AU2002314061B2 - DNA molecules that code for enzymes involved in starch synthesis, vectors, bacteria, transgenic plant cells and plants containing said molecules - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): HOECHST SCHERING AGREVO GMBH Invention Title: DNA MOLECULES THAT CODE FOR ENZYMES INVOLVED IN STARCH SYNTHESIS, VECTORS, BACTERIA, TRANSGENIC PLANT CELLS AND PLANTS CONTAINING SAID MOLECULES The following statement is a full description of this invention, including the best method of performing it known to me/us: la DNA MOLECULES ENCODING ENZYMES INVOLVED IN STARCH SYNTHESIS, VECTORS, BACTERIA, TRANSGENIC PLANT CELLS AND PLANTS CONTAINING THOSE MOLECULES All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

The present invention relates to DNA molecules encoding enzymes which are involved in the starch synthesis of plants. These enzymes represent two different isotypes of the soluble starch synthase as well as a starch granulebound starch synthase. This invention furthermore relates to vectors, bacteria, as well as to plant cells transformed with the DNA molecules described and to plants regenerated from them. Also, processes for the production of transgenic plants are described which, due to the introduction of DNA molecules encoding soluble or starch granule-bound starch synthases, synthesize a starch which is modified as regards its properties.

With respect to its increasing significance which has recently been ascribed to vegetal substances as regenerative sources of raw materials, one of the objects of biotechnological research is to try to adapt vegetal raw materials to the demands of the processing industry.

In order to enable the use of modified regenerative raw materials in as many areas as possible, it is furthermore -ibimportant to obtain a large variety of substances. Apart from oils, fats and proteins, polysaccharides constitute the essential regenerative raw materials derived from plants. Apart from cellulose, starch maintains an important position among the polysaccharides, being one of the most significant storage substances in higher plants.

Besides maize, rice and wheat, potato plays an important role as starch producer.

The polysaccharide starch is a polymer made up of chemically homogenous basic components, namely the glucose molecules. However, it constitutes a highly complex mixture from various types of molecules which differ from each other in their degree of polymerisation and in the degree of branching of the glucose chains. Therefore, starch is not a homogenous raw material. One differentiates particularly between amylose-starch, a basically non-branched polymer made up of a-1,4-glycosidically branched glucose molecules, and amylopectin-starch which in turn is a complex mixture of various branched glucose chains. The branching results from additional a-l,6-glycosidic interlinkings. In plants which are typically used for starch production, such as, e.g., maize or potato, the synthesized starch consists of about 25% of amylose starch and of about 75% of amylopectin starch.

In order to enable as wide a use of starch as possible, it seems to be desirable that plants be provided which are capable of synthesizing modified starch which is particularly suitable for various uses. A possibility of providing such plants is apart from breeding in the specific genetic modification of the starch metabolism of starch-producing plants by means of recombinant DNA techniques. However, a prerequisite therefor is to identify and to characterize the enzymes involved in the starch synthesis and/or the starch modification as well as to isolate the respective DNA molecules encoding these enzymes.

The biochemical pathways which lead to the production of starch are basically known. The starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissues these are the chloroplasts, in photosynthetically inactive, starchstoring tissues the amyloplasts.

The most important enzymes involved in starch synthesis are starch synthases as well as branching enzymes. In the case of starch synthases various isotypes are described which all catalyze a polymerization reaction by transferring a glucosyl residue of ADP-glucose to a-l,4-glucans. Branching enzymes catalyze the introduction of a-1,6 branchings into linear a-1,4glucans.

Furthermore, it is discussed that other enzyme activities, such as hydrolytic or phosphorolytic activities, are involved in the synthesis of starch (Preiss in Oxford Survey of Plant Molecular and Cell Biology, Oxford University Press, Vol. 7 (1991), 59- 114). It can furthermore not be precluded that the "R enzyme", or 3 the so-called disproportionizing enzyme, and the starch phosphorylases also are involved in starch synthesis, although these enzymes so far have been connected with the degradation of starch.

Starch synthases may be divided up in two groups: the granulebound starch synthases (GBSS), which are mainly present bound to starch granules but also in soluble form, and the soluble starch synthases (SSS). Within these classifications, various isotypes are described for various species of plants. These isotypes differ from each other in their dependency on primer molecules (so-called "primer dependent" (type II) and "primer independent" (type I) starch synthases).

So far only in the case of the isotype GBSS I its exact function during starch synthesis has been successfully determined. Plants in which this enzyme activity has been strongly or completely reduced, synthesize starch free of amylose (a so-called "waxy" starch) (Shure et al., Cell 35 (1983), 225-233; Visser et al., Mol. Gen. Genet. 225 (1991), 289-296; WO 92/11376); therefore this enzyme has been assigned a decisive role in synthesizing amylose-starch. This phenomenon is also observed in the cells of the green alga Chlamydomonas reinhardtii (Delrue et al., J.

Bacteriol. 174 (1992), 3612-3620). In the case of Chlamydomonas it was furthermore demonstrated that GBSS I is not only .involved in the synthesis of amylose but also"has a certain influence on amylopectin synthesis. In mutants which do not show any GBSS I activity a certain fraction of the normally synthesized amylopectin, exhibiting long chain glucans, is missing.

The functions of the other isotypes of the granule-bound starch synthases, particularly GBSS II, and of the soluble starch synthases are so far not clear. It is assumed that soluble starch synthases, together with branching enzymes, are involved in the synthesis of amylopectin (see, Ponstein et al., Plant Physiol. 92 (1990), 234-241) and that they play an important role in the regulation of starch synthesis rate.

For potato, the isotypes GBSS I, GBSS II, as well as two or three isotypes of the soluble starch synthases, which so far have not been characterized further, have been identified (Ponstein et al., Plant Physiol. 92 (1990), 234-241; Smith et al., Planta 182 (1990), 599-604; Hawker et al., Phytochemistry 11 (1972), 1287- 1293). Also for pea a GBSS II could be found (Dry et al., The Plant Journal 2,2 (1992), 193-202).

A cDNA encoding GBSS I from potato as well as a genomic DNA have already been described (Visser et al., Plant Sci. 64 (1989), 185- 192; van der Leij et al., Mol. Gen. Genet. 228 (1991), 240-248).

So far, no nucleic acid sequences encoding further granule-bound starch synthases or one of the soluble starch synthase isotypes from potato, have been reported.

Soluble starch synthases have been identified in several other plant species apart from potato. Soluble starch synthases have for example been isolated in homogeneous form from pea (Denyer and Smith, Planta 186 (1992), 609-617) and maize (WO 94/09144).

In the case of pea it was found that the isotype of the soluble starch synthase identified as SSS II is identical with the granule-bound starch synthase GBSS II (Denyer et al., Plant J. 4 (1993), 191-198). In the case of other plant species the existence of several SSS-isotypes was described by means of chromatographic methods, as for example in the case of barley (Tyyneld and Schulman, Physiologia Plantarum 89 (1993) 835-841; Kreis, Planta 148 (1980), 412-416), maize (Pollock and Preiss, Arch. Biochem. Biophys. 204 (1980), 578-588) and wheat (Rijven, Plant Physiol. 81 (1986), 448-453). However, DNA sequences encoding these proteins have so far not been described.

A cDNA encoding a soluble starch synthase so far has only been described for rice (Baba et al., Plant Physiol. 103 (1993), 565- 573).

In order to provide possibilities for modifying any desired starch-storing plant in such a way that they will synthesize a modified starch, respective DNA sequences encoding the various isotypes of granule-bound or soluble starch synthases have to be identified.

Therefore, it was the object of the present invention to provide DNA molecules especially from potato- encoding enzymes involved in starch biosynthesis and by means of which genetically modified plants may be produced that show an elevated or reduced activity of those enzymes, thereby prompting a modification in the chemical and/or physical properties of the starch synthesized in these plants.

This object has been achieved by the provision of the embodiments described in the claims.

The invention therefore relates to DNA molecules encoding starch synthases, particularly such DNA molecules encoding the granulebound starch synthases of the isotype II, as well as DNA molecules encoding soluble starch synthases.

The present invention particularly relates to DNA molecules encoding proteins with the biological activity of a granule-bound starch synthase of the isotype II (GBSSII) or a biologically active fragment of such a protein, such molecules preferably encoding proteins having the amino acid sequence indicated under Seq ID No. 8. Particularly, the invention relates to DNA molecules having the nucleotide sequence indicated under Seq ID No. 7, preferably molecules comprising the coding region indicated under Seq ID No. 7.

The subject matter of the invention are also DNA molecules encoding a GBSSII and the sequence of which differs from the nucleotide sequences of the above-described DNA molecules due to the degeneracy of the genetic code.

Furthermore, the invention relates to DNA molecules encoding GBSSII and hybridizing to any of the above-described

DNA

molecules. Such DNA molecules preferably are derived from starchstoring plants, particularly from dicotyledonous plants, and particularly preferred from potato.

The GBSSII proteins encoded by the DNA molecules according to the invention preferably have a molecular weight of 85±5 kD. GBSSII proteins are mainly present bound to starch granules, however, they may also be present in soluble form.

Furthermore, the invention relates to DNA molecules encoding proteins with the biological activity of a soluble starch synthase of the isotype B (SSSB) or a biologically active fragment of such a protein, with such molecules preferably encoding proteins having the amino acid sequence indicated under Seq ID No. 10. In particular, the invention relates to DNA molecules having the nucleotide sequence indicated under Seq ID Furthermore, the present invention relates to DNA molecules encoding SSSA and hybridizing to any of the above-described

DNA

molecules.

The SSSA protein preferably has an apparent molecular weight of about 120 to 140 kD, particularly of about 135 kD, in SDS gel electrophoresis.

In this invention the term "hybridization" signifies hybridization under conventional hybridizing conditions, preferably under stringent conditions as described for example in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). DNA molecules hybridizing to the DNA molecules according to the invention can basically be derived from any organism prokaryotes or eukaryotes, particularly from bacteria, fungi, algae, plants or animal organisms) which possesses such DNA molecules. Preferably, they originate from monocotyledonous or dicotyledonous plants, in particular from useful plants, and particularly preferred from starch-storing plants.

DNA molecules hybridizing to the molecules according to the invention may be isolated, from genomic or from cDNA libraries from various organisms.

The identification and isolation of such DNA molecules from plants or other organisms may take place by using the DNA molecules according to the invention or parts of these DNA molecules or, as the case may be, the reverse complement strands of these molecules, by hybridization according to standard methods (see, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

NY).

As a probe for hybridization, DNA molecules may be used which exactly or basically contain the nucleotide sequences indicated under Seq ID No. 7, 9 or 11 or parts thereof. The fragments used as hybridization probe may also be synthetic

DNA

fragments which were produced by means of the conventional

DNA

synthesizing methods and the sequence of which is basically identical with that of a DNA molecule according to the invention.

After identifying and isolating the genes hybridizing to the DNA sequences according to the invention, the sequence has to be determined and the properties of the proteins encoded by this sequence have to be analyzed.

The molecules hybridizing to the DNA molecules of the invention also comprise fragments, derivatives and allelic variants of the above-described DNA molecules which encode one of the proteins described above. Thereby, fragments are defined as parts of the DNA molecules, which are long enough in order to encode one of the described proteins. In this context, the term derivatives means that the DNA sequences of these molecules differ from the sequences of the above-mentioned DNA molecules at one or more positions and that they exhibit a high degree of homology to these DNA sequences. Hereby, homology means a sequence identity of at least 40%, in particular an identity of at least preferably of more than 80% and still more preferably a sequence identity of more than 90%. The deviations occurring when comparing with the above-described DNA molecules might have been caused by deletion, substitution, insertion or recombination.

Moreover, homology means that functional and/or structural equivalence exists between the respective DNA molecules or the proteins they encode. The DNA molecules, which are homologous to the above-described DNA molecules and represent derivatives of these DNA molecules, are generally variations of these molecules, that constitute modifications which exert the same biological function. These variations may be naturally occurring variations, for example sequences derived from other organisms, or mutations, whereby these mutations may have occurred naturally or they may have been introduced by means of a specific mutagenesis.

Moreover, the variations may be synthetically produced sequences.

The allelic variants may be naturally occurring as well as synthetically produced variants or variants produced by recombinant DNA techniques.

The proteins encoded by the various variants of the DNA molecules according to the invention exhibit certain common characteristics. Enzyme activity, molecular weight, immunologic reactivity, conformation etc. may belong to these characteristics as well as physical properties such as the mobility in gel electrophoresis, chromatographic characteristics, sedimentation coefficients, solubility, spectroscopic properties, stability; pH-optimum, temperature-optimum etc.

Significant characteristics of a starch synthase are: i) their localization within the stroma of the plastids of plant cells; ii) their capability of synthesizing linear a-1,4-linked polyglucans using ADP-glucose as substrate. This activity can be determined as shown in Denyer and Smith (Planta 186 (1992), 606- 617) or as described in the examples.

