0 OC for one hr (mix once a while). Centrifuge at 12000 rpm for 30 min at 4 0 C. Repeat the step once.

The pellet is redissolved in 90 mL hot water by heating in boiling water bath. Insoluble materials are immediately removed by centrifugation at room temperature. Add 10 mL butanol to the supernatant, stay at 0 oC for 1 hr and centrifuge at 12000 rpm for 30 min at 4 OC. Repeat the step once. The amylose precipitate is redissolved in 90 mL hot water by heating, and 10 mL butanol are added to the solution. After storing at 40 OC on ice for one hour, it is centrifuged at 4 OC for 30 min. Repeat the step once. The pellet is redissolved in 100 mL 10% butanol by heating. The amylose is stored at 0 OC and precipitated by centrifuging at 12000 rpm for min at 4 OC. The pellet is washed with 25 mL methanol 3 times and with acetone once. Dry over silica gel.

EXAMPLE 7 Enzyme assays mL of supernatant were used to assay STS and SBE activities as previously described (Preiss) with minor modification. The reaction mixture for STS contained 100 mM Bicine buffer, 10 mg/mL glycogen, 0.5 mg/mL BSA, 0.5 M sodium citrate, 25 mM potassium acetate, mM GSH, 3 mM [14C]ADPGlc (500 dpm/nmol) and enzyme in a final volume of 0.1 mL.

The reaction was carried out at 25 0 C for 15 min and terminated by boiling for 2 min. The unincorporated [14C]ADPGlc was separated with Dowex anion exchange column (200-400 mesh, Sigma Chemical One unit of activity is defined as 1 nmol Glc incorporated into the a-glucan per min at 25 0 C. SBE activity was determined by phosphorylase stimulation assay.

One unit of activity is defined as 1 mmol Glc incorporated into the a-glucan per min at 30 0

C.

Example 8 Enzyme purification :0 For the recombinant SS purification, the cell pellet was resuspended in sonication buffer mM Tris-acetate, pH 7.5, 10 mM EDTA, and 5 mM DTT; 7 ml buffer per gram of cell mass), and cells were lysed using a Fisher 550 Sonic Dismembrator with 5 x 1 min. bursts with sec. intervals. The homogenate was centrifuged at 96 00g for 30 minutes. SSI in the supernatant was then precipitated by slowly adding neutralized saturated ammonium sulfate to saturation. After stirring on ice for an additional 50 minutes, proteins were collected by centrifugation at 12700g for 45 minutes. The protein pellet was then redissolved in buffer A mM Tris, pH 7.5, 1 mM EDTA, and 5 mM DTT) containing 0.1 M KCI and dialyzed against the same buffer, with one change of buffer. After dialysis, the sample was centrifuged at 1 3 000g for 20 minutes to remove insoluble materials. The resulting supernatant was loaded onto an amylose affinity column pre-equilibrated with dialysis buffer, and the flow through was collected. The column was washed with 10 column volumes of buffer A containing 0.1 M KC1, and then with buffer A containing 0.5 M KC1 and 0.5 M maltose, collecting fractions during both washes. The active fractions were pooled and dialyzed overnight against buffer A, with one change of buffer. The next day, the amylose column sample was filtered and applied to a mono Q 5/5 FPLC column (Pharmacia). After washing with buffer A, a 20 ml 0-0.4 M KCI gradient was employed. The active fractions were electrophoresed on an 8% SDS-PAGE gel (31) to determine the purity of SSI in those fractions; the fractions which were apparently homogeneous were pooled and concentrated using a Centricon-30 spin column (Amicon).

o Table 1. Expression of maize starch synthases in Escherichla coil BL2I (DE3).

-Plasrnids; Maize starch synthase. N-terminus Protein Specific Activities* ee mtL (unitsling Protein)gne(mlL pET21 a Native plasmid 1.8 0.009 PEXS-3a SSIla-2 GENVMNV1W 2.8 0.069 (SEQ ID NO: I).

pEXS-3c SSIla AEAEAGGKD 2.8 0.28 (SEQ ID NO:2).

ODEXS-ld SS1-3 MSIVFVTGEA 3.0 0.23 (SEQ ID N03).

PEX(S-8 SSI-2 GDLGLEPEG 1.9 0.097 (SEQ H) NO:4) CVAELSREG 1.2 0.043 20 (SEQ ID pEXS-9 SSlb GSVGAALRSY 1.8 0.515 pEXS-9a SSIIb-2 MNWVVASEC 2.6 0.36 (SEQ ID NO:7) pEXS-wx GBSS (waxy) ASAGMNWFV 2 0.033 p: 5 SIwN GBSS(2) MNVVFVGAEM 2.2 0.32 (SEQ MDNO:9) Oeunit activity is defined as one lmol glucose incorporated into a-1 ,4 glucan per minute at ousing 5 mg/mL glycogen as primer.

0.00 0 0 0 0 0 *0 0 0 00* *0 Table 2. Properties of polysaccharides synthesized Plasmid Protein STS activity BE activity (Mg/mi) (u/mg protein) (u/mg protein) pExsCA pExsC-9 pExsC-3a 13.3 .0015 pExsC-8 12.6 pExsC-wx 15.2 pExsC-l-ll pExs9 7.84 pExsC-I-lI pExs3a 13.61 pExsC-l-Il pExs8 11.95 pExsC-l-Il pExslO0 8.9 pExsC-l-II pExswx 11.7 pExsC-l-Il pExsA1 11 .0032 0.002 0.08 0.011 0.042 .0094 .007 0.13 In E. coi.

Imax (nm) 580 585 600 580 600 480 530 525 500 450

DP

700 1007 983 435 836 2333 3616 1689 3174 2970 CL Yield (mg dry wt/g wet cell) 10.6 3.3 35.8 4.1 53 0i1 1.56 3.33 3.65 5.4 31.8 15.6 19 22 17.5 16.6 14.8 7.4 9.1 36 131 24.5 33.8 4.48 475 3940 14 28.9 S S

S

S**

S *5 S S S S S S S S S S S *5 Table 3. Properties listed by degree of DP of polysaccharides synthesized In E coil.

Plasmid Protein STS activity BE activity Imax DP CL Yield ce )(Mg/mL) (u/mg protein) (u/mg protein) (nm) mg dry wtlg wet pExsC-I-II pExsAl pExsC-l-II pExs3a 13.61 pExsC-1-11 pExsl 0 8.9 pExsC-I-ll pExswxl 1.7 pExsC-l-lI pExs9 pExsC-I-ll pExs8 pExsC-9 pExsC-3a pExsC-wx pExsCA pExsC-8 0.13 4.48 475 0.011 .0094 .007 1.56 3.65 5.4 530 500 450 7.84 11.95 0.08 0.042 4.71 3.33 13.3 15.2 .0015 0.002 480 525 585 600 600 580 580 3940 14 3616 22 3174 16.6 2970 14.8 2333 19 1689 17.5 1007 35.8 983 53 836 15.6 700 10.6 435 31.8 131 4.1 9.1 3.3 7.4 28.9 36 24.5 33.8 12.6 .0032 S S S S S S S S 55 S S S S S. 55 Table 4. Properties listed by degree of Xmax of polysaccharides synthesized in E. coil.

Plasmid Protein STS activity BE activity Xmax DP CL Yield cl)(Mg/mL) (u/mg protein) (u/mg protein) (nm) (mg dry wtlg wet pExsC-3a 13.3 .0015 600 983 53 pExsC-wx 15.2 0.002 600 836 15.6 9.1 pExsC-9 585 1007 35.8 4.1 pExsCA 580 700 10.6 3.3 pExsC-8 12.6 .0032 580 435 31.8 7.4 pExsC-I-1l pExs3a 13.61 0.011 1.56. 530 3616 22 36 pExsC-1-ll pExs8 11.95 0.042 3.33 525 1689 17.5 131 pExsC-1-1I pExsIO 8.9 .0094 3.65 500 3174 16.6 24.5 pExsC-I-ll pExs9 7.84 0.08 4.71 480 2333 19 pExsC-l-11 pExsAI 11 0.13 4.48 475 3940 14 28.9 pExsC-1-ll pExswx 11.7 .007 5.4 450 2970 14.8 33.8 i..

S

S

S S e S S S S S S .5 S S S S S S S. Table 5. Properties listed by degree of CL of polysaccharldes synthesized In E coil.

Plasmid Protel (Mg/rr cell) pExsC-3a 13.3 pExsC-9 pExsC-8 12.6 pExsC-i-Ii pExs3a 13.61 pExsC-l-lI pExs9 7.84 pExsC--II pExs8 11.95 pExsC-l-Il pExslO 8.9 pExsC-wx 15.2 pExsC-1-Il pExswx 11.7 pExsC-l-Il pExsAl 11 pExsCA n STS activity BE activity Xmax DP CL Yield QL (u/mg protein) (u/mg protein) (nm) mg dry wtlg wet .0015 .0032 0.011 0.08 0.042 .0094 0.002 .007 0.13 1.56 4.71 3.33 3.65 5.4 4.48 600 585 580 530 480 525 500 600 450 475 580 983 1007 435 3616 2333 1689 3174 836 2970 3940 700 53 35.8 31.8 22 19 17.5 16.6 15.6 14.8 14 10.6 36 131 24.5 9.1 33.8 28.9 3.3 Table 6. Purification Tables/or SSI-1, SSI-2, and SSI-3.

ssI-' Homogenate Supernatant 0-40% (Na 2

SO

4 Amylose column monoQ column volume (1114 630 570 48 17 0.27 total mg protein 4347 2622 494 2.6 0.26 activity U/mg 0.018 0.020 0.058 5.03 12.2 total units 76.2 53.0 28.7 11.3 purification (fold) 3.2 279 0~ 3.2 677 SSI-2 Homogenate Supernatant 0'40% 94H4) 2

SO

4 Amylose column monoQ column volume (111) 380 320 48 17.5 total mng activity protein U/mg 2797 0.0356 2118 0.0340 466 0.133 1.2 22.6 total Units 99.6 72.0 61.8 26.5 purification (fold) 3.7 634 1.0 0.325 17.2 5.6 483 SSI-3 Homogenate Supernatant 0-40% 2 S0 4 Amylose column monoQ column volume (ml[) 1300 1100 237 63 3.6 total mg protein 16770 9790 2204 30 3.1 activity U/mg 0.23 0.31 1.5 22.4 total Units 3900 3080 3294 668 purification (fold) 1 1.3 97 132 30.5 93 Notes: Assays performed during the course of purification contained 10 mg/mI glycogen and 3 mM 4 C]-ADPGlc. Assays were performed at room temperature in the presence of 0.5 M citrate. 1 Unit 1 pLrnol [U_ 14 C]-glucose transferred per min.

Table 7. Primer Kinetics for SSI enzymes Amylopectin SSI-3 SSI-2 ssI-1 citrate F, Vm2X 240 45 26.3 0.5 230 50 33.4 2.1 68 3 9.94 0.18 *999 99 9~9.

9 *9 9 *99999 .9 .9 9 9 .9.

150 t 22.5 t 0.6 120 t 7.62 t 0.99 citrate Km 230 60

V

1 1 13.2 0.3 Glycogen SSI-3 9 .9 .9 999~ .9 9 .9 9.

.9 citrate citrate

IV,,,

43.4 2.5 41.4 2.9 SSI-2 45.6 ±3.3 45.5 ±1.5 ssI-1 39.0 2.2 26.1 1.4 .99 '9 9 <9 9. .9 Notes: Assays were performed at 37*C as described in the Materials and Methods. Data are expressed as the average of three independent determinations along with the standard deviation. K. are expressed as l.Lg/ni primer and are in 4.mol/min/mg protein. ADPGlc =3 mMy in all assays.

'Because saturating glycogen concentrations could not be obtained, a standard 20 mg/mI glycogen was used to compare enzyme rates for that primer.

Table 8. ADPGc KEne ir STSI enzymeS. Assays and data evaluation are as in Table II K. are expressed as mM ADPGc and V. are in grna1/nin/mng protein. 5 mg/nil amylopectin was used as primer for all assays.

STSI-3 STSI-2 STSI-1 citrate K. 0.33 0.07 V. 26.4 1.4 citrate: K. 0.62 0.04 V. 14.7 1.3 0.32 0.02 32.6 0.8 0.25 ±0.04 11.7± 0.7 0.18 ±0.02 18.0 0.24 0.02 6.38 0.88 Table 9. Purification Tables for SSI~a enzymes. Assays for SSMa-2 purification contained 10 mg/nil glycogen and 1.5 mM [U- 14 C]-ADPGlc (both are at saturating concentrations). Assays for SS~a-1 purification contained 5 mg/mI amylopectin and 3 inM

[U_

1 4 CJ-ADPGlc. Assays were performed at room temperature in the presence of 0.5 M citrate. 1 U 1 AZmol [TJ-' 4 C]-glucose transferred per min.

4.

9* SSI~a-2 volume (m11) 300 total mg activity protein U/mg 1620 0.0216 Supernatant total Units 34.8 25.4 purification (fold)

I

4* *6 C. 4 4 4 0-40% (INH4 SO 4 Amylose column monoQ column 20 9.3 0.991 9.3 53 419 0.0606 2.8 45.9 222 0.9 0.94 4.81 SSna-1 volume (ml') 335 total mig activity protein U/ng 2613 0.28 total Units 737 purification (fold) 1 Supernatant 0-40% (NM)IS0 4 Amylose, column monoQ column 47 427 0.96 409 25 11.5 8.04 1.0 4.8 9.10 92 28.7 44 32.5 Table 10. Primer Kinetics for SSIIa enqmes. Assays were performed as described in the Materials and Methods. Data are expressed as the average of three independent determinations along with the standard deviation. K. are expressed in pg/ml and Va. are in pmol/min/mg protein. ADPGIc 3 mM in all assays. *NA not applicable; enzyme cannot be saturated by primer under these conditions.

Amylopectin SSIIa-2 o too:*, oq 0 0

C

so* oO S *s 0

O

citrate 27 0 C Km- V37°C K 37-C Km 153 22 7.82 0.63 133 18 SSIIa-1 182 38 24.1 153 64 41.1 0.2 Vmu 15.4 0.6 C C

SU

5 S C c'

S

CC

0o See.

citrate 27 0 C K, 234 30 Vmaz 37°C Km 4.31 0.32 1350 220 7.84 0.25 404 33 10.5 0.3

-NA'

VMax -NA' Glycogen SSIIa-2 SSIIa-1 citrate 27°C K 50.7 3.8 162 17 Vmax 5.53 0.44 14.2 0.7 37°C K 76.9 7.8 350 11 V=ai 11.3 0.7 31.6 0.8 Table 11. ADPGlc AKintcs for SSI~a enzmes. Assays and data evaluations are as in Table IL Concentration of primer in each case was saturating for each enzyme and was determined by the experiments detailed in Table IL. K. are expressed as mM ADPGlc and V.are In lLmol/min/ing protein. *NA not applicable, as the enzyme cannot be saturated by primer under these conditions.

with amylopectin as primer SSIIa-2 SSI~a-1 citrate 27 0 C K 1 m Vms 1 37-C Km 0.17 0.04 4.83 0.42 0.28 0.01 0.48 0.09 23.0 0.83 0.08 49. 1 12.6 VMs 1 11.4 0.6 citrate 27 0 C Km 0.27 0.02 VmZ: 4.87 ±'02 0.46 0.06 12.1 0.8

-NA*

-NA*

37 0 C Km

VMILZ

0.28 ±0.005 7.86 ±0.53 with glycogen as primer with lvcozen SSIIa-2

SSII

citrate 270C Km 0.16 t 0.03 0.19 0.02 Vmau 4.41 ±t 0.21 17.1 0.7 37"C Km 0.15 0.03 0.37 1 0.04 VmZ% 7.60 0.94 40.1 1.7 Table 12. Purificadon Tables for SSI~h-2 and SSIDb-1. Assays performed during the course of purification contained 10 mgfml glycogen and 3 mM [U- 1 'C]ADPGlc. Assays were performed at room temperature in the presence of 0.5 M citrate. 1 IT 1 ILmol [U- 4 CjglUcose transferred per min.

SSIIb-2 Supernatant 0-40% (NIL4) 2

SO

4 Amylose column monoQ column volume (ml1) 890 190 13 6.6 total mg protein 9256 2660 31.2 16.3 activity U/mg 0.48 1.24 50.6 56.8 total Units 4450 3306 1573 939 purification fold 1 2.6 105 118 SSI~b-1 Supernatant 0-40% (N h)S0 4 Amylose column monoQ column volume (111) 365 56 80 0.6 total mg protein 2336 436 10.4 0.28 activity U/mg 0.64 2.35 50.2 60.6 total units 1533 1030 521 17.6 purification fold 1 3.7 78 94 44 Table 13. Kinetics for SSIfb enzymes. Assays were performed at 37*C as described in the Materials- and Methods. Data are expressed as the average of three independent determinations along with the standard deviation. For ADPGIc kinetics, 4. are expressed in muM ADPGlc. For primer kinetics, 4. are expressed as mg/mi primer, and 3 mM ADPGlc were used in the assays. V.

are in j.Lmol min7 1 mg' protein.

ADPGlc Kinetics SSIMb-2

SIM-

with glycogen Kmn vila' with amylopectin 0.32 0.04 130 6 0.71 ±0.01 76.8 ±3.2 0.32 ±0.03 90.9 ±4.2 0.40 ±0.02 72.8 ±2.8 V maX Primer Kinetics SSIIb-2 SSIIb-1 glycogen Km 0.36 0.02 0.43 0.02 79.5 3.3 VMax amylopectin 120 3 Km 0.26 ±0.04 VMax 84.5 ±2.4 0.074± 0.008 67.9 1.7 Table 14. Compariso n of kinetic data for expressed SS's. Data for SSI and SSIIa are form Imparl-Radosevich et al, 1998; Impari-Radosevich Li P, McKean Al, Keeling PL, and Guan HP, submitted for publication. Kn for ainylopectin and glycogen are epxressed in mg/mi; for ADPGlc are in mM and were determined in the presence of amylopectin and M citrate. are in p.mol min'lmg-. The K. for glycogen for SSI could not be determined as saturating concentrations of glycogen could not be reached for this enzyme.

Kinetic Parameter K. for amylopectin K, for glycogen for ADPGlc (with amylopectin) (with glycogen) 0.15 0.077 SSI-3* SSI-1 SSI~a-2a SSI~a-1 SST~b-2a SST~b-1 0.18 22.5 39.0 .0.32 84.5 120 'denotes N-terminally truncated form of SS, while any SS with the designation SS-1 is the full length version of the Ss.

C Table 15. The starch synthase activities of the chimerical enzymes.

Generation of chimerical enzymes of maize starch synthase: the recombination of N-terminal extensions with C-terminal catalytic domains of starch synthase The gene coding for N-terminal extensions of STSI, STSIIa and STSIIb were inserted, in the same or reverse orientation of original N-terminal DNA sequence, in front of the C-terminal catalytic domains of WX2, STSIla and STSIIb, respectively. The chimerical enzymes were expressed in E.coli, and the activities were assayed.

WX2 STSIIa STSIIb C-catalytic domain C-catalytic domain C-catalytic domain STSI N1-WX2 N1-C2 N1-C3 N-extension NRA 9.0 6.6 39.7 89.2 NRA STSHa N2-WX2 N2-C2 N2-C3 N-extension 9.2 11.2 213.8 8.7 232.5 NRA STSIlb N3-WX2 N3-C2 N3-C3 N-extension NRA NRA 11.2 NRA 400.5 12.0 N: STSI N-terminal extension; N2: STSIIa N-terminal extension; N3: STSIIb N-terminal extension; WX2: WX2 C-terminal catalytic domain; C2: STSIIa C-terminal catalytic domain; C3: STSIIb C-terminal catalytic domain.

the N-terminal extensions were inserted in front of the C-terminal catalytic domains in same orientation; the N-terminal extensions were inserted in front of the C-terminal catalytic domains in reverse orientation.

Starch synthase enzyme activity: nmol/min mgprotein.

