MXPA00004586A - I(dull1) coding for a starch synthase and uses thereof - Google Patents

Abstract

The maize gene i(dull1) (i(du1)) of the present invention is a determinant of the structure of endosperm starch. Mutations of i(du1) affect the activity of at least two enzymes involved in starch biosynthesis, namely the starch synthase, SSII, and the starch branching enzyme, SBEIIa. i(Du1) codes for a predicted 1674 residue protein, and is expressed with a unique temporal pattern in endosperm but is undetectable in leaf or root. The size of the i(Du1) product and its expression pattern match precisely the known characteristics of maize SSII. The i(Du1) product contains two different repeated regions in its unique amino terminus, one of which is identical to a conserved segment of the starch debranching enzymes. The cDNA provided for in the present invention encodes SSII, and mutations within this gene affect multiple

Description

D lll CODIFICATION FOR A SYNTHETIC STARCH AND THE USES OF THE SAME BACKGROUND OF THE INVENTION Reference to the related application This application claims the benefit of the series of the United States No. 08 / 968,542, filed on November 12, 1997.

Legend of federal funding This invention was produced in part using funds in accordance with USDA grant number 96-35300-3779. Consequently, the federal government has certain rights in this invention.

FIELD OF THE INVENTION The present invention relates, in general, to the biochemistry of carbohydrates. More specifically, the invention relates to the biosynthesis of starch and the enzyme (s) involved.

Description of the Related Art Starch, the most important carbohydrate reserve in vegetable storage tissues comprises the amylose glucose and amylopectin homopolymers. Amylose consists mainly of linear chains of glucose residues with a- (1-4) bonds, whereas amylopectin is a highly branched glucan with a specific "clustered" distribution of α- (1-6) -glucoside bonds (ie say, branched links) connecting linear chains (French, 1984; Manners, 1989). Despite the relatively simple chemical structure of amylopectin, very little is known about the enzymatic processes responsible for the formation of highly specific and complex branching patterns in this polysaccharide. The biosynthesis of amylose and amylopectin involves activities of four groups of enzymes, each of which comprises multiple isozymes. These enzymes are ADPG pyrophosphorylases (AGPase), starch ribbons (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE) (Preiss, 1991, Hannah et al., 1993, Martin and Smith, 1995; Pan, 1995; Ball et al., 1996; Preiss and Sivak, 1996; Smith et al., 1996). These enzymatic steps can explain all the chemical bonds in the starch, however, the specific functions of the individual isozymes in the formation of specific branching patterns in amylopectin and the determination of the structure and properties of the starch granules remain unknown . The analysis of maize mutants with endosper or abnormal phenotypes has contributed greatly to the understanding of starch synthesis (Shannon and Garwood, 1984, Nelson and Pan, 1995) and facilitated the identification of multiple genes coding for the Biosynthetic enzymes of starch. The cloned genes whose products are considered to be directly included in starch biosynthesis are waxy (wx), which codes for the granule-bound starch synthase (GBSSI (Shure et al., 1983; Klósgen et al., 19'86), amylose extender (ae), which codes for SBEIIb (Fisher et al., 1993; Stinard et al., 1993), shrunken2 (sh2) and bri ttle2 (bt2), which code for the large and small subunits of AGPase, respectively ( Bae et al., 1990; Bhave et al., 1990), and sugaryl (sul), which codes for SDBE SU1 (James et al., 1995) .The strategy of labeling the transposon was used to determine that the phenotype of the Abnormal endosperm of the wx, ae or sul mutants result from primary defects in GBSSI, SBEIIb or SU1, respectively, and this approach remains the most efficient way to identify genes such as dulll (dul), in which the primary defect can not be associated with a specific enzyme deficiency Mutations (dul) define a gene with a very important function in the synthesis of starch, as indicated by the extensive structural analysis of starch from the mutant endosperms dul, and by the effects of these mutations when combined with other genetic deficiencies in the biosynthetic enzymes of starch (Shannon and Garwood, 1984; Nelson and Pan, 1995). The reference mutation dul-jRef "was first identified as a recessive modifier of sul-Ref" and sul-amylaceous (sul-am) (Mangelsdorf, 1947). Mutations of dul, when they are homozygous in non-mutant backgrounds, give rise to mature seeds with a dull, vitreous and somewhat opaque appearance known as the "dull phenotype". However, the expression of this phenotype depends on the particular genetic background (Mangelsdorf, 1947, Davis et al., 1955). The total content of carbohydrates and starch in sweet-ripe mutant seeds is slightly lower than normal (Creech, 1965; Creech and McArdle, 1966). The apparent amylose content in starch of mutant dul is slightly or very high compared to the normal depending on the genetic background (Shannon and Garwood, 1984), although the properties of the polysaccharides in the apparent amylose fraction are practically unchanged (Dvonch et al., 1951). Approximately 15% of the starch in mutant endosperms dul is in the form known as "intermediate material", which is distinguished from amylose and amylopectin by the properties of its complex starch-iodine (Ang et al., 1993b). Analysis of the combined amylopectin / intermediary material fractions indicates that the starch of the mutant dul has the highest degree of branching among a wide variety of normal and mutant seeds tested (Inouchi et al., 1987; Ang et al., 1993a Wang et al., 1993b). The starch granules from the dul mutants appear to have normal physical structure and properties, although some abnormal granules are found in the mutant endosperm (Shannon and Garwood, 1984). Despite these subtle effects exerted by the single mutation, the alleles of dul when combined with other mutations that affect the synthesis of starch give rise to a wide range of more severe alterations (Shannon and Garwood, 1984; Nelson and Pan, 1995 ). Mutations of dul have been examined in combination with mutations wx, ae, sul and sugary2 (su2), and in all cases the double mutant seeds contained more soluble sugars and less total starch than when any of the mutations were present alone. In many cases the double mutations also produce polysaccharide forms that are different from the starch found in any of the individual mutant seeds. These pleiotropic effects indicate that Dul's product affects many aspects of starch biosynthesis in corn endosperm, however, without knowing the identity of this protein, it is difficult to assess its specific functions.

Consistent with pleiotropic gene effects, dul mutations cause reduced activity in the endosperm of two apparently unrelated biosynthetic starch enzymes, SSII starch synthase and the branching enzyme SBEIIa (Boyer and Preiss, 1981). SSII is one of the two activities of enzymatically distinct starch synthase identified in the soluble fraction of the maize endosperm; the in vitro activity of SSII requires an exogenous glucan initiator, and its molecular weight was determined in different studies as 95 kD or 180 kD (Boyer and Preiss, 1981; Mu et al., 1994). In the same way, SBEIIa is one of the three known SBE isozymes in endosperm cells (Boyer and Preiss, 1978b, Fisher et al., 1993, Fisher et al., 1995, Gao et al., 1997). There are some possibilities to explain the double biochemical effects of dul mutations. Dul can code for a protein that regulates the expression or activity of both SSII and SBEIIa. Otherwise, dul may code for either of these two enzymes, and deficiency in one enzyme may also affect the second enzyme due to a direct physical interaction or mediated by the substrate. DU1 encoded for a starch synthase, as indicated by the great similarity of its amino acid sequence deduced for potato SSIII, and by the substantial similarity between the C terminal residues of DUl and a large group of phylogenetically diverse starch and glycogen synthases. Particularly surprising are the two regions that together comprise more than half of the DUl sequence deduced from 1674 residues, which share very high similarity of 51% and 73%, respectively, with the corresponding regions of the potato SSIII sequence. Within an extension of 450 amino acids in the C terminal of DUl, almost 30% of the best aligned residues are identical in comparisons to a wide variety of starch and glycogen synthases, suggesting the location of a domain within DUI that provides activity of -1, 4-glucosyltransferase. The starch synthase encoded by DUl is the soluble isozyme identified biochemically as SSII (Ozbun et al., 1971; Boyer and Preiss, 1981). The deduced molecular weight of DUl including a potential transient peptide, 188 kD, coincides closely with that of 180 kD reported for mature SSII lacking a transient peptide (Mu et al., 1994). The difference in size of approximately 8 kD may be due to the transient peptide present in the deduced DUl sequence. The specific tissue expression pattern of Dul mRNA also coincides with the expression pattern of SSII. The Dul transcripts were not detectable in sheets by gel absorption analysis of RNA or RT-PCR analysis, corresponding to this fact, no detectable SSII activity was present in leaf extracts (Dang and Boyer, 1988). In addition, the activity of SSII, together with that of SBEIIa, was greatly reduced in the endosperm of mutant dul (Boyer and Preiss, 1981). Therefore, it appears that the sweet locus of corn encodes the soluble SSII starch synthase, the counterpart of potato SSIII. This characterization of DUl implies that the phenotypic effects of dul mutations, including changes in starch structure, deficiencies of two starch biosynthetic enzymes and genetic interactions with mutations ae, sul r su2 and wx, all result directly or indirectly from the alteration of SSII. The reduction of SBEIIa activity in the endosperm of the mutant dul may result from the deficiency of SSII due to the physical interaction between the two enzymes. A direct physical association of SSII and SBEIIa is indicated by the observation that the peak activities of SSII and SBEIIa always coincide in the same fractions in the DEAE-cellulose column (Boyer and Preiss, 1978a, Boyer and Preiss, 1981; Boyer, 1988). Thus, SSII and SBEIIa can function together in vivo in the form of a single multienzyme complex. The loss of the intact enzyme complex due to the reduction of SSII in the mutant dul endosperm may give rise to normally rapid proteolytic production of SBEIIa, or prevent the accumulation of the enzyme by some other mechanism. Otherwise, the expression of the Sbe2a gene in the dul mutant endosperm can be inhibited as a more indirect consequence of the SSII deficiency, for example through the reduction of a transcription inducer or elevation of a repressor. Although the dul -Ref mutation indirectly affects the expression of other starch biosynthetic genes (Giroux et al., 1994), it actually causes an increase in gene expression more than the reduction observed for SBEIIa. Furthermore, considering that large glucose polymers are expected to be the substrate and product of DUI, inactivation of Sbe2b expression by a transcription mechanism seems unlikely. Thus, the above hypothesis may explain the deficiency of SBEIIa in the endosperm of the mutant dul. The broad impact of the combination of dul mutations with different sul alleles on seed phenotype and starch synthesis (Cameron, 1947; Shannon and Garwood, 1984) can be explained by SU1 SDBE also interacting closely with SSII in live, perhaps in the same enzyme complex with SBEIIa. This proposed association of SBEIIa and SU1 in a multi-enzymatic complex is consistent with the simultaneous branching and debranching actions proposed during the synthesis of amylopectin by SBE and SDBE (James et al., 1995, Nelson and Pan; Ball et al., nineteen ninety six).

Thus, the prior art is deficient in the understanding of the complex association of the enzymes involved in the synthesis of starch and in the cloning genes corresponding to these enzymes. The present invention meets this need and desire in the art.

COMPENDIUM OF THE INVENTION To illustrate the function of the dul locus in starch biosynthesis, a strategy was used labeling the transposon to isolate the gene and describe its polypeptide product. The present invention reports the labeling of the dul locus with a Mutator transposon (Mu), the cloning and characterization of a portion of the gene, and the sequence of an almost complete length cDNA (SEQ ID NO: 1). The amino acid sequence deduced from this cDNA indicates that Dul codes for a 186 kD polypeptide extremely similar to SSIII, a potato tubular starch synthase (Abel et al., 1996; Marshall et al., 1996). Dul's expression pattern was also characterized. Taken together, these characterizations indicate that Dul most likely codes for SSII from the maize endosperm. In addition, the Dul product contains unique sequence features in its amino terminus that can mediate direct interactions with other biosynthetic enzymes of starch.

An object of the present invention is to provide an enzyme with which to regulate the production of starch and with which to produce altered and novel forms of starch. In one embodiment of the present invention, a cDNA corresponding to the maize dulll gene is provided. In yet another embodiment of the present invention, there is provided an expression vector containing the dulll sequence with which to produce the enzyme starch synthase in transgenic plants or other prokaryotic or eukaryotic organisms. In still another embodiment of the present invention, there is provided: (1) cDNA having the nucleotide sequence comprising nt 120 to nt 1221 of SEQ ID NO: 1, the coding sequence of the first 368 amino acids of DUl; (2) cDNA having the nucleotide sequence comprising nt 655 to nt 1221 of SEQ ID NO: 1, the sequence coding for amino acids 180 to 168 of DUl; (3) cDNA having the nucleotide sequence comprising nt 565 to nt 816 of SEQ ID NO: 1, said sequence encoding amino acids 150 to 233 of DUl; (4) the cDNA having the nucleotide sequence comprising nt 1369 to nt 1944 of SEQ ID NO: 1, the sequence encoding amino acids 418 to 609 of DUl; (5) the cDNA having the nucleotide sequence comprising nt 1 to nt 1437 of SEQ ID NO: 1, the sequence encoding amino acids 1 to 440 of DUl; (6) the cDNA having the nucleotide sequence comprising nt 1438 to nt 2424 of SEQ ID NO: 1, the sequence encoding amino acids 441 to 769 of DUl; (7) the cDNA having the nucleotide sequence comprising nt 2425 to nt 3791 of SEQ ID NO: 1, the sequence encoding amino acids 769 to 1225 of DUl. Other aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These modalities are provided for the purpose of description.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings have been included herein so that the aforementioned characteristics, advantages and objectives of the invention are clear and can be understood in detail. These drawings are part of the specification. It should be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered as limiting the scope thereof. Figure 1 shows the isolation of mutations of dul.

Figure 1A shows the crossing scheme. The specific lines of corn used in this procedure are mentioned below. The designation of the dul allele -M "indicates a putative recessive mutation at the dul locus caused by the insertion of a Mu transposon. Figure IB shows the phenotype of the dull mutant.The cob shown was obtained by self-pollination of a heterozygous -R2370: zMul / Dul. Dull seeds and wild type seeds are present at approximately Mendelian frequency of 1: 3, respectively. Figure 2 shows co-segregated BamHI genomic DNA fragment containing Mal with dul -R2370:: Mul. Figure 2A shows the detection of genomic DNA fragments containing MuI. The genomic DNA digested with BamHI from the seedlings grown from the non-mutant segregation (1: 1) and the sister seeds dull was separated on a 1% agarose gel, absorbed and probed with the internal Mlul fragment of 960 bp of Muí cut from the pásmid pMJ9 (Barker et al., 1984). Figure 2 demonstrates the structure of the cloned 2.0 kb BamHI fragment. The striped bars indicate the position of Mui as revealed by the nucleotide sequence of the cloned fragment. The position of the 500 bp F500 probe fragment is indicated, and the figure is drawn to scale. The restriction sites are indicated for BamHI (B) and Notl (N). Figure 2C demonstrates detection in genomic DNA of restriction fragments homologous to the cloned fragment. The analysis is the same as that shown in Figure 2A, except that the band was hybridized with an on-chain probe generated by PCR using the F500 fragment shown in Figure 2B, as the template. Figure 3B shows the isolation of a cDNA clone dul of almost complete length. Figure 3A shows the identification of the genomic fragments containing flanking regions of the MuI element in the cloned 2.0 kb BamHI fragment. The genomic DNA digested with EcoRI and Xbal from sweet mutants -R2370:: Mul / dul -Ref and sister non-mutant seedlings dul / dul -Ref was probed with F500 fragment. Figure 3B shows an illustration of the procedure for cloning Dul's cDNA of almost complete length. The BE1300 genomic fragment was cloned by PCR with nested primer as detailed below. The wild-type counterpart of the original cloned BamHI fragment (indicated by crossed frames with dashes) was shown to be part of an EcoRI fragment of 6.0 kb in Figure 3A. A population of EcoRI genomic fragments of approximately 6.0 kb was ligated to pBluescript SK + (dotted lines). The ligation mixture was used to amplify a 2.0 kb fragment by the dul-spl and T3 primers. The BE1300 fragment was then amplified from the 2.0 kb fragment by the dul-sp4 and T3 primers. The position of Mui insertion in dul -R2370:: Mul is indicated by the asterisk. The positions of the PCR primers used for amplification of the fragment are indicated. The restriction sites are indicated for EcoRI (E) and BamHI (b). The nearly complete length cDNA diagram represents the continuous sequence from the three superimposed cDNA fragments. The solid arrow indicates the location and the 5 '-3' direction of the coding sequence of dul. The partial intron, exon structure was deduced by comparing the available genomic sequence with the cDNA sequence. Figure 4 shows the physical alteration of the locus cloned in plants carrying dul-R2649. The genomic DNA digested with Sali from the seedlings grown from the mutant dul-R2649 / dul-Ref and the non-mutant sister seeds Dul / dul-Ref was absorbed and probed with cDNA insert from pMgflO. Figure 5 shows the Dul gene that has a unique expression pattern. Figure 5A shows the gel absorption analysis of RNA from the total RNAs of the developing endosperm. Total RNAs extracted from the endosperm of W64A seeds harvested at different stages of development and from dul-JRef and mutant seeds dul-R2370:: Mul harvested at 20 DDP, were fractionated on a formaldehyde-agarose gel, absorbed and probed by the cDNA insert in pMgdAa. The minor charge differences were calibrated by hybridization of the 26S rRNA in the same absorption, separated from the cDNA probe to a cDNA probe from tomato rRNA. The size of the transcript was calculated using an RNA size standard (GibcoBRL). Figure 5B shows the level of the relative steady state of the Dul transcript in developing endosperm. The radioactivity of the hybridized transcripts for the Dul cDNA probe was analyzed using Phosphorimager, quantified using the ImageQuant program and expressed as the percentage of the maximum signal intensity over the same range (relative level) after calibration of smaller load differences. The data represents the average of three replicates of the analysis with standard error less than 10%. Figure 5C shows the RT-PCR analysis. The DNA fragments amplified from the total RNAs by RT-PCR using primers dul-F3 and dul-RI were separated on an agarose gel and visualized by staining with ethidium bromide. RNA endosperm (En) and embryo (Em) were tissue collected 22 DDP. The strip designated "-control" is from the same sample as the strip In, except that the RNA was pre-treated with RNAase A before amplification. The RNAs of the dul mutants indicated were obtained from the endosperm collected 22 DDP. Figure 6 shows the amino acid sequence of DUl is more similar to that of potato SSIII. Figure 6A shows the alignment of the primary sequence. The amino acid sequences deduced from dul and SSIII from potato (GenBank accession number X95759) are aligned. The solid directional arrows indicate the positions of the three SBE superrepetitions of 60 amino acids, and the dotted arrows denote individual copies of the SBE repeat. The dashed arrows indicate the positions of the three repeat units that constitute the repetition of 85 residues. The two-way arrows marked with Roman numerals indicate the positions of the blocks of the correspondingly designated conserved sequences, identified in the glucan synthase family (Preiss and Sivak, 1996). Figure 6B shows DUI domains. Similarity in the records between each segment of DUl and SSIII are shown under each region. The "catalytic domain" indicates the DUI region similar in amino acid sequence to the a- (1- »4) -glucosyltransferases in general. "SSIII / DU1 homology domain" indicates the region specifically shared by DUl and SSIII among known proteins. "Specific region DUI" indicates the portion of DUI that is unique in the sequence of amino acids between known proteins. Figure 7 shows the repeats in the single amino terminal DUl. Figure 7A shows the alignment of the SBE superrepetitions. The numbers refer to the positions of the residues within the coding sequence of the DUI. Each SBE superrepetition of 60 residues comprises six copies of the SBE repeat unit of 10 amino acids (indicated by the arrows). The degree of sequence conservation between each SBE repeat descends towards the C terminal of each of the SBE repeats. Figure 7B shows the alignment of the selected copies of the SBE repeat and the conservation of the M box within the branching enzymes. In the first grouping the numbers refer to the position within the coding sequence of the DUI. The residues within the boxes are identical to the consensus sequence of the SBE repeat. The arrows indicate the sequence of the box M (DQSIVG). The sequence of the M box is almost completely conserved in the members of the SBEI family, including the SBEI of corn (GenBank access No. D11081), SBEII of pea (GenBank access No. X80010), SBEI of wheat (GenBank access No. Y12320). The sequence of the M box is also well preserved, with the substitution of two residues of similar properties, in the members of the SBEII family and glycogen synthases, including corn SBEIIa (Gao et al., 1997), corn SBEIIb (GenBank). access No. L08065), pea SBEI (GenBank access No. X80009), human liver glycogen synthase (GenBank access No. D29685) and glycogen synthases from S. cerevisiae (the GLC3 product; GenBank access No. M76739). The numbers of the residues refer to the first enzyme in each group. The arrows indicate the presence of the sequences of the M box or related sequences. The asterisks indicate conserved residues that in the amylolytic enzymes of determined structure are known as part of the active site. Figure 7C shows the conservation of the repeat sequence of 28 amino acids. The three repeats within the region of 85 residue repeats were best aligned to show the pattern of sequence conservation between the two portions of the basic repeat unit of 28 residues. The numbers refer to the positions within the coding sequence of DUl. Figure 8 shows the expression of DU1C in E. coli. The gene expression of the T7 promoter of the indicated pásmid was induced in the E. coli cells in the exponential phase. The total soluble used were fractionated by SDS-PAGE and the specific proteins containing the S-tag sequence (specified by the pET-pásmid) were detected by the S-protein AP conjugate. Strip 1: pET-32b; lane 2: pHC6 (DU1C in pET-32b); lane 3: pET-29b; Strip 4: pHC5 (DU1C in pET-29b). The asterisks indicate polypeptides of approximately the predicted size from the pirasmid and the Dul cDNA sequence, which are present only when the coding region of DU1C is contained within the pyramid. Figure 9 shows the immunological detection of DUl and SSI in seed extracts. Figure 9A: total soluble extracts from 20 DDP seeds of homozygote with genetic background W64A for the indicated allele were fractionated by SDS-PAGE and probed with anti-DUIN or anti-SSI. In each strip an equal amount of protein was loaded. "dul-? f5" indicates the dul allele -R4059. The asterisk indicates full-length DUl. Figure 9B: Extracts of non-mutant W64A and 20 DDP seeds collected from congenic dul-Ref mutant seeds were separated into pellets (ie, 10000 x g package) and total soluble fractions (ie, 10000 x g supernatant). Equal volumes of each fraction were separated by SDS-PAGE, so that each pair of strips was standardized to the fresh weight of the seed. Samples were probed with anti-DUIN or anti-SSI, as indicated. Figure 9C: Total soluble extracts of W64A seeds grown at different times after pollination, as indicated, were analyzed by SDS-PAGE and immunosorbent analysis using anti-DUIN or anti-SSI. Figure 10 shows immunoagglutination of SS activity. Total soluble extracts of seeds collected from the genotype indicated 20 DDP were treated with preimmune serum, or saturating amounts of the indicated antiserum, and the residual activity of the SS was assayed after removal of the immune complexes. The dul-Ref mutant was in the genetic background W64A. The SS activity that remained after treatment with preimmune serum was defined as 100%. These values were 7.0 nmol min -1 mg-1 for W64A, 12.9 nmol mm-1 mg-1 for the mutant dul-.Ref ", and 16.4 nmol min" mg ~ for Oh43. Figure 11 shows the specific identification of SS isozymes. Figure HA: zymogram of SS activity. Proteins in total soluble endosperm extracts were separated based on molecular weight by SDS-PAGE and then allowed to renature in the gel. The SS substrates were provided throughout the gel, and the positions of the glucan synthesis were detected by iodine staining. Two congenital stains in the genetic background W64A were analyzed, one carrying the non-mutant allele Dul and the other containing dul -Ref (indicated as vdul-). Two SS activities are evident in the non-mutant endosperm, one of which is Absent from the Dul-Ref extract Figure 11B: Immunosorption analysis Proteins in duplicates of the gene shown in panel A were transferred to nitrocellulose paper and probed with the indicated antiserum A polypeptide of the same mobility and genetic specificity as the activity of the larger SS is recognized by anti-DUIN, while a protein of the same mobility as the activity of the smaller SS is recognized by anti-SSI.