The DNA molecules according to the invention may basically originate from any organism expressing the proteins described, preferably from plants, particularly from starch-synthesizing or starch-storing plants. These plants may be monocotyledonous but also dicotyledonous plants. Particularly preferred are, e.g., cereals (such as barley, rye, oats, wheat, etc.), maize, rice, pea, cassava, potato, etc.

Furthermore, the invention relates to vectors, especially plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the above-mentioned DNA molecules of the invention.

In a preferred embodiment the DNA molecules contained in the vectors are linked to DNA elements that ensure the transcription and synthesis of a translatable RNA in prokaryotic and eukaryotic cells.

The expression of the DNA molecules of the invention in prokaryotic cells, in Escherichia coli, is interesting insofar as this enables a more precise characterization of the enzymatic activities of the enzymes encoding these molecules. In particular, it is possible to characterize the product being synthesized by the respective enzymes in the absence of other enzymes which are involved in the starch synthesis of the plant cell. This makes it possible to draw conclusions about the function, which the respective protein exerts during the starch synthesis within the plant cell.

Moreover, it is possible to introduce various mutations into the DNA molecules of the invention by means of conventional molecular-biological techniques (see, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), whereby the synthesis of proteins with possibly modified biological properties is induced. By means of this it is on the one hand possible to produce deletion mutants, in which DNA molecules are produced by continuing deletions at the or the 3'-end of the encoding DNA-sequence. These DNA molecules may lead to the synthesis of correspondingly shortened proteins. Such deletions at the 5'-end of the nucleotide sequence make it possible, for example, to identify amino acid sequences which are responsible for the translocation of the enzyme in the plastids (transit peptides). This allows for the specific production of enzymes which due to the removal of the respective sequences are no longer located in the plastids but within the cytosol, or which.

due to the addition of other signal sequences are located in other compartments.

On the other hand, point mutations might also be introduced at positions where a modification of the amino acid sequence influences, for example, the enzyme activity or the regulation of the enzyme. In this way, mutants with a modified Km-value may be produced, or mutants which are no longer subject to the regulation mechanisms by allosteric regulation or covalent modification usually occurring in cells.

Furthermore, mutants may be produced exhibiting a modified substrate or product specificity such as mutants that use ADPglucose-6-phosphate instead of ADP-glucose as substrate.

Moreover, mutants with a modified activity-temperature-profile may be produced.

For the genetic manipulation in prokaryotic cells the DNA molecules of the invention or parts of these molecules may be integrated into plasmids which allow for a mutagenesis or a sequence modification by recombination of DNA sequences. By means of standard methods (cf. Sambrook et al., 1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA) base exchanges may be carried out or natural or synthetic sequences may be added. In order to connect the DNA fragments, adapters or linkers may be attached to the fragments. Moreover, use can be made of manipulations which offer suitable restriction sites or which remove superfluous DNA or restriction sites. Wherever use is made of inserts, deletions or substitutions, in vitro mutagenesis, "primer repair", restriction or ligation may be used. For analyzing use is usually made of a sequence analysis, a restriction analysis or further biochemicomolecularbiological methods.

In a further embodiment the invention relates to host cells, in particular prokaryotic or eukaryotic cells, which contain a DNA molecule of the invention as described above or a vector of the invention. These are preferably bacterial cells or plant cells.

Furthermore, the proteins encoded by the DNA molecules of the invention are the subject-matter of the invention as well as methods for their production whereby a host cell of the invention is cultivated under conditions that allow for a synthesis of the protein and whereby the protein is then isolated from the cultivated cells and/or the culture medium.

It was found that by making available the nucleic acid molecules of the invention it is now possible by means of recombinant

DNA

techniques to interfere with the starch metabolism of plants in a way so far impossible and to modify it in such a way that a starch is synthesized which, is modified, compared to the starch synthesized in wild-type plants, with respect to its physico-chemical properties, especially the amylose/amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the pastification behavior, the size and/or the shape of the starch granule. Soluble starch synthases, play, a central role in the regulation of the synthesis rate of starch. There is the possibility of increasing the yield of genetically modified plants by increasing the activity of these enzymes or by making mutants available which are no longer subject to cell-specific regulation schemes and/or different temperature-dependencies with respect to their activity. The economic significance of the chance to interfere with the starch synthesis, namely of potato plants, is obvious: In Europe, for example, potato is one of the most important plants for producing starch apart from maize and wheat. About 20% of the starch produced in Europe per year is obtained from potatoes.

Furthermore, potato starch exhibits some advantageous properties as compared to starch from maize or wheat, such as, a low protein and lipid content as well as relatively large starch granules and phosphate content. Therefore, if possible, potato starch is preferably used.

Therefore, it is possible to express the DNA molecules of the invention in plant cells in order to increase the activity of one or more starch synthases. Furthermore, the DNA molecules of the invention may be modified by means of methods known to the skilled person, in order to produce starch synthases which are no longer subject to the cell-specific regulation mechanisms or show modified temperature-dependencies or substrate or /product specificities.

The synthesized protein may in principle be located in any desired compartment within the plant cell. In order to locate it within a specific compartment, the sequence ensuring the localization in the plastids must be deleted and the remaining coding regions optionally have to be linked to DNA sequences which ensure localization in the respective compartment. Such sequences are known (see, Braun et al., 1992, EMBO J.

11:3219-3227; Wolter et al., 1988, Proc. Natl. Acad. Sci. USA 846-850; Sonnewald et al., 1991, Plant J. 1:95-106).

Thus, the present invention also relates to transgenic plant cells containing a DNA molecule of the invention, this DNA molecule being linked to regulatory DNA elements, which ensure the transcription in plant cells, especially with a promoter which is heterologous with respect to the DNA molecule.

By means of methods known to the skilled person the transgenic plant cells can be regenerated to whole plants. Thus, the plants obtained by regenerating the transgenic plant cells of the invention are also the subject-matter of the present invention.

A

further subject-matter of the invention are plants which contain the above-described transgenic plant cells. The transgenic plants may in principle be plants of any desired species, they may be monocotyledonous as well as dicotyledonous plants. These are preferably useful plants, such as cereals (rye, barley, oats, wheat etc.), rice, maize, peas, cassava or potatoes.

The invention also relates to propagation material of the plants of the invention, fruits, seeds, tubers, cuttings etc.

Due to the expression or, as the case may be, additional expression of a DNA molecule of the invention, the transgenic plant-cells and plants of the invention synthesize a starch which compared to starch synthesized in wild-type plants, nontransformed plants, is modified, in particular with respect to the viscosity of aqueous solutions of this starch and/or the phosphate content. Thus, the starch derived from transgenic plant cells and plants according to the invention is the subject-matter of the present invention.

A further subject-matter of the invention are transgenic plant cells, in which the activity of a protein according to the invention is reduced when compared to non-transformed plants. It was found that plant cells exhibiting a reduced activity of a protein of the invention synthesize a starch having modified chemical and/or physical properties as compared to that of wildtype plant cells.

The production of plant cells with a reduced activity of a protein of the invention may for example be achieved by using the DNA molecules of the invention. Possibilities are the expression of a corresponding antisense-RNA,, of a sense-RNA for achieving a cosupression effect or the expression of a correspondingly constructed ribozyme, which specifically cleaves transcripts encoding a protein of the invention.

Preferably, an antisense RNA is expressed to reduce the activity of a protein of the invention in plant cells.

For this purpose, a DNA molecule can be used which comprises the complete sequence encoding a protein of the invention, including possibly existing flanking sequences as well as DNA molecules, which only comprise parts of the encoding sequence whereby these parts have to be long enough in order to prompt an antisenseeffect within the cells. Basically, sequences with a minimum length of 15 bp, preferably with a length of 100-500 bp and for an efficient antisense-inhibition, in particular sequences with a length of more than 500 bp may be used. Generally DNA-molecules are used which are shorter than 5000 bp, preferably sequences with a length of less than 2500 bp. Preferably, use is made of DNA molecules that are homologous with respect to the plant species to be transformed.

Use may also be made of DNA sequences which are highly homologous, but not completely identical to the sequences of the DNA molecules of the invention. The minimal homology should be more than about 65%. Preferably, use should be made of sequences with homologies between 95 and 100%.

The transgenic plant cells of the invention can be regenerated to whole plants by means of methods known to the skilled person.

Thus, plants containing the transgenic plant cells of the invention are also the subject-matter of the present invention.

These plants generally are plants of any species, i.e., monocotyledonous and dicotyledonous plant. Preferably these plants are useful plants, especially starch-storing plants such as cereals (rye, barley, oats, wheat, etc.), rice, maize, peas, cassava or potatoes. The invention also relates to propagation material of the plants of the invention, such as fruit, seeds, tubers, cuttings, etc.

Due to the reduction of the activity of one of the proteins of the invention, the transgenic plant cells and plants of the invention synthesize a starch which is modified, compared to the starch from non-transformed plant cells or plants, in its chemical and/or physical properties. This starch exhibits for example a modified viscosity of its aqueous solutions and/or a modified phosphate content.

Thus, starch derived from the above-mentioned transgenic plant cells and plants is also the subject-matter of the invention.

The' starches of the invention may be modified according to techniques known to the skilled person; in unmodified as well as in modified form they are suitable for use in foodstuffs or nonfoodstuffs.

Basically, the possibilities of uses of the starch can be subdivided into two major fields. One field comprises the hydrolysis products of starch which mainly include glucose and glucan components obtained by enzymatic or chemical processes.

They serve as starting materials for further chemical modifications and processes such as fermentation. In this context, it might be of importance that the hydrolysis' process can be carried out simply and inexpensively. Currently, it is carried out substantially enzymatically using amyloglucosidase.

It is thinkable that costs might be reduced by using lower amounts of enzymes for hydrolysis due to changes in the starch structure, increased surface of the grain, improved digestibility due to less branching or a steric structure, which limits the accessibility for the used enzymes.

The other area in which starch is used due to its polymer structure as so-called native starch, can be subdivided into two further areas: i. Use in foodstuffs Starch is a classic additive for various foodstuffs, in which it essentially serves the purpose of binding aqueous additives and/or causes an increased viscosity or an increased gel formation. Important characteristic properties are flowing and sorption behavior, swelling and pastification temperature, viscosity and thickening performance, solubility of the starch, transparency and paste structure, heat, shear and acid resistance, tendency to retrogradation, capability of film formation, resistance to freezing/thawing, digestibility as well as the capability of complex formation with, inorganic or organic ions.

2. Use in non-foodstuffs The other major field of application is the use of starch as an adjuvant in various production processes or as an additive in technical products. The major fields of application for the use of starch as an adjuvant are, first of all, the paper and cardboard industry. In this field, the starch is mainly used for retention (holding back solids), Sfor sizing filler and fine particles, as solidifying substance and for dehydration. In addition, the advantageous properties of starch with regard to stiffness, hardness, sound, grip, gloss, smoothness, tear strength as well as the surfaces are utilized.

2.1 Paper and cardboard industry Within the paper production process, a differentiation can be made between four fields of application, namely surface, coating, mass and spraying.

The requirements on starch with regard to surface treatment are essentially a high degree of brightness, corresponding viscosity, high viscosity stability, good film formation as well as low formation of dust. When used in coating the solid content, a corresponding viscosity, a high capability.

to bind as well as a high pigment affinity play an important role. As an additive to the mass rapid, uniform, loss-free dispersion, high mechanical stability and complete retention in the paper pulp are of importance. When using the starch in spraying, corresponding content of solids, high viscosity as well as high capability to bind are also significant.

2.2 Adhesive industry A major field of application is, for instance, in the adhesive industry, where the fields of application are subdivided into four areas: the use as pure starch glue, the use in starch glues prepared with special chemicals, the use of starch as an additive to synthetic resins and polymer dispersions as well as the use of starches as extenders for synthetic adhesives. 90% of all starch-based adhesives are used in the production of corrugated board, paper sacks and bags, composite materials for paper and aluminum, boxes and wetting glue for envelopes, stamps, etc.

2.3 Textile and textile care industry Another possible use as adjuvant and additive is in the -'production of textiles and textile care products. Within the textile industry, a differentiation can be made between the following four fields of application: the use of starch as a sizing agent, as an adjuvant for smoothing and strengthening the burring behavior for the protection against tensile forces active in weaving as well as for the increase of wear resistance during weaving, as an agent for textile improvement mainly after quality-deteriorating pretreatments, such as bleaching, dying, etc., as a thickener in the production of dye pastes for the prevention of dye diffusion and as an additive for warping agents for sewing yarns.

2.4 Building industry The fourth area of application of starch is its use as an additive in building materials. One example is the production of gypsum plaster boards, in which the starch mixed in the thin plaster pastifies with the water, diffuses at the surface of the gypsum board and thus binds the cardboard to the board. Other fields of application are admixing it to plaster and mineral fibers. In ready-mixed concrete, starch may be used for the deceleration of the sizing process.

Ground stabilization Furthermore, the starch is advantageous for the production of means for ground stabilization used for the temporary protection of ground particles against water in artificial earth shifting. According to state-of-the-art knowledge, combination products consisting of starch and polymer emulsions can be considered to have the same erosion- and incrustation-reducing effect as the products used so far; however, they are considerably less expensive.