The residue glycogen synthase activity ofBL21(DE3) is 2.6nmolmin mg protein.

NRA--No recombinant available.

47 The photographs listed in the figures 42 and 21attempt to show the visual differences that are present into the starches as compared to those known in the art.

Description of the starch Corn starch is a milky, slightly thickened gel which is slightly if at all flowable.

Rice starch forms two levels the upper level is a thickened syrup like consistency more flowable then corn starch (less thick then corn starch) opaque milky color (more translucent o then corn starch in this level) and a lower level which is a very white glob not transmitting S much light through this bottom level of material. This lower level is formed in a very thick o mass and does not appear flowable.

Corn amylopectin is slightly less white then the top level of rice starch and is a very slightly opaque milky color (more translucent then corn starch) slightly less flowable then the rice top level.

Potato dextrin is the most transparent almost appearing clear but is still opaque white and it is very flowable appearing only slightly less flowable then water.

Waxy Maize starch will flow very slowly and has the consistency of honey. The color is very opaque transmitting little light and the color is only slightly less light then corn starch.

SSI starch made from plasmid pExs-8 has two distinct levels. The top level appears clear and slightly thicker then the flowability of water. The bottom level appears as a precipitate. This sample resembles the ornaments that contain little figures and plastic flakes resembling snowflakes. Like those ornaments when turned upside down the sample appears to be falling snow. However the flakes in this sample appear to be slightly gummy and appear in the first moments of level mixing to form a opaque white liquid.

SSI starch made from a host containing the following two plasmids pExsC BEI BEII and pExs8 is not as clear as the top level ofpExs-8 and appears slightly less thick then pExs-8.

48 It has even more flowability then does Potato Dextrin.

SSIIb starch made from a host containing the following two plasmids pExsC BEI BEII and pExs-9 is not as clear as the top level ofpExs-8 and appears slightly less thick then pExs-8.

It has even more flowability then does Potato Dextrin.

WAXY starch made from a host containing the following two plasmids pExsC BEI BEII and pExs-wx is not as clear as the top level of pExs-8 but seems to have a few tiny thread like chains that settle to the bottom and when mixed give the material a slightly more white color and appears slightly less thick then pExs-8. It has even more flowability then does Potato Dextrin.

SSII starch made from a host containing the following two plasmids pExsC BEI BEII and pExs-3a is the color of corn starch and maybe slightly whiter but not as white as the bottom level of pExs8 and definitely transmitting more light through and has the flowability characteristic of pExs-8 when mixed.

*.e .lgA starch appears to have a very slight precipitate and is comparable in color to corn amylose pectin and ExsC BEI BEII and pExs-wx. And the flowability is between corn amylose and pExsC BEI BEII pExs-wx.

The samples of polysaccharides listed above form groups generally according to color as follows: waxy maize starch and corn starch and pExsC BEI BEII pExs3a and pExsc8 are the whitest group. The flowability characteristics of this group are fairly diverse. With corn starch a lump and Waxy maize starch only slightly flowable and pExsC BEI BEII and pExs- 3a and pExsC-8 more like water then syrup. The second group contains corn amylopectin and pExsC BEI BEII pExs-wx and pExsC BEI BEII and pExs-Al which are less white and clearer.

The flowability of corn amylopectin is less then the other two members of this group but it is still similar. The last group is the least white and thus the clearest. This group includes pExsC BEI BEII and pExs-8, potato dextrin, pExsC BEI BEII and pExs-10, pExsC BEI BEII and pExs-9. The flowability of this group is also similar to each other.

Plant Hosts The following plasmids have been transformed into rice plants. The sequence for the mutant glgC gene is shown in Figure 46. The plasmids are made substantially in a similar manner as described above for the production of bacterial plasmid. Clearly the plasmid maps shown in figures 25-41 and this application and the listed short protocols allow the ordinarily skilled person in the art to make the present plasmids. The following combinations of plasmids have been transformed into rice plants. Additionally combinations of plasmids including the combination that includes all of the maize genes SSI, SSII, SSII, BEI, BEII, and GBSS in one *99999 host or alternatively in two host that are then crossed to form a hybrid having the entire complement of up regulated starch genes are being developed. Clearly the ordinarily skilled person in the art could have placed the sequences in the antisense positions to down regulate these genes to the extent that maize genes will down regulate the partial homologous rice genes. The first group of transgenic are groupl, including rice transformants (transformed by microprojectile bombardment) containing MSTSI-2 (pExs52) and glgC 3 (pExs66), MSTSIIa-2 and glgC 3 (pExs53 and pExs56). The second group of rice transformants contains MSTSIIa and glgC 3 (pExs54 and pExs56). The third group of transformation contain: transgenic MSTSIIb and glgC 3 (pExs 61 and pExs 66); transgenic 6 Maize wx and glgC 3 pExs65 and pExs66). Additionally, glgA and glgB and glgC are combined and transformed into rice. This last transformant is combining the rice plants starch pathway with the gene encoding for ADPG pyrophosphorylase and the bacterial genes. The combination of the plasmids encoding for at least one of the following enzymes, SSI, SSIIa, SSIIb, SSIII, Debranching enzymes, BEI, BEII, GBSS (wx)and some or all of the bacterial starch genes is also useful. There ae presently over 300 transformants in the greenhouse.The T1 transgenic rice plants have been screened and characterized (Figure 56, 57). 12 plants have successfully expressed maize SSI-2 in rice seeds.

21 plants have successfully expressed maize SSIIb in rice seeds. We are currently screening rice plants down regulated the rice SS expression by cosuppression and have 400 T2 plants in the greenhouse.

Maize Starch Synthase and its Mutant Forms.

In order to characterize the multiple forms of maize starch synthase, the genes coding for the full length SS and its N-terminally truncated forms were expressed in E.coli,. The recombinant enzymes were purified and kinetically characterized. We have demonstrated that different isoforms and its truncated forms all have distinct properties (Table 6-14, Figure 58- 63). The specific activities (Vmax) of the purified maize SSI-1, SSI-2, and SSI-3 were 22.5, 33.4 and 26.3 :mol glc/min/mg of protein respectively, Our results have clearly indicated that the catalytic center of SSI is not located in its N-terminal extension. However, N-terminal truncation decreased the enzyme affinity for amylopectin, with the K, for amylopectin of the truncated SSI-3 being about 60%-90% higher than that of the full length SSI-1. The effects of S' N-terminal truncation of SSIIa depend upon the assay conditions used. For both SSIIa-1 and SSIIa-2, the of each enzyme increased 2-fold upon raising assay temperature from 27 NC to 37 NC (Tables II and III). However, the effect of temperature on ADPGlc affinity was S. different for SSIIa-1 and SSIIa-2. For the truncated SSIIa-2, the K. for ADPGlc was not affected by raising temperature. In contrast, the K of ADPGlc for the putative full length SSIIa-1 increased 2 fold upon raising the assay temperature from 27 NC to 37 NC (Table III).

Interestingly, the truncated SSIIa-2 exhibited a lower Km for ADPGlc than SSIIa-1 did in all assay conditions used in this study except that they showed similar Km values for ADPGlc when glycogen was used as a primer at 27EC. Although N-terminal truncation of SSIIa appears to lower the Km for ADPGlc under most assay conditions, it also must be noted that the maximal velocity of the truncated SSIIa-2 is decreased by about 2-4 fold when compared to SSIIa-1. The truncated SSIIb-2 was found to be more temperature stable than the longer SSIIb- 1 in the presence of citrate, while little difference was observed in their pH activity profiles.

While the putative full length SSIIb-1 showed a similar Vm, using amylopectin or glycogen as a primer, the N-terminally truncated SSIIb-2 showed a 40% increase in Vm, using glycogen compared with amylopectin as a primer. N-terminal truncation of SSIIb increased its Vm, by with amylopectin as a primer. We also demonstrated that kimeric enzymes of maize starch synthase (combining the C-terminal domain of SS with different N-terminal sequences of SS or unrelated sequences would produce a functional enzyme with SS activity and altered properties) (Table Conclusions, Ramifications, and Scope Accordingly, it can be seen that, according to the invention, The starch genes can produce new and altered starch in either host, plant or bacteria. Additionally, polysaccahrides very similar to corn starch can be produced in a bacterial host.

Although the description above contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention. Various other embodiments and ramifications are possible within it's scope. For example, different combinations of the Sfl plasmids in either host for the production of useful plant and useful grain and useful

S

polysaccahrides.

Thus the scope of the invention should be determined by the appended claims and their legal equivalents, rather than by the examples given. All references cited herein are incorporated herein in their entirety by reference.

S*

Page(s)MlV 1 are claimspages they appear after the sequence listing 52 SEQUENCE- LISTING <110> Guan, H-anping Keeling, Peter L.

<120> PLANT LIKE STARCHES AND THE METHOD OF MAKING THEM IN

HOSTS

<130> 2461-52 <140> 69/402,254 <141> 1999-10-01 <150> PCT/US98/06660 <151> 1998-04-03 *<150> 60/042,939 <151> 1997-04-04 *<160> 77 <170> Patentjn Ver. 2.1 *<210> 1 <211> <212> PRT <213> Zea mays <400>1u Asn Val Met Asn Val Ile Val Val 1 5 1 <210> 2 *<211> 9 <212> PRT <213> Zea mays <400> 2 Ala Glu Ala Glu Ala Gly Gly Lys Asp 1 <210> 3 <211> <-212> PRT <213> Zea mays <400> 3 Met Ser Ile Val Phe Val Thr Gly Glu Ala 1 5 <210> 4 <211> 9 <212> PRT <213> Zea mays <400> 4 Gly Asp Leu Gly Leu Glu Pro Glu Gly 1 <210> <211> 9 <212> PRT <213> Zea mays 53 <400> Cys Val Ala Glu Leu Ser Arg Glu Gly 1 <210> 6 <211> <212> PRT <213> Zea mays <400> 6 Gly Ser Val Gly Ala Ala Leu Arg Ser Tyr 1 5 <210> 7 <211> :<212> PRT <213> Zea mays <400> 7 Met Asn Val Val Val Val Ala Ser Glu Cys 5 <210> 8 <211> <23 T mays <400> 8 Ala Ser Ala Gly Met Asn Val Val Phe Vai 1 5 <210> 9 <211> <212> PRT Zea mays <400> 9 Met Asn Val Val Phe Val Gly Ala Glu Met 1 5 <210> <211> 8 <212> PRT <2.13> Zea mays <400> Ala Ala Ala Pro Ala Gly Glu Glu 1 <210> 11 <211> 11 <212> PRT <213> Zea mays <400> 11 Cys Val Ala Glu Leu Ser Arg Glu Gly Pro Ala 1 5 <210> 12 <211> 12 <212> PRT <213> Zea mays <400> 12 Met Asn Val Val Val Val Ala Ser Glu Cys Ala Pro 1 5 <210> 13 <211> <212> PRT <213> Zea mnays <400> 13 Ala Ser Ala Gly met Asn Val Val Phe Val Gly Ala Glu Met Ala 1 5 10 1 <210> 14 <211> 11 <212> PRT <213> Zea mays <400> 14 Met Asn Val Val Phe Val Gly Ala Glu Met Ala 1 5 <210> <211> 13 <212> PRT <213> Zea mays <400> Met Ser Ile Val Phe Thr Gly Glu Ala Ser Pro Tyr Ala *1 5 <210> 16 <212> PRT <213> Zea mays <400> 16 Cys Val Ala Glu Leu Ser Arg Asp Leu Gly Leu Glu Pro Glu Gly 1 5 10 <210> 17 <211> <212> PRT <213> zea mays <220> <221> MODRES <222> (2) <223> Ser or Thr <400> 17 Lys Xaa Gly Gly Leu 1 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 18 caagaatgct gcgggagtc 19 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 19 Saagtcgacat atgtgcgtcg cggagctgag cag 33 <210> <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gggccccata tgagcattgt ctttgtaacc gg 32 <210> 21 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer S<400> 21 ctcgggccca tatgggggag aatgttatga a 31 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 22 gaggcatcaa tgaacacaaa gtcg 24 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 23 gaagggcccc atatggctga ggctgaggcc gggggcaag 39 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 74 ttggatccat atgggagctg cggttgcatt ggg 33 <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> cctgcgggct ctggcttcac c 21 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 26 Sttggatccat atgaacgtcg tcgtggtggc ttc 33 <210> 27.

*<211> 23 <212> DNA o<213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 27 gcataccatg gaacctcaac age 23 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 28 ggtaccatat gaacgtcgtc ttcggcg 27 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence 57 <220> <223> Description of Artificial Sequence: primer <400> 29 gacaggcccg tagatcttct cc 22 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> ttggtaccat atggccagcg ccgccggcat gaacg 9 <210> 31 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 31 gaagatctgg cagggacctg cacac S<210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 32 ggactagtgc attatcgctc ctgtttat 28 <210> 33 <211> 1488 <212> DNA <213> E. coli <220> <221> CDS <222> (join(1..1431, 1435..1488)) <220> <223> glgA <400> 33 atg cag gtt tta cat gta tgt tea gag atg ttc ccg ctg ctt aaa acc 48 Met Gin Val Leu His Val Cys Ser Glu Met Phe Pro Leu Leu Lys Thr 1 5 10 ggc ggt ctg get gat gtt att ggg gca tta ccc gca gca caa ate gca 96 Gly Gly Leu Ala Asp Val Ile Gly Ala Leu Pro Ala Ala Gln Ile Ala 25 gac ggc gtt gac get cgc gta ctg ttg cct gca ttt ccc gac att cgc 144 Asp Gly Val Asp Ala Arg Val Leu Leu Pro Ala Phe Pro Asp Ile Arg 40 cgt ggc gtg Arg Gly Val acc gat gcg cag gta gta tcc cgt cgt Thr Asp Ala Gin Val Val Ser Arg Arg gat acc tec gcc Asp Thr Ser Ala 0*

S

0 0 *0 0 4* WS 0 0~

S

S

*4 0 0 0e a..

0 9*IS gga Gly c tg Leu ga t Asp c tg Leu cgt Arg geg Ala 145 cac His ate Ile gga Gly acg Thr gee Ala 225 ctt Leu acg Thr aaa Lys gac Asp aaa Lys ac Thr ggg Giy ect Pro 130 tat Tyr aac Asn caa Gin caa Gin geg Ala 210 tac Tyr tcc Ser gac Asp geg Aia ga t Asp 290 gg t Gly aac As n tgg Trp 115 ga t Asp c tg Leu cta Leu t tg Leu ate Ile 195 gtc Val1 gg t Gly gge Gly tta Leu gaa Giu 275 aaa Lys etc Leu cat atc His Ile att gac Ile-Asp acg Thr gcg Ala tta Leu 100 gtt Val1 gtg Val1 gcg Ala gc C Ala cca Pro 180 tct Ser ag t S er a tg Met gta Va I c tg Leu 260 aa t- Asn g tg Val1 ga t Asp c tg Leu ceg Pro ttt Phe ggg Gly gtg Val1 gcg Ala tat Tyr 165 tgg Trp ttc Phe cca Pro gaa Glu ccg Pro 245 ttg Leu aag Lys C cg Pro teg Ser ttc Phe etc Leu cat His gaa Giu gcg Ala 135 ggg Gly ggc Gly ttc Phe aag Lys tac Tyr 215 c tg Leu ggc Gly teg Ser cag Gin ttt Phe 295 c tg Leu gg t Gly tat Tyr ace Thr a tg Met 120 cac His egt Arg a tg Met ttt Phe gee Ala 200 get Ala t tg Leu gtg Val1 eg t Arg tea Ser 280 gca Ala gaa Glu eat Hi s ga t Asp gac Asp 105 gee Ala gac Asp ceg Pro ttt Phe aat As n 185 gg t Gly cgc Arg eaa Gin gac Asp tac Tyr 265 eaa Gin g tg Val1 gee Al a tac aao: Tyr Asfl cgt eeg Arg Pro 90 aae gta Asn Val age ggg Ser Gly tgg cat Trp His geg aag Ala Lys 155 tat gea Tyr Ala 170 att cat Ile His etg tac Leu Tyr gag atc: Giu Ile cag egt Gin Arg 235 gag aaa Giu Lys 250 ace ego Thr Arg ate goa Ile Ala gtg age Val Ser tea ceg Ser Pro gtt Vai agt Ser egt Arg gac Asp 125 ggc Gly g tg Val eac His e tg Leu gee Ala 205 gaa Glu ege Arg tgg Trp acg Thr gga Gly 285 c tg Leu te t Ser ggc at t Gly ile ceg tat Pro Tyr ttt geg Phe Ala 110 oca tte Pro Phe ctt geg Leu Ala ttt act Phe Thr atg aat Met Asn 175 gaa tte Glu Phe 190 gat cac Asp His ceg cag Pro Gin gaa ggg Glu Gly agt eca Ser Pro 255 ttg -gaa Leu Glu 270 tee aag Ser Lys ace age Thr Ser teg gag Ser Giu 720 305 310 ggc ggg cag ctg gcg cta Gly Gly Gin Leu Ala Leu 325 ggt ttc ctt gcg gcg gca Gly Phe Leu Ala Ala Ala 340 att ggc-tat cac gaa gca Ile Gly Tyr His Giu Ala 355 gtc att ctg gtg ccc agc Val Ile Leu Val Pro Ser 370 tat gga tcg aag tac ggt Tyr Gly Ser Lys Tyr Gly 385 390 ctt gct gat acg gtt tct Leu Ala Asp Thr Val Ser 405 gtc gcc aat ggg ttt atc Val Ala Asn Gly Phe Ile 420 cgg act att cga cgt gct Arg Thr Ile Arg Arg Ala 43 cgg ttt gtg caa cgt cag Arg Phe Val Gin Arg Gin 450 gcg gcg aag. tcg tac cgt Ala Ala Lys Ser Tyr Arg 465 470 gga aac gcc tac atg aat Gly Asn Ala Tyr Met Asn 480 485 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 148 ccg ttt aca tat tca.

Pro Phe Thr Tyr Ser 490 tcg ccc acg ctt Ser Pro Thr Leu 495 <210> 34 <211> 495 <212> PRT <213> E. coli <223> g1gA <400> 34 Met Gin Val Leu His Val Cys Ser Giu Met Phe Pro Leu Leu Lys Thr 1 5 10 Gly Gly Leu Ala ASP Val Ile Gly Ala Leu Pro Ala Ala Gin Ile Ala 25 Asp Gly Val Asp Ala Arg Val Leu Leu Pro Ala Phe Pro Asp Ile Arg 40 Arg Gly Val Thr Asp Ala Gin Val Val Ser Arg Arg Asp Thr Ser Ala 55 Gly His Ile Thr Leu Leu Phe Gly His Tyr Asn Gly Val Gly Ile Tyr Ile Asp Ala Pro 0 0.0 0 o Go..

000.