Figure 12 shows the SS activity in total soluble seed extracts. The total soluble extracts of seeds of the genotype indicated 20 DDP collected were tested for SS activity in the presence or absence of the exogenous initiator (10 mg / ml of glycogen) and 0.5 M of citrate, as indicated. The dul-Ref mutant was in the genetic background W64A.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, techniques of molecular biology, microbiology and traditional recombinant DNA can be employed within the skills in the art. These techniques are explained more extensively in the literature. See, for example, Maniatis, Fritsh & Sambrook, "Molecular Cloning: A Laboratory Manual (1982);" DNA Cloning: A Practical Approach "vol. I and II (DN Glover ed., 1985);" Oligonucleotide Synthesis "(MJ Gait ed., 1984);" Nucleic Acid Hybridization "[BD Hames &SJ Higgins eds. (1985)];" Transcription and Translation "[BD Hames &SJ Higgins eds. (1984)];" Animal Cell Culture "[RI Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)], B. Perbal, "A Practical Guide to Molecular Cloning" (1984) .Therefore, if they appear in the present, the following terms must have the definitions that are established later.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) in its single-stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule and does not limit it to any of the particular tertiary forms. Thus, this term includes double-stranded DNA that is found, inter alia, in linear DNA molecules (eg, restriction fragments), viruses, plasmids and chromosomes. In the description the structure in the present in accordance with the normal convention of giving only the sequence in the 5 'direction along the strand of non-transcribed DNA (ie, the strand having a sequence homologous to the mRNA). A "vector" is a replicon, such as a piásmido, phage or cosmid, to which another DNA segment can be linked to carry out the replication of the attached segment. A "replicon" is any genetic element (eg, pyramid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; that is, capable of replication under its own control. An "origin of replication" refers to those DNA sequences that participate in the synthesis of DNA. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "operably linked" and "under control" of transcriptional and translational control sequences in a cell when the RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. In general, expression vectors containing the promoter sequences that facilitate efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector usually contains an origin of replication, promoter (s), terminator (s), as well as specific genes that are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to the means known in the art to obtain optimal cell growth. A "coding sequence" of DNA is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of the appropriate regulatory sequences. The limits of the coding sequence are determined by an initiation codon at the 5 '(amino) terminal and a translation antisense codon at the 3' (carboxyl) terminus. A coding sequence may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) MK, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3 'for the coding sequence. A "cDNA" is defined as a DNA copy or complementary DNA and is a product of a reverse transcription reaction of an mRNA transcript. The transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators and the like, which are responsible for the expression of a coding sequence in a host cell. A "cis element" is a sequence of nucleotides, also called a "consensus sequence" or "motif", that interacts with other proteins that can activate or deactivate the expression of a specific gene locus. A "signal sequence" can also be included with the coding sequence. This sequence encodes a N-terminal signal peptide for the polypeptide, which communicates with the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating the transcription of a coding sequence downstream (3 'direction). For purposes of defining the present invention, the promoter sequence is "bound at its 3 'terminus by the transcription initiation site and extended upstream (5' direction) to include the minimum number of bases or elements necessary to initiate the Transcription at detectable levels above the background Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. but not always, "TATA" boxes and "CAT" boxes The prokaryotic promoters contain Shine-Dalgarno sequences in addition to consensus sequences -10 and -35 The term "oligonucleotide" is defined as a molecule composed of two or more deoxyribonucleotides, preferably more than three, its exact size will depend on multiple factors that, in turn, will depend on the function and final use of the the oligonucleotide The term "initiator" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digestion or synthetically produced, that is capable of acting as a point of initiation of the synthesis when it is placed under conditions in which the synthesis of an extension product of the primer is induced, which is complementary to a strand of nucleic acid, that is, in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The initiator can be single-stranded or double-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the initiator will depend on multiple factors, including temperature, the source of the initiator and the method used. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer usually contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the sequence of the initiator does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached at the 5 'end of the primer, with the remainder of the primer sequence being complementary to the strand. Otherwise, non-complementary bases or longer sequences may be interposed in the primer, provided that the sequence of the primer has sufficient complementarity with the sequence or hybridizes thereto and thereby forms the template for the synthesis of the extension. As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to enzymes that cut double-stranded DNA at or near a specific nucleotide sequence. A cell has been "transformed" or "transfected" with exogenous or heterologous DNA when this DNA has been introduced into the cell. The transforming DNA may or may not be integrated (covalently linked) in the cell genome. In prokaryotic, yeast and mammalian cells, for example, the transforming DNA can be maintained in an episomal element such as a vector or piásmido. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA is integrated into a chromosome so that it is inherited by the daughter cells through the replication of the chromosome. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones composed of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of growing stably in vi tro over multiple generations. An organism, such as a vegetable or animal that has been transformed with exogenous DNA is called "transgenic". As used herein, the term "host" means that it includes not only prokaryotes but also eukaryotes such as' yeast, plant and animal cells. A recombinant DNA molecule or gene encoding a corn starch synthase enzyme of the present invention can be used to transform a host using any of the techniques commonly known to those skilled in the art. A preferred embodiment is the use of vectors containing coding sequences for the gene encoding a corn starch synthase enzyme of the present invention for the purposes of prokaryotic transformation. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells and, more preferably, plant cells such as Arabidopsis thaliana and Tobaccum nicotiana. Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%), and more preferably at least about 90% or 95%) of the nucleotides correspond to the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using the standard software available in sequence data banks, or in a low Southern hybridization experiment, for example, stringent conditions as defined by this specific system. The definition of suitable hybridization conditions is within the skill of the artisan. See, for example, Maniatis et al., Supra; DNA Cloning, vols. I & II, supra; Nucleic Acid Hybritization, supra. A "heterologous" region of DNA construction is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the largest molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, the coding sequence is a construct where the coding sequence itself is not found in nature (eg, a cDNA where the genomic coding sequence contains introns, or synthetic sequences having different codons from the native gene). Allelic variations or mutational events that occur naturally do not give rise to a heterologous region of DNA as defined herein. In addition, the invention also includes fragments (e.g., antigenic fragments or enzymatically functional fragments) of the enzyme corn starch synthase. As used herein, "fragment," when applied to a polypeptide, will usually be at least 10 residues, most commonly at least 20 residues, and most preferably at least 30 residues (e.g., 50) of length, but smaller than the complete sequence, intact. Fragments of the enzyme starch synthase can be generated by methods known to those skilled in the art, for example, by enzymatic digestion of the naturally occurring or recombinant starch synthase protein, by recombinant DNA techniques using an expression vector that code for a defined fragment of the starch synthase or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of starch synthase (for example, binding to an antibody specific for starch synthase, or exhibiting partial enzymatic or catalytic activity) can be assessed by the methods described herein. Purified fragments of the starch synthase or antigenic fragments of starch synthase can be used to generate new starch regulatory enzyme using multiple functional fragments from different enzymes, as well as to generate antibodies, using standard protocols known to those skilled in the art. It is possible to use a standard Northern blot assay to find out the relative amounts of starch synthase mRNA in a cell or tissue obtained from plants or other transgenic tissue, in accordance with the traditional Northern hybridization techniques known to those of ordinary skill in the art. technique. Otherwise, it is possible to use a standard Southern blot assay to confirm the presence and number of copies of the starch synthase gene in transgenic systems, in accordance with the traditional Southern hybridization techniques known to those skilled in the art. Northern blot and Southern blot techniques utilize a hybridization probe, eg, radiolabeled corn starch synthase cDNA, containing full length, single-stranded DNA having a sequence complementary to SEQ ID NO: 1 or a fragment of this DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 100 consecutive nucleotides in length). The probe for DNA hybridization can be labeled by any of the many different methods known to those skilled in the art. The most commonly used brands for these studies are radioactive elements, enzymes, chemicals that emit fluorescence when exposed to ultraviolet light, and others. Different fluorescent materials are known and can be used as markings. These include, for example, fluorescein, rhodamine, auramine, Texas Red, blue AMCA and Lucifer Yellow. A material for particular detection is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. The proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope can be selected from H, C, P, S, Cl, 51 C "o, 57C_o, 58C" o, 59.F., e, 90-Y, 125tI, 131.I., and 186_Re. The enzyme labels are in the same useful way, and can be detected by any of the currently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with source molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes that can be used are known and used in these methods. Preferred are peroxidase, b-glucuronidase, b-D-glucosidase, b-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090, 3850,752 and 4,016,043 are referred to as examples for their description of alternative marking materials and methods. As used herein, the term "metabolism" is defined as the sequence of reactions catalyzed by enzymes in which a molecule is degraded to simpler products or synthesized from simple precursors. The present invention is directed to a cDNA corresponding to the gene coding for the corn starch synthase enzyme. That is, the present invention provides an isolated cDNA having the sequence shown in SEQ ID NO: 1 encoding a corn starch synthase enzyme. The present invention is also directed to an expression vector containing this cDNA or fragments or derivatives thereof operably linked to a promoter allowing the expression of this cDNA. Such an expression vector can be used to transfect a host cell to produce desired amounts of the corn starch synthase enzyme. The present invention is also directed to a starch synthase protein or fragments or derivatives thereof, wherein the protein has a molecular weight of about 180 kDa, a maximum level of transcription in the endosperm at 12 days after pollination, a C-terminal region possessing α-1,4-glucosyltransferase catalytic activity, and an N-terminal region containing the amiloplast targeting peptide and repeat motifs comprising, but not limited to box M (SEQ ID NO: 9). In another embodiment, the present invention also provides an antibody directed to the corn starch synthase polypeptide, or fragments thereof. In yet another embodiment, the present invention is directed to a transgenic plant, wherein the transgene is an expression vector containing the cDNA corresponding to the corn starch synthase gene. In another aspect, the present invention is directed to a method of producing starch, comprising the steps of: transforming a cell with the vector described herein, and extracting and purifying the starch using the methods described in the present and well-known specification by an expert in the art. This method can be used together with cells carrying additional mutations in genes involved in the synthesis and / or metabolism of starch, synthesis and / or glucose metabolism, synthesis and / or glycogen metabolism and synthesis and / or carbohydrate metabolism. In another aspect, the present invention is directed to a method of using N-terminal "arms" of DUl expressed in transgenic plants for the purpose of binding other proteins to alter the function or activity of those proteins. The amino acid residues 1225 that are N-terminal for the DUI catalytic domain (residues 1226 to 1674) define a region termed the "N-terminal arm of DU1". This region contains the characteristics that suggest that the entire arm or specific portions thereof are involved in interactions with other proteins. The N-terminal arm of DUl can be expressed in its integrity in transgenic plants to bind one or more proteins that interact with different portions of the arm. In addition, the specific regions of the N-terminal arm of DUl can be expressed in transgenic plants to bind proteins that associate only with these regions. Portions representative of the N-terminal arm of DUI that can be expressed in transgenic plants include: (1) the nucleotide sequence containing nt 120 to nt 1221, which code for the first 368 amino acids of DUl. This region of the protein binds specifically to the isoform of the branching enzyme, SBEIIa; (2) the nucleotide sequence comprising from nt 655 to nt 1221, which codes for amino acids 180 to 368 of DUl. This region of the protein can function to activate the transcription of a reporter gene in combination with the DNA binding domain from the transcriptional activator Gal4 of Saccharomyces cerevisiae; (3) the nucleotide sequence comprising nt 565 to nt 816, which codes for amino acids 150 to 233 of DUl. This region of the protein consists of 85 residues that form three cascade repetitions of 28 residues each; (4) the nucleotide sequence comprising nt 1369 to nt 1944, which codes for amino acids 418 to 609 of DUl. This region of the protein consists of 180 residues that form three hierarchical repetitions in a cascade of 60 residues each. Each of the three repeats of 60 residues is termed "SBE superrepetition". Each SRE superrepetition is composed of six 60-residue cascade repeats, which are referred to as "SBE repetition". The designation "SBE" in the name manifests the fact that the repeater unit is similar to a sequence found in all SBE. The nature of the 180-residue repetition suggests that it is involved in a specific DUI function; (5) the nucleotide sequence comprising nt 1 to nt 1437, which codes for amino acids 1 to 440 of DUI. This region of the protein is unique for DUI, indicating that its function is specific for DUI; (6) the nucleotide sequence comprising nt 1438 to nt 2424, which codes for amino acids 441 to 769 of DUl. This region of the protein has approximately 15% identity with the corresponding region of potato SSIII; (7) the nucleotide sequence containing nt 2425 to nt 3791, coding for amino acids 769 to 1225 of DUl. This region of the protein is immediately N-terminal for the catalytic domain, and has approximately 51% identity with the corresponding domain in the potato SSIII enzyme. In another aspect, the present invention is directed to a method of using the full-length DUI, a N-terminal arm of DU1, portions of the N-terminal arm of DU1 or catalytic domain of DU1 as fusion proteins to purify these regions of polypeptides or to identify proteins or other factors that interact with these polypeptide regions. The full length DUI comprising residues 1 to 1674, or the DUl catalytic domain comprising residues 1226 to 1674, or the N terminal arm of the DUl comprising residues 1 to 1225, or portions of the N terminal arm of DUl (described above ) can be cloned into translation vectors for the purpose of expressing fusion proteins. The fusion proteins would include a peptide or peptide tag for affinity purification to allow convenient detection or purification of the expressed DUl polypeptides, facilitated by binding of the peptide tag region to an affinity resin or matrix. After binding the fusion protein to the affinity matrix, the protein or whole cell extracts from plant tissues could then be incubated with the mixture, with the result that the proteins or other factors that physically interact with the expressed region of the DUl They would also be united. An example of this, an expression description of a DUl portion in the pET expression vector is provided below. In another aspect, the present invention is directed to a nucleic acid sequence comprising the Dul promoter. the promoter Dulll directs the expression of the Dulll gene within a period of time of development and within the specific tissues of the corn plant. The gel absorption analysis of RNA indicates that Dulll is highly expressed in developing maize endosperm, starting approximately 12 days after pollination (DDP) and continuing through at least 32 DDP. This analysis also shows that Dulll is expressed slightly in the maize embryo and in the corn spikelet tissue at approximately 20 DDP. These results were confirmed by RT-PCR analysis, which showed that the specific fragments of Dulll were amplified from the reverse transcribed total RNA isolated from the endosperm, embryo and tissues of the developing corn spikelet, but not from the leaves or root tissues. Thus, the characteristics of the Dulll promoter ensures that Dulll is expressed in the reproductive tissues of the plant during the period in which the starch is synthesized, but is not expressed in the vegetative tissues. In accordance with the teachings described herein, a person having ordinary skill in this technique will easily be able to determine the sequences of the Dul promoter. In another aspect, the present invention is directed to an amino acid sequence comprising a polypeptide fragment (transient peptide) that targets the DUI protein for the corn amyloplast. The amino acid sequence of the DUI polypeptide predicts a 71 amino acid transient peptide with a predicted cleavage site (VKVA_A) after amino acid 71. This cleavage site is similar to the consensus sequence V / IXA / C-A reported for the transient peptides of chloroplasts. In addition, the predicted cleavage site of DUl has an arginine residue at position -10, which is also a characteristic consistent with the transient peptides of chloroplasts. The DUl protein is enriched in the stromal fraction of the corn amiloplast, strongly indicating that it is specifically directed to the amyloplast by means of a transient peptide. In another aspect, the present invention is directed to an expression vector wherein the cDNA fragment of SEQ ID NO: 1 is selected from the group consisting of nucleotide 120 to nucleotide 1221 of SEQ ID NO: 1, nucleotide 655 to nucleotide 1221 of SEQ ID NO: 1, nucleotide 656 to nt 816 of SEQ ID NO: 1, nucleotide 1369 to nucleotide 1944 of SEQ ID NO: 1, nucleotide 1 to nucleotide 1437 of SEQ ID NO : 1, nucleotide 1438 to nucleotide 2424 of SEQ ID NO: 1, and nucleotide 2425 to nucleotide 3791 of SEQ ID NO: 1. The present invention is also directed to a transgenic plant, wherein the transgene is the vector previously described. In another aspect, the present invention is also directed to a fusion construct, comprising part or all of the corn starch synthase enzyme DNA fused to the DNA encoding an affinity purification peptide. The present invention is also directed to the fusion protein expressed by such fusion constructs. In another aspect, the present invention is also directed to an antisense nucleotide sequence, wherein the sequence is antisense to the cDNA of the present invention or fragments thereof. In addition, the present invention is directed to an expression vector containing this antisense nucleotide sequence operably linked to the elements that allow the expression of the antisense nucleotide sequence and to a transgenic plant, wherein the transgene is this vector. In another aspect, the present invention is also directed to the starch extracted from a transgenic plant as described herein. The following examples are provided for the purpose of illustrating the different embodiments of the invention and are not intended to limit the present invention in any way: EXAMPLE 1 Nomenclature, plant materials mutation isolation dul The nomenclature follows the normal format of genetics for maize (Bebáis et al., 1995). The alleles begin with a capital letter indicating a functional form, that is, not mutant of the gene (for example, Dul). Unspecified mutant alleles are indicated by hyphens that do not follow the designation (for example, dul-). Gene products are indicated by capital letters, not in italics (for example, - DUl). Transcripts and cDNAs are indicated, by the non-italicized gene symbol (for example, Dul). The standard lines used were the Fl B77 / B79 or Q66 / 67 hybrids, products of four inbred lines that have no background or Mutator activity background. The Mu-active parents used in the isolation scheme of the mutant were described by Robertson (1978). The inbred corn line W64A was used for the detection of the Dul transcript in seeds and other tissues.