2.6 Use of starch in plant protectives and fertilizers Another field of application is the use of starch in plant protectives for the modification of the specific properties "of these preparations. For instance, starches are used for improving the wetting of plant protectives and fertilizers, for the dosed release of the active ingredients, for the conversion of liquid, volatile and/or odorous active ingredients into microcristalline, stable, deformable substances, for mixing incompatible compositions and for the prolongation of the duration of the effect due to a reduced disintegration.

2.7 Drugs, medicine and cosmetics industry Starch may also be used in the fields of drugs, medicine and in the cosmetics industry. In the pharmaceutical industry, the starch may be used as a binder for tablets or for the dilution of the binder in capsules. Furthermore, starch is suitable as disintegrant for tablets since, upon swallowing, it absorbs fluid and after a short time it swells so much that the active ingredient is released. For qualitative reasons, medicinal flowance and dusting powders are further fields of application. In the field of cosmetics, the starch may for example be used as a carrier of powder additives, such as scents and salicylic acid. A relatively extensive field of application for the starch is toothpaste.

2.8 Starch as an additive in coal and briquettes The use of starch as an additive in coal and briquettes is also thinkable. By adding starch, coal can be quantitatively agglomerated and/or briquetted in high quality, thus preventing premature disintegration of the briquettes.

Barbecue coal contains between 4 and 6% added starch, calorated coal between 0.1 and Furthermore, the starch is suitable as a binding agent since adding it to coal and briquette can considerably reduce the emission of toxic substances.

2.9 Processing of ore and coal slurry Furthermore, the starch may be used as a flocculant in the processing of ore and coal slurry.

2.10 Starch as an additive in casting Another field of application is the use as an additive to process materials in casting. For various casting processes cores produced from sands mixed with binding agents are needed. Nowadays, the most commonly used binding agent is bentonite mixed with modified starches, mostly swelling starches.

The purpose of adding starch is increased flow resistance as well as improved binding strength. Moreover, swelling starches may fulfill more prerequisites for the production process, such as dispersability in cold water, rehydratisability, good mixability in sand and high capability of binding water.

2.11 Use of starch in rubber industry In the rubber industry starch may be used for improving the technical and optical quality. Reasons for this are improved surface gloss, grip and appearance. For this purpose, the starch is dispersed on the sticky rubberized surfaces of rubber substances before the cold vulcanization. It may also be used for improving the printability of rubber.

2.12 Production of leather substitutes Another field of application for the modified starch is the production of leather substitutes.

2.13 Starch in synthetic polymers In the plastics market the following fields of application are emerging: the integration of products derived from starch into the processing process (starch is only a filler, there is no direct bond between synthetic polymer and starch) or, alternatively, the integration of products derived from starch into the production of polymers (starch and polymer form a stable bond) The use of the starch as a pure filler cannot compete with other substances such as talcum. This situation is different when the specific starch properties become effective and the property profile of the end products is thus clearly changed. One example is the use of starch products in the processing of thermoplastic materials, such as polyethylene. Thereby, starch and the synthetic polymer are combined in a ratio of 1 1 by means of coexpression to form a 'master batch', from which various products are produced by means of common techniques using granulated polyethylene. The integration of starch in polyethylene films may cause an increased substance permeability in hollow bodies, improved water vapor permeability, improved antistatic behavior, improved anti-block behavior as well as improved printability with aqueous dyes. Present disadvantages relate to insufficient transparency, reduced tensile strength as well as reduced extensibility.

Another possibility is the use of the starch in polyurethane foams. Due to the adaptation of starch derivatives as well as due to the optimization of processing techniques, it is possible to specifically control the reaction between synthetic polymers and the starch's hydroxy groups. The results are polyurethane films having the following property profiles due to the use of starch: a reduced coefficient of thermal expansion, decreased shrinking behavior, improved pressure/tension behavior, increased water vapor permeability without a change in water acceptance, reduced flammability and cracking density, no drop off of combustible parts, no halides and reduced aging. Disadvantages that presently still exist are reduced pressure and impact strength.

Product development of film is not the only option. Also solid plastics products, such as pots, plates and bowls can be produced by means of a starch content of more than 50%. Furthermore, the starch/polymer mixtures offer the advantage that they are much easier biodegradable.

Furthermore, due to their extreme capability to bind water, starch graft polymers have gained utmost importance. These are products having a backbone of starch and a side lattice of a synthetic monomer grafted on according to the principle of radical chain mechanism. The starch graft polymers available nowadays are characterized by an improved binding and retaining capability of up to 1000 g water per g starch at a high viscosity. The fields of application of these super absorbers have extended over the last few years and they are used mainly in the hygiene field, in products such as diapers and sheets, as well as in the agricultural sector, in seed pellets.

What is decisive for the use of the new starch modified by recombinant DNA techniques are, on the one hand, structure, water content, protein content, lipid content, fiber content, ashes/phosphate content, amylose/amylopectin ratio, distribution of the relative molar mass, degree of branching, granule size and shape as well as crystallization, and on the other hand, the properties resulting in the following features: flow and sorption behavior, pastification temperature, viscosity, thickening performance, solubility, paste structure, transparency, heat, shear and acid resistance, tendency to retrogradation, capability of gel formation, resistance to freezing/thawing, capability of complex formation, iodine binding, film formation, adhesive strength, enzyme stability, digestibility and reactivity.

The production of modified starch by genetically operating with a transgenic plant may modify the properties of the starch obtained from the plant in such a way as to render further modifications by means of chemical or physical methods superfluous. On the other hand, the starches modified by means of recombinant

DNA

techniques might be subjected to further chemical modification, which will result in further improvement of the quality for certain of the above-described fields of application. These chemical modifications are principally known to the person skilled in the art. These are particularly modifications by means of heat treatment acid treatment oxidation and esterification leading to the formation of phosphate, nitrate, sulfate, xanthate, acetate and citrate starches. Other organic acids may also be used for the esterification: formation of starch ethers starch alkyl ether, O-allyl ether, hydroxylalkyl ether, 0carboxylmethyl ether, N-containing starch ethers, P-containing starch ethers and S-containing starch ethers.

formation of branched starches formation of starch graft polymers.

In order to express the DNA molecules of the invention in senseor antisense-orientation in plant cells, these are linked to regulatory DNA elements which ensure the transcription in plant cells. Such regulatory DNA elements are particularly promoters.

The promoter may be selected in such a way that the expression takes place constitutively or in a certain tissue, at a certain point of time of the plant development or at a point of time determined by external circumstances. With respect to the plant the promoter may be homologous or heterologous. A suitable promoter for a constitutive expression is, the 35S RNA promoter of the Cauliflower Mosaic Virus. For a tuber-specific expression in potatoes the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) or a promoter which ensures expression only in photosynthetically active tissues, the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) may be used. For an endosperm-specific expression the HMG promoter from wheat, or promoters from zein genes from maize are suitable.

Furthermore, a termination sequence may exist which serves to correctly end the transcription and to add a poly-A-tail to the transcript which is believed to stabilize the transcripts. Such elements are described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged as desired.

According to the invention, it is basically possible to produce plants in which only the activity of one isotype of the SSS or the GBSS II is modified, and also plants in which the activities of several starch synthase forms are simultaneously modified.

Thereby, all kinds of combinations and permutations are thinkable.

By modifying the activities of one or more isotypes of the starch synthases in plants, a synthesis of a starch modified in its structure is brought about.

By increasing the activity of one or more isotypes of the starch synthases in the cells of the starch-storing tissue of transformed plants such as in the potato tuber or in the endosperm of maize or wheat, increased yields may be the result.

24 Since the DNA sequence encoding the GBSS I from potato is already known (Visser et al., Plant Sci. 64 (1989), 185-192),

DNA

sequences encoding all starch synthases so far identified in potato are available. This allows for the identification of the function of the individual isotypes in the starch biosynthesis as well as for the production of genetically modified plants in which the activity of at least one of these enzymes is modified.

This enables the synthesis of starch with a modified structure and therefore with modified physico-chemical properties in the plants manipulated in such a way.

The DNA molecules of the invention may be used in order to produce plants in which the activity of the starch synthases mentioned is elevated or reduced and in which at the same time the activities of other enzymes involved in the starch biosynthesis are modified. Thereby, all kinds of combinations and permutations are thinkable. For example, DNA molecules encoding the SSS proteins or GBSS II may be introduced into plant cells according to the process described above in which the synthesis of endogenous GBSS I-proteins is already inhibited due to an antisense-effect (as described in Visser et al., Mol. Gen. Genet.

225 (1991), 289-296), or in which the synthesis of the branching enzyme is inhibited (as described in W092/14827).

If the inhibition of the synthesis of several starch synthases in transformed plants is to be achieved, DNA molecules can be used for transformation, which at the same time contain several regions in antisense-orientation controlled by a suitable promoter and encoding the corresponding starch synthases. Hereby, each sequence may be controlled by its own promoter or else the sequences may be transcribed as a fusion of a common promoter.

The last alternative will generally be preferred as in this case the synthesis of the respective proteins should be inhibited to approximately the same extent.

Furthermore, it is possible to construct. DNA molecules in which apart from DNA sequences encoding starch synthases other DNA sequences are present encoding other proteins involved in the starch synthesis or modification and coupled to a suitable promoter in antisense orientation. Hereby, the sequences may again be connected up in series and be transcribed by a common promoter. For the length of the individual coding regions used in such a construct the above-mentioned facts concerning the production of antisense-construct are also true. There is no upper limit for the number of antisense fragments transcribed from a promoter in such a DNA molecule. The resulting transcript, however, should not be longer than 10 kb, preferably 5 kb.

Coding regions which are located in antisense-orientation behind a suitable promoter in such DNA molecules in combination with other coding regions, may be derived from DNA sequences encoding the following proteins: granule-bound starch synthases (GBSS I and II), other soluble starch synthases (SSS I and II), branching enzymes (Ko8mann et al., Mol. Gen. Genet. 230 (1991) 39-44), debranching enzymes (R enzymes), disproportionizing enzymes (Takaha et al., J. Biol. Chem. 268 (1993), 1391-1396) and starch phosphorylases. This enumeration merely serves as an example. The use of other DNA sequences within the framework of such a combination is also thinkable.

By means of such constructs it is possible to inhibit the synthesis of several enzymes at the same time within the plant cells transformed with these molecules.

In order to prepare the integration of foreign genes into higher plants a high number of cloning vectors are at disposal, containing a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples for such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence may be integrated into the vector at a suitable restriction site. The obtained plasmid is used for the transformation of E. coli cells. Transformed E. coli cells are cultivated in a suitable medium and subsequently harvested and lysed. The plasmid is recovered. As an analyzing method for the characterization of the obtained plasmid DNA use is generally made of restriction analysis, gel electrophoresis and other biochemico-molecularbiological methods. After each manipulation the plasmid DNA may be cleaved and the obtained DNA fragments may be linked to other DNA sequences. Each plasmid DNA may be cloned into the same or in other plasmids.

In order to integrate DNA into plant host cells a wide range of techniques are at disposal. These techniques comprise the transformation of plant cells with T-DNA by using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation medium, the fusion of protoplasts, the injection and the electroporation of DNA, the integration of DNA by means of the biolistic method as well as further possibilities.

In the case of injection and electroporation of DNA into plant cells, there are no special demands made to the plasmids used.

Simple plasmids such as pUC derivatives may be used. However, in case that whole plants are to be regenerated from cells transformed in such a way, a selectable marker gene should be present.

Depending on the method of integrating desired genes into the plant cell, further DNA sequences may be necessary. If the Ti- or Ri-plasmid is used, for the transformation of the plant cell, at least the right border, more frequently, however, the right and left border of the Ti- and Ri-plasmid T-DNA has to be connected to the foreign gene to be integrated as a flanking region.

If Agrobacteria are used for the transformation, the DNA which is to be integrated must be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. Due to sequences homologous to the sequences within the T-DNA, the intermediate vectors may be integrated into the Ti- or Ri-plasmid of the Agrobacterium due to homologous recombination. This also contains the vir-region necessary for the transfer of the T-DNA.

Intermediate vectors cannot replicate in Agrobacteria. By means of a helper plasmid the intermediate vector may be transferred to Agrobacterium tumefaciens (conjugation.,.. Binary vectors may replicate in E. coli as well as in Agrobacteria. They contain a selectable marker gene as well as a linker or polylinker which is framed by the right and the left T-DNA border region. They may be transformed directly into the Agrobacteria (Holsters et al. Mol.

Gen. Genet. 163 (1978), 181-187). The Agrobacterium acting as host cell should contain a plasmid carrying a vir-region. The vir-region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The Agrobacterium transformed in such a way is used for the transformation of plant cells.

The use of T-DNA for the transformation of plant cells was investigated intensely and described sufficiently in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.