Asp Leu Arg Ala 145 His Ile Giy Thr Ala 225 Leu Thr Lys Asp Lys 305 Gly Gly Ile Val1 Tyr 385 Leu Val Thr Gly Pro 130 Tyr Asn Gin Gin Ala 210 Tyr Ser Asp Ala Asp 290 Gly Gly Phe Gly Ile 370 Gly Al a Al a Asn Trp 115 Asp Leu Leu Leo Ile 195 Val1 Gly Gly Leo Glu 275 Lys Leu Gin Leu Tyr 355 Leo Ser Asp As n *Leu 100 Val1 Val1 Ala Ala Pro 180 Ser Ser Met Val Leo 260 Asn Val Asp Leu Al a 340 His Val1 Lys Thr Gly 420 Phe Gly Val Al a Tyr 165 Trp Phe Pro Glu Pro 245 Leu Lys Pro Ser Al a 325 Ala Giu Pro Tyr Val1 405 Phe His Val1 Ala His Arg 150 Gln Ser Leu Thr Gly 230 Asn Ala Arg Leu Val 310 Leu Ala Ala Ser Gly 390 Ser I IleI Leu Tyr Asp Arg 90 His Thr Asp Asn 105 Glu Met Ala Ser 120 Ala His Asp Tr-p 135 Gly Arg Pro Ala Gly Met Phe Tyr 170 Phe Phe Asn Ile 185 Lys Ala Gly Leu 200 Tyr Ala Arg Glu 215 Leo Leu Gin Gln Gly Val Asp Glu 250 Ser Arg Tyr Thr 265 Gin Ser Gin Ile 280 Phe Ala Val Val 295 Leu Giu Ala Ser Leu Gly Ala Gly 330 Ala Glu Tyr Pro 345 The Ser His,.Arg 360 krg Phe Giu Pro 375 Phr Leu Pro Leo .sp Cys Ser Leu 410 'he Glu Asp Ser 425 Pro Val1 Gly His Lys 155 Ala His Tyr Ile Arg 235 Lys Arg Ala Ser Pro 315 Asp Gly Ile Cys Val1 395 Glu Asn Arg Asp 125 Gly Val1 His Leu Ala 205 Glu Arg Trp Thr Gly 285 Leo Ser Val1 Val1 Gly 365 Leu Arg Leu Trp Phe 110 Pro Leu Phe Met Glu 190 Asp Pro Glu Ser Leo 270 Ser Thr Ser Leo Gly 350 Gly Thr Thr Ala Ser 430 Ala Phe Ala Thr As n 175 Phe His Gin Gly Pro 255 Glu Lys Ser Glu Gin 335 Val1 Ala Gin Gly Asp 415 Leu Leu Trp Pro Val 160 Asp Asn Ile Phe Arg 240 Glu Asp Val1 Gin Gin 320 Glu Gin Asp Leo Gly 400 Gly Leo Gly Ser Pro Tyr His 61 Arg Thr Ile Arg Arg Ala Phe Val Leu Trp Ser Cys Pro Pro Leu Trp 435 440 445 Arg Phe Val Gin Axg Gin Ala Met Ala Met Asp Phe Gly Trp Gin Val 450 455 460 Ala Ala Lys Ser Tyr Arg Glu Leu Tyr Tyr Arg Ser Lys Phe Ser Gly 465 470 475 480 Asn A19 Tyr Met Asn Ala Pro Phe Thr Tyr Ser Ser Pro Thr Leu 485 490 495 <210> <211> 2361 <212> DNA <213> E. coli <220> <223> glgB <220> <221> CDS <222> (join(l. .2184, 2188. .2280, 2284. .2361)) <400> atg tcc gat cgt atc gat aga gac gtg att aac gcg cta att gca ggc Leu Ile Ala Gly cca aac gcc Pro Asn Ala 145 cgt cgg gtc tcg Arg Arg Val Ser 150 gtg gtt ggg caa Val Val Gly Gin 155 ttc aac tac tgg Phe Asn Tyr Trp gac 480 Asp 160 ggt cgc Gly Arg ctg ttt Leu Phe att gat Ile Asp gaa gcg Giu Ala 210 gaa aag Giu Lys 225 gcg cca Ala Pro acc gac Thr Asp gtg cct Val Pro att aac Ile Asn 290 ctg tat Leu Tyr 305 ttc att Phe Ile gtg cca Val Pro ggc acg Gly Thr gao tgg Asp Trp 370 ttc ctc Phe Leu 385 gcg ctg Ala Leu cgt aaa Arg Lys cg t Arg atc Ile gcc Ala 195 caa Gin gtt Val1 atc Ile aac Asn tat Tyr 275 gag Giu gcg Ala ga t Asp ggc Gly aac Asn 355 aac Asn gtc Val1 cgc Arg gag Glu cac His cct Pro 180 aat Asn a tg Met gta Val tct Ser aat Asn 260 gc t Ala cat His cca Pro gcc Ala cac His 340 t tg Leu acg Thr gg t Gly g tc Val1 ggg Gly 420 ccg Pro 165 ggg Gly ggc Gly cgc Arg cag Gin att Ile 245 ttc Phe 'aaa Lys ccc Pro acc Thr gca Ala 325 ttc Phe tat Tyr c tg Leu aac Asn ga t Asp 405 gag Glu ogt aaa gag Arg Lys Glu 170 ggt cag Cco Gly Gin Leu 185 ctg aag tcco Leu Lys Ser gcg tot Ctt Ala Ser Leu cgc aaa aaa Arg Lys Lys 235 cac otg ggt His Leu Gly 250 tac cgc gag Tyr Arg Giu 265 ttt aoc cac Phe Thr His agt tgg ggt Ser Trp Giy ggt act Cgc Gly Thr Arg 315 ggt ctg aao Gly Leu Asn 330 gac ttt gog Asp Phe Ala 345 gat cog cgt Asp Pro Arg tat ggt cgc Tyr Gly Arg tgg att gaa Trp Ile Giu 395 tca atg att Ser Met Ilie 410 aac gaa ttt Asn Glu Phe 425 ggC Gly aaa Lys cct Pro 205 tgo Cys aat As n tgg Trp gcc Ala gaa Glu 285 cag Gin gao Asp att.

Ile gcc Ala ggc Gly 365 gaa Glu ttt.

Phe cgc Arg ggg Gly 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 ctt gaa gcg att gaa ttc ttg cgt Leu Glu Ala Ile Glu Phe Leu Arg 435 440 aat acc aac cgt att. ctt Asn Thr Asn Arg Ile Leu 445 ggt gag Gly Glu gag Glu gg t Gly t tg Leu 490 aaa Lys ccg Pro cgC Arg tat Tyr aac As n 570 tgg Trp o tg Leu gaa Glu aaa Lys gaa Glu 650 cgc Arg gat Asp tct acc Ser Thr 460 ggC ttc Gly Phe tac atg Tyr Met acc ttc Thr Phe tog oat Ser His 525 ocg ggo Pro Gly 540 tgg atg Trp Met ttt goC Phe Ala ctg ttg Leu Leu cgc gat Mrg Asp 605 gat ttt Asp Phe 620 ogc tog Arg Ser atc gtt Ile Val ggc ata Gly Ile atg Cac Met His 685 ga t Asp tgg Trp aag Lys ggg Gly 510 ga t Asp gao Asp tgg Trp cag Gin gaa Glu 590 ctg Leu gao Asp g tg Val1 gcc Ala aac Asri 670 tat Tyr 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 2064 2112 cat aaa His Lys otg gtg Leu Val 630 gat aaa Asp Lys 645 ocg ogt Pro Arg gaa ato Glu Ile agt aat goa ggc aat ggc ggc acg gta cac ago gat gag att gcc agc Ser Asn Ala Gly Asn Gly Gly Thr Val His Ser Asp Glu Ile Ala Ser 64 690 695 700 cac ggt cgt cag cat tca cta agc ctg acg cta. cca ccg ctg gcc act 2160 His Gly Arg Gin His Ser Leu Ser Leu Thr Leu Pro Pro Leu Ala Thr 705 710 715 720 atc toig ctg gtt cgg gag gca gaa tga cac aac tcg cca ttg gca aac 2208 Ile Trp Leu Val Arg Glu Ala Giu His Asn Ser Pro Leu Ala Asn 725 730 735 ccg ctc-ccc tcg gcg cgc att acg acg gtc agg gcg tca act tca cac 2256 Pro Leu Pro Ser Ala Arg Ile Thr Thr Val Arg Ala Ser Thr Ser His 740 745 750 ttt tct ccg ctc atg ccg agc ggg tag aac tgt gtg tct ttg acg cca 2304 Phe Ser Pro Leu Met Pro Ser Giy Asn Cys Val Ser Leu Thr Pro *755 760 765 *acg gcc agg aac atc gct atg act tgc cag ggc aca gtg gcg aca ttt 2352 Met Ala Arg Asn Ile Ala Met Thr Cys Gin Gly Thr Val Ala Thr Phe 770 775 780 ggc acg gtt 2361 785 <210> 36 <211> 785

PRT

<213> E. coli <223> gigB <400> 36 Met Ser Asp Arg Ile Asp Arg Asp Val Ile Asn Ala Leu Ile Ala Gly *1 5 10 His Phe Ala Asp Pro Phe Ser Val Leu Gly Met His Lys Thr Thr Ala 25 Gly Leu Giu Val Arg Ala Leu Leu Pro Asp Ala Thr Asp Val Trp Val 40 Ile Giu Pro Lys Thr Gly Arg Lys Leu Ala Lys Leu Glu Cys Leu Asp 55 Ser Arg Gly Phe Phe Ser Gly Val Ile Pro Arg Arg Lys Asn Phe Phe 70 75 Arg Tyr Gln Leu Ala Val Val Trp His Gly Gin Gin Asn Leu Ile Asp 90 Asp Pro Tyr Arg Phe Gly Pro Leu Ile Gin Giu Met Asp Ala Trp Leu 100 105 110 Leu Ser Glu Gly Thr His Leu Arg Pro Tyr Giu Thr Leu Gly Ala His 115 120 125 Ala Asp Thr met Asp Gly Val Thr Gly Thr Arg Phe Ser Val Trp Ala 130 135 140 Pro Asn Ala Arg Arg Val Ser Val Val Gly Gin Phe Asn Tyr Trp Asp 145 150 155 160 Gly Arg Arg His Pro Met Arg Leu Arg Lys Glu Ser Gly Ile Trp Glu 165 170 175 Leu Phe Ile Pro Gly Ala His Asn 180 Gin Leu Tyr Lys Tyr Glu Met 190 a.

a a *aa.

a *aa.

Ile Glu Giu 225 Aia Thr Val1 Ile Leu 305 Phe Val1 Gly Asp Phe 385 Ala Arg Leu Gin Gly 465 Trp Pro Tyr Asp Aia 210 Lys Pro Asp Pro Asn 290 Tyr Ile Pro Thr Trp 370 Leu Leu Lys Giu Val1 450 Val1 As n Val1 Asn Aia 195 Gi1n Val1 Ile Asn Tyr 275 Glu Ala Asp Gly Asn 355 Asn Val Arg Giu Ala 435 Ser Ser Leu Tyr Tyr Asn Met Val1 Ser Asn 260 Ala His Pro Ala His 340 Leu Thr Gly Val Gly 420 Ile Gly Arg Gly Arg 500 Thr Gly Arg Gin Ile 245 Phe Lys Pro Thr Ala 325 Phe Tyr Leu Asn Asp 405 Glu Glu Ala Pro Trp 485 Gin Glu Asn Pro Thr 230 Tyr Trp Trp Phe Arg 310 His Pro Glu Ile Ala 390 Ala Trp Phe Val1 Gin 470 Met Tyr As n Leu Giu 215 Glu Glu Leu Met Asp 295 Arg Ala Thr His Tyr 375 Leu Val Ile Leu Thr 455 Asp His His Phe Arg 200 Thr Giu Val Ser Gly 280 Gly Phe Ala Asp Ser 360 Asn Tyr Ala Pro Arg 440 Met Met Asp His Val1 Ser Asp Leu I e 220 Lys Ala 235 Gly Ser Glu Leu His Leu Gly Tyr 300 Arg Asp 315 Asn Val Ala Leu Arg Glu Arg Arg 380 Glu Arg 395 Ile Tyr Phe Gly Asn Arg Glu Ser 460 Leu Gly 475 Asp Tyr Leu Thr Leu Ser Pro Tyr Ala PhE 205 Cys Gly Leu Prc Asn Gin Phe Asr 24C Trp Arg Arg HiE 255 Ala Asp Gin LeL 270 Glu Leu Leu Prc 285 Gin Pro Thr Gl Asp Phe Arg Tyx 32C Ile Leu Asp Trg 335 Ala Giu Phe AsF 350 Gly Tyr His Gin.

365 Giu Val Ser Asn Phe Gly Ile Asp 400 Arg Asp Tyr Set 415 Gly Arg Giu Asn 430 Ile Leu Gly Glu 445 Thr Asp Phe Pro Phe Trp Tyr Lys 480 Met Lys Leu Asp 495 Phe Gly Ile Leu 510 His Asp Glu Val 525 515 520 Val His Gly Lys Lys Ser Ile Leu ASP Arg Met Pro Gly Asp Ala Trp 530 535 540 Gin Lys Phe Ala Asn Leu Arg Ala Tyr Tyr Gly Trp Met Trp Ala Phe 545 550 555 560 Pro Gly Lys Lys Leu Leu Phe Met Gly Asn Glu Phe Ala Gin Gly Arg 565 570 575 Glu Trp Asn His Asp Ala Ser Leu Asp Trp His Leu Leu Glu Gly Gly 580 585 590 Asp Asn Trp His His Gly Val Gin Arg Leu Val Arg Asp Leu Asn Leu 595 600 605 Thr Tyr Arg His His Lys Ala Met His Glu Leu Asp Phe Asp Pro Tyr 610 615 620 Gly Phe Giu Trp Leu Val Val Asp Asp Lys Giu Arg Ser Val Leu Ile *625 630 635 640 Phe Val Arg Arg Asp Lys Giu Gly Asn Giu Ilie Ile Val Ala Ser Asn Pe645 650 655 Ph Thr Pro Val Pro Arg His Asp Tyr Arg Phe Gly Ile Asn Gin Pro *660 665 670 Gly Lys Trp Arg Glu Ile Leu Asn Thr Asp Ser Met His Tyr His Gly 675 680 685 Ser Asn Ala Gly Asn Gly Gly Thr Val His Ser Asp Glu Ile Ala Ser 690 695 700 His Gly Arg Gin His Ser Leu Ser Leu Thr Leu Pro Pro Leu Ala Thr 705 710 715 720 Sle Trp Leu Val Arg Giu Ala Giu His Asn Ser Pro Leu Ala Asn Pro 725 730 735 Leu Pro Ser Ala Arg Ile Thr Thr Val Arg Ala Ser Thr Ser His Phe *740 745 750 Ser Pro Leu Met Pro Ser Gly Asn Cys Val Ser Leu Thr Pro Met Ala 755 760 765 Arg Asn Ile Ala Met Thr Cys Gin Gly Thr Val Ala Thr Phe Gly Thr 770 775 780 Val1 785 <210> 37 <211> 150 <212> PRT <213> Zea mays <400> 37 Met Ala Ala Lys Met Leu Ala Leu Phe Ala Leu Leu Ala Leu Cys Ala 1 5 10 Ser Ala Thr Ser Ala Thr His Ile Pro Gly His Leu Pro Pro Val Met 25 Pro Leu Gly Thr Met Asn Pro Cys Met Gin Tyr Cys Met Met Gin Gin 40 Gly Leu Ala Ser Leu Met Ala Cys Pro Ser Leu Met Leu Gin Gin Leu Ala Leu Pro Leu Gin Thr Met Pro Val Met Pro Gin Met Thr Pro Asn Met Ser Pro Leu Met Pro Ser Met Met Ser Pro Met Val Leu Cys Asp Ala 115 Ser Met Met Ser Ile Met Met Pro Gin Cys His 1110 Pro ?he Met Val Ser Gin Ile Leu Gin Gin Gin Phe Asn 130 Pro Met Ala Met Ile Pro Pro Met Leu Gin Gin Pro 4 4* Vai Gly Ala Ala <210> 38 <211> 2562 <212> DNA <213> Zea mnays 4* .4 4 .4 4* <400> 38 aagcttgcta tatacactct ctttacttga catgtgcgca aaacatatag atataaaatg ttcggttgct ttcccgtogt tgtggtgtaa a tgcaogaog tggtacatat tccgtataca atactcaaaa atatttgtat ccgttccgat atatattaat ggcttatccc tgtctttgtt gcctcagtcg oagcoaaga t cccatat too agtactgcat agcaactgtt ctaacatgat tgatgtcgca aacagoagtt agoaaooott gttgacatgc tttttatatc aatatatttt atatatataa atattatggg aatataagtc atatatotag aaaaactaat caatataatt oaaaaaaagg gaatgcataa gataccatcg tgagcgattt ctttctttcc gtatacaagg ggaaaaggaa atgacatacc ttoatatato acgaaaaagg tgttotcttc agtgttctta tagcccattg aacgaaao tg attatccgtt agtccaagta tttgatgttt ctgatttgaa atttgtcatg ctgaaatcag ctttccaact gcctgcatac caoatato ta gcttgcattg agggoac ttg ga tgoaaoag ggccttaccg gtcaccattg aataatgatg accattcatg tgttggtgct catogog tg t to tcocta to agaoc ttaaa aacacataca acggagggag tggtttttca gtgcagcatc cgtccgttgt aatgtaatat agaagatcaa gtaatgattt gatgttcgat aaaaatactc ttaatgttga atgcgataca acaaatatga atcatatttg agatattaaa tacgagttga ggctagcgag ggtgatttct ggagaagggg gtggt ttaaa catattcgaa ttaagtatcc aagattttaa t tatgaaaao ctttgctaaa ttagagaagc ttcagttctg atctataaat ctatactata ttcgctctoc coaccagtca ggg t tgooa o ttoagacga a tgatgooga ccacaatgtc ttcaacccaa goattotaga gactcattat catttttgca ttgtatgtct ttaagtattg tactttatta atcaatoaat cg tgcacga t cggtcaccta gactcotoct ggtaaataaa gtgtotcttt ggtaatacaa ctatcaattg tttcoocttt cacatcagct tgtggccatc atatcataaa gtgataagaa aaaggagtc gtggtagaat cacggccca t agagatatag gtagtagagg tttgatccgt gatocatatc tatatattta aaatatggaa aataccttta ctacaaattt caagcgccag aggacctgct ctctaggaag tagctctttg tgccattggg gc ttgatggc tgccagtgat.

goatgatgtc ac tgogaogo tggooatgac tagaaatat t taacaataaa aagoccatta atattcaaaa tatgaatcta gtagattaca atatattaoc cgtaaaagaa taaagattoa tttgaattac ggcattttgt atattttttt tgatgoctta ttcgaaagt t gttagatgtt agtcctaatgi acattoocaa aataaat" ta caaa tat tac aacccctttt gtgagagtgt ctgtgatata atctacgtga tttgtcatta ataagagggo ggatctt,,at aactttatag cgattaatct caaggcatcrt gaccaaatgc aaatogttto agatcaatcg caaggacacc tgoaagcgco taccatgaac gtgtccgtcc gatgccaoag accaatgg tc cgto tcgcag gattocacoc tgtgttgtat acaagtttcc tcot ttao tc gaatgacaa.

ttaaaatgct ttgttatttt atgtccaaac gcagtcaogg tgaagagaac ttaggaataa gagaaaaca: tattcacgtg gctcogagag ctttgtctoa cftttgzgtta atgocaccga taaoaa tgao ttatcaaagt attttatctt :tatagcttg tgcgcgtgga gogactcata tttgcacgtg aaggt~gtaaa taagatctaa ccgta--ccgt gaactcgata oagtotatat tgtgcagatt cgagtcatco :ocatczaoat cagtoca tog aoogocatgg aotagtgoga ooatgoatgo otgatgotgo atga tgaogo :tgocgagoa attatgotgo atgt tottao ogaataatga tottattato cctaagtooo aaatotagao aaaaogao ta oto tat toca attttgaatt tgttggtooo caaaataagg oataagoaaa ggaagoataa aat ttaoata cttogaatga zgcatgggca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 68 atgtacctct atttataggg acggtgcgac gtacaaattt gtataaaatt atatttttat 2460 tcccaaatcc tatgcatatg tgtcggggac cataattagg ggtaccctca aggctcctaa 2520 ttctcagctg gtaaccccat cagcataaag ctgcaaaggc ct 2562 <210> 39 <211> 1141 <212> DNA <213> Zea mays <400> 3'9 aagcttgcta tatacactct ctttacttga catgtgcgca aaacatatag atataaaatg ttcggttgct ttcccgtcgt tatgtggtgt tgatgcacga aatggtacat aatccgtata gtatactcaa taatatttgt atccgttccg ttatatatta ccggcttatc attgtctttg cggcctcagt ctttctttcc gtacacaagg ggaaaaggaa atgacatacc ttcatatatc acgaaaaagg tgttctcttC agtgttctta aatagcccat cgaacgaaac atattatccg caagtccaag aatttgatgt atctgatttg atatttgtca atctgaaatc ccc tttccaa ttgcctgcat cgcacatatc ttaatgttga atgcgataca acaaatatga atcatatttg agatattaaa tacgagt tga ggctagcgag ggtgatttct tgggagaagg tggtggttta ttcatattcg tattaagtat ttaagatttt aattatgaaa tgctttgcta agttagagaa ctttcagttc acatctataa tactatacta tttccccttt cacatcagct tgtggccatc atatcataaa gtgataagaa aaaggagtcc gtggtagaat cacggcccat ggagaga tat aagtagtaga aatttgatcc ccgatccata.