The mutant alleles dul -R2197, dul -R2339, dul -R2649r dul -R2370:: Mul r dul -R4059 and dulR-11 78 [sicj were identified from the ears of self-pollinated Fl plants 87-2197-9, 87-2339- 2, 87-88-2649-11, 87-2370-20, 82-4059-23, 89-1178-3, respectively (Figure 1A). The inclusion of the letter JR in the names of the allele indicates that the raw materials originally are from the laboratory of Dr. DS Robertson, and the inclusion of the term Muí in the name of the alle alle -R1270:: Mul indicates that this transposon has been definitively identified within the mutant gene. The stock number XlOA of the Maize Genetics Cooperation Stock Center (Urbana, IL), homozygous for the reference allele dul -Ref, was used for complementation tests to generate segregating populations (Figure 1A).

EXAMPLE 2 Cloning The methods used for the extraction of genomic DNA and the gel absorption analysis of DNA were as described in (James et al., 1995). Most probes were labeled with 32P by the normal random priming method (Boehringer Mannheim, Indianapolis, IN). The 2.0 kb BamHI fragment containing MuI and co-secreted with Dul -R2370:: Mul was isolated from a library expressing? ZAPII selected by size constructed from genomic DNA digested with Ba HI from a plant dul -R2370:: Mul / dul -Ref essentially as described (James et al., 1995) and subcloned into pBluescript SK + to form the pásmid pJW3.

The F500 fragment (Figure 2B) was amplified for use as a probe by PCR from pJW3 using the dul-spl primers (5 '-GTACAATGACAACTTTATCCC-3') (SEQ ID NO: 2) and dul-sp2 (5 '-CATTCTCACAAG-TGTAGTGGACC-3') (SEQ ID NO: 3). The single-stranded F500 probe, labeled with 32P, was generated by PCR using the du-spl primer [sic] and the gel-purified BamHI fragment from pJW3 as a template according to Konat et al. (1994). For the PCR amplification of a longer genomic fragment by superimposing the flanking sequence of the MuI element on the Ba HI protein of 2.0 kb, fragments of selected size were prepared from 80 μg of genomic DNA digested with EcoRI from sister wild type plants (Dul / dul -Ref, see Figure 1A) fractionated in a 0.5% preparative agarose gel. Five fractions of the EcoRI fragments were isolated by electroelusion (Sambrook et al., 1989) from consecutive gel slices by forking the 6.0 kb size marker, and verified for the presence of the MuI flanking sequences in the BamHI fragment. cloned, original by PCR using the dul-spl and dul-sp2 primers. The aliquots of two fractions containing the highest amounts of the target fragment were ligated to pBluescript SK + linearized with EcoRI and 1 μl of each ligation mixture was used directly for PCR amplification of the region that overlaps the cloned BamHI fragment using the primer dul-spl or dul-sp2 in combination in pairs with the primer T3 or T7 in pBluescript SK +. A fragment of approximately 2.0 kb amplified by the initiator pair dul-spl T3 was confirmed to contain the BamHI fragment by amplification of the subsequent PCR using the primers dul-spl and dul-sp2, and was used as a template for another round of PCR using the dul-sp4 nested primer (Figure 3A) (5'-GTCGTAGGAATCGTCACTCG-3 ') (SEQ ID NO: 4) and the T3 primer. The specifically amplified 1.3 kb fragment was polished with T4 DNA polymerase, digested with EcoRI to remove the sequence of the remaining vector and then cloned the EcoRV and EcoRI sites of pBluescript SK + to form the pMgla pásmid.

EXAMPLE 3 Detection of the cDNA library Randomly initiated maize endosperm cDNA libraries in? Gtll were provided by Dr. Karen Cone (University of Missouri, Columbia, MO). Normal procedures were followed for the preparation of phage elevations, phage amplification and purification in a single plate (Ausubel et al., 1989; Sambrook et al., 1989). The phage elevations were hybridized at 65 ° C for 16-18 hours for probes labeled with 32P-dCTP by the method of random initiation with washing under very strict conditions as described by Church and Gilbert (1984). The cDNA inserts in phage clones were subcloned in pBluescript SK + or pBluescript KS + from the phage DNAs prepared by the izard DNA purification kit (Promega). Purified phage cDNA inserts were characterized with respect to their length by direct PCR amplification from phage disrupted using two primers, λ30 (5'-ATTGGTGGCGACGA-CTCCTG-3 ') (SEQ ID NO: 5) and? l356 (5'-GTGTGGGGGTGATGGCTTCC-3 ') (SEQ ID NO: 6), located 19 bp proximal to the EcoRI cloning site in the left arm and 281 bp distant from the EcoRI site in the LacZ' region of the right arm in the phage DNA? gtll, respectively. an aliquot of the purified, homogeneous phage (1 μl of a phage suspension 1 x 1010 pfu / μl) was disrupted in 20 μl of optimal PCR buffer (10 M Tris-HCl, pH 9.2, 1.5 mM MgCl2, 25 M of KCl) containing 0.2 μM each of the two primers and 0.2 mM each of the four dNTPs for 15-20 minutes at 96 ° C, and then directly used for PCR amplification of the cDNA inserts commonly as follows: 94 ° C for 4 minutes, one cycle (addition of one unit of Taq DNA polymerase at the end); 10 cycles of 58 ° C for 45 seconds, 72 ° C for 0.5 to 3 minutes (depending on the size of the insert) and 94 ° C for 45 seconds; 20 cycles of 61 ° C for one minute, 72 ° C for 0.5 to 3 minutes (depending on the size of the insert) and 94 ° C for one minute; and a cycle of 61 ° C for 5 minutes and 72 ° C for 7 minutes. The lengths of the cDNA inserts were determined by gel electrophoresis of 5-10 μl of the PCR products.

The detection of the cDNA library was as follows: in the first round, approximately 340 positive signals were obtained in the primary detection of approximately 0.5 x 10 pfu using the BE1300 fragment as a probe. The longest cDNA insert between 15 most purified and characterized sections was 3.2 kb in length (nt 2577 to nt 5782 in the almost complete length sequence). This insert was subcloned as two EcoRI fragments in the plasmids pMg271L and pMg271S containing the 2.7 kb cDNA at the end of the 0.5 kb cDNA at the 3 'end, respectively. In the second round, the 0.5 kb EcoRI / Scal fragment at the 5 'end of the 2.7 kb cDNA insert in pMg271L and the 0.5 kb EcoRI fragment from pMg271S were used separately as probes in the primary detection of 1.5 x 10 additional pfu ufp. The longest insert identified by the 5 'end probe in one of 24 purified and characterized phage sections, 4.3 kb in length, was subcloned into the piMamido pMg6Aa. The pMg271S probe identified an approximately 4.0 kb cDNA insert containing an EcoRI fragment at the 3 'end of 0.67 kb that overlapped with and extended into fragments of the original cloned 3' end. The 1.4 kb portion of the 3 'end of this 4.0 kb cDNA insert was amplified by PCR directly from the purified phage and cloned as a BamHI / HindIII fragment in pMgt6-2M. The original, EcoRI terminal site was mutated to a HindIII site during PCR amplification to facilitate subsequent reconstruction of the complete cDNA. The 240 bp BamHI fragment at the 5 'end of the cDNA in pMg6Aa was then used as a probe for the primary detection of another 1.0 x 106 pfu in the third round. Among 19 purified and characterized phage clones, the cDNA insert that overlapped with the insert in pMg6Aa and containing the longest extension at the 5 'end of approximately 1.5 kb in length was subcloned into the pMgflO. The continuous sequence of three cDNA fragments superimposed on the plasmids pMgflO, pMg6Aa and pMgt6-2M represents the almost complete length cDNA sequence (Figure 3B). The nucleotide sequences were obtained using an automated ABI Prism sequencing system (Perkin Elmer) at the Nucleic Acids Sequencing and Synthesis Facility at Iowa State University, using double-stranded pyramid templates. All nucleotide sequences were confirmed by analysis of both strands, computational analyzes were performed using the isconsin package (Genetics Computer Group, Madison, I) and the Lasergene software package (DNASTAR Inc., Madison, I).

EXAMPLE 4 Analysis of RNA gel absorption and RT-PCR The extraction of total RNA from different W64A inbred corn tissues and the gel absorption analysis of RNA were essentially as described (Gao et al., 1996). The radioactivity of the hybridized transcripts for the Dul cDNA probe was analyzed and quantified using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and expressed as the percentage of the maximum signal intensity over the same band (relative level or RL% ). Minor load differences between samples in each band were calibrated using a tomato cDNA probe hybridizing to the 26S rRNA in the appropriate band to normalize the intensity of the Dul mRNA signal. The RT-PCR assay used the Titan RT-PCR system (Boehringer Mannheim) following the manufacturer's instructions. The two primers used were dul-F3 (5'-ATAAATGTGTGGCGT-GGACT-3 ') (SEQ ID NO: 7) and du-Rl (5 -CGTTCCTTGTCATTGTCCAC-3) (SEQ ID NO: 8) encompassing the cDNA region of 934 bp from nt 3997 to nt 4930. The total RNA (1 μg) of different samples were used as templates. To distinguish the RT-PCR amplification of mRNA from the PCR amplification of residual genomic DNA, potential, total RNA from one of the samples (endosperm 22 DBP) was treated with RNase A (100 ng / ml) for 10 minutes at 37 ° C before use as a template. The RT-PCR products were analyzed on a 1% agarose gel, then absorbed and hybridized using the pMg6Aa cDNA insert as the probe to confirm the identity of the product.

EXAMPLE 5 Identification of mutations dul- Node mutants were identified in plants obtained from parental lines containing a transposable element system Mu by the strategy delineated in Figure 1A. normal non-Mu lines were pollinated by active Mu plants, and the resulting progeny Fl were self-pollinated. Six cobs Fl containing seeds with the dull phenotype were found at a frequency of approximately 25%, as illustrated in Figure IB. Plants grown from dull seeds were crossed to standard lines to generate supposed Dul / dul heterozygous seeds. These were grown to maturity and crossed with dul -Ref / dul -Ref test plants, giving rise to a 1: 1 segregating population of dull and normal sister seeds for each of the six putative Dul alleles induced by Mu. Thus, in all cases the dull phenotype is a single gene trait conditioned by a mutation that is most likely allelic for dul -Ref. Sweet-novel mutations are called dul -R2370:: Mul, dul -R2339r dul -R2649, dul -R4059, dul -R2197 and dul -R1178.

EXAMPLE 6 Cloning and characterization of the genomic losi-geni It was found that a specific Mui transposon co-segregates with the dull phenotype among the progeny of a sweet heterozygote -R2370:: Mul / Dul. The heterozygous parent was crossed to a homo-sweet dul-Ref, generating ears containing approximately 50% dull seeds (dul-R2370:: Mul / dul - Ref) and 50% of normal seeds (Dul / dul-Ref). The genomic DNAs were extracted from germinated seedlings of 35 seeds of each type, digested with BamHI, and subjected to analysis by gel absorption using the Mlul fragment. [sic] 960 bp internal of Muí as a probe. Figure 2A shows the representative data of these analyzes; a fragment containing Mui of 2.0 kb was detected in all the plants analyzed carrying dul-R1270:: Mul, but not in the plants lacking this allele. The 2.0 kb MuI-containing genomic DNA fragment co-segregating with the mutant dull phenotype was cloned by detecting a fractionated genomic library by size, prepared from a heterozygote dul -R2370:: Mul / dul -Ref in the vector expressing ? ZAPII, using an internal MuI fragment as a probe. Figure 2B shows the structure of the cloned fragment. As expected, the nucleotide sequence of this fragment revealed two direct repeats of 9 bp (5 '-GTGAGAATG-3') flanking a MuI element. Figure 2C illustrates a subsequent DNA gel absorption analysis confirming that the fragment containing the cloned MuI was obtained from the genomic range co-secreting with dul -R1270:: Mul. The F500 single-stranded probe, which is adjacent to the MuI element (Figure 2B) detected a fragment of approximately 0.62 kb in all the plants of the segregating population and also a fragment of approximately 2.0 kb specific for plants obtained from dull seeds (dul -2370: : Mul / dul -Ref). 27 seeds of each type were characterized. The size difference of 1.4 kb indicates that the larger 2.0 kb BamHI fragment most likely arises from the insertion of a 1.4 kb MuI element within the 0.62 kb region delineated by these two BamHI sites. Taken together, these data indicate that the fragment containing cloned Mui is located within the locus dul or tightly bound thereto.

Another support for this conclusion is shown in Figure 3A, which illustrates DNA gel absorption analysis of other restriction fragments using the F500 fragment as a probe. The difference in size of 1.4 kb, indicating a MuI insertion, was also observed between the EcoRI fragment of 6.0 kb detected in plants Dul / dul-Ref and plants dul-2370:: Mul / dul-Ref and the fragment kb found specifically in these last. Due to the allelic variation two different Xbal fragments were detected in Dul / dul-Ref plants of the segregating population. In sister plants carrying dul-2370: Mul the smallest of these two fragments, approximately 3.0 kb in size, was invariably replaced by a larger fragment of 1.4 kb [sic]. The genomic DNAs used in these two analyzes were obtained from eight dull seeds and eight normal seeds. In all cases, the difference of 1.4 kb between the largest fragment detected only in carrier plants of the mutant alle alle -2370:: Mul and the smallest fragment associated with the Dul wild-type allele is consistent with the insertion of this MuI element. having caused the mutation dul -. These data also revealed larger genomic fragments comprising the 2.0 kb cloned Ba HI fragment, and thus facilitated the isolation of the cDNA genes corresponding to Dul mRNA.

EXAMPLE 7 Dul Codes for a Transcript of at least 6027 bp To obtain additional coding sequence for the purpose of detecting an endosperm cDNA library, a larger geomic fragment superimposed on the cloned BamHI fragment of 2.0 kb was isolated from the genomic DNA type wild. As already described, an EcoRI fragment of 6.0 kb from wild-type genomic DNA contains flanking sequences of the MuI element in the original cloned fragment (Figure 3A). A 1.3 kb portion of this EcoRI fragment, designated BE1300, was cloned by PCR amplification with nested primer, on one side. Figure 3B illustrates that the BE1300 fragment extends from within the shorter MuI flanking region of the 2.0 kb BamHI fragment cloned, original to one of the terms of the 6.0 kb EcoRI fragment. The nucleotide sequence of the BE1300 fragment confirmed its superposition with the 2.0 kb BamHI fragment. The BE1300 fragment was then used as a probe to detect a? Gtll cDNA library of maize endosperm. A cDNA sequence of almost complete length of 6027 bp was obtained from three cDNA overlays (Figure 3B). These isolates were isolated from three consecutive rounds of detection of approximately 3 x 10 total pfu of phage. The piMamido pMg6Aa contains an internal 4.3 kb cDNA insert for the almost complete length cDNA (nt 1002 to nt 5367), and the cDNA inserts in plasmids pMgflO (nt 1 to nt 1657) and pMgt6-2M (nt 4433 a nt 6027) superimposed and extends the cDNA sequence in this central cDNA fragment at the 5 '3' ends, respectively (Figure 3B). The continuous sequence of these three cDNA fragments revealed a coding sequence initiated in ATG of 1674 codons (Figure 3B). Multiple antisense codes in all three reading frames at the 5 'end of the pMgflO cDNA insert indicates that the coding sequence begins with this fragment. The size of a DNA fragment amplified from total endosperm RNA by 3 'RACE indicates that the 3'_ end of the cloned cDNA is very close to the polyadenylation site (s) of the corresponding transcript. The cloned cDNA, therefore, is of almost complete length and contains the entire coding sequence. This conclusion was further supported by the detection of a 6 kb transcript in non-mutant endosperm RNA using the cDNA insert of pMg6Aa as a probe.