For transferring the DNA into the plant cells, plant explants may suitably be co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g.

pieces of leaves, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) whole plants may then be regenerated in a suitable medium which may contain antibiotics or biozides for the selection of transformed cells. The plants obtained in such a way may then be examined as to whether the integrated DNA is present or not. Other possibilities in order to integrate foreign DNA by using the biolistic method or by transforming protoplasts are known to the skilled person (cf., Willmitzer, 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise Rehm, G. Reed, A.

Pihler, P. Stadler, editors), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).

Once the introduced DNA has been integrated in the genome of the plant cell, it usually continues to be stable there and also remains within the descendants of the originally transformed cell. It usually contains a selectable marker which confers resistance against a biozide or against an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricine, etc.

to the transformed plant cells. The individually selected marker should therefore allow for a selection of transformed cells to cells lacking the integrated DNA.

The transformed cells grow in the usual way within the plants (see also McCormick et al., 1986, Plant Cell Reports 5: 81-84).

The resulting plants can be cultivated in the usual way and cross-bred with plants having the same transformed genetic heritage or another genetic heritage. The resulting hybrid individuals have the corresponding phenotypic properties.

Two or more generations should be grown in order to ensure whether the phenotypic feature is kept stably and whether it is transferred. Furthermore, seeds should be harvested in order to ensure that the corresponding phenotype or other properties will remain.

The plasmid pBinARHyg used in this invention was deposited with Deutsche Sammlung von Mikroorganismen (DSM) [German collection of microorganisms] in Brunswick, Federal Republic of Germany, as international recognized depositary authority in accordance with the stipulations of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on January 20, 1994 under deposit no. DSM 9505.

Abbreviations used bp base pair GBSS granule-bound starch synthase IPTG isopropyl B-D-thiogalacto-pyranoside SSS soluble starch synthase PMSF phenylmethylsulfonylfluoride VK full-length clone Media and solutions used in the examples: x SSC 175.3 g NaCI 88.2 g sodium citrate ad 1000 ml with pH 7.0 with 10 N NaOH Buffer A 50 mM Tris-HCl pH mM DTT 2 mM EDTA 0.4 mM PMSF glycerol 0.1% sodium dithionite Buffer B Buffer C x TBS x TBST Elution buffer Dialysis buffer Protein buffer 50 mM Tris-HCl pH 7.6 mM DTT 2 mM EDTA 0.5 M sodium citrate pH 7.6 mM Tris-HCl pH 7.6 mM DTT 2 mM EDTA 0.2 M Tris-HCl pH M NaCi 10 x TBS 0.1% (vol./vol.) Tween 25 mM Tris pH 8.3 250 mM glycine 50 mM Tris-HCl pH mM NaCi 2 mM EDTA 14.7 mM B-mercaptoethanol mM PMSF 50 mM sodium phosphate buffer pH 7.2 mM EDTA mM PMSF 14.7 mM B-mercaptoethanol Fig. 1 shows plasmid pSSSA The thin line corresponds to the sequence of pBluescript II The thick line represents the cDNA encoding the SSS A isotype from Solanum tuberosum. The restriction sites of the insert are indicated. The cDNA insert is ligated between the EcoR I and Xho I restriction sites of the polylinker of the plasmid.

The DNA sequence of the cDNA insert is indicated under Seq ID No.

1.

Fig. 2 shows plasmid pSSSB The thin line corresponds to the sequence of pBluescript II The thick line represents the cDNA encoding the SSS B isotype from Solanum tuberosum. The restriction sites of the insert are indicated. The cDNA insert is ligated between the EcoR I and Xho I restriction sites of the polylinker of the plasmid.

The DNA sequence of the cDNA insert is indicated under Seq ID No.

2.

Fig. 3 shows plasmid Structure of the plasmid: A fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21 (1980), 285-294) B fragment B: cDNA from Solanum tuberosum encoding soluble starch synthase; SSSA isotype; Xba I/Asp718 fragment from pSSSA, about 2.1 kb orientation with respect to the promoter: antisense C fragment C: nt 11748-11939 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846) Fig. 4 shows plasmid Structure of the plasmid: A fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21 (1980), 285-294) B fragment B: cDNA from Solanum tuberosum encoding soluble starch synthase; SSSB isotype; Xho I/Spe I fragment from pSSSB, about 1.8 kb orientation with respect to the promoter: antisense C fragment C: nt 11748-11939 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846) Fig. 5 shows plasmid pGBSSII The thin line corresponds to the sequence of pBluescript II The thick line represents the cDNA encoding the GBSS II isotype from Solanum tuberosum. The restriction sites of the insert are indicated. The cDNA insert is ligated between the EcoR I and Xho I restriction sites of the polylinker of the plasmid.

The DNA sequence of the cDNA insert is indicated under Seq ID No.

3.

Fig. 6 shows plasmid Structure of the plasmid: A fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21 (1980), 285-294) B fragment B: cDNA from Solanum tuberosum encoding granule-bound starch synthase; GBSS II isotype; Sma I/Asp 718 fragment from pGBSS II, about 1.9 kb orientation with respect to the promoter: antisense C fragment C: nt 11748-11939 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846) Fig. 7 shows a partial comparison of the amino acid sequences of prokaryotic glycogen synthases, granule-bound starch synthases and,/soluble starch synthases from various organisms.

a: glycogen synthase from E. coli b: GBSS I from barley c: GBSS I from wheat d: GBSS I from maize e: GBSS I from rice f: GBSS I from cassava g: GBSS I from potato h: GBSS II from pea i: GBSS II from potato k: SSS from rice 1: SSS A from potato m: SSS B from potato The marked regions (II) and (III) indicate three peptide sequences which are strongly conserved between the various starch synthases and glycogen synthases.

Fig. 8 shows activity gels of the soluble starch synthase isotypes from tuber extracts from wild-type and starch synthase "antisense" potato plants.

A) GBSS II "antisense" plant, lines 14 and 35, K wild-type plant B) SSS A "antisense" plant, lines 25 and 39 K wild-type plant C) SSS B "antisense" plant, lines 1 and 4, K wild-type plant jig each of the protein extracts were separated on a native gel and the activities of the synthase isotypes were determined in the citrate-stimulated mixture with 0.1% amylopectin as primer. The synthesized glucans were dyed with Lugol's solution.

The examples serve to illustrate the invention.

In the examples, the following methods were used: 1. Cloning methods Vector pBluescript II SK (Stratagene) was used for cloning in E.

coli.

For plant transformation, the gene constructs were cloned into the binary vector pBinAR Hyg (DSM 9505).

2. Bacterial strains For the Bluescript vector and for the pBiriAR Hyg constructs the E. coli strain DH5a (Bethesda Research Laboratories, Gaithersburg, USA) was used. F6r the in vivo excision the E. coli strain XLl-Blue was used.

The transformation of the plasmids in the potato plants was carried out using the Agrobacterium tumefaciens strain C58C1 pGV2260 (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777-4788).

3. Transformation of Agrobacterium tumefaciens The transfer of the DNA was carried out by direct transformation according to the method by Hbfgen Willmitzer (Nucl. Acids Res.

16 (1988), 9877). The plasmid DNA of transformed Agrobacteria was isolated according to the method by Birnboim Doly (Nucl. Acids Res. 7 (1979), 1513-1523) and was analyzed gel electrophoretically after suitable restriction digestion.

4. Transformation of potatoes Ten small leaves of a potato sterile culture (Solanum tuberosum L.cv. Desiree) were wounded with a scalpel and placed in 10 ml MS medium (Murashige Skoog, Physiol. Plant. 15 (1962), 473) containing 2% sucrose which contained 50 pl of a selectively grown overnight culture of Agrobacterium tumefaciens. After gently shaking the mixture for 3-5 minutes it was further incubated in the dark for 2 days. For callus induction the leaves were placed on MS medium containing 1.6% glucose, 5 mg/l naphthyl acetic acid, 0.2 mg/l benzyl aminopurine, 250 mg/l claforan, mg/l kanamycin, and 0.80% Bacto Agar. After incubation at and 3,000 lux for one week the leaves were placed for shoot induction on MS medium containing 1.6% glucose, 1.4 mg/1 zeatin ribose, 20 mg/l naphthyl acetic acid, 20 mg/l giberellic acid, 250 mg/l claforan, 50 mg/l kanamycin and 0.80% Bacto Agar.

Radioactive labeling of DNA fragments The DNA fragments were radioactively labeled using a DNA Random Primer Labelling Kit of Boehringer (Germany) according to the manufacturer's information.

6. Determination of the starch synthase activity The starch synthase activity was determined via the determination of the incorporation of 14 C glucose from ADP 14C glucose into a product insoluble in methanol/KCl as described by Denyer and Smith (Planta 186 (1992), 609-617).

7. Detection of soluble starch synthases in the native gel In order to detect the activity of soluble starch synthases by non-denaturing gel electrophoresis tissue samples of potato tubers were extracted with 50 mM Tris-HCl pH 7.6, 2 mM DTT, mM EDTA, 10% glycerol and 0.4 mM PMSF. Electrophoresis was carried out in a MiniProtean II chamber. (BioRAD). The monomer concentration of the gels having 1.5 mm thickness was 25 mM Tris-glycine pH 8.4 served as gel and running buffer. Equal amounts of protein extract were applied and separated for 2 hrs at 10 mA per gel.

The activity gels were subsequently incubated in 50 mM tricine NaOH pH 8.5, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 1 mM ADP glucose, 0.1% (wt./vol.) amylopectin and 0.5 M sodium citrate. The glucans formed were dyed with Lugol's solution.

8. Starch analysis The starch produced by the transgenic potato plants was characterized using the following methods: a) Determination of the phosphate content In potato starch some glucose units may be phosphorylated at the carbon atoms at positions C3 and C6. In order to determine the phosphorylation degree at the C6 position of the glucose 100 mg starch were hydrolyzed in 1 ml 0.7 M HC1 at 95°C for 4 hours (Nielsen et al., Plant Physiol. 105 111-117). After neutralization with 0.7 M KOH, 50 pil of the hydrolysate were subjected to a photometric-enzymatic test to determine the glucose-6-phosphate content. The alteration of the absorption of the test mixture (100 mM imidazole/HCl; 10 mM MgC1 2 0.4 mM NAD; 2 units glucose-6phosphate dehydrogenase from Leuconostoc mesenteroides; 30 0

C)

was measured at 334 nm.

b) Analysis of the side chain length distribution For an analysis of the side chains of the starch molecules 1 ml of a 0.1% starch solution was digested with about 1 unit isoamylase overnight at 37 0 C in 100 mM sodium citrate buffer, pH 4.0 Lee, Analytical Biochemistry 189 (1990), 151- 162). The individual glucan chains were separated via a complex gradient on HPLC (column PAl; elution. with 150 mM NaOH with sodium acetate gradients).

c) Determination of granule size The granule size was determined with a photosedimentometer of the "Lumosed" type by Retsch GmbH, Germany. For this purpose, 0.2 g starch were suspended in about 150 ml water and measured immediately. The program supplied by the manufacturer together with the photosedimentometer calculated the average diameter of the starch granules based on an average density of the starch of 1.5 g/1.

d) Pastification properties The pastification curves of the starch were recorded with a Viskograph E of Brabender oHG, Germany, or with a Rapid Visco Analyser, Newport Scientific Pty Ltd, Investment Support Group, Warriewood NSW 2102, Australia. When the Viskograph E was used, a suspension of 30 g starch in 450 ml water was subjected to the following heating regimen: heating up from 0 C to 96 0 C at 30/min, maintaining constant for 30. minutes, cooling off to 30 0 C at 30/min and maintaining constant for another 30 minutes. The temperature profile yielded characteristic pastification properties.

When the Rapid Visco Analyser was used, a suspension of 2 g starch in 25 ml water was subjected to the following heating regimen: suspending at 50 0 C for 50 s, heating up from 50°C to 0 C at 12 0 /min, maintaining constant for 2.5 minutes, cooling off to 50 0 C at 16.40/min and maintaining constant for another 2 minutes. The temperature profile yielded the maximum and the final viscosity as well as the pastification temperature.

Example 1 Identification, isolation and characterization of two cDNAs encoding the isotypes SSS B and GBSS II of the starch synthase from Solanum tuberosum Although SSS proteins have already been detected in a variety of plant species, inter alia in potato, and cDNA sequences have been described for SSS proteins from rice (Baba et al., supra), the purification of these proteins from potato or other plants as well as the identification of such DNA sequences has not been successful. The problem in isolating such DNA sequences resides in that the homogeneous purification of soluble starch synthases so far has not been successful due to technical reasons, although it has been attempted many times. The soluble synthases co-purify in all purification steps with the branching enzyme and other impurities. Therefore, these proteins have not been amenable to the detection of partial amino acid sequences. It is hence extremely difficult to identify cDNA sequences by hybridization to degenerate oligonucleotides derived from the amino acid sequence. For the same reasons, it is not possible to develop antibodies which specifically recognize these enzymes and thus could be used to screen expression libraries.