aatatatatt ac aaatatgg aaaatacctt gcctacaaat tgcaagcgcc ataggacctg tactctagga gttagatgtt agccaatg acattctcaa aataaattta caaatattac aacccctttt gtgagagtgt ccgtgatata agatctacgt ggtttgtcat gtataagagg tcggatcttt taaactttat aacgattaat tacaaggcat ttgaccaaat agaaatcgtt ctagatcaat agcaaggaca ctttgtgtta atgccaccga taacaatgac ttatcaaagt attttatctt ttatagcttg tgcgcgtgga gcgactcata gatttgcacg tagaggtgta gctaagatct atccgtatcc aggaactcga ttcagtc tat cttgtgcaga gccgagtcat tttcatctac cgcagtccat ccaccgccat 120- 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1141 <210> <211> 1328 <212> DNA <213> E. coli <220> <223> glgC3 <220> <221> CDS <222> (join(1. .1293, 1297. .1326)) <400> atg gft agt tta gag Met Val Ser Leu Glu 1 5 aag aac gat cac Lys Asn Asp His atg ttg gcg cgc cag ctg Met Leu Ala Arg Gin Leu cca ttg aaa Pro Leu Lys ctg aag gat Leu Lys Asp gtt gcc ctg ata Val Ala Leu Ile gcg gga gga cgt Ala Gly Gly Arg ggt acc cgc Gly Thr Arg cac ttc ggc His Phe Gly tta acc aat aag Leu Thr Asn Lys gca aaa ccg gcc Ala Lys Pro Ala ggt aag Gly Lys ttc cgc att atc Phe Arg Ile Ile ttt gcg ctg Phe Ala Leu tct aac Ser Asn tac cag Tyr Gin tgc atc aac tcc Cys Ile Asn Ser ggg Gly atc cgt cgt atg ggc gtg atc acc cag Ile Arg Arg Met Gly Val Ile Thr Gin 70 tcc cac act Ser His Thr gtg cag cac att cag cgc ggc tgg tca ttc ttc aat gaa gaa atg aac Val Gin His Ile Gin Arg Gly Trp Ser Phe Phe Asn Glu Giu Met Asn 90 gag ttt gtc gat ctg ctg cca gca cag cag aga atg aaa ggg gaa aac 336 Giu Phe Val Asp Leu Leu Pro Ala Gin Gin Arg Met Lys Gly Giu Asn 100 105 110 tgg tat cgc ggc acc gca gat gcg gtc aco caa aac ctc gac att atc 384 Trp Tyr Arg Gly Thr Ala Asp Ala Val Thr Gin Asn Leu Asp le Ile 115 120 125 cgt cgt tat aaa gcg gaa tac gtg gtg atc ctg gog ggc gac cat atc 432 Arg Arg Tyr Lys Ala Giu Tyr Val Val Ile Leu Ala Gly Asp His Ile 130 135 140 tac aag caa gac tac tcg cgt atg Ott arc gat cac gtc gaa aaa ggc 480 *Tyr Lys Gin Asp Tyr Ser Arg Met Leu Ile Asp His Val Giu Lys Gly 145 150 155 160 gta cgt tgt acc gtt gtt tgt atg cca gta ccg att gaa gaa gcc too 528 Arg Cys Thr Vai Val Cys Met Pro Val Pro Ile Glu Giu Ala Ser :165 170 175 gca ttt ggc gtt atg gcg gtt gat gag aao gat aaa act atc gaa ttc 576 AlaPheGlyValMetAlaValAspGiu Asn Asp Lys Thr Ile Giu Phe 180 185 190 gtg gaa aaa cct gct aac ccg ccg tca atg ccg aac gat cog ago aaa 624 Vai Giu Lys Pro Ala Asn Pro Pro Ser Met Pro Asn Asp Pro Ser Lys *195 200 205 *tot ctg gcg agt atg ggt atc tao gto ttt gao goc gao tat ctg tat 672 Ser Leu Ala Ser Met Gly Ile Tyr Val Phe Asp Ala Asp Tyr Leu Tyr 210 215 220 *gaa ctg ctg gaa gaa gac gat cgc gat gag aac too ago cac gao tztt 720 S.Giu Leu Leu Giu Giu Asp Asp Arg Asp Giu Asn Ser Ser His Asp Phe 09*225 230 235 240 ggc aaa gat ttg att ccc aag ato aco gaa goc ggt ctg gcc oat gcg 768 *Gly Lys Asp Leu Ile Pro Lys Ile Thr Giu Ala Gly Leu Ala Tyr Al a Go.245 250 255 cac cog tto cog oto oct tgc gta caa too gao oog gat goc gag C0:2 816 His Pro Phe Pro Leu Ser Cys Val Gin Ser Asp Pro Asp Ala Giu Pro 260 265 270 tao tgg cgc gat gtg ggt acg ctg gaa got tao tgg aaa gog aac otc 864 Tyr Trp Arg Asp Val Giy Thr Leu Giu Ala Tyr Trp Lys Ala Asn Le_ 275 280 285 gat ctg goc tot gtg gtg gao aaa otg gat atg tao gat ogo aat tgz:91- Asp Leu Ala Se: Vai Val Asp Lys Leu Asp Met Tyr Asp Arg Asn Trpt 290 295 300 oca att ogo aco tao aat gaa tca toa cog oca gog aaa too gtg oag 960 Pro Ile Arg Thr Tyr Asn Giu Ser Leu Pro Pro Al1a Lys Phe Val G I n 305 310 315 32-0 gat ogo too ggt ago cac ggg atg aco Ott aac toa ctg gtc too cgc 100 8 Asp Arg Ser Giy Ser His Gly Met Thr Leu Asn Ser Leu Vai Ser Glv, 325 330 335 ggt tgt gtg ato too ggt tog gtg gtg gtg cag too gtt otg tto tog 1056 Gly Cys Vai Ile Ser Gly Ser Val Val Val Gin Ser Val Leu Phe Ser 340 345 350 gtt Val1 gaa Glu 370 og t Arg gaa Glu acg Thr tgc Cys tgc aac Cys Asn 360 tcg tgc Ser Cys gaa ggc Glu Gly tat cgt Tyr Arg aag tta Lys Leu 425 gtt cag Val Gin ttg tta Leu Leu gtc atc Val Ile aac gca Asn Ala 400 gtg ctg Val Leu 415 cga Arg 1104 1152 1200 1248 1293 1328 <210> 41 <211> 441 <212> PRT <213> E. coli <223> glgC3 <400> 41 Met Val Ser Leu Glu Lys Asn Asp His Leu Met Leu Ala Arg Gin Leu 1 5 10 Pro Leu Gly Gly Val1 Glu Trp, Arg Tyr 145 Val1 Ala Ser Leu Arg Arg Ile Asp 100 Gly Lys Asp Thr Val1 180 Val Glu Lys Pro Ala Asn Pro Pro Ser Met Pro Asn Pr Se Ly Pro Ser Lys 195 Ser Glu 225 Gly His Tyr Asp Pro 305 Asp Gly Arg Pro Asp 385 Glu Val1 Arg Leu Ala 210 Leu Leu Lys' Asp Pro Phe Trp Arg 275 Leu Al a 290 Ile Arg Arg Ser Cys Val Val Arg 355 Glu Val 370 Arg Ala Glu Asp Thr Arg Phe Tyr 435 200 Gly Ile Tyr 215 Asp Asp Arg 230 Pro Lys Ile Ser Cys Val Gly Thr Leu 280 Val. Asp Lys 295 Asn Giu Ser 310 His Gly met Gly Ser Val Ser Phe Cys 360 Gly Arg Ser 375 Ile Pro Glu 390 Arg Phe Tyr Leu Arg Lys Val Gin Arg 440 Tyr His Aia Al a 270 Al a Arg Phe Val1 Leu 350 Val Cys Giu 1ie Glu 430 <210> 4 2 <211> 1328 <212> DNA <213> E. coli <220> <223> glcC <220> <221> CDS <222> (join .1293, 1297 .1326)) <400> 42 atg gtt agt tta gag aag aac gat cac tta atg ttg gcg cgc cag ctg Met Val Ser Leu Glu Lys Asn Asp His Leu Met Leu Ala Arg Gin Leu 1 5 10 cca Pro ct~g Leu gg t Gly ggg Gly 65 g tg Val1 gag Glu ,gg Trp cgc Arg tac Tyr 145 gca Ala gca Ala gtt Val1 tct Ser gaa Giu 225 ggC Gly cac His ttg Leu aag Lys aag LyT atc Ilie cag Gin ttt Phe tat Tyr cg t Arg 130 aag Lys cgt Arg ttt Phe gaa Glu c tg Leu 210 c tg Leu aaa Lys z cg Pro aaa Lys ga t Asp ttc Phe og t Arg cac His gtc Val1 cgc Arg 115 tat Tyr caa Gin tgc Cys ggC Gly aaa Lys 195 gcg Ala c tg Leu ga t Asp ttc Phe tct Ser tta Leu cgc Arg cgt Arg att Ile ga t Asp 100 ggc Gly aaa Lys gac Asp acc Thr gtt Val 180 Cct Pro agt Ser gaa Glu ttg Leu ccg Pro 260 gtt Val1 ac Thr att Ile a tg Met cag Gin 85 c tg Leu acc Thr geg Ala tac Tyr gtt Val1 165 a tg Met gc t Ala a tg Miet gaa G1u att Ile 245 ztC Leu gcc Ala aa t Asn atc Ile ggc Gly 70 cgc Arg ctg Leu gca Ala gaa Gl u tcg Ser 150 gc t Al a gcg Ala aac As n gg t Gly gac Asp 230 ccc Pro tct Ser c tg Leu aag Lys gac Asp 55 gtg Val ggc Gly cca Pro ga t Asp tac Tyr 135 cgt Arg tgt Cys gtt Val ccg Pro atc Ile 215 ga t Asp aag Lys tgc Cys a La Ile cga Arg 40 ttt Phe a tc Ile tgg Trp gca Al a gcg Ala 120 g tg Val a tg Met a tg Met ga t Asp C cg Pro 200 t ac Tyr cgc Arg atc Ile gta Val1 ctg Leu 25 *gca Ala gcg *Ala acc Thr tca Ser cag Gin 105 gc Val1 g tg Val1 ctt Leu cca Pro gag Glu 185 tca Ser gtc Val1 gat Asp acc Thr caa Gin 265 gca Al a aaa Lys C tg Leu cag Gin t tc Phe 90 cag Gin acc Thr atc Ile atc le gta Va I 170 aac Asn a tg Met ttz Phe gag gaa 250 tc Ser gga Gly gcc Al a aac Asn cag Gin aa t Asn a tg Met aac Asn gcg Al a 140 cac H i s att Ile aaa Lys aac Asn gcc Ala 220 tCC Ser gg t Gly ccg Pro cg t Arg gta Val1 tgc Cys tcc Ser gaa Giu aaa Lys ctc Leu 125 ggc Gly gtc Val1 gaa Giu att Ile ga t Asp 205 gac Asp agc Ser c tg Leu ga t Asp gg t Gly cac His atc Ile cac His gaa Glu ggg Gly 110 gac Asp gac Asp gaa Giu gaa Glu atc Ile 190 ccg Pro tat Tyr cac His gcc Ala gcc Ala 270 acc Thr t tc Phe aac Asn act Thr a tg Met gaa Gi u att Ile cat His aaa Lys gcc Ala 175 gaa Glu agc Ser c tg Leu gac Asp tat Tyr 255 gag Glu cgc Arg ggc Gly tcc Ser c tg Leu aac Asn aac As n atc Ile atc Ile ggc Gly 160 tcc Ser t tc Phe aaa Lys tat Tyr ttt Phe 240 gcg Ala ccg Pro tac tgg cgc gat gtg Tyr Trp Arg Asp Val ggt acg ctg gaa gct tac tgg aaa gcg aac ctc Gly Thr Leu Giu Ala Tyr Trp Lys Ala Asn Leu gat ctg gee tet gtg gtg ccg gaa ceg Asp Leu Ala Ser Val Val Pro Giu Leu 290 295 eca att egc ace tac aat gaa tea tta Pro Ile Arg Thr Tyr Asn Giu Ser Leu 305 310 gat ege tee ggt age cac ggg aeg ace Asp Arg Ser Gly Ser His Gly Met Thr 325 ggt tgt gtg ate tee ggt teg gtg geg Gly Cys Val Ile Ser Gly Ser Val Val 340 345 ege gtt ege gtg aat tea tte tge eac Arg Val Arg Val Asn Ser Phe Cys Asn 355 360 ceg gaa gta tgg gta ggt ege teg tge Pro Giu Val Trp Val Gly Arg Ser Cys 370 375 gat cgt get tgt gtt ate ceg gaa gge Asp-Arg Ala Cys Val Ile Pro Glu Gly 385 390 gag gaa gat gea egt egt tte tat egt Giu Glu Asp Ala Arg Arg Phe Tyr Arg 405 gta aeg ege gaa atg eta egg aag tta Vai Thr Arg Giu Met Leu Arg Lys Leu 420 425 tac gat ege aat tgg Tyr Asp Arg Asn Trp 300 geg aaa tte gtg eag Ala Lys Phe Val Gin 320 tea etg gtt tee gge Ser Leu Val Ser Gly 335 tee gtt ctg tte teg Ser Val Leu Phe Ser 350 tee gee gta ttg tta Ser Ala Val Leu Leu 365 ege ege tge gte ate Arg Arg Cys Vai Ile 380 att ggt gaa aae gca Ile Gly Glu Asn Ala 400 gaa gge ate gtg etg Glu Gly Ile Val Leu 415 aaa eag gag ega Lys Gin Giu Arg 430 912 960 1008 1056 1104 1152 1200 1248 1293 1328 taa tge agg ttt tac atg tat gtt eag aga tgt tt Cys Arg Phe Tyr Met Tyr Val Gin Arg Cys 435 440 <210> 43 <211> 441 <212> PRT <213> E. coli <223> gleC <400> 43 Met Val Ser L 1 Pro Leu Lys S, Leu Lys Asp L, Gly Lys Phe A: Gly Ile Arg A: Val Gin His I.

Leu. Ala Gly Arg Ala Val Asn Cys Gin Ser Asn Giu 9* Glu Trp Arg Tyr 145 Ala Ala Val1 Ser Glu 225 Gly His Tyr Asp Pro 305 Asp Gly Arg Pro Asp 385 Giu Val1 Arg Phe Tyr Arg 130 Lys, Arg Phe Glu Leu 210 Leu Lys Pro Trp Leu 290 Ile Arg Cys Val1 Giu 370 Arg Giu Thr Phe Val1 Arg 115 Tyr Gin Cys Gly Lys 195 Ala Leu Asp Phe Arg 275 Ala Arg Ser Val1 Arg 355 Val1 Ala Asp Arg Tyr 435 Asp 100 Gly Lys Asp Thr Val1 180 Pro Ser Giu Leu Pro 260 A sp Ser Thr Gly Ile 340 Val1 Trp Cys Ala Giu 420 Met Leu Thr Al a Tyr Val1 165 Met Ala Met Giu Ile 245 Leu Val Val Tyr Ser 325 Ser Asn Val1 Val1 Arg 405 Met Tyr Leu Ala Giu Ser 150 Ala Ala Asn Gly Asp 230 Pro Ser Gly Val1 Asn 310 His Gly Ser Gly Ile 390 Arg Leu Val1 Gin Arg Thr Gin Ile Leu Ile Asp Val Pro 170 Asn Asp Met Pro Phe Asp Glu Asn 235 Glu Ala 250 Ser Asp Ala Tyr Asp Met Pro Pro 315 Leu Asn 330 Val Gin Ile Asp Arg Leu Met Val 395 Ser Giu 410 Gly His Gly 110 Asp Asp Giu Giu Ile 190 Pro Tyr His Ala Aia 270 Al a Arg Phe Val1 Leu 350 Val1 Cys Glu Ile Giu 430 <210> 44 <211> 1328 <212> DNA <213> E. coli <220> <223> gigCwt <220> <221> GDS <222> (join(l..1293, 1297..1326)) <400> 44 a tg Met 1 cca Pro c tg Leu ggt Gly ggg Gly gtg Val1 gtt agt Val Ser ttg aaa Leu Lys aag gat Lys Asp aag ttc Lys Phe 50 atc cgt Ile Arg cag cac Gin H-is ttt gtc Phe Val tat cgc Tyr Arg 115 cgt tat Arg Tyr 130 aag caa Lys Gin cgt tgc Arg Cys ttt ggc Phe Gly gaa aaa Giu Lys 195 tta gag Leu Giu 5 tct gtt Ser Val 20 tta acc Leu Thr cgc att Arg Ile cgt atg Arg Met att cag Ile Gin gat ctg Asp Leu 100 ggc acc Gly Thr aaa gcg Lys Ala gac tac Asp Tyr acc gtt Thr Val 165 gtt atg Val Met 180 cct gct Pro Ala aag aac gat cac tta atg ttg gcg cgc cag ctg His c tg Leu 25 gca Ala gcg Ala acc Thr tca Ser cag Gin 105 gtc Val1 g tg Val1 ctt Leu cca Pro gag Giu 185 tca Ser gga Gly ccg Pro tct S er tac Tyr ttc Phe aga Arg caa Gin c tg Leu gat Asp 155 ccg Pro ga t Asp C cg Pro gga cgt Gly Arg gcc gta Ala Val aac tgc Asn Cys cag tcc Gin Ser aat gaa Asn Giu atg aaa Met Lys aac ctc Asn Leu 125 gcg ggc Ala Gly 140 cac gtc His Val att gaa Ile Giu aaa att Lys Ile aac gat Asn Asp Met Leu Ala Arg Gin ggt acc Gly Thr cac ttc His Phe atc aac Ile Asn cac act His Thr gaa atg Glu Met ggg gaa Gly Glu 110 gac att Asp Ile gac cat Asp His gaa aaa Giu Lys gaa gcc Glu Ala 175 atc gaa Ile Giu 190 ccg agc Pro Ser 205 tct ctg gcg agt atg ggt atc tac gtc ttt gac gcc gac tat ctg tat Ser Leu Ala Ser Met Gly Ile Tyr Val Phe Asp AlaAs TrLe Tr 210 220 Asp Tyr LeU Tyr gaa Giu 225 ggc Gly cac His tac Tyr ga t Asp cca Pro 305 ga t Asp gg t Gly cgc Arg ccg Pro ga t Asp 385 gag Glu gta Val1 taa ctg ctg gaa Leu Leu Giu aaa gat ttg Lys Asp Leu ccg Etc ccg Pro Phe Pro 260 tgg cgc gat Trp Arg Asp 275 ctg gcc tct Leu Ala Ser 290 att cgc acc Ile Arg Thr cgc tcc ggt Arg Ser Gly tgt gtg atc Cys Val Ile 340 gtt cgc gtg Val Arg Val 355 gaa gta tgg Giu Val Trp 370 cgt gct tgt Arg Ala Cys gaa gat gca Giu Asp Ala acg cgc gaa Thr Arg Glu 420 tgc agg ttt gaa gac gat cgc gat gag aac tc Glu Asp Asp Arg Asp Giu Asn Se 230 235 att ccc aag atc acc gaa gcc gg Ile Pro Lys Ile Thr Glu Ala Gi1 245 250 ctc tct tgc gta caa tcc gac cc Leu Ser Cys Val Gin Ser Asp Pr 265 gtg ggt acg ctg gaa gcE tac tg Val Gly Thr Leu Giu Ala Tyr Tr: 280 gtg gtg ccg aaa ctg gat atg Val Val Pro Lys Leu Asp Met TY 295 tac aat gaa tca. tta ccg cca gc, Tyr Asn diu Ser Leu Pro Pro Al 310 315 agc cac ggg atg acc cct aac tc Ser His Gly Met Thr Leu Asn Se.