EXAMPLE 8 Verification of the cloned cDNA as a product of the locus dul The physical characterization of another alle alle independently isolated, dul -R4059, indicates that the cloned cDNA is encoded by the dul locus, rather than by a different gene tightly bound to dul. The genomic restriction fragments of Dul-R4059 / Dul-Ref and Dul / Dul -Ref sisters plants (Figure 1A) were analyzed by DNA gel absorption analysis using the piMamid cDNA insert pMgflO as the probe. As illustrated in Figure 4, a 6.6 kb Salí fragment was invariably detected in all dul-R4059 carrier plants, in addition to the 5.2 kb fragment which was also the only signal obtained from Dul / dul-Ref plants. The displacement in the 1.4 kb size in the Sali fragment associated with dul-R4059 has probably resulted from the insertion of a Mui element. This alteration is different from that associated with dul -R2370:: Mul, because the probe that detects this polymorphism does not identify any abnormal fragment in mutant dul -R4059 (data not shown). He "The fact that two independent genomic rearrangements in the same gene coincide with the appearance of the dull phenotype is most likely explained by the Mui insertions being the causal agents of the dul- mutations." Therefore, in cloned cDNA it is most likely encoded by Dul. The structure of dul -R2370:: Mul is consistent with this conclusion.Figure 3B shows the intron / exon structure deduced by comparing the sequences of the cloned cDNA and the genomic fragments.The inscription of Mui into the 2.0 kb BamHI fragment. cloning is within an exon, and thus is expected to break the integrity of the transcript corresponding to the cDNA cloned in the endosperm of dul -R2370:: Mul. As predicted, the steady state levels of the transcripts that hybridize to the cloned cDNA in dul -R2370:: Mul and other dulos mutant endosperms were drastically reduced compared to the non-mutant endosperm of the same stage of development. Figure 5A shows these results for dul -R2370:: Mul and the dul -Ref endosperm as determined by the gel absorption analysis of RNA using a portion of the cloned cDNA as a probe, and similar data were obtained for dulos endosperms -R2339 and dul -R2197. Residual transcripts in the dulos-R2339 endosperm or dul -2370:: Mul mutants were approximately 1.4 kb larger than normal (Figure 5A) possibly resulting from transcriptional reading through the inserted MuI element. The residual transcripts that hybridize to the cloned cDNA were normal in mutant endosperms dul -Ref and dul -R2197 (Figure 5A and data not shown). In summary, four dul-independent mutant alleles including dul -Ref are associated with disruption of the transcript detected by the cDNA probe, providing definitive confirmation that Dul codes for the cloned cDNA. The Dul transcripts were not completely eliminated in the endosperm of any of the dul-examined mutants, common to many maize mutations that affect the biosynthesis of endosperm starch (Giroux et al., 1994; James et al., 1995; et al., 1996); Figure 5C shows that the residual Dul transcripts, although possibly non-functional, were clearly detectable in the endosperm of three dul-independent mutants by the more sensitive RT-PCR method, confirming the results of the gel absorption of RNA.

EXAMPLE 9 ul has a unique spatial and temporal expression pattern Gel absorption analysis of endosperm total RNA from inbred W64A collected on different days after pollination (DBP) revealed a unique temporal expression pattern of a 6.0 kb transcript hybridizing to Dul cDNA (Figure 5A). The Dul transcripts were not detected in the endosperm collected in 7 DBP. The transcript level was highest in endosperms in the early development stage of about 12 BPD, at which time other synthetic starch genes, such as Sbel, Sbe2b, Bt2, Sh2 and Wx in the same endogamous 64A have little or no expression (Gao et al., 1996). The steady state level of the Dul transcripts declined gradually over time, contrary to other synthetic starch genes that increase expression as the endosperm develops (Gao et al., 1996). The lowest Dul transcript level, only about 40% of the maximum, was found in the endosperm of 22-26 DBP, which has the highest rate of starch synthesis (Jones et al., 1996). The level of transcription of Dul reached approximately 62% of the maximum in the most mature endosperm of 32 DBP seeds. The transcription of Dul was also detected in other reproductive tissues, specifically of embryo and spikelet (most likely in pollen). Very low mRNA levels were scarcely detectable by gel absorption analysis of the total RNAs of these tissues. The presence of Dul transcripts was clearly demonstrated, however, by the more sensitive RT-PCR analysis (Figure 5C). The expected 940 bp cDNA fragment was amplified from total RNA extracted from embryos or spikelet; this fragment was not amplified from Total RNA digested with RNase from the endosperm of 22 DBP (Figure 5C), indicating that it was amplified from mRNA instead of residual contaminating genomic DNA. The gel absorption analysis of DNA using a Dul cDNA probe confirmed that the 940 bp fragment is amplified from the Dul mRNA. The additional fragment of approximately 400 bp non-hybrid to the Dul cDNA probe, and thus is a non-specific amplification product. The Dul transcripts were not detectable by the RT-PCR analysis on total leaf and root RNAs (Figure 5C). These data suggest that the enzyme encoded by Dul is specialized for the synthesis of storage starch in reproductive organs but is not involved in the production of transient starch in the leaves.

EXAMPLE 10 Pul codes for a putative starch synthase with conserved characteristics The amino acid sequence deduced from the cloned cDNA indicates that Dul codes for a starch synthase. The longer open reading frame of the Dul cDNA sequence continues to encode a polypeptide, designated DUl, of 188 kD including a transient peptide amyloplast potential. Sequence similarity searches found that the deduced amino acid sequence of DUI is very similar to that of SSIII potato starch synthase (Abel et al., 1996; Marshall et al., 1996) among all proteins in the bases of public data.

Figure 6 shows the alignment of the deduced amino acid sequences of DUl and SSIII, and indicates three discrete regions with different degrees of similarity between the two proteins. The C-terminal regions, over an extension of 645 amino acids (residues of DUl 1029 to 1674), share the highest degree of similarity in the alignment; 73% of the aligned residues are identical in these sequences with only a single space of an amino acid. In the central regions of DUl and SSIII, corresponding to DUl 770-1028 residues, 51% of the 259 aligned residues are identical with no spaces in the alignment. This central region was defined by a sudden decrease in the degree of similarity between short extensions of the amino acid sequence of DUl and SSIII as the alignment is examined along the lengths of the two proteins. The remaining N terminal region of DUl (residues 1 to 769) does not have any significant similarity with that of potato SSIII, nor for any polypeptide sequence available in the databases. An extension of 440 residues in relation to SSIII is present in the N terminal of DUl. Another comparison of the deduced amino acid sequence of DUI with the starch synthases and cloned glycogen synthases of different species indicates that part of the C terminal region is likely to provide a catalytic activity of a-1,4-glucosyltransferase. An extension of 450 amino acid residues near the C terminus of DUl is substantially similar to the corresponding amino acid sequence near the C terminal of very distinct types of a-1,4-glucosyltransferase, including the glycogen synthases of E. coli (Genbank access No. P09323), yeast (Genbank accession nos. M60919 and M65206), and human liver (Genbank Access No. S70004), starch synthase linked to GBSSI and GBSSI pea granule (Genbank Accession No. X88789 and X88790), and GBSSI of corn (Genbank Access No. X03935). The degree of conservation of the sequence in these alignments increases towards the C terminals. As an example, 28% of the 438 aligned C terminal residues are the same in DUl glycogen synthase and E. coli, and 67% of the 48 aligned DUI residues from position 1550 to 1597 are identical to the corresponding region of the E. coli enzyme without gaps in the alignment (data not shown). The three blocks of sequences are located within this region of DUI that are highly similar to the conserved regions identified by comparison of glycogen synthase from E. coli with GBSSI from a wide variety of plant species (Figure 6A) (Preiss and Sivak, nineteen ninety six) . The substantial conservation of the amino acid sequence in the C-termini of this phylogenetically divergent group of α-1,4-glucosyltransferase suggests that this region of DUl most likely constitutes the complete catalytic domain for such enzymatic activity. This speculation is furtsupported by the observation that the central regions of DUl and SSIII, in which 51% of the amino acids are the same, have no significant similarity to any of the otglycogen synthases or cloned starch synthases. This exclusive sequence conservation, tfore, is expected to define functions belonging only to a subgroup of vegetable starch synthases represented by SSIII and DUI. It is expected that the unique sequence of 769 residues in the N terminal of DUl contains an amiloplast orientation peptide and defines unique functions for this enzyme.

EXAMPLE 11 Two groups of repeats in the single N terminal region of DUl Figures 6A and 7 show two distinct groups of repeats comprising a total of 180 and 85 amino acids, respectively, which were identified in the unique N terminal region of DUl by analysis of graph of points intra sequences. The largest group of 180 residues (positions 418-597) is a hierarchical repetition. This sequence contains three 60-residue cascade repeats called the "SBE superrepetition" each of which in turn consists of six 10-residue cascade repeats called "the SBE repeat" (Figure 7A); These names manifest the fact that the repeater unit is similar to a sequence found in all SBE. This two-level repeat structure was deduced from the pattern of sequence conservation among the 18 SBE repeats, that is, each individual SBE repeat is more similar to the two repeats located 60 or 120 distant residues (Figure 7A). Furthermore, within the individual SBE super-repetitions, each individual SBE repeat is always more similar to the preceding repetition in the N-terminal direction compared to the one following it. These patterns of sequence similarity strongly indicate a hierarchical repetition process involving the duplication of the SBE superrepetition as a unit, instead of 18 individual repetition events. Each SBE repetition consists of two "semi-repetitions", of 6 and 4 residues, respectively, as can be deduced from: 1) the different degrees of conservation of the sequences exhibited by the first and second semi-repetitions among all the SBE repeats, and 2 ) the presence of 4 residues between two complete SBE repeats (Figure 6A); residues 414-417) probably resulting from an unequal crossing mechanism (Smith, 1976; Lewin, 1997).

The nature of the 180-residue repeat suggests that this is involved in a specific DUI function, the SBE repeats that begin each super repeat SBE are more similar to each other at the SBE repeats at any of the other five positions in the superrepetition (Figure 7A ). This suggests that these three SBE repeats were subjected to the highest selection pressure and thus can represent a functional domain. Conversely, if the first SBE repeats were not important to function, then mutations must accumulate in these sequences at the same rate that appeared in other positions of the SBE superrepetition, which is not the case. The consensus sequence between these three conserved SBE repeats is DQSIVG (SEQ ID NO: 9) in the first semi-repetition, designated as "M box" and SHKQ (SEQ ID NO: 10) in the second semi-repetition. When the sequence of the M-box was searched for known polypeptides, a single type of enzyme containing an exact match was found, namely the members of the SBEI family. As illustrated in Figure 7B, the sequence of box M is invariant in SBEI of corn, SBEII of pea, SBEI of wheat, RBEI of rice and SBEI of potato. Box M is well preserved, with substitutions of two residues of similar properties producing the sequence DQALVG (SEQ ID NO: 11) in the corresponding region of the members of the SBEII family including SBEIIa and SBEIIb of corn, SBEI of pea, RBEIII of rice, SBEII from wheat and SBE2.1 and SBE2.2 from Arabidopsis (Figure 7A). The DQALVG sequence is also present in glycogen branching enzymes from yeast and humans (Figure 7B). The smallest group of 85-residue repeats in the N-terminal DUI (amino acid 150-2339) is composed of three 28-residue cascade repeats (Figure 6A and 7C) .The basic repetition unit also consists of two halves of 12 and 16 residues each, which again is very likely to have evolved through imperfect cascading duplications through the unequal crossing mechanism.This conclusion was supported by the different degree of conservation of the sequence of the two semi-repetitions between the three repeats The first semi-repetition is highly conserved in the first and third copies of the 28-residue repeat, while the second half is more conserved in the first and third copies of the repeats (Figure 7C). The following four lines of evidence support the conclusion that the cloned genomic locus is a portion of the dul gene. First, the cloned genomic interval is within or tightly bound to the dul locus, because it co-segregates with the dull phenotype in 70 progeny plants.

Second, two mutations of dul arise coincidentally with insertions of 1.4 kb at different positions in the cloned transcription unit, one of which is known as a MuI element located within an exon. Third, the transcript that hybridizes to the cloned cDNA is drastically reduced to the same extent in dul-Ref endosperm and three dul mutants independently isolated. In two of these mutants associated with MuI insertions in dul, the residual transcript is 1.4 kb larger than the wild-type mRNA, consistent with the insertion of a MuI element in an exon. Fourth, the genetic codes cloned for a putative starch synthase, consistent with the fact that the mutants dul - are greatly reduced in the activity of soluble SSII starch synthase. Assuming that Dul's transcript level reflects enzymatic activity, these observations suggest that DUl is involved in starch biosynthesis at a very early chronological step, possibly closely associated with the initiation event. The conservation of the sequence of the M-box, the supposed first semiregition within the amplified SBE repeat, specifically in the starch and glycogen branching enzymes of phylogenetically very divergent species is particularly surprising considering that the SBE and SSII act in a concerted biosynthetic pathway . The sequence of the M-box, therefore, may be a basic structural motif for a particular function shared by all these enzymes, possibly including glucan binding, protein-protein interaction or serving as regulatory sites. In addition, many consensus sites for N-glycosylation and phosphorylation were found within these repeats, suggesting that these may serve as regulatory sites. The total group of repeats can form a helix-turn-helix structure, reminiscent of the helix-turn-helix DNA-binding motifs in many transcription factors (Mitchell and Tjian, 1989). Considering the helical architecture of DNA and glucan polymers bound to a- (1-4), the repeats of 85 residues can mediate the binding of SSII and proteins associated with the growing glucan chains. Thus, the present invention is directed to an isolated cDNA having the sequence shown in SEQ ID NO: 1 encoding a corn starch synthase II enzyme. Ordinarily, a person with ordinary skill in this art can construct an expression vector containing this cDNA, or functional fragments thereof, operably linked to the elements that allow the expression of the cDNA. In addition, it would be possible to transfect a host cell with this vector.

The present invention is also directed to a corn starch synthase II enzyme encoded by this cDNA. The present invention is also directed to a polypeptide encoding a starch synthase-II protein, wherein the protein has a molecular weight of about 180 kDa, a maximum level of transcription in the endosperm at 12 days after pollination, a C terminal region possessing a-1, 4-glucosyltransferase catalytic activity and an N-terminal region containing the aminoplast targeting peptide and repeating motifs comprising, but not limited to, box M (SEQ ID NO: 9). In one embodiment, the protein has the amino acid sequence shown in SEQ ID NO: 12). The present invention is also directed to an antibody directed to the polypeptide herein described or functional fragments thereof. In a separate modality, a person with ordinary skills in this technique could manipulate a plant to create a transgenic plant, having as the transgene the vector described above. By using this technology it is possible to produce starch, comprising the steps of: transforming a cell with the vector described here; and extract and purify the starch. Preferably, the cells carry a mutation. Representative examples of the useful mutations include a gene that codes for an enzyme involved in starch synthesis, starch metabolism, glucose synthesis, glucose metabolism, glycogen synthesis, glycogen metabolism, carbohydrate synthesis and metabolism of carbohydrates The manipulation of the enzymatic machinery of starch production in higher plants can be used to create starch forms that have specific branching patterns and specific chain lengths. The properties of chain length and / or degree of branching confer specific characteristics on the starch as well as swelling properties, polarity, water retention, clarity, ability to disperse pigments and freeze-thaw. The production of designed or manipulated starches with defined and predictable properties is expected to be useful for a variety of specific industrial and food applications. The alteration of DUI starch synthase activity through the transgenic approaches mentioned below can be used to create novel forms of starch with length and / or branching patterns of the chains that differ from traditional starches. For example, it is possible to modify the starch in transgenic plants by over-expression of the DUl starch synthase. Secondly, it is possible to modify the starch in transgenic plants by reducing or eliminating the expression of DUl starch synthase, by: 1) the introduction of DUl in the antisense orientation, or 2) the co-suppression of DUl resulting from overexpression of the transgen of DUl. Overexpression of DUl starch synthase. Third, it is possible to modify the starch in transgenic plants by introducing an altered Dul sequence, thereby producing an altered DUl protein. In four, it is possible to modify the starch in transgenic plants by introducing a polypeptide fragment of the DUl protein, or by introducing a polypeptide fragment of the DUI protein in the antisense orientation, or by introducing a fragment of altered DUl protein polypeptide. In addition, it is possible to modify the production of glycogen in transformed bacterial and / or yeast cells by the expression of DUl starch synthase. The expression of DUl can be placed under the control of constitutive or inducible promoters. It is possible to propagate the transgenic plants to produce a starch form described with specific characteristics, or to cross the transgenic plants with plants in different genetic backgrounds or having different genetic traits to produce additional altered starch forms. These starches can be marketed for their unique characteristics to different industries; for example, as additives for food or beverages, or as processing agents in the manufacture of paper or textiles. Also, someone with a license could grow yeast or recombinant bacteria manipulated to express the starch synthase on a large scale to produce an altered glucan that could have industrial utility. Also provided by the present invention are fragments of polypeptides comprising regions of the DUI starch synthase recognized by an antibody specific for a DUI determinant. A polypeptide comprising a DUl fusion protein can be prepared by one skilled in the art as an antibody reactive with the DUI protein or fragments of polypeptides. One skilled in the art could also prepare a transgenic plant comprising a genome that includes a foreign DNA sequence encoding the DUl protein under the control of its own promoter or other promoter; or including a sequence encoding modified DUI to produce altered DUl activity.

EXAMPLE 12 Construction of expression plasmids Piásmido pHCl was constructed as an intermediate in the generation of the antigens used to increase anti-DUIN and anti-DUlF; this piásmido contains the entire coding region of Dul cDNA delineated by two EcoRI sites, one located immediately upstream of the putative initiation codon and the second 225 pb downstream of the termination codon. A 1.5 kb fragment was amplified by PCR from the pMgflO clone of Dul cDNA (Gao et al., 1998) using HCpl and M13F primers. The HCpl (5'-AAACCCGGGAATTCGATGGAGATGGTCCTACG-3 ') contains the Smal and £ coRI sites located upstream of the putative initiation codon (the restriction sites and the initiation codon are underlined), and M13F is located downstream of the inserted cDNA. the non-coding strand The amplified fragment was dissociated at the Smal site of the primer and the unique Agel site within the cDNA sequence. This fragment was cloned into the Smal and Agel sites of pMglO-6, which contains the Dul cDNA extending from 125 bp upstream of the putative initiation codon__ to the downstream coRI site. The resulting pyramid is pHCl. The piásmido pHC2 expresses a fusion protein containing the glutathione-S-transferase protein (GST) of Schistosoma japonicum at its N-terminus and residues 1-648 of DUl at its C terminal; this polypeptide was used as the DU1N antigen. The pHC2 was constructed by cloning the JScoRI-SalI fragment from pHCl into pGEX4T-3 (Pharmacia) digested with the same enzymes. The pHC4 piásmido expresses a fusion protein containing thioredoxin in its N terminal and full length DUl in its C terminal; this protein was used as the DU1F antigen. The pHC4 was constructed by cloning the EcoRI fragment from pHCl into pET-32b (+) (Novagen). Plasmids pHC5 and pHC6 express the C terminal region of DU1 (DU1C) in E. coli. The Dul cDNA from codon 1226 to the 1675 termination codon was amplified by PCR using pHCl as the template. The upstream initiator was HCp2 (5'-GCAGAATTCGATGCACA-TTGTCCAC-3f), which places an .EcoRI site adjacent to codon 1226 (the EcóRI site and codon 1226 are underlined). The downstream initiator was M13F. The amplified fragment digested with EcoRI was cloned into pET-29b (+) and pET-32b (+) (Novagen) to form pHC5 and pHC6, respectively. The sequence of the entire DU1C insert and the binding with the T7 promoter was determined in clones with correct restriction maps. Two amino acid substitutions were found in relation to the cDNA sequence, Q for H at position 1281, and N for K at position 1294. None of these residues is a conserved region in the SS plant.