The prerequisite for the isolation of DNA sequences encoding SSS proteins from potato by hybridization to heterologous probes encoding the soluble starch synthases from other plant species is that there is sufficiently high homology and at the same time no significant homologies to other encoding DNA sequences. In the case of the only heterologous DNA sequence from rice available (Baba et al., supra), however, it was known that it has high homologies to the granule-bound starch synthases from rice as.

well as to GBSS I and therefore presumably also to GBSS II from potato. Due to these high homologies to GBSS I and II crosshybridizations occur to GBSS I and II cDNAs when screening cDNA libraries. The identification of cDNAs which encode SSS proteins can therefore only be achieved by differential screening. This, however, requires the availability of cDNA sequences for GBSS I and II proteins from potato. cDNA sequences encoding GBSS I from potato, however, have not been available so far.

In the following, the isolation of a cDNA encoding a soluble starch synthase from potato is described.

For this purpose, a DNA fragment from a cDNA from rice encoding a soluble starch synthase (Baba et al., 1993, Plant Physiol.

103:565-573) was amplified using the polymerase chain reaction.

The following oligonucleotides were used as primers: Oligonucleotide 1: 5'-ACAGGATCCTGTGCTATGCGGCGTGTGAAG-3' (Seq ID No. 14) Oligonucleotide 2: 5'-TTGGGATCCGCAATGCCCACAGCATTTTTTTC-3' (Seq ID No. The fragment resulting from PCR was 1067 bp long. This DNA fragment was later on used as heterologous probe for the identification of cDNA sequences from potato encoding soluble starch synthases.

For the preparation of a cDNA library, poly(A mRNA was isolated from potato tubers of the potato variety "Berolina". Starting from the poly(A mRNA cDNA was prepared according to the method of Gubler and Hoffmann (1983, Gene 25:263-269) using an Xho I oligo d(t) 18 primer. This cDNA was first provided with an EcoR I linker and then digested with Xho I and ligated in a specific orientation into a lambda ZAP II vector (Stratagene) which had been digested with EcoR I and Xho I.

500,000 plaques of a thus constructed cDNA library were screened for DNA sequences which are homologous to the heterologous probe of rice using said probe. Since the probe from rice used strongly cross-hybridizes to various sequences from potato, a direct identification of cDNA molecules encoding soluble starch synthases was not possible. From homology comparisons it was known that the cDNA encoding the SSS protein from rice has a high homology to the GBSS I cDNA already isolated from potato. Since GBSS I and GBSS II exhibit high homologies in other organisms, it could be presumed that the probe from rice would also exhibit a high homology to GBSS II sequences from potato. In order to make an ,identification of cDNA sequences possible which encode a soluble starch synthase from potato, it was therefore necessary to have sequences available encoding GBSS I and II from potato.

DNA sequences encoding GBSS I from potato had already been described, however, none encoding GBSS II from potato. Therefore, a cDNA was isolated encoding the GBSS II from potato.

For this purpose, granule-bound proteins from potato starch were isolated. The isolation was carried out by electroelution in an elution device which was constructed in analogy to the "Model 422 Electro-Eluter" (BIORAD Laboratories Inc., USA) but had a substantially greater volume (about 200 ml). 25 g dried starch were dissolved in elution buffer (final volume 80 ml). The suspension was heated in a water bath to 70-80 0 C. 72.07 g urea were added (final concentration 8 M) and the volume was filled up with elution buffer to give 180 ml. The starch was dissolved under constant stirring and developed a glue-like consistency.

The proteins were electroeluted overnight from the solution using the elution device (100 V; 50-60 mA). The proteins eluted were carefully removed from the device. Suspended matter was removed by short centrifugation. The supernatant was dialyzed 2-3 times for one hour each at 4 0 C against dialysis buffer. Then, the volume of the protein solution was determined. The proteins were precipitated by adding ammonium sulfate (90% final concentration) while constantly stirring the solution at 0°C. The proteins precipitated were sedimented by centrifugation and dissolved in protein buffer.

The proteins isolated were used to prepare polyclonal antibodies from rabbits which specifically detect granule-bound proteins.

With the help of such antibodies a cDNA expression library was then screened by standard methods for sequences encoding the granule-bound proteins. The expression library was prepared as described above.

Positive phage clones were purified further using standard techniques. By way of the in vivo excision method E. coli clones were obtained from positive phage clones which contain a doublestranded pBluescript plasmid exhibiting the respective cDNA insert. After ascertaining the size and the restriction pattern of the inserts suitable clones were analyzed further. A clone cGBSSII was identified as a clone encoding the GBSSII protein.

From this clone, plasmid pGBSSII (Fig. 5) was isolated and its cDNA insert was determined by standard techniques by the didesoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA 84 (1977), 5463-5467). The insert is 1925 bp long and is merely a partial cDNA sequence. The nucleotide sequence is indicated under Seq ID No. 5. Sequence comparisons showed that this DNA sequence, too, in various sites exhibited high homologies to the cDNA from rice encoding soluble starch synthase. Therefore, these sequences hybridize to the probe from rice when the cDNA library is screened.

The insert of this plasmid was later on used as probe in the screening of a cDNA library from potato to identify sequences encoding GBSS II proteins.

When screening the expression library with the polyclonal antibodies which are directed to the granule-bound proteins clones were isolated besides the clone cGBSSII that exhibited the cDNA inserts encoding GBSS I from potato. From one of these clones, cGBSSI, plasmid pGBSSI was isolated and the sequence of the cDNA insert was determined. This sequence substantially corresponded to the known DNA sequences encoding GBSSI from potato (Visser et al., Plant Sci. 64 (1989), 185-192; van der Leij et al., Mol. Gen. Genet. 228 (1990), 240-248). This cDNA insert, obtained in plasmid pGBSS I, was therefore later on used as probe when screening a cDNA library from potato tubers in order to identify sequences encoding the GBSS I proteins.

The above-described cDNA library from potato was first screened for sequences encoding GBSS I or GBSS II from potato. For this purpose, the phage plaques were transferred to nitrocellulose filters, the DNA was denatured by NaOH treatment, the filters were neutralized and the DNA was fixated on the filters by heat treatment. The filters were prehybridized for 2 hours at 42 0 C in 0.25 M NaHP0 4 pH 7.2, 0.25 M NaC1, 7% SDS, 1 mM EDTA, formamide, 10% PEG. Then the filters were hybridized overnight at 42 0 C in 0.25 M NaHP0 4 pH 7.2, 0.25 M NaCl, 7% SDS, 1 mM EDTA, formamide, 10% PEG after the respective radioactively labeled probe had been added. As probe on the one hand the cDNA insert from plasmid pGBSSII was used and one the other hand the cDNA insert from plasmid pGBSSI.

The filters were subsequently washed 2 x 30 min in 0.1 x SSC, SDS at 65 0 C and exposed on X-ray films.

In a parallel procedure, filters of the same cDNA library were hybridized under the same conditions as described for GBSS I and GBSS II with the radioactively labeled cDNA probe derived from rice. The washing of the filters was carried out in this case for 2 x 30 min at 40 0 C with 2 x SSC, 0.5% SDS. Phage clones that did not hybridize to GBSS I or GBSS II from potato but to the rice cDNA were purified further using standard techniques. By way of the in vivo excision method E. coli clones were obtained from positive phage clones, which contain a double-stranded pBluescript plasmid exhibiting the respective cDNA insert. After ascertaining the size and the restriction pattern of the inserts suitable clones were subjected to a sequence analysis.

Example 2 Sequence analysis of the cDNA insert of plasmid pSSSB Plasmid pSSSB (Fig. 2) was isolated from an E. coli clone obtained according to Example 1 and its cDNA insert was determined by standard techniques using the didesoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insert is 1758 bp long and represents a partial cDNA. The nucleotide sequence is indicated under Seq ID No. 3.

The corresponding amino acid sequence is depicted under Seq ID No. 4.

Example 3 Isolation of the full-length cDNA encoding the GBSS II isotype of the granule-bound starch synthase from Solanum tuberosum A leaf-specific cDNA expression library from Solanum tuberosum L.

cv. D&siree (KoBmann et al., Planta 186 (1992), 7-12) was screened for full-length clones by standard techniques using hybridization to a 5' fragment of the cDNA insert of plasmid pGBSS II (1.9 kb). As a result, plasmid pGBSS II-VK could be isolated that contains a cDNA insert having a length of about 2.8 kb.

Example 4 Sequence analysis of the cDNA insert of plasmid pGBSS II-VK Plasmid pGBSS II-VK was isolated from the E. coli clone obtained according to Example 3 and its cDNA insert was determined by standard techniques using the didesoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci.. USA 74 (1977), 5463-5467). The insert is about 2.8 kb long. The nucleotide sequence is indicated under Seq ID No. 7 and comprises besides flanking regions the entire coding region for the GBSSII protein from potato. The molecular weight derived from the amino acid sequence of the protein is about 85.1 kD.

Example Isolation of the full-length cDNA encoding the SSS B isotype of the soluble starch synthase from Solanum tuberosum A leaf-specific cDNA expression library from Solanum tuberosum L.

cv. Desire (Ko8mann et al., Planta 186 (1992), 7-12) was screened for full-length clones by standard techniques using hybridization to a 5' fragment of the cDNA insert of plasmid pSSS B (1.6 kb). As a result, plasmid pSSS B-VK could be isolated that contains a cDNA insert having a length of about 2.3 kb.

Example 6 Sequence analysis of the cDNA insert of plasmid pSSS B-VK Plasmid pSSS B-VK was isolated from the E. coli clone obtained according to Example 5 and its cDNA insert was determined by standard techniques using the didesoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insert is about 2.3 kb long. The nucleotide sequence is indicated under Seq ID No. 9 and comprises besides flanking regions the entire coding region for the B isotype of the soluble starch synthase from potato. The molecular weight derived from the amino acid sequence of the protein is about 78.6 kD.

Example 7 Identification, isolation and characterization of a cDNA encoding the SSS A isotype of the soluble starch synthase from Solanum tuberosum A sequence comparison between the sequences encoding soluble and granule-bound starch synthase from plants known so far (Fig. 7) showed that there are three strongly conserved regions between the various proteins (regions (II) and (III) in Figure 7).

In order for a soluble starch synthase from potato to be isolated, these three regions were selected to generate polyclonal peptide antibodies. For this purpose, three synthetic polypeptides having the following amino acid sequences were prepared: Peptide 1: NH 2 -PWSKTGGLGDVC-COOH (Seq ID No. 16) Peptide 2: NH 2 -PSRFEPCGLNQLY-COOH (Seq ID No. 17) Peptide 3: NH 2 -GTGGLRDTVENC-COOH (Seq ID No. 13) These peptides were coupled to the KLH carrier (keyhole limpet homocyanin) and then used to prepare polyclonal antibodies in rabbits (Eurogentec, Seraing, Belgium).

The resulting antibodies were designated as follows: anti-SS1 polyclonal antibody against peptide 1 anti-SS2 polyclonal antibody against peptide 2 anti-SS3 polyclonal antibody against peptide 3.

The antibodies were examined for their specificity with partially purified soluble starch synthase from potato.

The purification of the soluble starch synthases was carried out as follows: kg potatoes were processed in 2 1 buffer A. After removal of the starch by centrifugation at 1000 g for 5 min the protein extract was bound to DEAE-FastFlow column material (Pharmacia LKB)(equilibrated with buffer After washing the column with a five-fold column volume of buffer B, bound proteins were eluted with 300 mM NaC1 in buffer B. The eluted proteins were collected fractionwise and fractions having a high starch synthase activity were pooled. The pooled fractions were desalted by chromatography on a gel filtration column (G25) which was equilibrated with buffer B. 1 volume sodium citrate, 50 mM Tris-HCl pH 7.6, 2.5 mM DTT, 2 mM EDTA were added to the eluate. The protein solution was applied to an amylose resin column (AR column) equilibrated with buffer C. The column was washed with the 20-fold column volume of buffer C. Bound proteins were then eluted with buffer B.

The fractions exhibiting high starch synthase activity were pooled and desalted by gel filtration on a G25 column.

The fractions having high starch synthase activity were applied to a. MonoQ column equilibrated with buffer B. The column was washed with a five-fold column volume of buffer B. Bound proteins were eluted using a linear NaC1 gradient of 0-300 mM and pooled fractionwise.

The analysis of the fractions for their starch synthase activity and for their molecular weight was carried out using various methods: a) analysis of the fractions on a native polyacrylamide gel b) analysis of the fractions on a denaturing SDS polyacrylamide gel and subsequent silver staining c) determination of the synthase activity by incorporation of radioactively labeled ADP glucose (Amersham, UK) in newly synthesized starch d) analysis of the fractions in a Western blot.

For a Western blot analysis, 50 gg, 5 Ag and 0.5 pg protein of a protein crude extract were electrophoretically separated on an SDS polyacrylamide gel along with 15 pg protein of the fractions eluted from the DEAE FastFlow column, 10 gg protein of the factions eluted from the AR column and 3 ig protein of the fractions eluted from the MonoQ column. The proteins were transferred onto a nitrocellulose membrane using the semidry ,electroblot method.