325 330 tcc ggt tcg gtg gtg gtg cag tc Ser Gly Ser Val Val Val Gin Se.

345 aat tca ttc tgc aac att gat tco Asn Ser Phe Cys Asn Ile Asp Se.

360 gta ggt cgc tcg tgc cgt ctg cg, Val Gly Arg Ser Cys Arg Leu Ar 375 38' gtt atE ccg gaa ggc acg gtg at Val Ile Pro Giu Gly Met Val Ii, 390 395 cgt cgt Etc tat cgt tca gaa ga, Arg Arg Phe Tyr Arg Ser Glu G1, 405 4 atg cta cgg aag Eta ggg cat aa Met Leu Arg Lys Leu Gly His Ly: 425 Eac atg tat gtt cag aga tgt tt 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 129:1.

1328 Cys Arg Phe Tyr Met Tyr Val Gin Arg Cys 435 4,10 <210> <211> 441 <212> PRT <213> E. coli <223> glgCwt <400> 9

S

9* *9 S *5 S. S S S 4* 555.

S

*5 S S 5**S

S

S.

*5*S

S

Met Val Ser Leu Giu Lys Asn 1 Pro Leu Lys Ser Val Ala Leu Leu Lys Asp Leu Thr Asn Lys Gly Lys Phe Ag Ile Ile Asp 5(7 55 Giy Ile Arg Arg Met Giy Val 70 Vai Gin His Ile Gin Arg Gly Giu Phe Val Asp Leu Leu Pro 100 Trp Tyr Arg Gly Thr Ala Asp 115 Arg Arg Tyr Lys Aia Giu Tyr .130 135 Tyr Lys Gin Asp Tyr Ser Arg 145 150 Aia Arg Cys Thr Vai Aia Cys 165 Ala Phe Giy Vai Met Aia Vai 180 Val Giu Lys Pro Aia Asn Pro 195 Ser Leu Aia Ser Met Giy Ile 210 215 Giu Leu Leu Giu Giu Asp Asp 225 230 Giy Lys Asp Leu Ile Pro Lys 245 His Pro Phe Pro Leu Ser Cys 260 Tyr Trp Arg Asp Val Giy Thr 275 Asp Leu Aia Ser Vai Val Pro 290 295 Pro Ile Arg Thr Tyr Asn Giu 305 310 Asp Arg Ser Giy Ser His Giy 325 Giy Cys Val Ile Ser Giy Ser 340 Asp Ile Arg 40 Phe Ile Trp Ala Ala 120 Vai Met Met Asp Pro 200 Tyr Arg Ile Val1 Leu 280 Lys Ser Met Vali Met Giy Pro Ser Tyr Phe Arg Gin Leu Asp 155 Pro Asp Pro Asp Asn 235 Ala Asp Tyr Met Pro 315 Asn Gin Leu Ala Gly Arg Ala Val Asn Cys Gin Ser Asn Glu Met Lys Asn Leu 125 Ala Gly 140 His Val Ile Giu Lys Ile Asn Asp 205 Ala Asp 220 Ser Ser Gly Leu Pro Asp Trp Lys 285 Tyr Asp 300 Ala Lys Ser Leu Ser Val 345 350 Arg Val Arg Val Asn Ser Phe Cys Asn Ile Asp Ser Ala Vai Leu Leu 355 Pro Giu Val 370 Asp Arg Ala 385 Giu Giu ASP Val Thr Arg Arg Phe Tyr 435 Trp Vai Giy Arg 375 Cys Val Ile Pro 390 Ala Arg Arg Phe 405 Giu Met Leu Arg 420 Met Tyr Val Gin 365 Cys Arg Leu Arg Arg Cys Vai Ile 380 Giy Met Vai Ilie Gly Giu Asn Ala 395 400 Arg Ser Giu Giu Gly Ile Vai Leu 410 415 Leu Giy His Lys Gin Giu Arg Cys 425 430 Cys 99@@ 0@ 9 C..e 9 99 9* 9 4.

999*9* 9. 9* 9 9

C

9* 9 9@ 99 <210> 46 <211> 1915 <212> DNA <213> Zea mays <220> <221> CDS <222> (join(i. i85, 1819. .1914)) *9 9 C

*O

9c~9 9 99 .9 99 9 4.

*999 9 9C99 *9 94 9 9.

C 9 9999 <400> 46 atg gcg got otg Met Aia Ala Leu 1 ggc gtc ccg gao Giy Val Pro Asp agg ggg gco cgg Arg Giy Ala Arg ago gcg ogo gcg Ser Ala Arg Ala ttoc aag ggc agc Ser Lys Gly Ser tto gtc ggo gc Phe Val Giy Ala gac gtc oto ggo Asp Val Leu Giy 100 gto atg gtc gtc Vai Met Val Val 115 aco agc gtc gt~g Thr Sbr Val Vai 130 agg tto tto cac Arg Phe Phe His aog tog cag otic gto Val1 ogo Arg gao Asp oag Gin ago Ser ago Ser a tg Met oag Gin gga Giy g tg Val1 goa aog Ala Thr ggo goo Giy Ala aog cto Thr Leu oag gog Gin Ala goc ggo Ala Gly aag ac Lys Thr gcc gog Ala Aia tac aag Tyr Lys gac ggg Asp Giy 140 gac ogc Asp Arg 145 cac cca His Pro tao ggg Tyr Giy ago ctg Ser Leu aac aac Asn Asn 210 gto tgc Val Cys 225 aac tac Asn Tyr atc cac Ile His gag ctg Glu Leu ggc tac Gly Tyr 290 ggg atc Gly Ile 305 gag gag Glu Giu atg cgc Met Arg gag tgg Giu Trp tog acg Ser Thr 370 gag gtc Giu Val 385 ggc agg Gly Arg cog oag o tg Leu cot Pro ota Leu 195 aac Asn aac As n cag Gin aao As n aac As n 275 gag Giu otc Leu otc Leu oto Leu gao Asp 355 go o Ala ggg Giy o tg Leu ctc ttc Phe gto Val1 180 tgo Cys ooa Pro gao Asp too Ser ato Ile 260 otc Leu aag Lys gag Giu ato Ile aco Thr 340 coo Pro gtg Vali cto Leu gaa Giu atg o tg Leu 165 got Ala cag Gin tao Tyr tgg Trp oao His 245 too S er cog Pro 000 Pro gc Aia too S er 325 ggo Giy ago Ser gag Giu cog Pro gag Giu 405 gag 150 gag Giu aga Giy gga Giy ttc Phe cac His 230 ggo Giy tac Tyr gag Giu g tg Vali gao Asp 310 ggC Gly ato Ile agg Arg gc Ala g tg Val1 390 cag Gin a tg agg Arg aog Thr goa Ala too Ser 215 ac c Thr ato Ile cag Gin aga Arg gaa Giu 295 agg Arg ato Ile aco Thr gao Asp aag Lys 375 gao Asp aag Lys gtg gtt Val1 gac Asp Ott Leu 200 gga Gly ggc Giy tao Tyr ggc Gly ttc Phe 280 ggo Gly gto Val1 gcc Ala ggc Gly aag Lys 360 9gg Al a cgg Arg ggo Gly gag gag gag Glu Glu cag ctg Gin Leu 190 ato otg Ile Leu 205 gao gtc Asp Val tao cto Tyr Leu aco got Thr Ala tco gao Ser Asp 270 gat tto Asp Phe 285 tgg atg Trp Met coo tao Pro Tyr.

ctc gao Leu Asp atg gac Met Asp 350 aag tao Lys Tyr 365 gog ctg Ala Leu gtg gog Val Ala gog gco Ala Ala gtt ctg 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 Pro Gin Leu Met Giu Met Val Giu Asp Val Gin Ile Val Leu Leu Gly 420 aeg Thr ttc Phe cac His 465 gag Giu tgc Cys aag Lys gag Giu aag Lys 545 ate Ile e tg Leu gag Giu egg Arg ate Ile 425 atg Met g tc Val1 gtg Val1 eag Gin etc Leu 505 etc Leu gee Ala gag Giu ee t Pro gge Gly 585 aac Asn ege Arg tta Leu gag aag Giu Lys cZtg geg Leu Ala ege tte Arg Phe 480 aeg cee Thr Pro 495 gaa gge Giu Gly gte gtg Val Val gee ate Ala Ile tge atg Cys Met 560 aae gtg Asn Val 575 gge gag Giy Glu aga gtt Arg Val tgg gac Trp Asp t 430 1344 1392 1440 1488 1536 1584 1632 1680 172 8 1776 1824 1872 19215 <210> 47 <211> 637 <212> PRT <213> Zea mays <400> 47 Met Ala Ala Leu Ala Thr Ser Gin Leu Val Ala Thr Arg Ala Gly Leu 1 5 10 Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gin Gly Leu Arg Gly Ser Ala Ser Lys Phe Val Asp Val Val Met Thr Ser 130 Arg Phe 145 His Pro Tyr Gly Ser Leu Asn Asn 210 Val Cys 225 Asn Tyr Ile His Giu Leu Gly Tyr 290 Gly Ile 305 Giu Giu Met Arg Giu Trp Ser Thr 370 Ala Arg Gly Gly Leu Val1 115 Val1 Phe Leu Pro Leu 195 Asn Asn Gin Asn Asn 275 Giu Leu Leu Leu Asp 355 Ala Leu Ser Ala Ala Gly Met Thr Gly Ala Asn Lys Asp 125 Gly Tyr 140 Arg Val Thr Glu Asn Gin Arg Ile 205 Arg Asp 220 Cys Tyr Lys Thr Phe Ser Phe Asp 285 Asn Trp 300 Ser Pro Giu Leu Gly Met Val Lys 365 Giu Ala 380 Giu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Val Ala Phe Ile 385 390 395 400 Gly Arg Leu Glu Giu Gin Lys Gly Pro Asp Val Met Ala Ala Ala Ile 405 410 415 Pro Gin Leu Met Glu Met Val Glu Asp Val Gin Ile Val Leu Leu Gly 420 425 430 Thr Giy Lys Lys Lys Phe Giu Arg Met Leu Met Ser Ala Giu Glu Lys 435 440 445 Phe Pro Giy Lys Val Arg Ala Val Val Lys Phe Asn Ala Ala Leu Ala 450 455 460 His His Ile Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg Phe 465 470 475 480 Glu Pro Cys Gly Leu Ile Gin Leu Gin Gly Met Arg Tyr Gly Thr Pro *485 490 495 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr Ile Ile Giu Gly 500 505 510 Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp Cys Asn Val Val *.:515 520 525 Giu Pro Ala Asp Val Lys Lys Val Ala Thr Thr Leu Gin Arg Ala Ile 530 535 540 Lys Val Val Gly Thr Pro Ala Tyr Giu Giu Met Val Arg Asn Cys Met 545. 550 555 560 Ile Gin Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Giu Asn Val 565 570 575 Leu Leu Ser Leu Gly Val Ala Gly Gly Giu Pro Gly Val Glu Gly Glu *580 585 590 Glu Ile Ala Pro Leu Ala Lys Giu Asn Val Ala Ala Pro Arg Val Arg 595 '600 605 Pro Ala Gly Pro Leu Ile Ser Arg Val Val Gin Arg Cys Trp, Asp Ile 610 615 620 Phe Leu Tyr Met Leu Phe Arg Leu Cys Asp Met Asp Lys 625 630 635 <210> 48 <211> 2423 <212> DNA <213> Zea mays <220> <221> CDS <222> (join(i. .2094, 2098. .2103, 2107. .2304, 2 308. .2421)) <400> 48 atg ccg ggg gca atc tct tcc tcg tog tog gct ttt ctc ctc ccc gtc 48 Met Pro Gly Ala Ile Ser Ser Ser Ser Ser Ala Phe Leu Leu Pro Vai 1 5 10 gcg tcC too tcg ccg cgg cgc agg cgg ggc agt gtg ggt gct gct ctg 96 Ala Ser Ser Ser Pro Arg Arg Arg Arg Gly Ser Val Gly Ala Ala Leu cgc Arg egg Arg ccg Pro cag Gin tca Ser gca Al a cep Pro aga Arg 145 gc t Ala get Ala agg Arg gte Val ctt LeuC 225 cacc His cgg Arg gaa g Glu 'v teg Ser gge Gly gce Ala gcg Al a ect Pro atg Met gtg Val 130 gaa Glu aga Arg gat Asp gag Glu gtc lal 210 ;ga fly :gt ~rg ~at ~sp ~tt z ral 9) tac Tyr ceg Pro ggg Gly ggc Gly ccc Pro caa Gin 115 tcc Ser ate Ile ccg Pro gca Ala gat Asp 195 p tg VIal gat Asp ptt la 1 :ta .,eu ~ct rhr ggc tac Gly Tyr Cct cag Pro Gn ggc gaa Gly Glu gct gtt Ala Val aat cct Asn Pro 100 aac gga Asn Gly gga ccc Gly Pro gat gee Asp Ala gtg gaa Val Glu 165 gct ccg Ala Pro 180 aat gaa Asn Glu gtg get Val Ala gte gtg Val Val atg gtc Met Val 245 ggt gta Gly Val 260 tat ttt Tyr Phe agc Sex gat Asp agc Ser 70 cag Gln t tg Leu acg Thr aaa Lys agt Ser 150 agc Ser get Ala cc t Pro tct Ser gt Gly 230 p tg VIal agg A.rg :ac His ggc Gly gga Gly 55 gag *Glu gc Gly aca Thr agt Ser gc t Ala 135 gcg Al a ata Ile aca Thr ggc Gly gaa Glu 215 gc t Ala ata Ile aga Arg tc t Ser ge Ala 40 gcg Ala gag Glu agc Ser tet Sex ggg Gly 120 ga t Asp gtg Val ggc Gly gat Asp cct Pro 200 tg t Cys t tg Leu cca Pro cgt Arg tac Tyr 25 gag Glu gcg Ala pea Ala acg Thr get Ala 105 pge Gly cat His aag Lys ate Ile geg Ala 185 t tg Leu get Ala eet Pro aga Arg tae Tyr 265 att Ile e tg Leu teg Ser geg Ala gee Ala 90 eeg Pro age Ser eea Pro eea Pro get Ala 170 geg Al a get Ala cet Pro aag Lys tat Tyr 250 aag Lys ga t Asp egg ttg Arg Leu pta cge Val .Arg 60 aag age Lys Ser 75 aag get Lys Ala aag eaa Lys Gln age geg Ser Ala tea get Ser Ala 140 gag ec Glu Pro 155 gaa ceg Glu Pro geg apt Ala Ser ppp ect Gly Pro tte tgc Phe Cys 220 get etg Ala Leu 235 gga gag Gly Glu gta get Val Ala gga gtt Gly Val gee Ala tee Ser gtg Val agt Ser age Ser 125 eet Pro gea Ala gtp Val get Ala aa t Asn 205 aag Lys gep Ala tat Tyr ga Gly gat Asp 285 pea Ala tee Ser pat Asp cap Gin 110 ace Thr gte Val ppt Gly pat Asp cc t Pro 190 p tg Val1 aca Thr app Arg gee Al a cap Gln 270 ttt Phe pep Al a teg Ser te t Ser ape gee Ala ace Thr pa t Asp PC t Al a 175 tat Tyr atg Me z gct Gly, aza A rg paa 2 pat As Z.

gta Val 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 cat tgg peg egg His Trp Ala Arg 280 pta paa gee ect ccc tte egg cac egg eac aat aat att tat ggg gga Val Giu Ala Pro Pro Phe Arg 290 295 His Arg His Asn Asn Ile Tyr Gly Gly 300 gaa Glu 305 gtt Val ggC Gly g tC Val cgc Arg gta Val 385 ttc Phe gcg Ala a tg Met ata Ile atg Met 465 tac Tyr aag Lys cca c Pro I atc E IleI aga Arg gag Glu aac Asn tat Tyr tc t Ser 370 gac Asp aaa Lys ggg Gly tgg Trp aac Asn 450 agc Ser acc rhr ;cc kla tg .eu Ltc le *ttg Leu *gtt *Val tta Leu cta Leu 355 g ~g Val gac Asp c tg Leu c tg Leu gag Giu 435 cag Gin gag Glu aac Asn gcc Ala atc Ile gcc Ala ga t Asp cca Pro gtt Vai 340 aag Lys ctt Leu ttc Phe tat Tyr aag Lys 420 c tg Leu aac Asn tgg Trp tac Tyr ctg Leu 500 9gg Giy gac %sp att Ile tgg Trp 325 t tc Phe gcc Ala gtg Val gtc Vai gac Asp 405 acg Thr aag Lys gac Asp aac Asn acg Thr 485 cag Gin ttc Phe gcg Ala t tg Leu 310 tat Tyr att Ile tat Tyr a ta Ile aa t Asn 390 aac Asn gca Ala act Thr tgg Trp, ccc Pro 470 t tc Phe cgg Arg atc Ile a tc Ile aag Lys gc t Al a gc t Ala tac Tyr cac His 375 ttt Phe att Ile gac Asp tcg Ser aag Lys 455 gc t Aia gag Giu cag Gin ggg Giy cac His 535 cgc Arg cca Pro aa t Asn cgg Arg 360 aac Asn gac Asp ggC Gly C99 Arg gaa Glu 440 c tg Leu g tg Val1 acg Thr c -,g Leu Cgg Arg 520 tgg Trp a tg Met tg t Cys ga t Asp 345 gac Asp att Ile ttg Leu ggg Gly gtg Vali 425 ggc Giy cag Gin gac Asp c tg Leu ggc Gly 5.05 c tg Leu atc Ile att ttg Ile Leu 315 Gly Giy 330 tgg cat Trp His aat ggt Asn Gly gct cat Ala His cct gaa Pro Giu 395 gat cac Asp His 410 gtg acc Val Thr ggg tgg Gly Trp ggc atc Gly Ile gtg cac Val His 475 gac acc Asp Thr 490 ctg cag Leu Gin gac cac Asp His gcg ggg Ala Gly ttc Phe act Thr acc Thr t tg Leu cag Gin 380 cac His agc Ser gtt Val1 Gly g tg Val 460 ctc Leu ggc Gly gtc Val1 cag Gin cag Gin 540 aag Lys tat Tyr ctt Leu 350 cag Gin cgt Arg a tc Ile gt t Vai aat Asn 430 cac His ggc Gly tcc Ser cgg Arg gac Asp 510 ggc Gly g tg Val 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1 526 1584 1632 1680 gtg Val1 545 atg ctg ggc acc ggg Met Leu Gly Thr Gly cgg gcc gac ctg gag Arg Ala Asp Leu Giu gac atg ctg cgg Asp Met Leu Arg 550 555 ttc gag tcg gag cac agc gac aag gtg cgc gcg tgg gtg ggg ttc tcg 1728 Phe Giu Ser Glu His Ser Asp Lys Val Arg Ala Trp Val Gly Phe Ser 565 570 575 gtg ccc ctg gcg cac cgc atc acg gcg ggc gcg gac atc ctg ctg atg 1776 Val Pro Leu Ala His Arg Ile Thr Ala Gly Ala Asp Ile Leu Leu Met 580 585 590 ccg tcg cgg ttc gag ccg tgc ggg ctg aac cag ctc tac gcc atg gcg 1824 Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gin Leu Tyr Ala Met Ala 595 600 605 tac ggg acc gtg ccc gtg gtg cac gcc gtg ggg ggg ctc cgg gac acg 1872 Tyr Gly Thr Val Pro Val Val His Ala Val Gly Gly Leu Arg Asp Thr *610 615 620 gtg gcg ccg ttc gac ccg ttc aac gac acc ggg ctc ggg tgg acg ttc 1920 Val Ala Pro Phe Asp Pro Phe Asn Asp Thr Sly Leu Gly Trp Thr Phe .625 630 635 640 *gac cgc gcg gag gcg aac cgg atg atc gac gcg ctc tcg cac tgc ctc 1968 Asp Arg Ala Glu Ala Asn Arg Met Ile Asp Ala Leu Ser His Cys Leu *645 650 655 acc acg tac cgg aac tac aag gag agc tgg cgc gcc tgc agg gcg cgc 2016 Thr Thr Tyr Arg Asn Tyr Lys Glu Ser Trp Arg Ala Cys Arg Ala Arg .:660 665 670 ggc atg gcc gag gac ctc agc tgg gac cac gcc gcc gtg ctg tat gag 2064.