EXAMPLE 13 Production of anti-DUIN and anti-DUlF To produce the DU1N antigen, one liter of exponential phase cultures of E. coli cells containing pHC2 were grown for two hours at 37 ° C in the presence of 0.1 mM IPTG. Cells were harvested by centrifugation and the pack (7g wet weight, 2 1 culture) was suspended in 100 ml of 140 mM NaCl, 2.7 mM KCl, 1.8 mM Na2HP04, 1.8 mM KH2P04, lmM PMSF, 0.01 mM Er64, EDTA 10 mM, 5 mM DTT, lysozyme 1 mg / ml; all subsequent treatments were at 0 ° C. The cells were used by sonification. The GST-DU1N fusion protein was purified by affinity using agarose beads, glutathione. The fusion protein was eluted in 100 mM Tris-HCl, pH 8.0, 120 mM NaCl, 20 mM glutathione. To produce the DUIF antigen, 0.5 1 of exponential phase cultures of E. coli cells containing pHC4 were grown for 1.5 hours at 37 ° C in the presence of 0.5 mM IPTG. Cells were harvested by centrifugation, suspended in 25 ml of 50 mM Tris-HCl, pH 7.0, 1 mM PMSF, 10 mM EDTA, 5 M DTT, 10% glycerol, 3% inhibitor cocktail 10 X proteinase (Sigma no. P2714), and divided by sonification. The Used ones were centrifuged to 10, 000 x g for 10 minutes, and the packs were dissolved by boiling for 10 minutes in sample buffer IX SDS-PAGE. A band greater than 200 kD was observed in SDS-PAGE that was specific for cells containing pHC4 and that reacted with anti-DUIN in immunoabsorption analysis. This protein, therefore, was identified as the DUIF antigen. The DUIF antigen band was cut from 6% polyacrylamide gels on a large scale, crushed to a powder and used for immunization. The antisera were increased in rabbits by normal procedures (Harlow and Lane, 1988). For initial immunization with the DU1N antigen, 300 μg of the protein was injected in complete Freund's adjuvant. Booster immunizations of 200 μg of the fusion protein were delivered three times at three week intervals. Immunization with DUIF followed a similar protocol except that approximately 50 μg of antigen was delivered in all four injections.

EXAMPLE 14 Expression of DU1C in E. coli Strains BL21 (DE3) from E. coli containing pHC5 or pHC6 were grown in LBK or LBA medium, respectively. The cultures that grew overnight were inoculated in fresh medium at a 1:10 dilution and grown at 37 ° C until the density was 0.8 A600 l. IPTG was added at 0.5 mM and the cultures were grown for 5 hours at 25 ° C. Cells were harvested by centrifugation, suspended in a 1/20 culture volume of the sonification buffer (50 mM Tris-HCl, pH 7.0, 10% glycerol, 10 mM EDTA, 5 mM DTT, 3% inhibitor cocktail 10X proteinase [Sigma no.

P8465]), and divided by sonification. The used ones were clarified by centrifugation in a microcentrifuge and the supernatants were used for the subsequent analyzes. For the detection of the S-tag sequences, the Rapid Assay S-tag kit (Novagen) was used to measure the activity of the reconstituted ribonuclease A.

EXAMPLE 15 Analysis of the gimogram The analysis of the zymogram was performed practically as described by Buleon et al. (1997) with some modifications. The endosperm of 3-4 seeds was frozen in liquid nitrogen, crushed to a fine powder and suspended by vortex in 50 M Tris-acetate, pH 8.0, 10 mM EDTA, 5 mM DTT (1 ml per gram of fresh weight of the seed) . The crude homogenate was clarified by centrifugation at 10,000 x g for 10 minutes at 4 ° C and the concentration of the protein in the supernatant was determined. Protein samples (225 μg) were boiled in SDS-PAGE buffer (65 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol) and loaded on an 8% acrylamide gel containing 0.1% glycogen . Electrophoresis was performed under denaturing conditions (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.1% SDS, 5 mM DTT) for three hours at 4 ° C to 80 V in a BioRad Mini-Protean II cell. The gel was washed four times for 30 minutes each time at room temperature in 40 M Tris-HCl, pH 7.0, 5 mM DTT to remove the SDS and allow the proteins to renaturate. The gel was then incubated in reaction buffer (Vicien 100 mM, pH 8.0, 0.5 M citrate, 25 mM potassium acetate, 0.5 mg / ml BSA, 5 mM ADPGlc, 5 M 2-mercaptoethanol, 20 mg / ml glycogen) for 36 hours at room temperature. Enzymatic activities were detected by the addition of iodine staining (0.2% iodine and 2% potassium iodide in 10 mM HCl) and the zymograms were photographed immediately.

EXAMPLE 16 Fractionation of corn seed extracts Seeds were harvested from developing ears, immediately frozen in liquid nitrogen and stored at -80 ° C. The frozen seeds were crushed in ice with a mortar and pestle in homogenization buffer (50 mM Tris-HCl, pH 7.0, 10% glycerol, EDTA M, 5 mM DTT, 1 mM PMSF, 50 μl per gram of 10X proteinase inhibitor cocktail tissue [Sigma no. P2714]; 2.5 ml / g of tissue). The homogenate was centrifuged at 10,000 x g for 10 minutes and the supernatant was used for SS tests and determination of protein concentration. To obtain the starch granules, the 10,000 x g package was vigorously vortexed in homogenization buffer and centrifuged again. The third wash package was suspended in homogenization buffer and used as the fraction of the starch granules.

EXAMPLE 17 Glucan synthase assays Glucan synthase assays were performed in icrocentrifuge tubes in a total volume of 0.1 ml. Standard reactions contained 100 mM Bicine-NaOH, pH 8.0, 5 M EDTA, 0.5 M sodium citrate, 0.5 mg / ml BSA, 10 mg / ml glycogen, 1 M ADP- [C] glucose (150 cpm / nmol) ( Amersham No. CFB144) and different amounts of the total soluble extract. The reactions were initiated by the addition of labeled ADPG, incubated for 30 minutes at 30 ° C and terminated by the addition of 1 ml of 75% methanol / 1% KCl. The incorporation of the radioactive label into glucan insoluble in methanol was determined according to Cao and Preiss (1996). All the tests were performed in duplicate or triplicate and the maximum variation observed was approximately 10%. Preliminary experiments showed that the amount of 1C incorporated in methanol precipitable glucan is linear with the amount of protein in the assay. further, approximately 10% of the 14C cpm in the assay was recovered in insoluble glucan. Thus, the tests were carried out under conditions of excess substrate. Some trials varied from the normal protocol by omission of glucan and / or sodium citrate. When glucan was omitted for the assay, it was added to the normal concentration after the reaction was interrupted by the addition of methanol.

EXAMPLE 18 Methods of immunosuppression ß immunoaggestion The concentrations of the protein were determined according to Bradford (1976). SDS-PAGE and the transfer of the protein from the gels to nitrocellulose fibers followed the normal methods (Sambrook et al., 1989). The primary antisera were anti-SSI (Mu et al., 1994) diluted 1: 1000 or 1: 3000, anti-DUIN diluted 1: 10,000 or 1: 75,000, and anti-DUlF diluted 1: 2000. The secondary antibody was goat anti-rabbit IgG-alkaline phosphatase conjugate (Bio-Rad Laboratories) diluted 1: 3000, which was detected using the BCIP / NBT reagent system (Bio-Rad Laboratories). The fusion proteins containing the S-labeled amino acid sequence were detected by the same procedure except that the protein S conjugate alkaline phosphatase (Novagen) diluted 1: 5000 was used in place of the primary antibody. The immunoagglutination experiments were performed as follows: the extracts of total soluble seeds were mixed with an equal volume of immune serum plus pre-immune serum. In all cases the total volume of serum added to the protein extracts was constant; the variable was the immune / preimmune serum ratio in the mixture. The solutions were incubated on ice for 90 minutes with light mixing every 10-15 minutes. Protein A-Sepharose CL-4B (Sigma) (1/10 volume of bead slurry / protein solution) was added. The mixtures were gently agitated continuously for 30 minutes, centrifuged for 10 minutes at 10,000 x g and the supernatants were assayed for SS activity. The packets were washed with buffer three times before the immunoabsorption analyzes of the precipitated proteins.

EXAMPLE 19 Reasons for conserved sequences in DUl and SSs Three blocks of conserved sequences identified above in the comparisons of the different X proteins and glycogen synthase of E. coli (GS) are all present in the C terminal region of DUl. This comparative analysis was extended to include 28 SS or GS sequences of 17 species. 33 residues are conserved in all 28 enzymes. Five motifs of conserved sequences were identified in addition to the three previously mentioned. The eight blocks of conserved sequences are called the I-VIII motifs, in the order from the N terminal to the C terminal; according to this notation, the motives I, VII and VIII correspond to the regions I, II and III, respectively, as previously designated. The conserved sequences are distributed in the 359 DUI residues between positions 1237 and 1595.

EXAMPLE 20 Recombinant protein Dül exhibits SS activity The sequence similarity of DUl with glucan synthases suggests that its C terminal region starting upstream of residue 1237 possesses SS activity. To test this hypothesis, the 449 terminal C residues (position 1226-1674, designated DU1C) were expressed in E. coli from the plasmids pHC5 or pHC6. These plasmids are based on the expression vector pET-29B (+) or pET-32b (+), respectively, and thus produce DU1C fusion proteins containing 35 or 167 residues derived from the N-terminal pi-amide. The expression of the fusion proteins was monitored by enzymatic and immunoabsorption analysis that detected the S-tag sequence present in these N-terminal extensions. Proteins of the expected sizes were expressed specifically when the coding region of DU1C was present (Figure 8). The increase in glucan synthase activity was observed in the total soluble extracts of E. coli cells expressing DU1C. Cells containing pHC5 or pHC6 were exposed to IPTG to induce the expression of the DU1C proteins and the total soluble extracts were tested for glucan synthase activity. Expression of DU1C caused approximately five times increase in SS activity compared to control cells lacking the corn coding region (Table 1). A similar increase also occurred in the reconstituted ribonuclease A activity conferred by the S-tag sequence of the N-terminal expression (data not shown). Almost identical results were obtained when DU1C was expressed in pET-29b (+) or pET-32b (+). The increase in activity relative to the endogenous level was relatively modest, although similar levels were also detected for zSSI expressed in E. coli. In addition, the level of activity of the recombinant enzyme observed for DU1C was comparable with that of potato GBSSII expressed in a similar system. These data provide direct evidence that DUI is a starch synthase and that its 449 terminal C residues are sufficient to provide this enzymatic activity.

TABLE 1 Starch synthase activity in total soluble E. coli extracts Piásmido Insert Specific activity nmol min "g% pET-29b { +) None 2.10 ± 0.29 100 pHC5 DUl-Cb 10.41 ± 1.93 496 pET-32b (+) none 2.88 ± 0.18 100 pHC6 DU1-C 14.19 ± 1.60 493 anmol of glucose incorporated min "1 mg" 1. The values indicate the mean ± of the standard error (n = 4) b units of the total activity obtained for the appropriate vector without adequate insert are assigned with a value of 1 ° pET- 29b (+) and pET-32b (+) are from Novagen, pHC5 and pHC6 are based on these two vectors, residues 1226-1674 of DUl.Genic expression was induced for five hours in E. coli cells in exponential phase transformed with the indicated pyramid.The total soluble extracts were tested for the activity of starch synthase in the presence of citrate initiator and glycogen.Some efforts were made to express full length DUI, however, in all cases the protein was not accumulated as judged in immunoassays b sorption Full-length DUI expressed from pHC4 was found exclusively in the soluble phase of E. coli cells. Growth in the medium shown above to increase the solubility of the recombinant SSs did not result in the expression of soluble DUl. Also unsuccessful were attempts to express full-length DUl as a fusion protein directed to the periplasmic space of E. coli (IBI FLAG expression system, Sigma No. E5769) or Saccharomyces cerevieiae as a putative cytosolic protein (pYES2; Invitrogen; ).

EXAMPLE 21 Immunological detection of DUI in seed extracts The detection of DUI in seed extracts revealed the apparent size of this SS, its pattern of temporal expression and its lack of association with starch granules. Anti-DUIN polyclonal antiserum was increased in rabbits against the 648 N terminal residues of DUl. This region of DUl is unique among the sequences of the known proteins so that it was expected that the anti-DUIN would react specifically with DUI and not with other SSs. Figure 9A shows that in the non-absorption analyzes the extracts of total soluble seeds (ie, the supernatant of 10,000 xg) of the anti-DUIN of non-mutant seeds detected a protein that was equal in apparent molecular weight greater than 200 kD. . This protein was absent in two different mutants of dul -. In seeds homozygous for the reference mutation, dul-Ref, a smaller immunoreactive protein was detected, whereas in seeds homozygous for the allele purportedly induced by the transposon dul-R4059, the protein was completely eliminated (Figure 9A). Identical results were obtained using a different antiserum, anti-DUlF, which was increased against full-length dul. Thus, anti-DU1N and anti-DUlF recognize DUl, the product of the dul gene. The zSSI protein of apparently 76 kD was also identified in immunoabsorption analysis of these same seed extracts, using anti-SSI antiserum (Figure 9A). The anti-DUIN did not recognize SSI, and the anti-SSI did not recognize DUl. In this test, therefore, both antisera react specifically with a different isozyme. It was found that DUl is located mainly in the soluble fraction of the seed extracts contrary to being associated with starch granules. Seeds harvested 20 DDP were fractionated into soluble fractions and granules. The identity of the fraction of the granules was varied by enrichment for zSSI (Figure 9B), which is known to be associated with the granules and be soluble. The amount of DUl present in the granule and soluble fractions was determined by immunoabsorption analysis of standardized protein samples based on the fresh weight of the seed. Contrary to zSSI, the anti-DUIN signal was found almost exclusively in the soluble fraction (Figure 9B), indicating that DU1 is not stably associated with the starch granules in endosperm 20 DDP. The pattern of temporal expression of DUl and SSI in the seeds at different times after pollination was monitored. DUl was detected first at 12 DDP and remained at a nearly constant level during the period of starch biosynthesis to at least 32 DDP (Figure 9C). The anti-SSI produced a signal in the 8 DDP seed extract (Figure 9C) indicating that in these tissue samples, zSSI was expressed earlier than DUI.

EXAMPLE 22 Immunoassay of SS activity in seed extracts Immunostaining experiments investigated the amount of SS activity in the endosperm provided by DUl and zSSI. The total soluble extracts of seeds harvested 20 DDP were tested with anti-DUIN, anti-DUlF, anti-SSI or pre-immune serum. The immune complexes were separated from the solution after binding to protein A-sepharose beads, and the residual SS activity in the supernatant was determined in the presence of citrate and the exogenous initiator. Preliminary experiments entitled the amount of serum; the following data were obtained under conditions of excess antibody. The non-mutant background extracts W64A or Oh43 were depleted of approximately 35-45% of their total SS activity by anti-DUI serum (Figure 10). Anti-SSI depleted 80% and 60% of total SS activity in the two genotypes, respectively. The treatment of the mutant extracts dul-with anti-DUI serum had almost no effect on the total activity of the SS, suggesting that the specific enzyme affected by these sera is specifically the one encoded by Dul. The treatment of the extracts of the sweet anti-SSI mutant exhausted almost all the SS activity. These data demonstrate that the vast majority of SS activity in the soluble fraction of endosperm 20 DDP is provided by a combination of zSSI and DUl.

EXAMPLE 23 Fractionation of SS activities in total endosperm extracts The SS activities present in endosperm 20 DDP were also correlated with the particular cloned cDNAs by a combination of zymogram, immunoabsorption and mutation analysis. These SS were fractionated by SDS-PAGE and detected by their activity on gels after renaturation of the protein. Two activity bands were observed, one greater than 200 kD and the other approximately 76 kD (Figure HA). The sizes of these isozymes correlate approximately with those predicted by the Dul cDNA and the Ssl cDNA, respectively. The immunoabsorption analysis of the same protein samples revealed that the isozyme > 200 kD reacted with the anti-DUIN, while the 76 kD isozyme reacted with anti-zSSI (Figure 11B). The extracts of the endosperm of the mutant dul - completely lacked the activity of the isozyme of > 200 kD. These results suggest that there are two major soluble SSs present in developing endosperm cells, and that one of these is DUl, the product of the dul gene and the other is zSS, and the product of the Ssl cDNA.