43 Proteins that were recognized by the antibodies anti-SS1, anti- SS2 or anti-SS3 were identified using the "Blotting detection kit for rabbit antibodies RPN 23" (Amersham, UK) according to the manufacturer's instructions.

Three parallel Western blot analyses were performed with the above-described polyclonal antibodies anti-SS1, anti-SS2 and anti-SS3. It was found that the antibody anti-SS1 specifically recognized GBSS I and GBSS II and that the antibody anti-SS2 exhibited no specificity. Only antibody anti-SS3 specifically recognized in the Western blot new proteins, particularly proteins with molecular weights of 120-140 kD, besides GBSS I and GBSS II.

Antibody anti-SS3 was then used to screen a cDNA library from potato tubers for sequences encoding the soluble starch synthases from potato. For this purpose, a cDNA library prepared as described in Example 1 was used. For an analysis of the phage plaques they were transferred onto nitrocellulose filters which were previously incubated for 30-60 min in a 10 mM IPTG solution and then dried on filter paper. The transfer was carried out for 3 hrs at 37 0 C. The filters were then incubated for 30 min at room temperature in block reagent and washed twice for 5-10 min in TBST buffer. The filters were shaken for 1 hr at room temperature or for 16 hrs at 4 0 C with the polyclonal antibody anti-SS3 in suitable dilution. Plaques expressing a protein that was recognized by antibody anti-SS3 were identified using the "Blotting detection kit for rabbit antibodies RPN 23" (Amersham, UK) according to the manufacturer's instructions.

Phage clones of the cDNA library expressing a protein that was recognized by antibody anti-SS3 were further purified using standard techniques. With the help of the in vivo excision method (Stratagene) E. coli clones were obtained from positive phage clones, which contain a double-stranded pBluescript II SK plasmid with the corresponding cDNA insert between the EcoRI and the Xho I restriction site of the polylinker. After ascertaining the size and the restriction pattern of the inserts a suitable clone was subjected to sequence analysis.

Example 8 Sequence analysis of the cDNA insert of plasmid pSSSA Plasmid pSSA (Fig. 1) was isolated from an E. coli clone obtained according to Example 7 and its cDNA insert was determined by standard techniques using the didesoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insert is 2303 bp long. The nucleotide sequence is indicated under Seq ID No. 1. The corresponding amino acid sequence is depicted under Seq ID No. 2.

A sequence analysis and a sequence comparison with known DNA sequences showed that the sequence depicted under Seq ID No. 1 is new and comprises a partial coding region encoding a protein having homology to starch synthases from various organisms. The protein encoded by this cDNA insert or by sequences hybridizing thereto is designated SSSA within this application.

This DNA sequence differs from the DNA sequence depicted under Seq ID NO. 2 which likewise encodes a soluble starch synthase from potato and could not be isolated from a cDNA library from potato tubers using the method described in Example 1.

Example 9 Isolation of the full-length cDNA encoding the SSS A isotype of the soluble starch synthase from Solanum tuberosum A leaf-specific cDNA expression library from Solanum tuberosum L.

cv. D&sirie (KoBmann et al., Planta 186 (1992), 7-12) was screened for full-length clones by standard techniques using hybridization to a 5' fragment of the cDNA insert of plasmid pSSSA (2.3 kb). As a result, a clone could be isolated that contains a cDNA insert that is about 1.86 kb longer in the region. The cDNA insert had an entire length of about 4.16 kb.

Example Sequence analysis of the cDNA insert of plasmid pSSSA-VK Plasmid pSSSA-VK was isolated from an E. coli clone obtained according to Example 9 and its cDNA insert was determined by standard techniques using the didesoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insert is about 4.16 kb long. The nucleotide sequence is indicated under Seq ID No. 11. The corresponding amino acid sequence is depicted under Seq ID No. 12. The molecular weight derived from the amino acid sequence of the SSSA protein is about 135 kD.

Example 11 Construction of plasmid p35S-anti-SSSA and introduction of the plasmid into the genome of potato plants From plasmid pSSSA a DNA fragment of about 2.1 kb was isolated using the restriction endonucleases Xba I and Asp 718 which comprises the coding region for the A isotype of the soluble starch synthase from potato, and was ligated into vector pBinAR Hyg (DSM 9505) which was digested with Xba I and Asp 718.

The insertion of the cDNA fragment results in an expression cassette which is composed of fragments A, B and C as follows (Fig. 3): Fragment A (529 bp) contains the 35S promoter of the Cauliflower mosaic virus (CaMV). The fragment comprises nucleotides 6909 to 7437 of the CaMV (Franck et al., Cell 21 (1980), 285-294).

Fragment B contains besides flanking regions the protein-encoding region of the A isotype of the soluble starch synthase from Solanum tuberosum. This region was isolated as Xba I/Asp 718 fragment from pSSSA as described above and was fused to the promoter in pBinAR Hyg in antisense orientation.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The size of plasmid p35S-anti-SSSA is about 13 kb.

The plasmid was transferred to potato plants using Agrobacteriamediated transformation as described above. Whole plants were regenerated from the transformed cells.

As a result of transformation the transgenic potato plants exhibited a reduced activity of A isotype of the soluble starch synthase (cf. Figure 8).

The starch produced by these plants differs from the starch synthesized by wild-type plants in its phosphate content, in the viscosity of aqueous solutions, its pastification properties and the mean granule size. The results are depicted in Table I.

The phosphate content of the starch produced in transgenic plants is at least 30%, preferably 50%, particularly 70% higher than that of the starch synthesized by the wild-type plants.

The final viscosity of the starch from SSS A "antisense" plants exhibits values that are at least 10%, preferably particularly 30% lower than those of the starch synthesized by wild-type plants.

The pastification temperature, the maximum viscosity and the mean granule size of the modified starch is clearly lower than that of the starch produced in wild-type plants (see Table I).

Table I Characteristics of the starch from wild-type and SSS A "antisense" potato plants Wild-type Line 25 Line 39 Phosphate content [nmol 8.50 0.4 14.61 0.3 14.54 0.2 mg 1 starch 1 Pastification temperature 69.5 67.4 66.2 Maximum viscosity [cP] 4044 3720 3756 Final viscosity at 50 0 C 3312 2904 2400 [cP] Mean granule size [pm] 29 24 27 Example 12 Construction of plasmid p35S-anti-SSSB and introduction of the plasmid into the genome of potato plants From plasmid pSSSB a DNA fragment of about 1.8 kb was isolated using the restriction endonucleases Xho I and Spe I which comprises the coding region for the B isotype of the soluble starch synthase from potato, and was ligated into vector pBinAR Hyg (DSM 9505) which was digested with Sma I.

The insertion of the cDNA fragment results in an expression cassette which is composed of fragments A, B and C as follows (Fig. 4): 47 Fragment A (529 bp) contains the 35S promoter of the Cauliflower mosaic virus (CaMV). The fragment comprises nucleotides 6909 to 7437 of the CaMV (Franck et al., Cell 21 (1980), 285-294).

Fragment B contains besides flanking regions the protein-encoding region of the B isotype of the soluble starch synthase from Solanum tuberosum. This region was isolated as Xho I/Spe I fragment from pSSSB as described above and was fused to the promoter in pBinAR Hyg in antisense orientation.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The size of plasmid p35S-anti-SSSB is about 13 kb.

The plasmid was transferred to potato plants using Agrobacteriamediated transformation as described above. Whole plants were regenerated from the transformed cells.

As a result of transformation the transgenic potato plants exhibited a reduced activity of B isotype of the soluble starch synthase (cf. Figure 8).

Example 13 Construction of plasmid p35S-anti-GBSS I and introduction of the plasmid into the genome of potato plants From plasmid pGBSS II a DNA fragment of about 1.9 kb was isolated using the restriction endonucleases Asp 718 and Sma I which comprises the coding region for the GBSS II isotype of the soluble starch synthase from potato. The ends of the fragment were filled in with the T4 polymerase and the fragment was ligated into vector pBinAR Hyg (DSM 9505) which was digested with Sma I.

The insertion of the cDNA fragment results in an expression cassette which is composed of fragments A, B and C as follows (Fig. 6): Fragment A (529 bp) contains the 35S promoter of the Cauliflower mosaic virus (CaMV). The fragment comprises nucleotides 6909 to 7437 of the CaMV (Franck et al., Cell 21 (1980), 285-294).

Fragment B contains besides flanking regions part of the proteinencoding region of the GBSS II isotype of the starch synthase from Solanum tuberosum. This region was isolated as Asp 718/Sma I fragment from pGBSS II as described above and was fused to the promoter in pBinAR Hyg in antisense orientation once the ends of the fragment had been filled in.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The size of plasmid p35S-anti-GBSS II is about 13 kb.

The plasmid was transferred to potato plants using Agrobacteriamediated transformation as described above. Whole plants were regenerated from the transformed cells.

As a result of transformation the transgenic potato plants exhibited a reduced activity of GBSS II isotype of the starch synthase (cf. Figure 8).

The starch produced by these plants differs from the starch synthesized by wild-type plants in its phosphate content, in the viscosity, its pastification properties and the mean granule size. The results are depicted in Table II.

Table II Characteristics of the starch from wild-type and GBSS II "antisense" potato plants Wild-type Line 14 Line 35 Line 44 Phosphate 6.99 0.19 4.52 0.2 4.13 0.06 3.76 0.12 content [nmol mg 1 starch Pastification 64.1 62.55 63.25 63.55 temperature Maximum 4057 2831 2453 2587 viscosity [cP] Final 2849 2816 2597 2587 viscosity at 0 C [cP] Mean granule 37 32 31 32 size [pm] 49 The phosphate content of the starch produced in transgenic plants is at least 35%, preferably 40%, particularly 45% lower than that of the starch synthesized by the wild-type plants.

The maximum viscosity of the starch from GBSS II "antisense" plants exhibits values that are at least 30%, preferably particularly 40% lower than those of the starch synthesized by wild-type plants.

The pastification temperature and the final viscosity of the modified starch is below that of the starch produced in wild-type plants. The mean granule size of the starch produced in transgenic plants is clearly smaller than that of wild-type starch.

Example 14 Overexpression of the soluble starch synthases SSS A and SSS B in E. coli For an overexpression of soluble starch synthases in E. coli strain G6MD2 was cultivated, which is a mutant which exhibits a deletion both in the gig and in the mal operon. Hence it possesses neither the glycogen synthase (glgA), the branching enzyme (glgB) and the AGPase (glgC) nor the amylomaltase (malQ), the maltodextrine phosphorylase (malP) nor the other proteins involved in the metabolization of maltose. Therefore, mutant G6MD2 is not capable of synthesizing glycogen via the ADP glucose pathway nor a-1,4 glucans starting from maltose.

Cells of this mutant were transformed with the cDNA clones pSSSA- VK and pSSSB-VK. The E. coli cells expressing starch synthases were broken up after 2 hrs induction with IPTG in 50 mM Tris-HCl pH 7.6, 10% glycerol, 2 mM EDTA, 2 mM DTT and 0.4 mM PMSF by ultrasonification. As a control, cells transformed with pBluescript were used. Intact cells and cell wall material were removed by centrifugation for 10 min at 13,000 g. Then, the protein concentration of the supernatant was determined. 100 4g protein extract were added to the reaction buffer (final concentration: 50 mM tricine NaOH pH 8.5, 25 mM potassium acetate, 2 mM EDTA and 2 mM DTT, 1 mM ADP glucose). For an examination of the citrate-stimulated reaction (primerindependent) the reaction buffer additionally contained 0.5 M sodium citrate, while the primer-dependent reaction was performed in the presence of 0.02% (wt./vol.) maltooligosaccharides (Glucidex 19; 1-30 glucose units). The reaction was carried out overnight at room temperature. The. synthesized glucans were detected via Lugol's solution and examined spectralphotometrically for further characterization.

Both the SSS A isotype and the SSS B isotype synthesized glucans in the primer-dependent reaction (absence of citrate). The absorption maximum of the glucan synthesized by SSS A was at 614 nm which corresponds to a glucan of about 150 glucose units. The glucan produced by SSS A absorbed at 575 nm, which points to the synthesis of short-chain glucans having a polymerization degree of about 50 glucose units.

In the primer-independent, citrate-stimulated, reaction SSS B isotype alone yielded a glucan which absorbed at 612 nm after dyeing with Lugol's solution. The SSS A isotype showed no activity in the primer-independent reaction and therefore did not synthesize any glucan.

The protein extracts from the cells transformed with pBluescript did not yield any products in any of the reactions.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The entire disclosure in the complete specification of our Australian Patent Application No. 56014/99 is by this cross-reference incorporated into the present specification.

SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Institut fuer Genbiologische Forschung Berlin GmbH STREET: Ihnestrasse 63 CITY: Berlin Country: Germany POSTAL CODE: 14195 TELEPHONE: (030) 8300070 TELEFAX: (030) 83000736 (ii) TITLE OF THE INVENTION: DNA-Molecules encoding enzymes involved in starch synthesis, vectors, bacteria, transgenic plant cells and plants containing these molecules (iii) NUMBER OF SEQUENCES: 17 (iv) COMPUTER-READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPA) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2303 base pairs TYPE: nucleotide STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum STRAIN: cv Berolina TISSUE TYPE: tuber tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in pBluescriptSKII+ (ix) FEATURE: NAME/FEATURE: CDS LOCATION:3..2033 OTHER INFORMATION: /funlction=~ "Polymerization of starch" /product= "Starch synthase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GO CAC GAG GTC AAA AAG CTT GTT AAA TCT GAG AGA ATA GAT GOT OAT His Glu Val Lys Lys Leu Val Lys Ser Giu Arg Ile Asp Oly Asp TOG TGG TAT ACA GAG GTT Trp Trp Tyr Thr Glu Val GTT ATT CCT Val Ile Pro CAG GCA CTT TTC TTG OAT Gin Ala Leu Phe Leu Asp TGG GTT TTT Trp Val Phe OAT GGT CCA CCC AAO CAT GCC ATT GCT Asp Gly Pro Pro Lys His Ala Ile Ala TAT OAT AAC Tyr Asp Asn A.AT CAC CGC CAA Asn His Arg Gin so GAC TTC CAT GCC ATT GTC CCC AAC CAC ATT CCO GAG Asp Phe His Ala Ile Val Pro Asn His Ile Pro Giu OAA TTA Oiu. Leu.

TAT TOG OTT GAG Tyr Trp Val Glu.

GAA CAT CAG ATC Giu His Gin Ile AAG ACA CTT CAG Lys Thr Leu Gin GAG AGA AGO CTT Oiu Arg Arg Leu

AGA

Arg GAA OCO OCT ATO Glu Ala Ala Met OCT AAG OTT GAA Ala Lys Val Oiu ACA OCA CTT CTO Thr Ala Leu Leu

AAA

Ly s 100 ACT GA.A ACA AAG Thr Giu. Thr Lys AGA ACT ATO AAA Arg Thr Met Lys TCA TTT Ser Phe 110 TTA CTG TCT CAG AAG CAT OTA OTA Leu Leu Ser Gin Lys His-Val Val 115 ACT GAG CCT CTT Thr Oiu Pro Leu OAT ATC CAA Asp Ile Gin 125 ACA OTA CTT Thr Val Leu OCT OGA AGC Ala Gly Ser 130 AOC OTC ACA OTT TAC TAT AAT CCC 0CC Ser Val Thr Val Tyr Tyr Asn Pro Ala A.AT OCT Asn Gly 145 AAA CCT GA.A. ATT Lys Pro Olu Ile

TG

Trp 150 TTC AGA TOT TCA Phe Arg Cys Ser

TTT

Phe 155 AAT COC TOG ACT Asn Arg Trp Thr

CAC

H is 160 COC CTO OCGT CCA Arg Leu Gly Pro CCA CCT CAG AAA Pro Pro Gin Lys

ATG

Met 170 TCG CCT OCT Ser Pro Ala GAA AAT Oiu Asn 175 TAT ATO Tyr Met 190 GOC ACC CAT GTC Gly Thr His Val OCA ACT GTG AAG OTT Ala Thr Vai Lys Val.

185 CCA TTG OAT GCA Pro Leu Asp Ala ATO OAT TTT GTA TTT TCC GAG AGA GAA OAT GOT GOG ATT TTT GAC AAT Met Asp Phe Val Phe Ser Glu Arg Giu Asp Gly Gly Ile Phe Asp Asn AAG AGC GGA Lys Ser Gly 210 ATG GAC TAT CAC Met Asp Tyr His ATA CCT GTG Ile Pro Val 215 205 TT T GGA GGA GTC OCT AAA Phe Gly Gly Val Ala Lys 220 GAA CCT Giu Pro 225 CCA ATG CAT ATT Pro Met His Ile

GTC

Val1 230 CAT ATT GCT GTC His Ile Ala Val ATO GCA CCA ATT Met Ala Pro Ile

GCA

Ala 240 AAG GTG GGA GGC Lys Val Gly Gly GOT GAT GTT OTT Gly Asp Val Val

ACT

Thr 250 AGT OTT TOC COT Ser Leu Ser Arg

GCT

Al a 255 OTT CAA GAT TTA Val Gin Asp Leu

AAC

Asn 260 CAT A.AT OTO GAT His Ann Val Asp ATC TTA CCT AAG Ile Leu Pro Lys TAT GAO Tyr Asp 270 815 TOT TTG AAO Cys Leu Lys TTT TOO GOT Phe Trp Oly 290

ATO

Met 275 A.AT AAT OTO AAG Asn Asn Val Lys O AC Asp 280 TTT COG TTT CAC Phe Arg Phe His AAA AAC TAO Lys Asn Tyr 285 GTG GAA GOT Val Giu Gly 000 ACT GAA ATA Oly Thr Oiu Ile

AAA

Lys 295 GTA TOO TTT OGA Val Trp Phe Gly

AAG

Lys 300 CTC TOO Leu Ser 305 OTO TAT TTT TTG Vai Tyr Phe Leu

GAG

Oiu 310 OCT CAA AAC GG Pro Gin Asn Oly TTT TOO AAA 000 Phe Ser Lys Oly

TOO

cy s 320 OTO TAT GOT TOT Val Tyr Gly Cys AAT GAT GOT GA.A Asn Asp Gly Oiu

OGA

Arg 330 TTT GOT TTC TTC Phe Gly Phe Phe 1007 CAC 000 GOT TTG His Ala Ala Leu

GAO

O iu 340 TTT OTT OTO CAA PheLeu Leu Gin OGA TTT AGT COO Oly Phe Ser Pro OAT ATC Asp Ile 350 2055 ATT CAT TOC Ile His Cys GAA CAA TAT Olu Oin Tyr 370 OAT OAT His Asp 355 TOO TOT AGT Trp Ser Ser

GOT

Ala 360 COT OTT OCT TG Pro Val Ala Trp OTO TTT AAG Leu Phe Lys 365 OTO TTO ACO Val Phe Thr 1103 1151 ACA CAC TAT GOT Thr His Tyr Gly

OTA

Leu 375 AGO AAA TOT COT Ser Lys Ser Arg ATA CAT Ile His 385 AAT OTT GA.A Asn Leu Oiu TTT 000 Phe Gly 390 OCA OAT OTC ATT Ala Asp Leu Ile 000 Gly 395 AGA OCA ATO ACT Arg Ala Met Thr 1199 1247 AAO GOA GAO AAA Asn Ala Asp Lys 400 GOT ACA ACA Ala Thr Thr 405 OTT TCA CCA Val Ser Pro

ACT

Thr 410 TAO TOA CAG GAO Tyr Ser Gin Olu TOT G-GA AAC OCT OTA ATT 000 COT CA.C OTT CAC AAO TTO CAT GOT ATA Ser Gly Asn Pro Vai Ile Ala Pro His Leu His Lys Phe His Gly Ile 1295 430 GTC AAT GO Val Asn Gly ATT CCC ATT Ile Pro Ile 450

ATT

Ile 435 CAC CCA GAT ATT TGG GAT Asp Pro Asp Ile Trp Asp CCT TTA AAC GAT AAC TTC 1343 Pro Leu Asn Asp Lys Phe 445 AAA ACA OCA Lys Thr Ala CCC TAC ACC TCA Pro Tyr Thr Ser

GAA

Giu 455 XAC GTT GTT GAA Asn Val Val Giu

GGC

Gly 460 1391 CCC AAG Ala Lys 465 GAA GCT TTO CAG Olu Ala Leu Gin

CGA

Arg 470 AAA CTT GGA CTC Lys Leu Gly Leu CAG GCT GAC CTT Gin Ala Asp Leu 1439 1487 CCT Pro 480 TWO GTA GGA ATT Leu Val Gly Ile

ATC

Ile 485 ACC CGC TTA ACT Thr Arg Leu Thr

CAC

His 490 CAG AAA GGA ATO Gin Lys Gly Ile

CAC

His 49S CTC ATT AAA CAT Leu Ile Lys His

OCT

Al a 500 ATT TGC CGC ACC Ile Trp Arg Thr

TTG

Leu 505 GAA CCC AAC GGA Glu Arg Asn Gly CAG OTA Gin Val 510 1535 GTC TTG CTT Val Leu Leu AAT TTG GCA Asn Leu Ala 530

OCGT

Gly 515 TCT GCT CCT CAT Ser Ala Pro Asp AGO GTA CAA AAC Arg Val Gin Asn GAT TTT OTT Asp Phe Val 525 GCA CGA CTC Ala Arg Leu 1583 1631 A.AT CAA TTG CAC Asn Gin Leu His

TCC

Ser 535 AAA TAT AAT GAC Lys Tyr Asn Asp

CC

Arg 540 TOT OTA Cys Leu 545 ACA TAT GAC GAG Thr Tyr Asp Giu OTT TCT CAC OTO Leu Ser His Leu TAT OCT GGT GOT Tyr Ala Gly Ala 1679 1727 TTT ATT CTA OTT Phe Ile Leu Val COT TCA Pro -Ser 565 ATA TTT GAG Ile Phe Glu

OCA

Pro 570 TOT OGA OTA ACA Cys Gly Leu Thr OTT ACO OCT ATO Leu Thr Ala Met TAT GOT TCA ATT CCA OTO OTO COT AAA Tyr Gly Ser Ile Pro Val Val Arg Lys 585 ACT GGA Thr Gly 590 1775 OGA OTT TAT Cly Leu Tyr CAA CAG TOT Gin Gin Cys 610

CAT

Asp 595 ACT GTA TTT OAT Thr Val Phe Asp OTT GAO CAT Val Asp His 600 GAO AAA GAO AGA OCA Asp Lys Oiu Arg Ala 1823 GOT OTT GAA OCA AAT OGA TWO AGO TTT Oly Leu Giu Pro Asn Gly Phe Ser Phe GAT GGA GCA CAT Asp Ciy Ala Asp 620 1871 GCT GGC GGA GTT GAT TAT GCT CTG AAT AGA GOT Ala Gly Gly Val Asp Tyr Ala Leu Asn Arq Ala 625 630 GAT OCT CGG GAT TGG TTC AAC TCT TTA TGC A Asp GJly Arg Asp Trp Phe Asn Ser Leu Cys Lys 640 645- 650 GAT TG-G TCT TGG AAC OGA CCT GCT CTT GAT TAT Asp Trp Ser Trp Asn Arg Pro Ala Leu Asp Tyr 660 665 OCT GCT AGA AAG TTA GAA TAGTTAGTTT GTGAGATGC Ala Ala Arg Lys Leu Glu 675 TTCACGAGAT CTGCAATCTG TACAGGTTCA GTGTTTGCGT TATATCAA.AG TATAAATCAA GTCTACACTG AGATCAATAG TCATTTTTTG TGCAACATAT GAA.AGAG OTT AGCCTCTAAT TTTGTTTTGG, GAAGAAATGA GAAATCAAAG GATCCAAALAT (2)'INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 677 amino acids ART: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: His Glu Val Lys Lys Leu Val Lys Ser Glu Arg 1 5 10 Trp Tyr Thr Glu Val Val Ile Pro Asp Gin AlaI 25 Val Phe Ala Asp Gly Pro Pro Lys His Ala le 40 His Arg Gln Asp Phe His Ala Ile Val Pro Asn I so Leu Tyr Trp Val Glu Glu Glu His Gin Ile Phe I 70 75 Glu Arg Arg Leu Arg Glu Ala Ala M4et Arg Ala I 90 CTC TCT GOT TGG TAO Leu Ser Ala Trp Tyr 635 CAG GTO ATG GAA CA-A Gin Val Met Glu Gin 655 TTG GAG OTT TAO OAT Leu Glu Leu Tyr His 670 ~T AGCAGA.AAAA 1919 1967 2015 2063 2123 2183 224] 2303 OTGGACAGOT TTTTATTTCC CAGACAGICO TOAGTTCATT A.ATGTAGTCA TTGATGATTA ACTCTGAAAA AAAAAAA Asp Phe Tyr Ile Thr ValI Gly Leu 3O Asp Pro Leu Glu Asp Asp As n Glu Gin Lys Trp Trp As n Glu G lu Thr Ala Leu Leu Lys Thr Glu Thr Lys Glu Arg Thr Met Lys Ser Phe Leu 100 105 110 Leu Gly Gly 145 Arg Thr Asp Ser Pro 225 Lys Gin Leu Trp Ser 305 Val Ala Se.

Sei 13( Ly Let His Phe Gly 210 Pro Val Asp Lys ly 290 lal 'yr la r Gin 115 r Ser 0 s Pro 1 Gly Val Val 195 Met Met Gly Leu Met 2 275 Gly Tyr I Gly C Leu G 3 Ly Va Glu Pro Arg 180 Phe Asp His Gly Asn 260 ksn Chr 'he :ys lu 40 s His 1 Thr 1 le Leu 165 Ala Ser Tyr Ile Leu 245 His Asn Glu Leu Ser 1 325 Phe I Va.

Va.