Gly Met Ala Glu Asp Leu Ser Trp Asp His Ala Ala Val Leu Tyr Glu 675 680 685 *gac gtg ctc gtc aag gcg aag tac cag tgg tga gcg aat taa ttg gcg 2112 Asp Val Leu Val Lys Ala Lys Tyr Gin Trp Ala Asn Leu Ala 690 695 700 acg cga cgc cgc tcc tgt cgc agg acc tgg acg tta ttt aga agg ctc 2160 *Thr Arg Arg Arg Ser Cys Arg Arg Thr Trp Thr Leu Phe Arg Arg Leu 705 710 715 ttc tcc ctg gcg gct Etg atg cgt gcg tcg cat ttg cgc cgg gcg gac 2208 Phe Ser Leu Ala Ala Leu Met Arg Ala Ser His Leu Arg Arg Ala Asp 720 725 730 ggg cga cgg tgg ttg gcc tac cgc cta cgt cgg ctg cgt gcc ctg gga 2256 Gly Arg Arg Trp Leu Ala Tyr Arg Leu Arg Arg Leu Arg Ala Leu Gly 735 740 745 750 att tgg gcg ggc acg atg atg cca ctg ggc acc ggg cgc ggg gta gta 2304 Ile Trp Ala Gly Thr Met Met Pro Leu Gly Thr Gly Arg Gly Val Val 755 760 765 tga tat gaa acc gac ggc gat gga gat gag gcg cat ggc att ttc cca 2352 Tyr Glu Thr Asp Gly Asp Gly Asp Giu Ala His Gly Ile Phe Pro 770 775 780 ctg ata aat ggg gag ttg tat gct act tta ata tcg cca ctc ctg tta 2400 Leu Ile Asn Gly Glu Leu Tyr Ala Thr Leu Ile Ser Pro Leu Leu Leu 785 790 795 gta ttt ata ttg atg gcg gec gc 2423 Val Phe Ile Leu Met Ala Ala 800 <210> 49 <211> 804 <212> PRT <213> Zea mays <400> 49 Met Pro Gly Ala Ile Ser Ser Ser Ser Ser Ala Phe Leu Leu Pro Val 1 5 10 Ala Ser Ser Ser Pro Arg Arg Arg Arg Gly Ser Val Gly Ala Ala Leu 25 Arg Ser Tyr Gly Tyr Ser Gly Ala Glu Leu Arg Leu His Trp, Ala Arg 40 *Arg Gly Pro Pro Gln Asp Gly Ala Ala Ser Val Arg Ala Ala Ala Ala 55 Pro Ala Gly Gly Glu Ser Glu Glu Ala Ala Lys Ser Ser Ser Ser Ser 70 75 **.Gin Ala Gly Ala Val Gin Gly Ser Thr Ala Lys Ala Val Asp Ser Ala *9:85 90 Ser Pro Pro Asn Pro Leu Thr Ser Ala Pro Lys Gln Ser Gin Ser Ala 100 105 110 Ala Met Gin Asn Gly Thr Ser Gly Gly Ser Ser Ala Ser Thr Ala Ala *115 120 125 Pro Val Ser Gly Pro Lys Ala Asp His Pro Ser Ala Pro Val Thr Lys 130 135 140 .*Arg Glu Ile Asp Ala Ser Ala Val Lys Pro Glu Pro Ala Gly Asp Asp 145 150 155 160 Ala Arg Pro Val Giu Ser Ile Gly Ile Ala Giu Pro Val Asp Ala Lys 165 170 175 **Ala Asp Ala Ala Pro Ala Thr Asp Ala Ala Ala Ser Ala Pro Tyr Asp 180 185 190 Arg Glu Asp Asn Glu Pro Gly Pro Leu Ala Gly Pro Asn Val Met Asn 195 200 205 Val Val Val Val Ala Ser Glu Cys Ala Pro Phe Cys Lys Thr Gly Gly .210 215 220 Leu Gly Asp Val Val Gly Ala Leu Pro Lys Ala Leu Ala Arg Arg Gly 225 230 235 240 His Arg Val Met Val Val Ile Pro Arg Tyr Gly Glu Tyr Ala Glu Ala 245 250 255 Arg Asp Leu Gly Val Arg Arg Arg Tyr Lys Val Ala Gly Gin Asp Ser 260 265 270 Glu Val Thr Tyr Phe His Ser Tyr Ile Asp Gly Val Asp Phe Val Phe 275 280 285 Val Glu Ala Pro Pro Phe Arg His Arg His Asn Asn Ile Tyr Gly Gly 290 295 300 Glu Arg Leu Asp Ile Leu Lys Arg Met Ile Leu Phe Cys Lys Ala Ala 305 310 315 320 Val Glu Val Pro Trp Tyr Ala Pro Cys Gly Gly Thr Val Tyr Gly Asp 325 330 335 87 Gly Asn Leu Val Phe Ile Ala Asn Asp Trp His Thr Ala Leu Leu Pro 340 345 350 0* be 9 b C S 0 I 00108 d .e 9 8 9O S 09 S 5 0 lOb..

0 .5 '9 0 0 'S 4 000.

0 *900 00 0 09 9SS~

C

Ala Vai Val1 Asp 405 Thr Lys Asp Asn Thr 485 Gin Phe Ala Thr His 565 His Giu Pro Asp Ala 645 Asn Tyr Ile Asn 390 Asn Ala Thr Trp Pro 470 Phe Arg Ile Ile Gly 550 Ser Arg Pro Val Pro 630 Asn Tyr Gly Leu His Gin 380 Giu His 395 His Ser Thr Val Trp Gly Ile Val 460 His Leu 475 Thr Gly Gin Val His Gin Gly Gin 540 Glu Asp 555 Ala Trp Ala Asp Gin Leu Gly Gly 620 Gly Leu 635 Ala Leu Arg Ala Gin Arg Ile Val Asn 430 His Gly Ser Arg Asp 510 Gly Val Leu Gly Leu 590 Ala Arg Trp His Arg 670 Ala Pro His 400 Ala Tyr Ile Asp Asp 48.0 Cys Val1 Asp Leu Arg 560 Ser Met Ala Thr Phe 640 Leu Arg Gly Met Ala 675 Glu Asp Leu Ser Glu Ap Le Ser Asp His Ala Ala Val Leu Tyr Glu 88 Asp Val Leu Val Lys Ala Lys Tyr Gin Trp Ala Asn Leu Ala Thr Arg .690 695 700 Arg Arg Ser Cys Arg Arg Thr Trp Thr Leu Phe Arg Arg Leu Phe Ser 705 710 715 720 Leu Ala Ala Leu Met Arg Ala Ser His Leu Arg Arg Ala Asp Gly Arg 725 730 735 Arg Trp Leu Ala Tyr Arg Leu Arg Arg Leu Arg Ala Leu Gly Ile Trp 740 745 750 Ala Gly Thr Met Met Pro Leu Gly Thr Gly Arg Gly Val Val Tyr Glu 755 760 765 *Thr Asp Gly Asp Gly Asp Giu Ala His Gly Ile Phe Pro Leu Ile Asn 770 775 780 Gly Giu Leu Tyr Ala Thr Leu Ile Ser Pro Leu Leu Leu Val Phe Ile 785 790 795 800 .Leu Met Ala Ala <210> <211> 2010 <212> DNA Zea mays <220> <221> CDS <222> (1)..(2010) *<400> gct gag gct gag gcc ggg ggc aag gac gcg ccg ccg gag agg agc ggc 48 000. Ala Glu Ala Glu Ala Gly Gly Lys Asp Ala Pro Pro Giu Arg Ser Gly 1 5 10 0 gac gcc gcc agg ttg ccc cgc gct cgg cgc aat gcg gtc tcc aaa cgg 96 :.:oOAsp Ala Ala Arg Leu Pro Arg Ala Arg Arg Asn Ala Val Ser Lys Arg o o20 25 agg gat cct ctt cag ccg gtc ggc cgg tac ggc tcc gcg acg gga aac 144 Arg Asp Pro Leu Gin Pro Val Gly Arg Tyr Gly Ser Ala Thr Gly Asn 40 acg gcc agg acc ggc gcc gcg tcc tgc cag aac gcc gca ttg gcg gac 192 Thr Ala Arg Thr Gly Ala Ala Ser Cys Gin Asn Ala Ala Leu Ala Asp 55 gtt gag atc aag tcc atc gtc gcc gcg ccg ccg acg agc ata gtg aag 240 Val Glu Ile Lys Ser Ile Val Ala Ala Pro Pro Thr Ser Ile Val Lys 70 ttc cca gcg. ccg ggc tac agg atg atc ctt ccc tct ggg gac ata gcg 288 Phe Pro Ala Pro Gly Tyr Arg Met Ile Leu Pro Ser Gly Asp Ile Ala 90 ccg gag act gtc ctc cca gcc ccg aag cca czg cat gaa tcg cct gcg 336 Pro Glu Thr Val Leu Pro Ala Pro Lys Pro Leu His Giu Ser Pro Ala 100 105 110 gtt gac gga gat tca aat gga att gca cct czt aca gtt gag cca tta 384 Val Asp Gly Asp Ser Asn Gly Ile Ala Pro Pro Thr Val Giu Pro Leu 115 120 125 gta cag Val Gin 130 gag gcc act tgg gat ttc aag Giu Ala Thr Trp Asp Phe Lys aaa tac atc Lys Tyr Ile 135 140 0 00 0 cc t Pro 145 tc t Ser aa t Asn aaa Lys gcg Ala tat Tyr 225 gga Gly gac Asp a ta Ile tgc Cys tgc Cys 305 gca Ala atg Met ggc Gly tac Tyr gac Asp ttt Phe gtt Val1 aca Thr aga Arg 210 gtg Val cag Gin tt Phe tat Tyr aag Lys 290 tac Tyr ctc Leu cag Gln zg t Arg ztt Leu 370 gaa Giu gaa Giu a tg Met ggt Gly 195 aga Arg gaa Giu gac Asp g tg Val1 ggg Gly 275 gtt Val1 gga Gly c tg Leu tac Tyr ggt Gly 355 caa Gin gcg Ala cat His aac Asn 180 gg t Gly gga Giy gcc Ala c ta Leu t tc Phe 260 gga Gly gc t Ala ga t Asp cct Pro act Thr 340 cct Pro ca t Hiis aag Lys tat Tyr 165 g tg Val ctt Leu cat His ttt Phe gaa Giu 245 att Ile agt Ser gtt Val1 gga Gly gt t Val1 325 cgc Arg gta Val1 ttc Phe ga t Asp 150 ggg Gly atc Ile gga Gly cgt Arg ga t Asp 230 gtg Vali ga t Asp agg Arg gag Giu aa t Asn 310 tat Tyr tcc Ser ga t Asp gag Glu ga t Asp gac Asp gtg Val gat Asp gtt Val 215 atg Met aac Asn gcc Ala cag Gin gtt Vali 295 t tg Leu c tg Leu gtc Val gaa Giu c tg Leu 375 tcc Ser aat Asn gtg Val gtt Val 200 atg Met gga Gly tat Tyr cct Pro gaa Giu 280 cc t Pro g tg Val1 aag Lys ctc Leu ttc Phe 360 tac Tyr ggt Gly ga t Asp t tg Leu tc t Ser ccc Pro 205 agg Arg tac Tyr att Ile cgt Arg a tg Met 285 tgc Cys ga t Asp gac Asp atc Ile t tg Leu 365 ggc Gly ga t Asp gcc Ala cca Pro 190 aag Lys tat Tyr aaa Lys ga t Asp caa Gin 270 att Ile gg t Gly tgg Trp cat His gcc Ala 350 cc t Pro gag Glu ggt Gly 160 gag Giu tgc Cys t ta Leu gac Asp gca Ala 240 gtc Val gac Asp ttt Phe gtg Val1 act Thr .320 tta Leu cag Gin cac His gcc Ala ttt gac gag Phe Asp Giu 432 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 atc ttt gcc gcg Ile Phe Ala Ala ctg aag atg gca Leu Lys Met Ala cgg gtg gtg act Arg Val Val Thr agc Ser C tC Leu aac Asn cgg Arg aag Lys 465 cgc Arg aag Lys gac Asp atg Met gtc Val 545 gtg Val tac Tyr ctc Leu ggg Gly agg Arg 625 c tc Letu gag Glu cgc ggc Arg Gly cac gac His Asp ggc atc Gly Ile 435 tcg gac Ser Asp 450 cgg cag Arg Gin gac gac Asp Asp ggc gtg Gly Val gtg cag Val Gin 515 ctg cag Leu Gin 530 ggg ttc Gly Phe ctg gtg Leu Vai gcg atg Ala Met agg gac Arg ASP 595 tgg act Trp Thr 610 cac tgc His Cys cag gcg Gin Ala ctc tac Leu Tyr gaa ggc Giu Gly atc aat Ile Asn gtg gac Val Asp 445 aca ctc Thr Leu 460 ctg ggc Leu Gly cgt ctg Arg Leu tgg atc Trp Ile gcc gac Ala Asp 525 aag gtg Lys Val 540 acg gcg Thr Ala ggg ctg Giy Leu cac gcc His Ala ggc gac Gly Asp 605 ctg atc Leu Ile 620 gag agc Glu Ser tgg gac Trp Asp tac cag Tyr Gin 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2010 660 665 670 <210> 51 <211> 670 <212> PRT <213> Zea mays <400> 51 Ala Glu Ala Glu Ala Gly Gly Lys Asp Ala Pro Pro Glu Arg Ser Gly 1 5 10 Asp Ala Ala Arg Leu Pro Arg Ala Arg Arg Asn Ala Val Ser Lys Arg 25 Arg Asp Pro Leu Gin Pro Val Gly Arg Tyr Gly Ser Ala Thr Gly Asn 35 40 Thr Ala Arg Thr Gly Ala Ala Ser Cys Gin Asn Ala Ala Leu Ala Asp S* 50 55 SVal Glu Ile Lys Ser Ile Val Ala Ala Pro Pro Thr Ser Ile Val Lys 65 70 75 Phe Pro Ala Pro Gly Tyr Arg Met Ile Leu Pro Ser Gly Asp Ile Ala 90 Pro Glu Thr Val Leu Pro Ala Pro Lys Pro Leu His Glu Ser Pro Ala 100 105 110 Val Asp Gly Asp Ser Asn Gly Ile Ala Pro Pro Thr Val Glu Pro Leu 115 120 125 *Val Gin Glu Ala Thr Trp Asp Phe Lys Lys Tyr Ile Gly Phe Asp Glu 130 135 140 Pro Asp Glu Ala Lys Asp Asp Ser Arg Val Gly Ala Asp Asp Ala Gly 145 150 155 160 Ser Phe Glu His Tyr Gly Asp Asn Asp Ser Gly Pro Leu Ala Gly Glu 165 170 175 Asn Val Met Asn Val Ile Val Val Ala Ala Glu Cys Ser Pro Trp Cys 180 185 190 Lys Thr Gly Gly Leu Gly Asp Val Val Gly Ala Leu Pro Lys Ala Leu 195 200 205 Ala Arg Arg Gly His Arg Val Met Val Val Val Pro Arg Tyr Gly Asp 210 215 220 Tyr Val Glu Ala Phe Asp Met Gly Ile Arg Lys Tyr Tyr Lys Ala Ala 225 230 235 240 Gly Gin Asp Leu Glu Val Asn Tyr Phe His Ala Phe Ile Asp Gly Val 245 250 255 Asp Phe Val Phe Ile Asp Ala Pro Leu Phe Arg His Arg Gin Asp Asp 260 265 270 Ile Tyr Gly Gly Ser Arg Gin Glu Ile Met Lys Arg Met Ile Leu Phe 275 280 285 Cys Lys Val Ala Val Glu Val Pro Trp His Val Pro Cys Gly Gly Val 290 295 300 Cys Tyr Gly Asp Gly Asn Leu Val Phe Ile Ala Asn Asp Trp His Thr a a *4 44 *4 4 '4 4 4* 4 4 44 4 4 *44* 4 *4*4 .4 4 .4.4 305 Ala Met Gly Tyr Asn 385 Ser Leu Ash Arg Lys 465 Arg Lys Asp Met Val1 545 Val1 Tyr Leu Gly Arg 625 Leu Giu Leu Leu Gin Tyr Arg Gly 355 Leu Gin 370 Ile Phe Arg Giy His Asp Gly Ile 435 Ser Asp 450 Arg Gin Asp Asp Giy Vai Vai Gin 515 Leu Gin 530 Giy Phe Leu Vai Aia Met Arg Asp 595 Trp Thr 610 His Cys Gin Ala Leu Tyr 310 Val Tyr 325 Arg Ser Vai Asp Phe Giu Ala Gly 390 Leu Trp, 405 Ile Arg His Gin Tyr Thr Lys Ala 470 Pro Leu 485 Ile Ile Val Met Leu Giu Val Pro 550 Pro Ser 565 Tyr Giy Vai Ala Asp Arg Asp Thr 630 Gly Met 645 Asp Val Leu Vai Giu Leu 375 Leu Giu Ser Giu Asn 455 Ala Leu Gly Leu Arg 535 Met Arg Thr Pro Ala 615 Tyr Ser Leu Lys Leu Phe 360 Tyr Lys Leu Asn Trp 440 Tyr Leu Gly Asp Gly 520 Giu Ala Phe Vai Phe 600 Giu Arg Gln Val Ala Tyr 330 Val Ile 345.