EXAMPLE 24 Increase in total SS activity in extracts of the sweet mutant The conclusion that dul specifies an SS disagrees with previous results indicating that the activity of soluble SS does not decrease in a dul-mutant. In this study it was reported that the activity of the total soluble SS increased approximately twice in extracts of dul -Ref mutants; this observation was repeated independently in the current study (Figure 12). The congenic strains were analyzed discarding differences in the genetic background as the explanation for the different total SS levels. A possible explanation for this phenomenon is that an SS different from DUl is overactive in dul- mutants. To test this possibility, SS activity in extracts of total soluble seeds was analyzed in the presence or absence of citrate and / or exogenous glucan initiator. These experiments were tried to differentiate between zSSI, which is known to be significantly stimulated by citrate and is independent of the exogenous initiator, and SSII which is dependent on the initiator and very independent of citrate. The primer-independent SS activity stimulated by citrate was increased by approximately 8-fold in extracts of dul-.Ref "mutants compared to non-mutant congenic extracts (Figure 12). Similar results were obtained for six of other mutants dul The immunoagglutination data described above indicate that the only SS remaining in sweet mutants is zSSI (Figure 10)., it seems that the activity of zSSI is increased in dul- mutants. The stimulation of zSSI activity can not be explained simply by the increase in enzymatic abundance, because the immunoabsorption analysis revealed that the level of zSSI was almost the same in extracts of dl -Ref mutants as in the non-mutant extracts (Figure 9A). To understand the mechanisms of starch biosynthesis, the SS isozymes active in each stage of endosperm development must be identified. Multiple soluble SS are present in the endosperm, as initially demonstrated by biochemical fractionation. Two peaks of activity were observed, designated SSI, which does not require exogenous glucan initiator and is stimulated by citrate, and SSII, which is dependent on the exogenous initiator and very insensitive to citrate. However, five different cDNA clones that code for SS are known, so it is necessary to correlate each enzymatic activity with a specific genetic element. The cDNA coding for zSSI was recently identified, however, the protein (s) responsible for the second activity of the SS has not been clearly assigned before this study. The zSSI is associated with an apparent 76 kD protein. The comparison of the sequences indicates that the cDNA for Ssl codes for this polypeptide, and this cDNA directs the expression of an active SS that is reactive immunologically crossed with zSSI. Thus, the genetic element responsible for the synthesis of zSSI is now identified. It is assumed that at least one additional protein also provides different SS activity in the soluble fraction, due to the enzymatic characteristics and apparent molecular weight of SSII. The detailed characterization of this second enzyme is absent because its purification has proved difficult. The dul gene was proposed to encode a soluble SS activity based in part on the facts that the mutant-lacking mutants lack SSII and that Dul codes for a similar protein in known SS sequences. This study confirms the identification of DUl as an active SS. The expression of the C terminal DUl correlated with the induction of SS activity, and the DUI-specific antibodies immunoagonized a significant portion of the enzyme present in seed extracts. In addition, an enzymatic activity of specific SS identified by zymogram analysis migrated on SDS-PAGE at the same rate as DUl and was absent in a dul-mutant. Taken together, these data identify a second genetic element that specifies a soluble SS. The SS activity of DUl resides within the 450 terminal C residues; the functions of the remaining 1224 residues must be determined. The inferences inferred from immuno-binding data assume that the anti-DUIN is specific for DUI. The immunological specificity was indicated by three observations. First, in the immunosorbent assay, the anti-DUIN did not detect the zSSI (and the anti-SSI did not detect DUl). Second, when the sweetener extracts were treated with anti-DUIN there was no decrease in residual SS activity, although the anti-SSI treatment of the same extracts almost completely reduced activity. Thus, the anti-DUIN does not neutralize the zSSI. Third, anti-DUI and anti-SSI immunoprecipitates were analyzed by immunoabsorption using both antisera; the anti-DUIN complexes did not contain zSSI and vice versa. Most likely, DUl and zSSI represent almost all soluble SS activity in developing seeds. In zymograms two enzymes were observed, and each of these could be correlated with DUl or zSSI. The mutation of dul completely eliminated the larger of the two SS, and the treatment of the extracts with anti-SSI eliminated almost all the remaining soluble SS activity. Although unlikely, the possibility remains that anti-SSI immunoagotes more than an isozyme. However, any of the additional isozymes would have to co-migrate with zSSI in the zymogram gels or they would not renature after SDS-PAGE. In addition, antibodies reactive with any of the known, known SS, zSSIIa or zSSIIb do not detect the polypeptides in soluble seed extracts of 20 DBP. Therefore zSSIIa and zSSIIb, provide activities when much lower in this stage of development, possibly explaining that the residual SS is not eliminated by anti-SSI and a sweet-combined mutation. The conservation of the evolutionary sequence of zSSIIa and zSSIIb with pea and potato SSII, however, suggests that despite their level of expression under these two enzymes they probably provide specific functions in starch biosynthesis. The present study suggests that DUl explains the enzymatic activity of SSII. There are two SS peaks in anion exchange chromatography and two enzymes in zymograms, which is explained very simply by direct correspondence. This correspondence is also indicated by the comparisons in molecular weight: the immunoabsorption indicates that DUl is greater than 200 kD, a native SSII in a study was estimated with 180 kD. The protein > 200 kD detected by anti-DUIN was not present in dul- mutants. What is more interesting, the analysis of the zymogram revealed the existence of an SS > 200 kD that is absent in aulmutants, as is also the case for the chromatography fraction for SSII. All these diverse observations can be explained by the identity between DUl and SSII. The assignment of DUl as a soluble protein of > 200 kD was supported in an independent study. A polypeptide of this size was absent from the purified stromal fraction of the amiloplast of a sweet mutant. This protein is most likely the same as the SS of > 200 kD and the anti-DUIN reactive protein of > 200 kD shown in the present as absent in seeds of dul-. Taken together, these data indicate that DUl, as expected, is located within the plastids. The proteolytically labile nature of DUl may explain the facts that the purification of native SSII has been problematic and different molecular weights of 180 kD and 90 kD have been reported. Immunosorbent assays usually detect DUI as a series of bands with the largest migration to > 200 kD (Figure 9), suggesting proteolytic degradation. Incubation of seed extracts lacking proteinase inhibitors at 0 ° C for about two hours gave rise to an almost complete loss of the full-length DUI immunoabsorption signal again indicating rapid proteolysis. The full-length DUI expressed in E. coli was also unstable, even when the cells were homogenized in the presence of protease inhibitors and lysed directly in SDS-PAGE loading buffer. This phenomenon can be an inherent property of DUl due to its large size, low pl of 4.74 and / or irregular load distribution (for example, residues 1-648 of DUl have a net charge of -50 and a pl of 4.45, while the DU1C fragment has a net charge of -2 and a pl of 7.30). The low total pl of DUl is compared with the values of 6.14-6.98 for other maize SSs. The amount of SS activity depleted by anti-DUl serum may underestimate the prevalence of DUl in vivo, again due to susceptibility to proteolysis. DUl and zSSI share the property that their mobility in SDS-PAGE is slower than predicted from their cDNA sequences. The Ssl cDNA predicts a 64 kD protein, while zSSI runs on gels at 76 kD. The cDNA of dul predicts a 188 kD protein, however, DUI in seed extracts runs significantly slower than the 200 kD marker. The anomalous migration in SDS-PAGE is considered an intrinsic property of zSSI and other SS. The same phenomenon can be applied to DUl, or it could be modified after translation. The elimination of DUl from the soluble fraction of the endosperm apparently causes some change that leads to greater zSSI activity. One possible explanation is that deficiency in DUl causes accumulation of a glucan not normally present, and this provides an efficient initiator for zSSI. This observation explains the fact that total SS activity is not reduced in sweet mutant extracts although a specific SS isozyme is absent. The comparison of DUl with other 27 SS or GS sequences identified conserved residues that can provide clues related to the enzymatic mechanisms of a- (l- »4) bond formation. 33 residues are conserved in all 28 sequences, suggesting that these are important for enzymatic function because they are located within the active site or are required for the maintenance of the catalytic structure. The motif I contains the conserved KT (S) GGL sequence in which it is considered that the plant and both glycines have specific functions in the catalysis. This motif is present in all known glycosyltransferases, as well as other enzymes known to bind ADPG, such as the BT1 transport protein of the amyloplast envelope. Enzymes of the SSIII class, including DUl, are unique among SSs because the second residue of motif I is a variant valine, and that the KTGGL sequence occurs in the VTII motif. The KTGGL sequence also occurs in motif VIII of some prokaryotic glycogen synthases. Although the function of motif VIII must be determined, these data suggest the possibility that in the SSIII class it is also in an ADPG binding site. The IV motif contains a conserved lysine residue that in E. coli GS is known to be involved in the catalysis. This lysine occurs in proximity to some other highly conserved residues in motif IV. The VII motif contains the only cysteine that is conserved in all 28 enzymes, which suggests that it is involved in the binding of ADPG. Chemical modification studies indicated that a cysteine residue mediates the binding of ADPG in GS from E. coli. In this study, cysteine was also involved in glucan binding, however, other starch binding enzymes, such as BE and DBE, do not contain a conserved cysteine. Thus, the cysteine residue in the motif VII can be part of the ADPG binding site. Finally, arginine conserved in motif V is proposed to be involved in the binding of starch. All starch-binding enzymes of the α-amylase superfamily, including BE and DBE, contain a conserved arginine followed by a hydrophobic residue. Chemical modification studies indicated that an arginine is involved in glucan binding by corn BE's, so that this function is also suggested for the arginine of motif V in the glucan synthases. At present the reason why multiple soluble SS are used in storage starch biosynthesis is not known. DUl clearly is distinct from zSSI in that it is located almost completely within the soluble phase of the endosperm cells, whereas zSSI is abundant in the granule and soluble fractions (Figure 9B). The fact that the sweet mutations alter the structure of the starch indicates that DUl provides a specific function (s) that can not be compensated by zSSI. In the same way, severe reduction of potato SSIII by antisense RNA expression causes significant changes in granule structure that can not be compensated by the activity of the remaining soluble SS. Although the specific functions of each soluble SS must be determined, the identification of the genetic sources of the two major isoforms in corn will provide new tools for these investigations. The following references were mentioned in the present: Abel, G.J.W. et al. (nineteen ninety six). Plant J. 10, 981-991. Ausubel, F.M. et al (1989). Current Protocols in Molecular Biology. (NY: John Wiley and Sons). Bae, J.M. et al. 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Wang, Y. -J. et al., (1993b). Cereal Chem 70, 171-179. Any of the patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. In addition, these patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. - A person skilled in the art will readily appreciate that the present invention will be well adapted to realize the objectives and obtain the purposes and advantages mentioned., as well as those objectives, purposes and advantages inherent to it. The present examples, together with the methods, procedures, treatments, molecules and specific compounds described herein are currently representative of the preferred embodiments, are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will be apparent to those skilled in the art, which are encompassed within the spirit of the invention as defined by the scope of the claims.