Pro Tyr Asp Pro Met Ala Lys Thr 410 Asp Trp 425 Asn Pro Ser Leu Gin Arg Phe Ile 490 Ala Met 505 Thr Gly His Pro His Arg Giu Pro 570 Pro Val 585 Asp Pro Ala Asn Lys Tyr Asp Leu 650 Lys Ala 665 315 Tyr His Me t Val Asp 395 Val Lys Lys Giu Giu 475 Giy Pro Arg Asn Ile 555 Cys Val Phe Lys Gly 635 Ser Lys Arg Asn Asp Gly 380 Arg Glu Ile Vai Thr 460 Leu Arg Trp Ala Lys 540 Thr Gly His Gly Leu 620 Giu Trp Tyr Asp His Ile Ala 350 Leu Pro 365 Gly Giu Vai Vai Gly Gly Asn Gly 430 Asp Vai 445 Leu Asp Gly Leu Leu Asp Ile Ala 510 Asp Leu 525 Val Arg Ala Gly Leu Asn Ala Val 590 Asp Ala 605 Ile Giu Ser Trp Asp His Gin Trp, 670 Gly 335 His Giu His Thr Trp 415 Ile His Ala Giu Gly 495 Gly Giu Gly Ala Gin 575 Gly Gly Ala Lys Aia 655 320 Leu Gin His Ala Val 400 Gly Val Leu Gly Vali 480 Gln Gln Arg Trp Asp 560 Leu Giy Leu Leu Ser 640 Ala <210> 52 <211> 1749 <212> DNA <213> Zea mays <220> <221> CDS <222> (1)..(1749) <400> 52 tgc gtc gcg gag ctg agc agg gag ggg ccc gcg ccg cgc ccg ctg cca 48 Cys Val Ala Giu Leu Ser Arg Giu Gly Pro Ala Pro Arg Pro Leu Pro 1 5 10 ccc geg ctg etg geg ccc ccg etc gtg ccc ggc ttc etc gcg ceg ccg 96 Pro Ala Leu Leu Ala Pro Pro Leu Val Pro Gly Phe Leu Ala Pro Pro 25 gec gag ccc acg ggt gag ccg gca tcg acg ceg ccg ccc gtg ccc gac 144 Ala Glu Pro Thr Gly Glu Pro Ala Ser Thr Pro Pro Pro Val Pro Asp 40 gee ggc ctg ggg gac etc ggt ctc gaa cct gaa ggg att get gaa ggt 192 Ala Gly Leu Gly Asp Leu Gly Leu Glu Pro Glu Gly Ile Ala Glu Gly 55 tce ate gat aac aca gta gtt gtg gca agt gag caa gat tet gag att 240 Ser Ile Asp Asn Thr Val Vai Val Ala Ser Glu Gln Asp Ser Glu Ile 70 75 gtg gtt gga aag gag caa get ega get aaa gta aca caa agc att gtc 288 Val Val Gly Lys Glu Gln Ala Arg Ala Lys Val Thr Gin Ser Ile Val 90 ttt gta ace ggc gaa gct tct ect tat gca aag tet ggg ggt cta gga 336 Phe Val Thr Gly Glu Ala Ser Pro Tyr Ala Lys Ser Gly Gly Leu Gly 100 105 110 gat gtt tgt ggt tca ttg cca gtt get ctt gct get cgt ggt cac cgt 384 Asp Val Cys Gly Ser Leu Pro Val Ala Leu Ala Ala Arg Gly His Arg 115 120 125 gtg atg gtt gta atg ccc aga tat tta aat ggt acc tee gat aag aat 432 Val Met Val Val Met Pro Arg Tyr Leu Asn Gly Thr Ser Asp Lys Asn 130 135 140 tat gca aat gca ttt tac aea gaa aaa cae att cgg att cca tge ttt 480 Tyr Ala Asn Ala Phe Tyr Thr Glu Lys His Ile Arg Ile Pro Cys Phe 145 150 155 160 gge ggt gaa cat gaa gtt acc tte ttc eat gag tat aga gat tca gtt 528 Gly Gly Glu His Glu Val Thr Phe Phe His Glu Tyr Arg Asp Ser Val 165 170 175 gac tgg gtg ttt gtt gat cat ccc tea tat cac aga ect gga aat tta 576 Asp Trp Val Phe Val Asp His Pro Ser Tyr His Arg Pro Gly Asn Leu 180 185 190 tat gga gat aag ttt ggt gct ttt ggt gat aat cag ttc aga tac aca 624 Tyr Gly Asp Lys Phe Gly Ala Phe Gly Asp Asn Gin Phe Arg Tyr Thr 195 200 205 etc ett tgc tat get gca tgt gag get cct ttg ate ctt gaa ttg gga 672 Leu Leu Cys Tyr Ala Ala Cys Glu Ala Pro Leu Ile Leu Glu Leu Gly #~44 see* -210 gga tat Gly Tyr 225 gcc agt Ala Ser gtt tat Val Tyr cag ggt Gin Gly gaa tgg Glu Trp, 290 cat gcc His Ala 305 gtg aca Val Thr gtc aca Val Thr aga aag Arg Lys tgg aac Trp Asn 370 gac ctc Asp Leu 385 ggt tta Gly Leu ttg gat Leu Asp ctc atg Leu Met gag ctt Giu Leu 450 ttt cgt Phe Arg 465 gcc ggc tat Tyr gtg Val gac Asp 260 gag Glu gga Gly gac Asp ga t Asp gc t Ala 340 gta Val gcc Ala gga Gly ata Ile cag Gin 420 gaa Giu ga t Asp tgg Trp gat 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 Ala Gly Cys Asp Ile Leu Leu Met Pro Ser Arg Phe Giu Pro Cys Gly 485 490 495 ctc aat cag cta tat gct atg cag tat ggc aca gtt cct gtt gtc cat 1536 Leu Asn Gin Leu Tyr Ala Met Gin Tyr Gly Thr Val Pro Val Val His 500 505 510 gca act ggg ggc ct-t aga gat acc gtg gag aac ttc aac cct ttc ggt 1584 Ala Thr Gly Gly Leu Arg Asp Thr Val Giu Asn Phe Asn Pro Phe Giy 515 520 525 gag aat gga gag cag ggt aca ggg tgg gca ttc gca ccc cta acc aca 1632 Giu Asn Giy Giu Gin Giy Thr Giy Trp Ala Phe Aia Pro Leu Thr Thr 530 535 540 gaa aac atg ttt gtg gac att gcg aac tgc aat atc tac ata cag gga 1680 *Giu Asn Met Phe Vai Asp Ile Ala Asn Cys Asn Ile Tyr Ile Gin Giy 545 550 555 560 aca caa gtc ctc ctg gga agg gct aat gaa gcg agg cat gtc aaa aga 1728 *Thr Gin Val Leu Leu Gly Arg Ala Asn Glu Ala Arg His Val Lys Arg *565 570 575 c tt cac gtg gga cca tgc cgc 1749 *Leu His Val Gly Pro Cys Arg 580 <210> 53 <211> 583 <212> PRT <213> Zea mays <400> 53 *.Cys Val Ala Giu Leu Ser Arg GluGly Pro Ala Pro Arg Pro Leu Pro *1 5 10 Pro Ala Leu Leu Ala Pro Pro Leu Vai Pro Gly Phe Leu Ala Pro Pro 25 Giu Pro Thr Giy Giu Pro Ala Ser Thr Pro Pro Pro Val Pro Asp 40 Ala Gly Leu Gly Asp Leu Gly Leu Giu Pro Giu Gly Ile Ala Glu Gly 55 Ser Ile Asp Asn Thr Val Val Val Ala Ser Giu Gin Asp Ser Giu Ile 70 75 Val Val Gly Lys Giu Gin Ala Arg Ala Lys Val Thr Gin Ser Ile Val 90 Phe Val Thr Gly Giu Ala Ser Pro Tyr Ala Lys Ser Gly Gly Leu Gly 100 105 110 Asp Val Cys Gly Ser Leu Pro Val Ala Leu Ala Ala Arg Gly His Arg 115 120 125 Val Met Val Val Met Pro Arg Tyr Leu Asn Gly Thr Ser Asp Lys Asn 130 135 140 Tyr Ala Asn Ala Phe Tyr Thr Giu Lys His Ile Arg Ile Pro Cys Phe 145 150 155 160 Gly Gly Giu His Giu Val Thr Phe Phe His Giu Tyr Arg Asp Ser Val 165 170 175 0 C0 000C

C

*CC

0000 Asp Tyr Leu Gly 225 Ala Val Gin Glu His 305 Val Val1 Arg Trp, Asp 385 Gly Leu Leu Glu Phe 465 Ala Leu Ala Glu 530 GiU Asn Met 545 Thr Gin Val Leu His Val 535 540 Val Asp Ile Aia Asn Cys Asn Ile Tyr Ile Gin Giy 550 555 560 Leu Gly Arg Ala Asn Gl. Al1a Arg His Vai Lys Arg 565 570 575 Pro Cys Arg S *5 54 <2i1> 870 <2i2> PRT <2i3> Zea mays <400> 54 Met Ala Phe Arg

I

Pro Arg Leu Thr Gly Leu Phe Leu Giy Ala Met Arg Giy Giu Asn Asp Ser Asp Giu Leu Ala Giy Val Ala 100 Pro Pro Ser Asp 115 Giy Tyr Lys Tyr 130 Arg Ser Asp Ile 145 Ser Tyr Giu Lys Arg Giu Trp Ala 180 Asn Asn Trp Asp 195 Val Trp Giu Ile 210 Pro His Gly Ser 225 Lys Asp Ser Ile Glu Tyr Tyr Tyr 305 Tyr Ser Gly Gly Thr 385 His Asn Trp Thr Asn 465 Tyr Val1 Val1 Ala Met 545 Cys Thr Leu Pro Phe 275 Thr Asn Ala Gly Pro 355 Leu Asp Gly Gly Leu 435 Met Asn Met Ile Asp 515 Lys Asp Thr Ala Arg 595 600 605 Lys Met Ile Arg Leu Ile Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu 99 610 615 620 Asn Phe Met Gly Asn Giu Phe Gly His Pro Giu Trp Ile ALsp Phe Pro 625 630 635 640 Arg Giy Pro Gin Arg Leu Pro Ser Gly Lys Phe Ile Prc Gly Asn Asn 645 -650 655 Asn Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Asp 660 665 670 Tyr LeU Arg Tyr His Giy Met Gin Giu Phe-Asp Gin Ala Met Gin His 675 680 685 Leu Giu Gin Lys Tyr Giu Phe Met Thr Ser Asp His Gin T-jr Ile Ser 690 -695 700 Arg Lys His Giu Giu Asp Lys Vai Ile Val Phe Giu Lys Gly Asp Leu 705 710 715 .720 *Val Phe Val Phe Asn Phe His Cys Asn Asn Ser Tyr Phe Asp Tyr Arg 725 730 735 I .le Gly Cys Arg Lys Pro Giy Val Tyr Lys Val Vai Leu Asp jSer Asp 740 745 750! Ala Giy Leu Phe Gly Giy Phe Ser Arg Ile His His Ala Ala'Giu His 755 760 765 *.Phe Thr Aia Asp Cys Ser His Asp Asn Arg Pro Tyr Ser Phe Ser Vai -770 775 780 Tyr Thr Pro Set Arg Thr Cys Vai Vai-Tyr Ala Pro Val. Gi-u Arg Giy 785 790 795 800 Thr Arg Cys Cys Ala Ala Cys Val Gly Leu Ser Met Gly Lys Thr Phe ~805 810 815 Phe Gin Asn Arg Gin Met His Ala Cys Met Leu Gin Gly Ser Asp Thr ,820 825 830 Ldu Ile Asp Ala Gly Lys Pro Met His Leu Ala Ala Leu Ser Ser Leu 835 840 845 Phe Ile Asp Leu Gin Gly Val Asn Thr Ser Phe. Arg Phe Ser Leu Lys 850 855 860 Lys Lys Lys Lys Lys Leu 865 870 <210> <211> 2640 <212> DNK <213> Zea mays <400> gcgagatggc gttccgggtt'tctggggcgg tgctcggtgg ggccgtaagg gctccccgac tcaccggcgg cggggagggt agtctagtct tccggcacac cggcctcttc ttaactcggg 120 gtgctcgagt tggatgttcg gggacgcacg gggccatgcg cgcggcggcc gcggccagga 180 aggcggtcat ggttcctgag ggcgagaatg atggcctcgc atcaagggct gactcggctc 240 aattccagtc ggatgaactg gaggtaccag acatttctga agagacaacg tgcggtgctg 300 gtgtggctga tgctcaagcc ttgaacagag ttcgagtggt ccccccacca agcgatggac 360 aaaaaatatt ccagattgac cccatgttgc aaggctataa gtaccatctt gagtatcggt 420 acagcctcta tagaagaatc cgttcagaca ttgatgaaca tgaaggaggc ttggaagcct 480 tctcccgtag ttatgagaag tttggattta atgccagcgc ggaaggtatc Acatatcgag 540 aatgggctcc tggagcattt tctgcagcat tggtgggtga cgtcaacaac tgggatccaa 600 100 atgcagatcg cagatggtac cagggataaa taccatatga cgcaacctaa cggaaccgaa aacttggata ttggatacca tgaagtcttt atagtcatgc attactttca atgggaactg ataagtttga tacaagtaac cagtggttta ccattggtga taggttttga aaagtgatga tagagaagtg t tgcgt t ttg ctcctaccat taggaggaga attttccaag gttatgacaa gtatgcaaga ctgatcacca gagatttggt gttgtcgaaa gatttagcag ggccatattc agtgatagcg ccttcttcca tcgatgctgg aaggtgtcaa tatgagcaaa atcacctatt gga ttcaat t tgggatttat acgaccaaaa gataaacaca caatgcagtg tgtaactaat gattgataga gtcaagtaat cagtggtcca ggaagtttta tggtttccgt atttacgggg cttgatgctg agatgttagt ctatcggatg aacttggaag tgtaacttat gttgatggac tgatcgtggg gggctatctt aggtccgcaa atgtcgtcga gtttgatcag gtatatttcc atttgtgttc gcctggggtg gatccatcac attctcggtt gggtactcgt aaaccggcag aaagcccatg ttaaacatag aatgagtttg cctcatggat ccagcctgga tatgatcctc tcattgcgga tatgtaaact caaataatgg ttttttgcgc gcaca tgagc actctggatg cgtggccatc agatttcttc tttgatggtg aacttcaatg gtaaatgatc ggaatgccta catatggctg atgggtgata gctgaaagtc aaggatatgc atagcattac aatttcatgg agacttccaa agatttgacc gcaatgcaac cggaaacatg aacttccact tataaggtgg gcagccgagc tatacaccaa tgc tgcgcgg atgcatgcat catctcgctg agttttcgtt gtgtttggga ctcgtgtaaa tcaagtactc ctgaagaggt tatatgaaac ttagggatga caatccaaga caagtagtcg ttggtttgct ggttgaatgg actggatgtg tctccaatgc tgacccccat agtattttgg taattcatgg catttgccct tggc tgacaa ttgtgcacac atgatcaagc atgatttcat ataagatgat gaaatgagtt gtggtaagtt tgggtgatgc atcttgagca aggaggataa gcaacaacag tcttggactc acttcaccgc gcagaacatg catgtgtggg gcatgctaca cgttgtcctc tttcgcttaa aatttttctg ggtgagaatg agtgcaggcc aaagtatgtg acatgtcgga agtcctccca gcactcatat ttttggtacc agttctcatg ttttgatggt ggattctcgc tagatggtgg gatgtacact ctttgccacc actttatcct tcctgttcac atggattgac actgacaaat attagtcggc ggccctcgat tagacttatc tggacatcct tattccaggg agactatctt aaaata tgaa ggtgattgtg ctattttgac cgacgc tgga cgactgttcg tgtcgtctat gctgtcgatg ataaggttc t tctatttata aaaaaaaaaa cctaacaatg 660 gatactccat 720 ccaggagaaa 780 ttcaggcatg 840 atgagtagcc 900 agaataaaaa 960 tatggaagct 1020 ccagaagatt 1080 gatgtggttc 1140 acagatacac 1200 ctatttaact 1260 ctcgaggaat 1320 caccacggat 1380 gatgtagatg 1440 gaggctgtaa 1500 gatggtgggg 1560 cttctcaagc 1620 aggaggtggt 1680 gacaagacta 1740 agaccttcaa 1800 acaatgggtt 1860 gaatggatag 1920 aataacaaca 1980 aggtatcatg 2040 ttcatgacat 2100 ttcgaaaagg 2160 taccgtattg 2220 ctatttggtg 2280 catgataata 2340 gctccagtgg 2400 tgaggaaaaa 2460 gatactttaa 2520 taagaccttc 2580 aaaaaactca 2640 <210> 56 <211> 776 <212> PRT <213> Zea mnays <400> 56 Ala Thr Val Gin 1 Glu Asp Lys Thr Met 5 Thr Ala Lys Asp His Leu Pro Ile Tyr Asp Leu Pro Lys Leu Glu Gly Asp Val Ile Phe Lys Lys Gly Ser Asp His Phe Arg Ile Giu Giu Asn Tyr Arg Met Arg Phe Leu Glu Giu Gly Ser Leu Giu Ser Phe Lys Gly Tyr Leu Lys Phe GlyIle Asn Thr Asn Giu Asp Gly Thr 70 Val Tyr Arg Glu Ala Pro Ala Ala Asn Gly Ala Asn 100 Ile Lys Ile Asp 115 Glu Ala Glu Leu Ile Giy Asp Phe Asn Asp Trp His Lys Met Glu Lys Phe Gly Val Trp Ser 110 Ala Ile Pro His Asn Ser 125 His Val Lys Gly Lys Pro 120 Lys Val Lys Phe Arg Phe Leu His Gly Gly Val Trp Val Asp Arg Ile 101 130 135 140 Pro Ala Leu Ile Arg Tyr Ala Thr Val Asp Ala Ser Lys Phe Gly Ala 145 150 155 160 Pro Tyr Asp Gly Val His Trp Asp Pro Pro Ala Ser Glu Arg Tyr Thr 165 170 175 Phe Lys His Pro Arg Pro Ser Lys Pro Ala Ala Pro Arg Ile Tyr Glu 180 185 190 Ala His Val Gly Met Ser Gly Glu Lys Pro Ala Val Ser Thr Tyr Arg 195 200 205 Glu Phe Ala Asp Asn Val Leu Pro Arg Ile Arg Ala Asn Asn Tyr Asn 210 215 220 Thr Val Gin Leu Met Ala Val Met Glu His Ser Tyr Tyr Ala Ser Phe 225 230 235 240 oo .o Gly Tyr His Val Thr Asn Phe Phe Ala Val Ser Ser Arg Ser Gly Thr 245 250 255 P Pro Glu Asp Leu Lys Tyr Leu Val Asp Lys Ala His Ser Leu Gly Leu 260 265 270 Arg Val Leu Met Asp Val Val His Ser His Ala Ser Asn Asn Val Thr 275 280 285 i.

Asp Gly Leu Asn Gly Tyr Asp Val Gly Gln Ser Thr Gin Glu Ser Tyr 290 295 300 Phe His Ala Gly Asp Arg Gly Tyr His Lys Leu Trp Asp Ser Arg Leu 305 310 315 320 Phe Asn Tyr Ala Asn Trp Glu Val Leu Arg Phe Leu Leu Ser Asn Leu 325 330 335 Arg Tyr Trp Leu Asp Glu Phe Met Phe Asp Gly Phe Arg Phe Asp Gly 340 345 350 Val Thr Ser Met Leu Tyr His His His Gly Ile Asn Val Gly Phe Thr 355 360 365 Gly Asn Tyr Gin Glu Tyr Phe Ser Leu Asp Thr Ala Val Asp Ala Val 370 375 380 Val Tyr Met Met Leu Ala Asn His Leu Met His Lys Leu Leu Pro Glu 385 390 395 400 Ala Thr Val Val Ala Glu Asp Val Ser Gly Met Pro Val Leu Cys Arg 405 410 415 Pro Val Asp Glu Gly Gly Val Gly Phe Asp Tyr Arg Leu Ala Met Ala 420 425 430 Ile Pro Asp Arg Trp Ile Asp Tyr Leu Lys Asn Lys Asp Asp Ser Glu 435 440 445 Trp Ser Met Gly Glu Ile Ala His Thr Leu Thr Asn Arg Arg Tyr Thr 450 455 460 Glu Lys Cys Ile Ala Tyr Ala Glu Ser His Asp Gin Ser Ile Val Gly 465 470 475 480 Asp Lys Thr Ile Ala Phe Leu Leu Met Asp Lys Glu Met Tyr Thr Gly 485 490 495 102 Met Ser Asp Leu Gin Pro Ala Ser Pro -Thr Ile Asp Arg Giy Ile Ala 500 505 510 Leu Gin Lys Met Ile His Phe Ile Thr Met Ala Leu Giy Giy Asp Gly 515 520 525 Tyr Leu Asn Phe Met Giy Asn Glu Phe Gly His Pro Giu Trp Ile Asp 530 535 540 Phe Pro Arg Giu Gly Asn Asn Trp Ser Tyr Asp Lys Cys Arg Arg Gin 545 550 555 560 Trp Ser Leu Val Asp Thr Asp His Leu Arg Tyr Lys Tyr Met Asn Ala 565 570 575 Phe Asp Gin Ala Met Asn Ala Leu Asp Giu Arg Phe Ser Phe Leu Ser 580 585 590, Ser Ser Lys Gin Ile Val Ser Asp Met Asn Asp Giu Glu Lys Val Ile 595 600 605 *Vai Phe.Giu Arg Gly Asp Leu Val -Phe Vai Phe Asn Phe His Pro Lys 610 615 620 Lys Thr Tyr Giu Giy Tyr Lys Va1 Gly Cys Asp Leu Pro Gly Lys Tyr 625 630 635 640 Arg Vai Ala Leu Asp Ser Asp Ala Leu. Val Phe Gly Gly His Giy Arg *645 650 655 I.-4Val Gly His Asp Val Asp His Phe Thr Ser Pro Giu Gly Val Pro Gly 660 665 670 Pro Giu Thr Asn Phe Asn Asn Arg Pro Asn Ser Phe Lys Vai Leu 4 .675 680 685 Pro Pro Arg Thr Cys Val Ala Tyr Tyr Arg Val Asp Giu Ala Gly 690 695 -700 Ala Gly Arg Arg Leu- His Ala Lys Ala Giu Thr Gly Lys Thr Ser Pro 705 -710 715 720 Aia Giu Ser Ile Asp Val Lys Ala'Ser Arg Ala Ser Ser Lys Giu Asp 725 730 735 Lys GlhaAla Thr Ala Giy Giy Lys Lys Gly Trp Lys Phe Ala Arg Gin 740 745 750 Pro-Ser Asp Gin Asp Thr Lys Trp Cys Leu Ile Thr Glu Gin Ala Gly 755 760 -765 Thr Ala Cys Ile Aia Phe Leu Giu 770 -775 <210> 57 <2ii> 2763 <212> DNA 23>Zea mays <400; 57 gjctgtgcctc gtgtcgccct cttcctcgcc gactccgctt ccgccgccgc ggcgctctcg ctcgcatgct-gatcgggcgg caccgc cggg gatcgcgggt ggcggcaatg tgcgcctgag tgtgttgtct gtccagtgca aggctcgccg gtcaggggtg cggaaggtca agagcaaatt cgccactgca gctactgtgc aagaagataa aactatggca -actgccaaag gcgatgtcga 240 ccatctcccc atatacgacc tggaccccaa gctggagata ttcaaggacc atttcaggta 300 103 I. f.

f. v* e

C.