LIST OF SEQUENCES < 110 > Myers, Alan M. James, Martha G. < 120 > Coding of dulll for a starch synthase and the uses thereof < 130 > D6036PCT < 140 > < 141 > 1998-11-12 < 150 > US 08 / 062,102 < 151 > 1997-11-12 < 160 > 36 < 210 > 1 < 211 > 6027 < 212 > DNA < 213 > corn < 220 > < 223 > sseeccuueenncciiaa ddee cDNA corresponding to the gene coding for the enzyme starch synthase DUl < 400 > 1 gaattcccta gttcagagaa agaaagaagt tgagaatgag aagcaagtga ggcgcgtttg 60 ctgggaagtg gttcttgtga ggtttaggag ttcaccc tc ttttcttccc cttctagaaa 120 tggagatggt cctacggtcg cagagccctc tctgccttcg gagtgggccg gtgctcattt 180 ttcgaccaac cgtcgcgggc ggaggagggg gcactcagtc tttgttgagg actaccagat 240 ttgcgagaag aagggtcatt cgatgcgttg tagcaagtcc aggttgtcct aataggaaat 300 ctaggacagc gtctcccaac gtaaaagtag ctgcttatag caactatgcg ccaagactcc 360 tcgttgagtc aagctccaag aagagcgaac accatgatag cagcagac &C cgtgaagaaa 420 ctattgatac atacaatggg ctgtcaggtt ctgatgcagc agaattgaca agtaatagag 480 atgtagaaat tgaagtggat ttgcagcaca ggaattgcca tttctgagga ggaaaagtat 540 atcattagga cgattaatgc gaaatggaaa cagtggatga agctgaggtc gaggaggata 600 agtttgaggt agatacctca ggaattgtat tgcgcaatgt tgcagttcgg gaagtggatc 660 caaaggatga acataatgct aaagatgtat ttgtggtaga ttcgtcagga actgcaccag 720 ataatgctgc agtggaggaa gtggtagatg aagctgaggt tgaagaggat atggttgatg 780 tggatatctt gggacttgac ttgaataatg caacgatcga ggaaattgat ttgatggaag 840 aggctttact ggagaa cttc gacgtggatt caccaggcaa tgcttctagt ggtcgaacct 900 atgggggtgt ggatgagttg ggtgagctgc cttcaacatc cgtggattgc atcgccatta 960 acggaaaacg tagaagtttg aagcctaagc ccttgccaat tgtcaggttc caggaacaag 1020 aacagatagt tttaagcatt gttgacgaag aagggttgat tgctagttca tgtgaagaag 1080 gccaaccggt ggtagattac gataagcaag aggaaaactc taccgctttc gatgaacaga 1140 agcaattaac tgatgatttc cctgaagaag geatatetat agtteaette cctgagccaa 1200 acaatgatat tgttggatcc tcaaaattct tggagcaaaa acaagaattg gatggttctt 1260 ataaacaaga tcgatcaacc actggattgc atgaacaaga tcagtctgtt gttagttcac 1320 acggacaaga taaatcaatt gttggtgtgc ctcagcaaat ccagtacaat gatcaateta 1380 ttgctggttc tcatagacaa gatcaatcaa ttgccggtgc acctgagcaa atccaatccg 1440 tataaaacca ttgctggcta aatca tc to ttgtaaacaa ttgttggttc catgaattga 1500 gcctaagaaa ttattcctga atcgaatcca tcatcagtta gatcaateta caatgaaata 1560 ttgttggttc tcacaaacaa gacaaatctg ttgttagtgt atccaatcca gcctgagcaa 1620 cagcaaacca ttgttagtca aatcaatcta ctgttgattc ttatagacaa gctgaatcaa 1680 ttattggtgt gcctgagaaa tcaccagtta gtccaatcca cgataaacta gaccagtcca 1740 tcttaaacaa ttgttggttc gatgagccta ttattagcgt atccaatcca gcctgagaaa 1800 ttgtcc atta cactaaacca aatcagtcta gcccaaacaa ttgttggctt caacaatcaa 1860 cgttgaacca ttgttcatat aaacagtcca tagatggttt gatetatcaa ccctaaacaa 1920 tcgttggtat ctccaatgag tttcaaacaa agcaactggc tactgttggg actcatgatg 1980 gattgcttat gaagggtgtg gaagctaagg agacatetca aaagactgaa ggggatacac 2040 ttcaggcaac gttcaatgtc gacaacttgt cacagaaaca ggaaggctta actaaagaag 2100 aacaattatt cagacgagat gagaaaatca atgatgaaga ccttgtgatg attgaagaac 2160 agaaagcat agccatgaat gaagaacaga cgattg ac cgaagaagac attccaatgg 2220 ctaaggttga gacaaggcca gataggaatt aatttttaca tctgctttct gaagaagaga 2280 tgaaaatgaa gttcatggga ttgaggctga gtgggaataa tgaacagtat gaagtcgatg 2340 agacatctat gtccactgaa caagatatcc aggaatcacc taatgatgat ttggatccac 2400 aagcactatg gagtatgctt caagagcttg ctgaaaaaaa ttattcgctg ggaaacaagt 2460 tgt tactta tccagatgta ttgaaagctg attcaacaat tgatetetat ttcaatcgtg 2520 atctatcagc tgtggccaat gagcctgatg tacttatcaa aggagcattc aatgggtgga 2580 tttcactgaa agtggagatt aaattgcaca agagegaget ggcaggggac tggtggtgct 2640 gcaaactata cat tcctaag gaatggactt caggcataca aaggacaca tgtgtttttt 2700 cggtatatga aaataataac aataatgatt tcgtgataca aatagaaagc accatggatg 2760 aaaatttatt tgaggatttc ttggctgaag aaaagcaacg agaacttgag aaccttgcaa 2820 atgaggaagc tgaaaggagg agacaaactg atgagcagcg gcgaatggag gaagaaaggg 2880 ccgcagataa agctgacagg gtacaagcca aggttgaggt agagaegaag aagaataaat 2940 tgtgcaatgt attgggttta gccagagctc ctgttgataa tttatggtac attgag cca 3000 tcacgactgg acaagaggct actgtcagat tgtattataa cataaaetca agaectetag 3060 ttcacagtac tgagatatgg atgcatggtg gctataacaa ttggattgat GGAC cct 3120 ttgctgaaag gcttgttcat cate gaca aagattgtga ttggtggttt gcagatgttg 3180 tcgtgcctga aagaacatat gtattggact gggtttttgc tgacggccca ccagggagtg 3240 caaggaatta tgacaacaat ggaggacatg attttcatgc tacccttcca aataacatga 3300 cbgaggaaga gtattggatg gaagaagaac aaaggatcta tacaaggctt caacaagaga 3360 ggagggaaag ggaggaggct attaaaagga aggctgagag aaatgcaaaa atgaaagctg 3420 agatgaagga aaagactatg agaatgttcc tggtttctca gaaacacatt gtttacaccg 3480 aaccacttga aatacatgct ggaactacta ttgatgtgct ttataatcct tctaatacag 3540 ttctaactgg aaagccagag gtttggtttc gatgttcctt taatcgttgg atgtatccag 3600 gtggggtgtt gccacctcag aagatggtac aagcagaaaa tggttcacac ctaaaagcaa 3660 oagtttacgt tocacgagat gcctatatga tggacttcgt tttctcggag tcagaagaag 3720 tgataacaga gtggaattta aatgggttag actatcatat tcctgttttt gggtcaattg 37B0 caaaggaacc acctatgcac attgtccaca ttgctgttga gatggca cca atcgcaaagg 3840 ttggaggtct tggtgatgtt gtcactagtc tttcacgtgc tgtgcaagat ttaggacaca 3900 atgtggaggt tattcttcca aagtacggtt gcttgaatct aagcaatgtc aagaatctac 3960 aaatccatca gagtttttct tggggtggtt ctgaaataaa tgtgtggcgt ggactagtcg 4020 aaggcctttg tgtttacttc ctggaacctc aaaatgggat gtttggagtc ggatatgtat 4080 atggcaggga cgatgaccgc cgatttggct tcttctgtcg ttctgctcta gagtttctcc 4140 tccaaagtgg atcttctccg aacataatac attgccatga ttggtcaagt gctcctgttg 4200 octggctaca caaggaaaac tacgcgaagt ctagcttggc aaacgcacgg gtggtattca 4260 ocatccacaa tcttgaattt ggagcgcatc atattggcaa agcaatgaga tattgtgata 4320 aagcaacaac tgtctctaat acatattcaa aggaagtgtc aggtcatggt gccatagttc 4380 ctcatcttgg gaaattct & t ggcattctca atggaattga tccggatata tgggatccgt 4440 acaatgacaa. ctttatcccg gtccac aca cttgtgagaa fcgfcggtfcgaa ggcaagaggg 4500 ctgctaagag ggcactgcag cagaagtttg ggttacagca aatcgatgtc cccgtcgtag 4560 tcgcctgaca gaatcgtcac gcccaaaagg ggatccacct gatcaagcat gcgattcacc 4620 acggaacgga gtacactcga caggtggttt tgcttggttc agcgccggac tctcgaatcc 4680 aagctgattt tgtcaacctg gcgaatacgc tccacggcgt aaaccatggg caagtgaggc 4740 tttccttgac ctacgacgag cctctctcgc atctgatata cgctggctct gacttcattc 4800 tggtcccatc tatatttgag ccttgcggcc taactcagct cgtcgccatg cggtatggaa 4860 ccatcccgat tgtccgcaag actggagggc tcttcgacac tgtcttcgat gtggacaatg 4920 acaaggaacg agcccgagat cgaggccttg agcccaacgg gtttagcttt gacggagctg 4920 atagcaacgg tgttgactac gcgctgaaca gggcgatctc agcttggttc gatgcccgga 5040 ctccctttgc gctggttcca aagagagtca tggagcagga ctggtcgtgg aaccgacctg 5100 ccctcgacta catcgagctc taccgttcag cgtccaaatt gtaataatcc aaacaacggc 5160 caatgtagtg tgttgtctgc aggtctcaga tgcagccatt cagcttttgc aggttcctgg 5220 gcattgctgt acagcctcct tgtctttagt tagctccatt ccccgaggag cacagtgcaa 5280 tca ttttttatcc gttatta tgcatagatt gtctcagtag aatgctttct tcgggcatgt 5340 atgtttgttt cctctgttgt tgaattctgg tgttaagtcg cgtataggaa tctacaggaa 5400 atgaaaaagt ccatttcctg cgtcaacctt ttagggctac catgcacatg agacctttca 546C agtgcaaaga atattaggac tagactacta gtatgtgaac tctatttttc caagagattt 5520 caatttttcc aatgaaaaat aaactaattt ttcttggaaa aatggaaatc ccttggaaaa 5580 atggggttcc caaactagec cgtagagtat agatcataga attggtctag tggttcctcg 5640 agagagaaaa aaacatagac ttttcttgtc atatgcttat ttaagtttat tttgtacaaa 5700 ctttgagaac cttcaaaaac accccaatgg ctggttaagt gaccagggaa ataaagagga 5760 tctataggga ggaatccccc gcctctctct cacagatgtt gcctagcacc ggccagcctc 5820 atccgtccag tggaattaag gttggttgcg aogacagccc tcaatggaa accaacctcg 5880 tgccccgtgc cgggatctac cttccttect caccaccacg ccgatctcac cttccatagg 5940 agcttcctat gcactgttac ctattatagg tacatgacat tgtacatctt tgtatgaact 6000 tacatcaatg ccaaaaatcc ggaattc 6027 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_union < 223 > dul-spl primer used to amplify the F500 fragment from pJW3 < 400 > 2 gtacaatgac aactttatcc c 21 < 210 > 3 < 211 > 23 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_union < 223 > initiator dul-sp2 ut fragment F500 from pJW3 < 400 > 3 cattctcaca agtgtagtgg acc 23 < 210 > 4 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_ union < 223 > initiator dul-sp4 ut cloned BamHI fragment < 400 > 4 gtcgtaggaa tcgtcactcg 20 < 210 > 5 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_union < 223 > initiator? 030 i EcoRI cloning site in left arm in phage DNA? gtll used to amplify cDNA inserts < 400 > 5 attggtggcg acgactcctg í 20 < 210 > 6 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_union < 223 > initiator? 356 or EcoRI cloning site in the LacZ 'region in the right arm in the phage DNA? gtll used to amplify cDNA inserts < 400 > 6 gtgtgggggt gatggcttc 20 < 210 > 7 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_ union < 223 > du-F3 initiator useful from Dul < 400 > 7 ataaatgtgt ggcgtggact 20 < 210 > 8 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > initiator_ union < 223 > 'Initiator du-Rl useful from Dul < 400 > 8 cgttccttgt cattgtccac 20 < 210 > 9 < 211 > 6 < 212 > PRT < 213 > corn < 220 > < 222 > REPE < 223 > sseeccuuence box M of six residues in the first semirepector of the sequence of 10 residues of the SBE repeat in DUl < 400 > 9 sp Gln Ser lie Val Gly 1 5 < 210 > 10 < 211 > 4 < 212 > PRT < 213 > corn < 220 > < 222 > REPE < 223 > sseeccuuence of four residues of the second semi-repetition of the repeat sequence SBE of 10 residues in DUl < 400 > 10 Ser His Lys Gln 1 < 210 > 11 < 211 > 6 < 212 > PRT < 213 > unknown < 220 > < 222 > REPETITION < 223 > sequence d < 400 > 11 sp Gln Ala Leu Val Gly 1 5 < 210 > 12 < 211 > 1674 < 212 > PRT < 213 > corn < 220 > < 223 > amino acid sequence deduced from starch synthase DUl < 400 > 12 Met Glu Met Val Leu? Rg Ser Gln Ser Pro Leu Cys Leu? Rg Ser 1 5 10 15 Gly Pro Val Leu He Phe Arg Pro Thr Val Wing Gly Gly Gly Gly 20 25 30 Gly Thr Gln Ser Leu Leu Arg Thr Thr Arg Phe Ala? Rg Arg? Rg 35 40 45 Val lie? Rg Cys Val Val? The Pro Pro Gly Cys Pro? Sn Arg Lys 50 55 60 Ser Arg Thr Ala Ser Pro Asn Val Lys Val Ala Ala Tyr Ser Asn 65 70 75 Tyr Ala Pro Arg Leu Leu Val Glu Ser Ser Ser Lys Lys Ser Glu 80 85 90 His His Asp Being Ser Arg His Arg Glu Glu Thr He Asp Thr Tyr 95 100 105 Asn Gly Leu Ser Gly Ser Asp Wing Wing Glu Leu Thr Ser Asn Arg 110 115 120 Asp Val Glu He Glu Val? Sp Leu Gln His He Ser ßlu Glu ßlu 125 130 135 Leu Pro Gly Lys Val Ser Xle Asn Wing Ser Leu Gly ßlu Met ßlu 140 145 150 Thr Val? Sp Glu Ala Glu Val Glu Glu? Sp Lys Phe Glu Val? Sp 155 160 165 Thr Ser ßly He Val Leu? Rg Asn Val? The Val Arg Glu Val Asp 170 175 180 Pro Lys Asp Glu Rls Asn Wing Lys? Sp Val Phe Val Val Asp Ser 185 190 195 Ser Gly Fhr? Pro? Asn? The? Val Glu Glu Val Val? Sp 200 205 210 ßlu? the Glu Val Glu Glu? sp Met Val? sp Val? sp He Leu Gly 215 220 225 Leu? Sp Leu Asn Asn? The Thr He Glu Glu He Asp Leu Met Glu 230 235 240 ßlu Ala Leu Leu Glu Asn Phe Asp Val Asp Ser Pro Gly Asn? la 245 250 255 Being Ser Gly Arg Thr Tyr Gly Gly Val Asp Glu Leu Gly Glu Leu 260 265 270 Pro Ser Thr Ser Val Asp Cys lie Wing He Asn Gly Lys Arg Arg 275 280 285 Ser Leu Lys Pro Lys Pro Leu Pro He Val Arg Phe Gln Glu Gln 290 295 300 ßlu Gln He Val Leu Ser He Val Asp Glu Glu Gly Leu He Ala 305 310 315 Ser Ser Cyß Glu ßlu ßly ßln Pro Val Val? Sp Tyr? Sp Lys GI? 320 325 330 Glu Glu? Sn Ser Thr Wing Phe? Sp Glu Gln Lys Gln Leu Thr Asp 335 340 345 ? Phe Pro ßlu Glu Gly He S r He Val His Phe Pro Glu Pro 350 355 360 ? sn? sn? sp Ha Val ßly Ser Ser Lys Phe Leu Glu Gln Lys Gln 365 370 375 ßlu Leu? sp Gly Ser Tyr Lys Gln? sp? rg Ser Thr Thr Gly Leu 380 385 390 Hiß Glu Gln? Sp Gln Ser Val Val Ser Ser His Gly Gln? Sp Lys 395 400 405 Ser He Val Val Val Gln Gln Gln Ha Gln Tyr? Sn? Sp Gln Ser 410 415 420 He Wing Gly Ser His Ring Gln Asp Gln Ser He Wing Gly Wing Pro 425 430 435 Glu Gln Ha Gln Ser Val? La Gly and He Lys Pro Asn Gln Ser 440 445 450 He Val Gly Ser Cys Lys Gln His Glu Leu He He Pro Glu Pro 455 460 465 Lys Lys He Glu Ser He He Ser Tyr? Sn Glu He? Sp Gln Ser 470 475 480 He Val Gly Ser His Lys Gln Asp Lys Ser Val Val Ser Val Pro 485 490 495 ßlu Gln He Gln Sar He Val Ser Rls Ser Lys Pro? sn Gln Ser 500 505 510 Thr Val? Sp Ser Tyr Arg Gln Wing Glu Ser Ha Ha ßly Val Pro 515 520 525 ßlu Lys Val Gln Ser Ha Thr Ser Tyr? sp Lys Leu? sp Gln Sar 530 535 540 He Val Gly Ser Leu Lys Gln? Sp Glu Pro He He Sar Val Pro 545 550 555 ßlu Lys He ßln Ser He Val His Tyr Thr Lys Pro? sn Gln Ser 560 565 570 Has Val Gly Leu Pro Lys Gln ßln Gln Ser He Val His He Val 575 580 585 ßlu Pro Lys Gln Sex He? s Gly Phe Pro Lys Gln? sp Leu Ser 590 595 600 He Val Gly He Ser Asn ßlu Phe ßln Thr Lys Gln Leu? Thr 605 610 615 Val Gly Thr His Asp Gly Leu Leu Met Lys Gly Val Glu Ala Lys 620 625 630 Glu Thr Ser Gln Lys Thr Glu Gly Asp Thr Leu Gln Wing Thr Phe 635 640 645 Asn Val Asp Asn Leu Ser Gln Lys Gln Glu Gly Leu Thr Lys Glu 650 655 660 Wing Asp Glu He Thr He He Glu Lys He? Sn? Sp Glu Asp Leu 665 670 675 Val Met He Glu Glu ßln Lys Sar Ha? The Met? Sn ßlu ßlu ßln 680 685 690 Thr Ha Val Thr ßlu ßlu? Sp He Pro Met? The Lys Val ßlu He 695 700 705 ßly Ha? sp Lys? the Lys Phe Leu His Leu Leu Sar ßlu Glu Glu 710 715 720 Sar Ser Trp Asp Glu? Sn Glu Val Gly Ha He Glu Wing Asp Glu 725 730 735 ßln Tyr ßlu Val? sp Glu Thr Ser Met Ser Thr Glu Gln? sp He 740 745 750 ßln ßlu Ser Pro Asn Asp? sp Leu? sp Pro ßln? the Leu Trp Ser 755 760 765 Mßt Leu Gln ßlu Leu Ala ßlu Lys? Sn Tyr Ser Leu Gly Asn Lys 770 775 780 Leu Phe Thr Tyr Pro? Sp Val Leu Lys? La? Sp Ser Thr He Asp 785 790 795 Leu Tyr Phe? Sn Arg? Sp Leu Ser? The Val? La? Sn Glu Pro Asp 800 805 810 Val Leu He Lys Gly Wing Phe Asn Gly Trp Lys Trp Arg Phe Phe 815 820 825 Thr Glu Lys Leu His Lys Ser Glu Leu Wing Gly Asp Trp Trp Cys 830 835 840 Cys Lys Leu Tyr He Pro Lys Gln? The Tyr? Rg Met? Sp Phe Val 845 850 855 Phe Phe? Sn Gly His Thr Val Tyr Glu? Sn? Sn? Sn? Sn? Sn? Sp 860 865 870 Phe Val He Gln He ßlu Ser Thr Met? Sp ßlu? Sn Leu Phe Glu 875 880 885 ? Phe Leu? ßlu ßlu Lys ßln? rg Glu Leu Glu? sn Leu? 890 895 900 ? sn Glu ßlu? the Glu? rg? rg? rg Gn Thr? sp Glu Gl? rg? rg 905 910 915 Met ßlu Glu Glu? Rg? The? La? Sp Lys? The? Sp? Rg Val Gln? The 920 925 930 Lys Val ßlu Val ßlu Thr Lys Lys? Sn Lys Leu Cys? Sn Val Leu 935 940 945 Gly Leu? La? Rg? The Pro Val? Sp? Sn Leu Trp Tyr He Glu Pro 950 955 960 He Thr Thr Gly Gln Glu? The Thr Val Arg Leu Tyr Tyr Asn He 965 970 975 ? sn Ser? rg Pro Leu Val Hls Ser Thr Glu He Trp Met His Gly 980 985 990 Gly Tyr Asn? Sn Trp He? Sp Gly Leu Ser Phe? The Glu Arg Leu 995 1000 1005 Val His His Asp Lys Asp Cys Asp Trp Trp Phe Wing Asp Val 1010 1015 1020 Val Val Pro Glu Arg Thr Tyr Val Leu Asp Trp Val Phe Wing Asp 1025 1030 1035 Gly Pro Pro Gly Ser? La? Rg? Sn Tyr Asp Asn? Sn Gly Gly His 1040 1045. 1050 ? sp Phe His? the Thr Leu Pro? sn? sn Met Thr ßlu Glu Glu Tyr 1055 1060 1065 Trp Met Glu Glu Glu Gln Arg He Tyr Thr? Rg Leu ßln ßln ßlu 1070 1075 1080 ? rg? rg Glu? rg Glu Glu? the He Lys? rg Lys? the Glu? rg Asn 1085 1090 1095 Wing Lys Mat Lys Wing Glu Met Lys Glu Lys Thr Met Arg Met Phß 1100 1105 1110 Leu Val Ser ßln Lys His Jim Val Tyr Thr Glu Pro Leu Glu He 1115 1120 1125 His Wing Gly Thr Thr He? Sp Val Leu Tyr? Sn Pro Ser? Sn Thr 1130 1135 1140 Val Leu Thr Gly Lys Pro Glu Val Trp Phe? Rg Cys Ser Phe? Sn 1145 1150 1155 ? rg Trp Met Tyr Pro Gly Gly Val Leu Pro Pro Gln Lys Met Val 1160 1165 1170 Gln? Glu? Sn Gly Ser His Leu Lys? The Thr Val Tyr Val Pro 1175 1180 1185 Arg Asp Wing Tyr Met Met Asp Phe Val Phe Ser Glu Ser Glu Glu 1190 1195 1200 Gly Gly He iyr Asp Asn Arg Asn Gly Leu Asp Tyr His He Pro 1205 1210 1215 Val Phe Gly Ser He Wing Lys Glu Pro Pro Met His He Val His 1220 1225 1230 I have Ala Val Glu Met the Pro He? The Lys Val ßly and Gly Leu ßly 1235 1240 1245 ? sp to the Val Thr Ser Leu Ser? rg? the Val Gln? sp Leu ßly His 1250 1255 1260 ? sn Val ßlu Val He Leu Pro Lys Tyr ßly Cys Leu? sn Leu Ser 1265 1270 1275 ? sn Val Lys? sn Leu Gln He His Gln Ser Phe Ser Trp Gly Gly 1280 1285 1290 Ser ßlu lie? Sn Val Trp Arg Gly Leu Val Glu Gly Leu Cys Val 1295 1300 1305 Tyr Phe Leu Glu Pro Gln Asn Gly Met Phe Gly Val Gly Tyr Val 1310 1315 1320 Tyr Gly? Rg? Sp? Sp Asp Arg Arg Phe Gly Phe Phe Cys Arg Ser 1325 1330 1335 Ala Leu Glu Phe Leu Leu Gln Ser Gly Ser Ser Pro Asn He He 1340 1345 1350 HIS Cys His Asp Trp Ser Ser Ala Pro Val Ala Trp Leu His Lys 1355 1360 1365 Glu? Sn Tyr? La Lys Ser Ser Leu? La? Sn Ala Arg Val Val Phe 1370 1375 1380 Thr He His Asn Leu Glu Phe Gly Wing His His lie Gly Lys Wing 1385 1390 1395 Met? Rg Tyr Cys Asp Lys Wing Thr Thr Val Ser Asn Thr Tyr Ser 1400 1405 1410 Lys Glu Val Ser Gly His Gly Wing He Val Pro His Leu Gly Lys 1415 1420 1425 Phß Tyr Gly He Leu Asn Gly He? Sp Pro? Sp Ha Trp? Sp Pro 1430 1435 1440 Tyr? Sn? Sp? Sn Phe He Pro Val His Tyr Thr Cys Glu? Sn Val 1445 1450 1455 Val Glu Gly Lys? Rg? The? The Lys? Rg? The Leu Gln ßln Lys Phe 1460 1465 1470 Gly Leu Gln Gln Ha? Sp Val Pro Val Val ßly He Val Val? Rr 1475 1480 1485 Leu Thr? The Gln Lys Gly He His Leu He Lys His? La He His 1490 1495 1500 Arg Thr Leu Glu Arg Asn Gly Gln Val Val Leu Leu Gly Ser Wing 1505 1510 1515 Pro Asp Ser Arg He Gln? The? Ep Phe Val? Sn Leu? The Asn Thr 1520 1525 1530 Leu His Gly Val Asn His Gly Gln Val? Rg Leu Ser Leu Thr Tyr 1535 1540 1545 ? Glu Pro Leu Ser His Leu He Tyr? the Gly Ser? sp Phe He 1550 1555 1560 Leu Val Pro Be He Phe Glu Pro Cys Gly Leu Thr Gln Leu Val 1565 1570 1575? The Met? Rg Tyr Gly Thr He Pro He Val? Rg Lys Thr Gly Gly 1580 1585 1590 Leu Phß? Sp Thr Val Phe? Ep Val? Sp? Sn? Sp Lys Glu? Rg? La 1595 1600 1605 ? rg? sp? rg Gly Leu Glu Pro? sn Gly Phe Ser Phe? sp Gly? la 1610 1615 1620 ? sp Ser? sn Gly Val? sp Tyr? la Leu? sn? rg? la He Ser Ala 1625 1630 1635 Trp Phe? Sp? La? Rg Ser Trp Phe His Ser Leu Cys Lys? Rg Val 1640 1645 1650 Met Glu Gln? Sp Trp Ser Trp? Sn? Rg Pro? La Leu? Sp Tyr He 1655 1660 1665 < 210 > 13 < 211 > 60 < 212 > . PRT < 213 > corn < 220 > < 221 > 418..477 < 222 > REPETITION < 223 > SBE superrepetition of the first 60 amino acid residues of the 180 amino acid repeating residues in DUl < 400 > 13 Asp Gln Ser He Wing Gly Ser His Arg Gln? Sp Gln Ser He? La 1 5 10 15 Gly Ala Pro Glu Gln He Gln Ser Val Ala Gly Tyr He Lys Pro 20 25 30 sn Gln Ser Ha Val Gly Ser Cys Lys Gln His Glu Leu He He 35 40 45 Pro Glu Pro Lys Lys He Glu Ser He He Ser Tyr Asn Glu He 50 55 60 < 210 > 14 < 211 > 60 < 212 > PRT < 213 > corn < 220 > < 222 > REPETITION < 221 > 478 .537 < 223 > - SBE superrepetition of the second 60 amino acid residues of the 180 amino acid repeat residues in DUl < 400 > 14 ? sp Gln Sar He Val Gly Ser His Lys Gln? sp Lys Ser Val Val 1 5 10 15 Ser Val Pro Glu Gln He Gln Be He Val Ser His Ser Lys Pro 20 25 30 ? sn Gln Sex Thr Val Pro Ser Tyr? rg Gln? the Glu Ser He He 35 40 45 ßly Val Pro Glu Lys Val Gln Ser He Thr Ser Tyr? sp Lys Leu 50 55 60 < 210 > 15 < 211 > 60 < 212 > PRT < 213 > corn < 220 > < 222 > REPETITION < 221 > 438 .597 < 223 > Superrepetition SBE of the third 60 amino acid residues of the repeating residues of 180 amino acids in DUl < 400 > fifteen Asp Gln Ser He Val Gly Ser Leu Lys Gln Asp Glu Pro He He 1 5 10 15 Ser Val Pro Glu Lys He Gln Ser He Val His Tyr Thr Lys Pro 20 25 30 ? sn Gln Ser He Val ßly Leu Pro Lys Gln Gln Gln Ser He Val 35 40 45 His He Val ßlu Pro Lys ßln Ser He? Sp Gly Phe Pro Lys Gln 50 55 60 < 210 > 16 < 211 > 10 < 212 > PRT < 213 > corn < 220 > < 221 > 478..587 < 222 > REPETITION < 223 > Repetition sequence SBE in DUl < 400 > 16 ln Ser lie Val Gly Ser His Lys Gln 5 10 < 210 > 17 < 211 > 10 < 212 > PRT < 213 > corn < 220 > < 221 > 438. .547 < 222 > REPETITION < 223 > Repetition sequence SBE in DUl < 400 > 17 ln Ser He Val Gly Ser Leu Lys Gln 5 10 < 210 > 18 < 211 > 10 < 212 > PRT < 213 > corn < 220 > < 221 > 448. < 222 > REPE < 223 > sseeccuuencia of the repetition SBE in DUl < 400 > . 1188 ln Ser He Val Gly Ser Cys Lys Gln 5 10 < 210 > 19 < 211 > 10 < 212 > PRT < 213 > corn < 220 > 1 < 221 > 568. .577 < 222 > REPETITION < 223 > Repetition sequence SBE in DUl < 400 > 19 ln Ser He Val Gly Leu Pro Lys Gln 5 10 < 210 > 20 < 211 > • 10 < 212 > PRT < 213 > corn < 220 > < 221 > 418. < 222 > REPE < 223 > sseeccuuencia of the repetition SBE in DUl < 400 > 2200 ln Ser He Wing Gly Ser His Arg Gln 5 10 < 210 > 21 < 211 > 10 < 212 > PRT < 213 > corn < 220 > < 221 > 428..437 < 222 > REPETITION < 223 > Repetition sequence SBE in DUl < 400 > 21 ln Ser He Wing Gly Ala Pro Glu Gln 5 10 < 210 > 22 < 211 > 10 < 212 > PRT < 213 > corn < 220 > < 221 > 404. < 222 > REPE < 223 > sseeccuuencia of the repetition SBE in DUl < 400 > 2222 ys Be He Val Val Val Gly Gln Gln 5. 10 < 210 > 23 < 211 > 10 < 212 > PRT < 213 > corn < 220 > < 22l > "598..607 < 222 > REPETITION < 223 > Repeat sequence SBE in DUl < 400 > 23 eu Ser He Val Gly Asn Glu Phe Gln 5 10 < 210 > 24 < 211 > 30 < 212 > PRT < 213 > corn < 220 > < 221 > 529,553 < 222 > REPETITION < 223 > sequence of the M box conserved in SBEI of corn < 400 > 24 Lys Cyß lie? Tyr? Glu Ser His? Ep Gln Ser He Val Gly 1 5 10 15 Asp Lys Thr He? The Phe Leu Leu Met? Sp 25 30 < 210 > 25 < 211 > 25 < 212 > PRT < 213 > < 220 > < 221 > 529,553 < 222 > REPETITION < 223 > sequence of the M box conserved in SBEII of pea < 400 > 25 Lys Cys Val Ser Tyr? Glu Ser Hls Asp Gln Sex He Val Gly 1 5 10 15 ? sp Lys Thr He? the Phe Leu Leu Met Asp 20 25 < 210 > 25 [sic] < 211 > 10 < 212 > PRT < 213 > < 220 > < 221 > 529.. 553 < 222 > REPETITION < 223 > sequence of the M box preserved in wheat SBEI < 400 > 26 Lys Cys He Wing Tyr Wing Glu Ser His Asp Gln Ser He Val Gly 1 5 10 15 sp lys tyr met wing phe leu leu met asp 20 25 < 210 > 27 < 211 > 25 < 212 > PRT < 213 > corn < 220 > < 221 > 572,596 < 222 > REPETITION < 223 > sequence of the M box conserved in corn SBEIa < 400 > 27 Lys Cys Val Thr Tyr Cys Glu Ser His? Sp Gln? The Leu Val Gly 1 5 10 15 ? sp Lys Thr He? the Phe Trp Leu Met? sp 20 25 < 210 > 28 < 211 > . 15 < 212 > PRT < 213 > corn < 220 > < 221 > 572,596 < 222 > REPETITION < 223 > sequence of the M box conserved in corn SBEIIb < 400 > 28 Lys Cys Val Thr Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly 1 5 10 15 < 210 > 29 < 211 > 25 < 212 > PRT < 213 > < 220 > < 221 > 572,596 < 222 > REPETITION < 223 > sequence of the box M preserved in pea SBEI < 400 > 29 Lys Cys Val Val Tyr Cys Glu Ser His? Sp Gln? The Leu Val Gly 1 5 10 15? Sp Lys Thr Met? The Phe Leu Leu Met? Sp 20 25 < 210 > 30 < 211 > 25 < 212 > PRT < 213 > < 220 > < 221 > 477..501 < 222 > REPETITION < 223 > box sequence M conserved in yeast GLC3 glycogen synthase < 400 > 30 Lys Val Val Ala Tyr Cys Glu Ser His? Sp Gln Ala Leu Val Gly 1 5 10 15? Sp Lys Ser Leu? The Phe Trp Leu Met? Sp 20 25 < 210 > 31 < 211 > 25 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > 477..501 < 222 > REPETITION < 223 > sseeccuueenncciiaa ddee M preserved in human liver glycogen synthase < 400 > 31 Lys Cys He Ala Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly 1 5 10 15 asp lys thr leu wing phe trp leu met asp 20 25 < 210 > 32 < 211 > 27 < 212 > PRT < 213 > corn < 220 > < 221 > 150..177 < 222 > REPETITION < 223 > second repeat of 28 amino acid residues of the repeat of 85 residues at the N terminal of DUl [sic] < 400 > 32 Glu Thr Val Asp Glu Ala Glu Val Glu Glu Asp Lys Phe Glu Val 1 5 10 15 ? sp Thr Ser Gly He Val Leu Arg? in Val? la Val? rg 20 25 < 210 > 33 < 211 > 29 < 212 > PRT < 213 > artificial sequence < 220 > < 221 > 178..205 < 222 > REPETITION < 223 > second repeat of 28 amino acid residues of the repeat of 85 residues at the N terminal of DUl < 400 > 33 Glu Val Asp Pro Lys Asp Glu His Asn Ala Lys Asp Val Phe Val 1 5 10 15 Val Asp Being Ser Gly Thr Ala Pro Asp Asn Ala Ala Val Glu 20 25 < 210 > 34 < 211 > 27 < 212 > PRT < 213 > corn < 220 > < 221 > 206. .233 < 222 > REPETITION < 223 > third repeat of 28 amino acid residues of the repeat of 85 residues at the N terminal of DUl < 400 > 34 Glu Val Val Asp Glu Ala Glu Val Glu Glu Asp Met Val Asp Val 1 5 10 15 ? sp He Leu Gly Leu? sp Leu? sn? sn? the Thr He 20 25 < 210 > 35 < 211 > 1230 < 212 > PRT < 213 > potato < 220 > < 223 > amino acid sequence deduced from potato starch synthase SSIII < 400 > 35 Met Asp Val Pro Phe Pro Leu His Arg Ser Leu Ser Cys Thr Ser 1 5 10 15 Val Ser Asn Ala He Thr His Leu Lys He Lys Pro He Leu Gly 20 25 30 Phe Val Ser His Gly Thr Thr Ser Leu Ser Val Gln Ser Ser Ser 35 40 45 Trp Arg Lys Asp Gly Met Val Thr Gly Val Ser Phe Ser He Cys 50 55 60 Wing Asn Phe Ser Gly Arg Arg Arg Arg Lys Val Ser Thr Pro Arg 65 70 75 Ser Gln Gly Be Pro Pro Lye Gly Phe Pro Pro Arg Lys Pro Ser 80 85 90 Gly Met Ser Thr Gln Arg Lys Val Gln Lye Ser Asn Gly? Sp Lys 95 100 105 Glu Ser Lys Ser Thr Ser Thr Ser Lys Glu Ser Glu Ser Ser? Sn 110 115 120 Gln Lys Thr Val Glu Ala Arg Val Glu Thr Ser Asp Asp Asp Thr 125 130 135 Lys Gly Val Val Arg Asp His Lys Phe Leu Glu Asp Glu Asp Glu 140 145 150 He Asn Gly Being Thr Lys Being He Being Met Being Pro Val Arg Val 155 160 165 Being Ser Gln Phe Val Glu Ser Glu Glu Thr Gly Gly Asp Asp Lys 170 175 180 Asp Ala Val Lys Leu Asn Lys Ser Lys Arg Ser Glu Glu Ser Gly 185 190 195 Phe He He Asp Ser Val He Arg Glu Gln Ser Gly Ser Gln Gly 200 205 210 Glu Thr Asn Ala Ser Ser Lys Gly Ser His Wing Val Gly Thr Lys 215 220 225 Leu Tyr Glu He Leu Gln Val Asp Val Glu Pro Gln Gln Leu Lys 230 235 240 Glu Asn Asn Wing Gly Asn Val Glu Tyr Lys Gly Pro Val Wing Ser 245 250 25b Lys Leu Leu Glu He Thr Lys Wing Ser Asp Val Glu His Thr Glu 260 265 270 Ser? Sn Glu He? Sp? Sp Leu? Sp Thr? Sn Ser Phe Phe Lys Ser 275 280 285 Asp Leu He Glu Glu Asp Glu Pro Leu Ala Wing Gly Thr Val Glu 290 295 300 Thr Gly Aep Ser Ser Leu Asn Leu Arg Leu Glu Met Glu Ala Asn 305 310 315 Leu Arg Arg Gln Wing He Glu Arg Leu Wing Glu Glu Asn Leu Leu 320 325 330 Gln Gly He Arg Leu Phe Cys Phe Pro Glu Val Val Lys Pro Asp 335 340 345 Glu Asp Val Glu He Phe Leu Asn Arg Gly Leu Ser Thr Leu Lys 350 355 360 Asn Glu Be Asp Val Leu He Met Gly Wing Phe Asn Glu Trp Arg 365 370 375 Tyr Arg Ser Phe Thr Thr Arg Leu Thr Glu Thr His Leu Asn Gly 380 385 390 Asp Trp Trp Ser Cys Lys He Hie Val Pro Lys Glu Wing Tyz Arg 395 400 405 Wing Asp Phe Val Phe Phe Asn Gly Gln Asp Val Tyr Asp Asn Asn 410 415 420 Aep Gly Asn Asp Phe Ser He Thr Val l, ye Gly Gly Met Gln He 425 43C 435 He Asp Phe Glu Asn Phe Leu Leu Glu Glu Lys Trp Arg Glu Gln 440 445 450 ßlu Lys Leu Wing Lys Glu Gln Wing Glu Arg Glu? rg Leu? Glu 455 460 465 Glu Gln Arg? Rg He Glu? Glu Lys? Glu He Glu Ala? Sp 470 475 480 ? rg? the Gln? the Lys Glu Glu Ala Wing Lys Lys Lys Lys Val Leu 485 490 495 ? rg Glu Leu Met Val Lys Wing Thr Lys Thr Arg Asp He Thr Trp 500 505 510 Tyr He Glu Pro Ser Glu Phe Lye Cys Glu Asp Lys Val Arg Leu 515 520 525 Tyr yr Asn Lys Ser Ser Gly Pro Leu Ser His Wing Lys Asp Leu 530 535 540 Trp He His Gly Gly Tyr Asn Asn Trp Lye Asp Gly Leu Ser He 545 550 555 Val Lye Lys Leu Val Lys Ser Glu Arg He Asp Gly Aep Trp Trp 560 565 570 Tyr Thr Glu Val Val He Pro Asp Gln Ala Leu Phe Leu Asp Trp 575 580 585 Val Phe Wing Asp Gly Pro Pro Lys His Wing He Wing Tyr Asp Asn 590 595 600 Asn His Arg Gln Asp Phe His Wing He Val Pro Asn His He Pro 605 610 615 Glu Glu Leu Tyr Trp Val Glu Glu Glu His Gln He Phe Lys Thr 620 625 630 Leu Gln Glu Glu Arg Arg Leu Arg Glu Wing Wing Met Arg Ala Lys 635 640 645 Val Glu Lys Thr Ala Leu Leu Lys Thr Glu Thr Lys Glu Arg Thr 650 655 660 Met Lys Ser Phe Leu Leu Ser Gln Lys His Val Val Tyr Thr Glu 665 670 675 Pro Leu Aep He Gln Wing Gly Be Ser Val Thr Val Tyr Tyr Asn 680 685 690 Pro Ala Asn Thr Val Leu Asn Gly Lys Pro Glu He Trp Phe Arg 695 700 705 Cys Ser Phe Asn Arg Trp Thr His Arg Leu Gly Pro Leu Pro Pro 710 715 720 ßln Lyß Met Sex Pro Wing Glu Aen Gly Thr His Val Arg Wing Thr 725 730 735 Val Lys Val Pro Leu Asp Wing Tyr Met Met Asp Phe Val Phe Ser 740 745 750 Glu Arg Glu Asp Gly Gly He Phe Asp Asn Lys Ser Gly Met Asp 755 760 765 Tyr His He Pro Val Phe Gly Val Val Lys Glu Pro Pro Met 770 775 780 His He Val His He Wing Val Glu Met Wing Pro Pro Wing Ala Lye Val 785 790 795 Gly Gly Leu Gly Asp Val Val Thr Ser Leu Ser Arg Ala Val Gln 800 805 810 Asp Leu Asn His Asn Val Asp He He Leu Pro Lys Tyr Asp Cys 815 820 825 Leu Lys Met Asn Asn Val Lys Asp Phe Arg Phe His Lys? Sn Tyr 830 835 840 Phe Trp Gly Gly Thr Glu He Lys Val Trp Phe Gly Lys Val Glu 845 850 855 Gly Leu Ser Val Tyr Phe Leu Glu Pro Gln? Sn Gly Leu Phe Ser 860 865 870 Lye Gly Cys Val Tyr Gly Cys Ser Asn Asp Gly Glu Arg Phe Gly 875 880 885 Phe Phe Cys His? The Wing Leu Glu Phe Leu Leu Gln Gly Ghe Phe 890 895 900 Ser Pro Asp He He Hie Cys His Aep Trp Ser Ser Ala Pro Val 905 910 915 Wing Trp Leu Phe Lys Glu Gln Tyr Thr Hie Tyr Gly Leu Ser Lys 920 925 930 Being Arg He Val Phe Thr He His Asn Leu Glu Phe Gly Wing Asp 935 940 945 Leu He Gly Arg Wing Met Thr Asn Wing Asp Lys Wing Thr Tr Val 950 955 960 Ser Pro Thr Tyr Ser Gln Glu Val Ser Gly Asn Pro Val He Wing 965 970 975 Pro His Leu His Lys Phe His Gly He Val Asn Oly He Asp Pro 980 985 990? Sp He Trp Asp Pro Leu Asn Asp Lys Phe He Pro He Pro Tyr 995 1000 1005 Thr Ser Glu Asn Val Val Glu Gly Lys Thr Ala Wing Lys Glu Wing 1010 1015 1020 Leu Gln Arg Lys Leu Gly Leu Lye Gln? La? Sp Leu Pro Leu Val 1025 1030 1035 Gly He He Thr? Rg Leu Thr His Gln Lys Gly He His Leu He 1040 1045 1050 Lys His? La He Trp Arg Thr Leu Glu Arg Asn Gly Gln Val Val 1055 1060 1065 Leu Leu Gly Ser? The Pro? Sp Pro Arg Val Gln? Sn? Sn Phe Val 1070 1075 1080 ? sn Leu Ala? sn Gln Leu His Ser Lys Tyr? sn Aep Arg? la? rg 1085 1090 1095 Leu Cys Leu Thr Tyr? Sp Glu Pro Leu Ser His Leu He Tyr Wing 1100 1105 1110 Gly Wing Asp Phe He Leu Val Pro Be He Phe Glu Pro Cye Gly 1115 1120 1125 Leu Thr Gln Leu Thr Ala Met Arg Tyr Gly Ser He Pro Val Val 1130 1135 1140 Arg Lys Thr Gly Gly Leu Tyr Asp Thr Val Phe Asp Val Asp His 1145 1150 1155 Asp Lys Glu Arg Wing Gln Gln Cys Gly Leu Glu Pro Asn Gly Phe 1160 1165 1170 Ser Phe Asp Gly Wing Asp Wing Gly Gly Val Asp Tyr Wing Leu Asp 1175 1180 1185 Arg Ala Leu Ser Wing Trp Tyr Asp Gly Arg Asp Trp Phß Asn Ser 1190 1195 1200 Leu Cys Lys Gln Val Met Glu Gln Asp Trp Ser Trp Asn Arg Pro 1205 1210 1215 Ala Leu Asp Tyr Leu Glu Leu Tyr His Ala Ala? Rg Lys Leu Gl? 1220 1225 1230 < 210 > 36 < 211 > 9 < 212 > DNA < 213 > artificial sequence < 220 > < 222 > repetition_unity < 223 > sequence of the nine base pairs flanking a MuI element in the cloned fragment < 400 > 35 [sic] gtgagaatg 9

Claims (29)

1. An isolated cDNA having the sequence shown in SEQ ID NO: 1 encoding a corn starch synthase enzyme.

2. An expression vector containing the cDNA of claim 1, or fragments thereof, operably linked to the elements that allow the expression of the cDNA.

3. A host cell transfected with the vector of claim 2.

4. A corn starch synthase enzyme encoded by the cDNA of claim 1.

5. A polypeptide encoding a starch synthase protein, or fragments or derivatives thereof, in where the protein has a molecular weight of approximately 180 kDa, a maximum level of transcription in endosperm at 12 days after pollination, a C-terminal region possessing catalytic activity of a-1, 4-glucosyltransferase, and an N-terminal region containing the amiloplast targeting peptide and repeat motifs comprising, but not limited to, box M (SEQ ID NO: 9).

6. The polypeptide of claim 5, wherein the protein has the amino acid sequence shown in SEQ ID NO: 12, or fragments or derivatives thereof.

7. and, antibody directed to the polypeptide of claim 5T or fragments of the polypeptide thereof.

8. A transgenic plant, wherein 31 transgene is the vector of claim 2.

9. A method of producing starch, comprising the steps of: transforming a cell with the vector of claim 2; and extract and purify the starch.

10. The method of claim 9, wherein the cells carry a mutation. The method of claim 10, wherein the mutation is selected from the group consisting of a gene encoding an enzyme involved in the synthesis of starch, starch metabolism, glucose synthesis, glucose metabolism, synthesis of glycogen, metabolism of glu X jeuo, .. carbohydrate synthesis and carbohydrate metabolism ?. . , 12. The cDNA of claim 1, wherein the cDNA has the sequence, of. nucleotides comprising nt 120 to nt 1221 of SEQ ID NO: 1, the sequence encoding the first 368 amino acids of DUl. 13. The cDNA of claim 12, wherein the cDNA: L? N the nucleotide sec- tion comprising nt 655 to nt 1211 d "the fefp ID NO: 1, the coding sequence for amino acids 180 to 368 of DUl . The cDNA of claim 12, wherein the cDNA has the nucleotide sequence comprising nt 565 to nt 816 of SEQ ID NO: 1, the coding sequence for amino acids 150 to 233 of DUl. The cDNA of claim 1, wherein the cDNA has the nucleotide sequence comprising nt 1369 to nt 1944 of SEQ ID NO: 1, the coding sequence for amino acids 418 to 609 of DUl. The cDNA of claim 1, wherein the cDNA has the nucleotide sequence comprising nt 1 to nt 1437 of SEQ ID NO: 1, the coding sequence for amino acids 1 to 440 of DUl. The cDNA of claim 1, wherein the cDNA has the nucleotide sequence comprising nt 1438 to nt 2424 of SEQ ID NO: 1, the coding sequence for amino acids 441 to 769 of DUl. 18. The cDNA of claim 1, wherein the cDNA has the nucleotide sequence comprising nt 2425 to nt 3791 of SEQ ID NO: 1, the coding sequence for amino acids 769 to 1225 of DUl. 19. The expression vector of claim 2, wherein the functional fragment of the cDNA of SEQ ID NO: 1 is selected from the group consisting of nucleotide 120 to nucleotide 1221 of SEQ ID NO: 1, nucleotide 655 to nucleotide 1221 of SEQ ID NO: 1, nucleotide 565 to nt 816 of SEQ ID NO: 1, nucleotide 1369 to nucleotide 1944 of SEQ ID NO: 1, nucleotide 1 to nucleotide 1437 of SEQ ID NO : 1, nucleotide 1438 to nucleotide 2424 of SEQ ID NO: 1 and nucleotide 2425 to nucleotide 3791 of SEQ ID NO: 1. 20. A transgenic plant, wherein the transgene is the vector of claim 19. 21 A fusion construction, consisting of the DNA of claim 1 fused to the DNA encoding a peptide for affinity purification. 22. A fusion protein expressed by the fusion construct of claim 21. 23. A fusion construct, the DNA of claim 12 comprising fusing DNA encoding a peptide for affinity purification. 24. A fusion protein expressed by the fusion construct of claim 23. 25. A fusion construct, comprising the DNA of claim 13 fused to DNA encoding a peptide for affinity purification. 26. A fusion protein expressed by the fusion construct of claim 25. 27. A fusion construct comprising the DNA of claim 14 fused to DNA encoding a peptide for affinity purification. 28. A fusion protein expressed by the fusion construct of claim 27. 29. A fusion construct comprising the DNA of claim 15 fused to the DNA encoding a peptide. for affinity purification. 30 *. A fusion tip expressed by the fusion construct of claim 29, 31 * A f fusion construct comprising the DNA of claim 16 fused to DNA encoding a peptide for affinity purification. 32. A fusion protein expressed by the fusion construct of claim 31. 33. A fusion construct comprising the DNA of claim 17 fused to DNA encoding a peptide for affinity purification. 34 A fusion protein expressed by the fusion construct of claim 33. 35. A fusion construct comprising the DNA of claim 18 fused to DNA encoding a peptide for affinity purification. 36. A fusion protein expressed by the fusion construct of the claims. 37. An antisense nucleotide sequence, where I sequence - <ss antisense to the c.DNA of claim 1 or fragments thereof. 38. An expression vector comprising the antisense nucleic acid sequence of claim 37 operably linked to the elements that allow for the expression of the antisense nucleotide sequence. 39. A transgenic plant, wherein the transgene is the vector of claim 38. 40. A DNA sequence comprising the DUI promoter. 41. Starch extracted from the transgenic plant of claim 8. 42. Starch extracted from the transgenic plant of claim 20. 43. Starch extracted from the transgenic plant of claim 39.