I. C.

ccgga tgaaa atctttttct tcgtgaatgg tggtgcaaac tgtcaaaggg tggagtatgg at ttggagc t taagcatcct gagtggtgaa catacgagca tgcttctttc agaggacctc tgttgtccat acaaagcacc tagtcggctg atattggttg gtatcatcac ggacacagc t cttgccagaa agttgatgaa gattgactac tttgactaac tattgttggc gtcagacttg tcacttcatc tggtcaccca cagacgacag tgaccaagcg cgtcagcgac tgttttcaat tgggaaatac tggccacgac cttcaacaac ttaccgtgta gacgtctcca ggaggcaacg taccaaatga agtcctgctc tgcaggcgac ataataatca cagtttgtat t tc agattcctag aaaggctatt gcacc tgctg cataagatgg aaacc tgcca gttgatcgta ccctatgatg cggccttcaa aagccagcag aataactaca gggtaccatg aaatatcttg agccatgcaa caagagtcct ttcaactatg gatgaattca catggtatca gtggatgcag gcaactgttg ggtggggt tg ctgaagaata aggagatata gacaaaacta cagcctgctt acaatggccc gaatggattg tggagccttg atgaatgcgc atgaacgatg ttccatccca agagtagccc gtggatcact cggccgaac t gacgaagcag gcagagagca gc tgg tggca agccacgagt tactggacta tggtgtctca gggatggatg gtacaggagc agcagaaagg tgaaatttgg cgcaggaggc agaagga taa tccctcacaa ttccagcatt gtgttcattg agcctgctgc taagcacata acacagttca tgacaaattt ttgataaggc gtaataatgt attttcatgc ctaactggga tgtttgatgg atgtggggtt ttgtttacat ttgctgaaga ggtttgacta aagatgactc ctgaaaaatg ttgcatttct cacctacaat ttggaggtga actttccaag tggacac tga tcga tgagag aggaaaaggt agaaaactta tggactctga tcacgtcgcc cgttcaaagt gggctggacg tcgacgtcaa agaaggga tg ccttggtgag gccgccgctg tcaccgagca gatggtgtgt agttcccgtc atcaattgaa gattaataca agagcttatt atttggtgtt ttccaaggtt gattcgttat ggatcctcct tccacgtatc tagggaattt gttgatggca ctttgcggtt acacagtttg cacagatggt gggaga taga ggtattaagg cttccgattc tactggaaac gatgcttgca tgtttcaggc tcgcctggca tgagtggtcg catcgcatat cctgatggac tgatcgaggg tggctacttg agaagggaac tcacttgcgg attttccttc tattgtcttt cgagggctac tgctc tggtc tgaaggggtg cctttctccg acgtcttcac agcttccaga gaagtttgcg gactggactg gcgcccttgg ggcaggcact attggctatc cagaataaaa gaaaatgagg aatgaggatg ggtgacttca tgg-:cgatca aaaz::tcgct gcgactgttg gcc-tctgaaa tacgaagccc gcagacaatg gttatggagc agcagcagat ggt t:gcgag ttaaatggct ggtt-atcata tttcttcttt gatggagtta taccaggaa t aaccatttaa atgccggtcc atggctatcc atgggtgaaa gctgagagcc aaggaaatgt attgcactcc aattttatgg aactggagct tacaagtaca ctttCgtcgt gaacgtggag aaagtgggat ttcggtggac ccaggggtgc ccccgcacct gcgaaagcag gctagtagca cggcagccat gctgccggcg aacggtcctt gcttgtatag tggctagacg aaaaacttgt gaagtcttga gaactgtata atgactggaa aaattgacca ttctacatgg atgcctctaa ggtacacatt atgtaggtat tgttgccacg attcgtacta caggcacacc ttctgatgga a tga tg ttgg aactttggga ctaacctgag catcaatgct atttcagttt tgcacaaact tttgccggcc ctgatagatg tagcgcatac atgatcagtc acactggcat aaaagatgat gaaatgagtt atgataaatg tgaatgcgtt caaagcagat atttagtttt gcgatttgcc a tggaagag t ccgaaacgaa gtgtggctta agacaggaaa aagaagacaa ccga tcaaga ccctgttagt tcctgtagct cttttctaga tgcatgtgcc tggggggttt 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2763

I.

<210> 58 <211> 153 <212> DNA <213> Zea mays <220> <221> CDS <222> <400> 58 atg gcg acg ccc tcg gcc gtg ggc gcc Met Ala Thr Pro Ser Ala Val Gly Ala 1 5 tgc ctc ctc ctc Cys Leu Leu Leu gcg cgg Ala Arg gcc gcc tgg Ala Ala Trp gcc gcc gtc ggc Ala Ala Val Gly cgg gcg cgc ccg Arg Ala Arg Pro cgg cgg ctc Arg Arg Leu agg gag ggg Arg Glu Gly cag cgc gtg ctg cgc cgc cgg tgc gtc gcg gag ctg Gin Arg Val Leu Arg Arg Arg Cys Val Ala Glu Leu ccc cat atg 104 Pro His Met <210> 59 <211> 51 <212> PRT <213> Zea mays <400> 59 Met Ala Thr Pro Ser Ala Val Gly Ala Ala Cys Leu Leu Leu Ala Arg 1 5 10 Ala Ala Trp Pro Ala Ala Val Gly Asp Arg Ala Arg Pro Arg Arg Leu 25 *Gln Arg Val Leu Arg Arg Arg Cys Val Ala Glu Leu Ser Arg Glu Gly 40 Pro His Met so <210> <211> 9 <212> PRT <213> Zea mays o

MODRES

-222> (7) <223> Arg or Asp *<400> 60 (o Cys Val Ala Glu Leu Ser Xaa Leu Gly e. 1 <210> 61 <211> 8 (o <212> PRT 0, <213> Zea mays <400> 61 Gly Glu Asn Val Met Asn Val Ile 1 <210> 62 <211> <212> PRT <213> Zea mnays <400> 62 Cys Val Ala Glu Leu Ser Arg Glu Gly Pro 1 5 <210> 63 <211> 8 <212> PRT <213> Zea mays <400> 63 Ser Ile Val Phe Val Thr Gly Glu 1 105 <210> 64 <211> 8 <212> PRT <213> Zea mays <400> 64 Ala Ser Ala Gly Met Asn Val Val 1 <210> <211> 8 <212> PRT <213> Zea mays <400> Ala Glu Ala Glu Ala Gly Gly Lys 1 <210> 66 <211> 8 <212> PRT <213> Zea mays <400> 66 :Gly Glu Asn Val Met Asn Val Ile 5 <210> 67 <211> 9 <212> PRT <213> Zea mays <400> 67 Gly Ser Val Gly Ala Ala Leu Arg Ser (9 I'9 to (9 9 99,99o 'o <210> 68 <211> 8 <212> PRT <213> Zea mays <400> 68 Ala Ala Ala Pro Ala Gly Glu Glu 1 <210> 69 <211> 8 <212> PRT <213> Zea mays <400> 69 Met Val Val Val Trp Ala Ser Glu 1 <210> <211> 798 <212> PRT <213> Zea mays 106 <400> Met Ala Phe Arg Val Ser Gly Ala Val Leu Gly Gly Ala Val Arg Ala 1 5 10 Pro Arg Leu Thr Gly Gly Gly Glu Gly Ser Leu Val Phe Arg His Thr 25 Giy Leu Phe Leu Thr Arg Gly Ala Arg Val Gly Cys Ser Gly Thr His 40 Gly Ala Met Arg Ala Ala Ala Ala Ala Arg Lys Ala Met Val Pro Glu 55 Giy Glu Asn Asp Gly Leu Ala Ser Arg Ala Asp Ser Ala Gin Phe Gin 70 75 ***Ser Asp Gilu Leu Giu Val Pro Asp Ile Ser Giu Glu Thr Thr Cys Gly 90 Ala Gly Val Ala Asp Ala Gin Ala Leu Asn Arg Val Arg Val Val Pro *100 105 110 Pro Pro Ser Asp Giy Gin Lys Ile Phe Gin Ilie Asp Pro Met Leu Gin ~*115 120 125 Gly Tyr Lys Tyr His Leu Glu Tyr Arg Tyr Ser Leu Tyr Arg Arg Ile 130 135 140 *Arg Ser Asp Ile Asp Giu His Giu Gly Gly Leu Glu Ala Phe Ser Arg *145 150 155 160 Ser Tyr Giu Lys Phe Gly Phe Asn Ala Ser Ala Glu Gly Ile Thr Tyr 165 170 175 4Arg Giu Trp Ala Pro Gly Ala Phe Ser Ala Ala Leu Val Gly Asp Val 180 185 190 Asn Asn Trp Asp Pro Asn Ala Asp Arg Met Ser Lys Asn Giu Phe Gly 195 200 205 *Val Trp Giu Ile Phe Leu Pro Asn Asn Ala Asp Gly Thr Ser Pro Ile 210 215 220 Pro His Gly Ser Arg Val Lys Val Arg Met Asp Thr Pro Ser Gly Ile 225 230 235 240 Lys Asp Ser Ile Pro Ala Trp Ile Lys Tyr Ser Val Gin Ala Pro Gly 245 250 255 Giu Ile Pro Tyr Asp Gly Ile Tyr Tyr Asp Pro Pro Giu Giu Val Lys 260 265 270 Tyr Val Phe Arg His Ala Gin Pro Lys Arg Pro Lys Ser Leu Arg Ile .275 280 285 Tyr Giu Thr His Val Gly Met Ser Ser Pro Giu Pro Lys Ile Asn Thr 290 295 300 Tyr Val Asn Phe Arg Asp Giu Val Leu Pro Arg Ile Lys Lys Leu Gly 305 310 315 320 Tyr Asn Ala Vai Gin Ile Met Ala Ile Gin Glu His Ser Tyr Tyr Gly 325 330 335 Ser Phe Gly Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe 340 345 350 107 9bb* S C S. S

C.

S. S C S~SS 5 9* C

S

S. S C C

C.

4 0 go** Gly Gly Thr 385 His Asn Trp Thr Asn 465 Tyr Val Val Ala Met 545 Cys Thr Leu Lys Asn 625 Arg Asn Tyr Leu Pro Giu 355 Leu Val Asp Gly Gly Pro Gly Asn 420 Leu Giu 435 Met Met Asn Giu Met Leu Ile Gly 500 Asp Gly 515 Lys Trp Asp Ile Thr Tyr Ala Phe 580 Arg Pro 595 Ile Arg Met Gly Pro Gin Tyr Asp 660 Arg Tyr 675 Gin Lys Leu Met Asn 390 Giy Giu Tyr Thr Phe 470 Asn Asp Val1 Asp His 550 Giu Leu Thr Ile Giu 630 Leu Cys Gly Glu Arg Ala His 365 His Ala Ser 380 Asp Thr His Asp Ser Arg Leu Ser Asn 430 Arg Phe Asp 445 Val Thr Phe 460 Val Asp Ala Leu Tyr Pro Thr Phe Ala 510 Met His Met 525 Asp Glu Thr 540 Arg Trp Leu Leu Val Gly Tyr Asp Phe 590 Gly Ile Ala 605 Gly Glu Gly 620 Trp Ile Asp Ile Pro Gly Leu Gly Asp 670 Gin Ala Met 685 His Gin Tyr 700 690 695 Arg Lys His Giu Giu Asp LYS Val Ile Val Phe Glu Lys Gly Asp Leu 108 705 Val Phe Val Phe Asn 725 Ile Gly Cys Arg Lys 740 Ala Gly Leu Phe Gly 755 Phe Thr Ala Asp Cys 770 Tyr Thr Pro Ser Arg 785 710 Phe His Cys Asn Asn 730 Pro Giy Vai Tyr Lys 745 Gly Phe Ser Arg Ile 760 Ser His Asp Asn Arg 775 Thr Cys Val Vai Tyr 790 715 720 Ser Tyr Phe Asp Tyr Arg 735 Val Val Leu Asp Ser Asp 750 His His Ala Ala Glu His 765 Pro Tyr Ser Phe Ser Val 780 Ala Pro Val Glu 795 0000 00 *0 0 00 00 0 0 000000 0 0~ @0 0 0 0 0* S 00 00 00 5 00 0.

0000 0 0000 00 @0 0 0 0 .000 0 0000 00 0 0 00 0000 0 000.

<210> 71 <211> 14 <212> PRT <213> Zea mays <400> 71 Arg Gly Thr Arg Cys Cys Ala Ala Cys Val Gly Leu Ser Met 1 5 <210> 72 <211> 16 <212> PRT <213> Zea mays <400> 72 Gly Lys Thr Phe Phe Gin Asn Arg Gin Met His Aia Cys Met Leu Gin 1 5 10 <210> 73 <211> 22 <212> PRT <213> Zea mays <400> 73 Gly Ser Asp Thr Leu Ile Asp Ala Gly Lys Pro Met His Leu Ala Ala 1 5 10 Leu Ser Ser Leu Phe Ile <210> 74 <211> 6 <212> PRT <213> Zea mays <400> 74 Asp Leu Gin Gly Val Asn 1 <210> 109 <211> 13 <212> PRT <213> Zea mays <400> Ser Phe Arg Phe Ser Leu Lys Lys Lys Lys Lys Lys Leu 1 5 <210> 76 <211> 431 <212> PRT <213> E. coli <223> glgC3 <400> 76 Met Val Ser Leu Glu Lys Asn Asp His Leu Met Leu Ala Arg Gin Leu 10O Pro Leu Lys Ser Val Ala Leu Ile Leu Ala Gly Gly Arg Gly Thr Arg *20 25 Leu Lys Asp Leu Thr Asn Lys Arg Ala Lys Pro Ala Val His Phe Gly 40 Gly Lys Phe Arg Ile Ile Asp Phe Ala Leu Ser Asn Cys Ile Asn Ser 55 Gly Ile Arg Arg Met Gly Val Ile Thr Gin Tyr Gin Ser His Thr Leu 70 75 Val Gin His Ile Gin Arg Gly Trp Ser Phe Phe Asn Glu Giu Met Asn 90 Glu Phe Val Asp Leu Leu Pro Ala Gin Gin Arg Met Lys Gly Giu Asn 100 105 110 *Trp Tyr Arg Gly Thr Ala Asp Ala Val Thr Gin Asn Leu Asp Ile Ile 115 120 125 Arg Arg Tyr Lys Ala Glu Tyr Val Vai Ile Leu Ala Gly Asp His Ile 130 135 140 Tyr Lys Gin Asp Tyr Ser Arg Met Leu Ile Asp His Val Giu Lys Gly 145 150 155 160 Val Arg Cys Thr Val Val Cys Met Pro Val Pro Ile Giu Giu Ala Ser 165 170 175 Ala Phe Gly Val Met Ala Val Asp Giu Asn Asp Lys Thr Ile Giu Phe 180 185 190 Val Giu Lys Pro Ala Asn Pro Pro Ser Met Pro Asn Asp Pro Ser Lys 195 200 205 Ser Leu Ala Ser Met Gly Ile Tyr Val Phe Asp Ala Asp Tyr Leu Tyr 210 215 220 Glu Leu Leu Giu Giu Asp Asp Arg Asp Giu Asn Ser Ser His Asp Phe 225 230 235 240 Gly Lys Asp Leu Ile Pro Lys Ile Thr Giu Ala Giy Leu Ala Tyr Ala 245 250 255 His Pro Phe Pro Leu Ser Cys Val Gln Ser Asp Pro Asp Ala Giu Pro 260 265 270 110 Tyr Trp Arg Asp Val Gly Thr Leu Giu Ala Tyr Trp Lys Ala Asn Leu 275 280 285 Asp Leu Ala Ser Val Val Asp Lys Leu Asp Met Tyr Asp Arg Asn Trp 290 295 300 Pro Ile Arg Thr Tyr Asn Giu Ser Leu Pro Pro Ala Lys Phe Val Gin 305 310 315 320 Asp Arg Ser Gly Ser His Gly Met Thr Leu Asn Ser Leu Val Ser Giy 325 330 335 Giy Cys Val Ile Ser Gly Ser Val Val Val Gin Ser Val Leu Phe Ser 340 345 350 S.*Arg Val Arg Vai Asn Ser Phe Cys Asn Ile Asp Ser Ala Val Leu Leu 355 360 365 Pro Giu Val Trp Val Giy Arg Ser Cys Arg Leu Arg Arg Cys Val Ile 370 375 380 S.Asp Arg Ala Cys Val Ile Pro Giu Gly Met Val Ile Gly Giu Asn Ala *385 390 395 400 Glu Giu Asp Ala Arg Arg Phe Tyr Arg Ser Giu Glu Gly Ile Val Leu 405 410 415 SVai Thr Arg Giu Met Leu Arg Lys Leu Gly His Lys Gin Giu Arg 420 425 430 *<210> 77 <211> <212> PRT <213> E. coli <223> gigC3 *<400> 77 Cys Arg Phe Tyr Met Tyr Val Gin Arg Cys 1 5

Claims (7)

1. A plant transformed with a nucleic acid encoding a mutant E. coli ADP glucose pyrophosphorylase having an amino acid sequence as set forth in SEQ ID NO: 76.

2. The plant of claim 1, wherein the plant is a dicot.

3. The plant of claim 2, wherein the dicot is selected from the group consisting of potato, sweet potato, taro, yam, lotus, cassava, peanut, pea, soybean, bean and chickpea.

4. The plant of claim 3, wherein the dicot is soybean.

The plant of claim 1, wherein the plant is a monocot. 0* S*

6. The plant of claim 5, wherein the monocot is selected from the group consisting-of rice, 10 corn, wheat, barley, oats, sorghum and milo.

7. The plant of claim 6, wherein the monocot is corn. S8. AAn isolated nucleic acid encoding a mutant E. coli ADP glucose pyrophosphorylase aving an amino acid sequence as set forth in SEQ ID NO: 76. Exseed Genetics, LLC 15 By their patent attorneys CULLEN CO. Date: 13-MaylO August 2005 COMS ID No: SBMI-01411146 Received by IP Australia: Time 14:32 Date 2005-08-10