Tr 15C Pro Thr Glu His Val 230 Gly Asn V'al Ile ;lu 310 \sn .eu 1 Va I Ty~ 13! Ph Prc Val Arg Ile 215 His Asp Val Lys Lys 295 Pro Asp Leu 1 Tyr 120 r Tyr 4 Arg Gin Lys Glu 200 Pro Ile Val Asp Asp i 280 Val 1 Gin Gly C Gin G 3 Ala P 360 Thi Asr Cys Lys Val 185 Asp Val Ala Vial Ile 265 ?he rrp ~sn lu ;ly '45 ro r Glu Pro Sen Met 170 Pro Gly Phe Val Thr 250 Ile Arg Phe Gly I Arg 1 330 Gly P Val A Pro Leu Asp Ile Gin Ala 125 Al Pht 15! Sel Leu Gly Gly Glu 235 Ser Leu Phe ;ly .eu 15 'he 'he la a As 14 a Asi 5 Prc 1 As 1 Ile Gly 220 Met Leu Pro His Lys 300 Phe Gly Ser Trp n Thr 0 n Aig Ala Ala Phe 205 Val Ala Sen Lys Lys 285 Val C Sen I Phe P Pro A 3 Leu P 365 Va Tr Gl Tyr 190 Asp Ala Pro Arg ryr 270 ksn lu ,ys he ~sp 'he I Le' Thi Asr 171 Met Asr Lys Ile Ala 255 Asp Tyr Gly Gly Cys 335 Ile Lys Asn His 160 i Gly Met Lys Glu Ala 240 Val Cys Phe Leu Cys 320 His Ile Glu His Cys His Asp Trp Ser Ser 355 Gin Tyr Thr His Tyr Gly Leu 370 375 Ser Lys Ser Arg Val Phe Thr Ile His Asn Leu Giu Phe Gly Ala Asp Leu Ile Gly Arg Ala Met Th 385 390 Ala dly Asn Pro Lys 465 Leu Ile Leu Leu Leu 545 Phe Thr Leu Gin Gly 625 Gly Asp Asn Gly Ile 450 Glu Val Lys Leu Ala 530 Thr Ile Ala Tyr Cy 610 Gly Arg Lys Pro Ile 435 Pro Ala Gly His Gly 515 Asr Tyr Leu Met Asp 595 Gly Val Asp Ala p Val 420 Asp Tyr Leu Ile Ala 500 Ser Gin Asp Val Arg 580 Thr Leu C Asp I Trp 1 Thi Il Prc Thr Gin lie 485 Ile Ala Leu Glu Pro 565 Tyr lal ;lu ryr ,he Thr 5 Ala Asp Ser Arg 470 Thr Trp Pro His Pro 550 Ser Gly Phe Pro Ala 630 Asn Val Prc Ile 0lu 455 Lys Arg Arg Asp Ser 535 Leu Ile Ser Asp Asn 6S Leu Ser Se! HiE Tr 44C Asr Leu Leu Thr Pro 520 Lys Sen Phe Ile Val 600 Gly Asn Leu r Prc Lei 42E As; Val Gly Thr Leu 505 Arg Tyr His Glu Pro 585 Asp Phe Arg Cys 0 Thr 410 His Pro Val Leu His 490 Glu Val Asn Leu Pro 570 Val His Ser I Ala I

E

Lys C 650 Ty Ly LeL Gle Lys 475 Gin Arg Gin Asp Ile 555 Cys Val ksp ?he .eu j35 In Sen Phe a Asn 1 Gly 460 Gin Lys Asn Asn Arg 540 Tyr Gly Arg I Lys C Asp C 620 Ser P Val M Gl Hi! As 1 441 Lys Ala Gly Gly Asp 525 Ala Ala Leu Lys ;lu 505 ;ly la let n Glu Gly 430 Lys Thr Asp ile Gin 510 Phe Arg Gly Thr Thr 590 Arg Ala I Trp I Glu G 6 Va 41 Il Phe Al Leu His 495 Va1 Val Leu Ala Gln 575 ;lY kla ~sp 'yr ;ln r Asn 400 1 Ser a Val s lie i Ala Pro 480 Leu Val Asn Cys Asp, 560 Leu Gly Gin Ala Asp 640 Asp Trp Ser Trp Asn Ang Pro Ala Leu Asp Tyr Leu Giu Leu Tyr His Ala 660 665 670 Ala Arg Lys Leu Glu 675 INFORM~ATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1758 base pairs ART: nucleotide STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: cONA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum STRAIN: cv. Berolina TISSUE TYPE: tuber tissue (vii) IMMEDIATE SOURCE: LIBRA.RY: cDNA-library in pBluescriptSKll+ (ix) FEATURE: NAME/FEATURE: CDS LOCATION:1. .1377 OTHER INFORMATION:/function= -Polymerization of starch" product= "Starch synthase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GGC

dly

I

ACG AGC AT GCT GTT-GAC CTT GAT GTG CGG GCC ACT GTC CAT TGC Thr Ser Asn Ala Val Asp Leu Asp Val Arg Ala Thr Val His Cys TTT GGT CAT Phe Gly Asp GTT GAT TGG Val Asp Trp CAG GAA GTA GCC TTC TAC CAT GAA TAC Gin Glu Val Ala Phe Tyr His Glu Tyr AGG GCA GGT Arg Ala Gly GTA TTT GTG Val Phe Val GAC CAC Asp His GGT GCA Gly Ala TCT TCT TAC CGC Ser Ser Tyr Arg CCT GGA ACG 144 Pro Gly Thr CCA TAT Pro Tyr so GGT GAT ATT TAT Gly Asp Ile Tyr TTT GGTGAT Phe Gly Asp CAG TTT CGC TTC Gin Phe Arg Phe

ACT

Thr TTG CTT TCT CAC Leu Leu Ser His OCA TGT GAA GC Ala Cys Glu Ala TTG GTT CTT Leu Val Leu CCA CTG Pro Leu- GGA GGG TTC ACT TAT GGA GAG AAG TGC TTG TTT CTC GCT AAT GAT TGC Gly Gly Phe Thr Tyr Gly Glu Lys Cys Leu Phe 90 Leu Ala Asn Asp Cys AAC GCT GCC Asn Ala Ala GGT GTT TAO Gly Vai Tyr 115

TTG

Leu 100 GTT CCT TTA CTT Val Pro Leu Leu GCG CCC AAG TAT Ala Ala Lys Tyr CGT OCT TAT Arg Pro Tyr 110 AAC ATT GCA Asn Ile Ala AAG GAT GCT CGT Lys Asp Ala Arg

AGT

Ser 120 ATT GTC GCA ATA Ile Val Ala Ile CAT CAG His Gin 130 GGA GTG GAG OCT Gly Val Giu Pro GTA ACC TAC AAT Val Thr Tyr Asn

A.AT

Asn 140 TTG GGT TTG OCT Leu Cly Leu Pro

OCA

Pro 145 CAA TGG TAT GGA Gin Trp Tyr Gly

GCA

Ala 150 OTT GAA TOO ATA Val Giu Trp Ile

TTT

Phe 155 CCC ACA TOO OCA Pro Thr Trp Ala

AGO

Arg 160 GCG CAT GOG OTT Ala His Ala Leu ACT GGT GAA ACA Thr Cly Glu Thr AAC OTT TTG AAA Asn Val Leu Lys COO GOA Gly Ala 175 ATA GOA GTT Ile Ala Val GAA ATA ACA Glu Ile Thr 195

GOT

Ala 180 GAT OGG ATA OTG Asp Arg Ile Leu OTT AGO CAG GGA Val Ser Gin Gly TAO TOA TGG Tyr Ser Trp 190 CTG TTG AGO Leu Leu Ser ACT OCT GAA GGG Thr Pro Giu Gly

GGA

Gly 200 TAT GGG OTA CAT Tyr Gly Leu His ACT AGA Ser Arg 210 CAG TOT GTT OTT Gin Ser Val Leu OCA ATT ACT AAT Oly Ile Thr Asn ATA CAT GTT AAT Ile Asp Val Asn

CAT

Asp 225 TGG AAC CC TOG Trp Asn Pro Ser ACA -GAT Thr Asp 230 GAG OAT ATO Glu His Ile

CT

Al a 235 TOG OAT TAO TOO Ser His Tyr Ser AAT GAO OTO TOO Asn Asp Leu Ser COT OCA AAG GTT Pro Cly Lys Val TOO A.AG ACT GAT Cys Lys Thr Asp OTG CAA Leu Gin 255 AAG GAA OTO Lys Glu Leu ATT OGA AGO Ile Oly Arg 275

GCO

Oly 260 OTT OCA ATT OGA Leu Pro le Arg CAT TOT OCA OTG Asp Cys Pro Leu ATT OGA TTT Ile Oly Phe 270 OTO TCA GCA Leu Ser Ala OTO GAO TAO CAG Leu Asp Tyr Gin

AAA

Lys 280 OCT OTT GAO ATA Gly Val Asp Ile ATT OCA Ile Pro 290 GAA OTT ATO CAC Giu Leu Met Gin CAT OTO CAA OTT Asp Val Gin Val

GTA

Val 300 ATG OTT OGA TOT Met Leu Gly Ser COT GAG AAA CAA TAT GAA GAO TOO ATO AGA CAT ACA GA.A AAT OTT TIT Gly 305 Glu Lys Gin Tyr Glu 310 Asp Trp Met Arg His Thr Glu Asn Leu 315 AAA GAC AAA TTT Lys Asp Lys Phe GCT TGG OTT GGA Ala Trp Val Gly AAT GTT CCA GTT Asn Val Pro Val TCT CAT Ser His -1-1-1 1008 AGG ATA ACA Arg Ile Thr CCG TGT GC Pro Cys Gly 355

GCA

Al a 340 GGA TG GAG ATA Gly Cys Asp Ilie

CTA

Leu 345 TTG ATG CCC TCA Leu Met Pro Ser AGA TTC GAA Arg Phe Glu 350 ACC ATA CCT Thr Ile Pro 1056 1104 TTA AAC CAA TTG Leu Asn Gin Leu GCA ATG AGA TAT Ala Met Arg Tyr ATT OTT Ile Val 370 CAT AGC ACG G His Ser Thr Gly

GGC

Gly 375 CTA AGA GAC ACA Leu Arg Asp Thr

CTG

Val1 380 AAG CAT TTT AAT Lys Asp Phe Asn .1152 1200

CCA

Pro 385 TAT GCT CAA GAA Tyr Ala Gin Gilu

GGA

Giy 390 AAA GOT GAA GOT Lys Giy Giu Gly GGG TGG ACA TTT Gly Trp Thr Phe CCT CTA ACG AGT Pro Leu Thr Ser AAO TTG TTT OAT Lys Leu Phe Asp

ACA

Thr 410 CTO AAO CTG OCG Leu Lys Leu Ala ATC AGO Ile Arg 415 ACT TAT ACA Thr Tyr Thr ATG GCA AGO Met Gly Arg 435

GAA

C iu 420 CAT AAG TCA TCT His Lys Ser Ser

TOO

Trp 425 GAO OGA TTG ATO A.AO AGA GOT Glu Gly Leu Met Lys Arg Gly 1248 1296 1344 GAC TAT TCC TG Asp Tyr Ser Trp

GAA

Giu 440 AAT GCA GCC ATT CAA TAT GAG CAA Asn Ala Ala Ile Gin Tyr Glu Gin 445 OTT TTC Val Phe 450 ACC TGG 0CC Thr Trp Ala TTT -ATA Phe Ile 455 GAT CCT CCA TAT Asp Pro Pro Tyr GTCAGATGAT TTATCAAGAA 1397 AGATTGCAAA CGGGATACAT CATTAAACTA TACOCAOAGC TTTTOOTGCT ATTAGCTACT GTCATTGGGC GCGGAATGTT TGTGGTTCTT TCTGATTCAO AGAGATCAAG TTAGTTCCAA AGACATGTAG CCTGCCCCTG TCTGTGATOA AGTAAAACTA CAAAGGCAAT TAGAAACCCA CCAACA.ACTO CCTCCTTTOG OAGAAGAGTG GAAATATGTA AAAAAGAATT TTGAGTTTAA TGTCAATTGA ATTAATTATT CTCATTTTTA AAAAAAACAT CTCATCTCAT ACAATATATA AAATTGATCA TOATTOATOC CCCCTAAAAA AAAAAAAA~ AAAAAA AAAAAAAA

A

1457 1517 1577 1637 1697 1757 1758 IN~FORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 459 amino acids ART: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gly Thr Ser Asn Ala Val Asp Leu Asp Val Arg Ala Thr Val His Cys 10 Phe Val1 Pro Thr Gly Asn Gly His Pro 145 Ala I le Glu S er Asp' 225.

Gly Asp Tyr Leu Gly Ala Val Gin 130 Gin His Ala Ile Arg 210 Trp *Asp Trp Gly Leu Phe Ala Tyr 115 Gly Trp Al a Val Thr 195 Gin Asn Ala Val Asp Ser Thr Leu 10.0 Lys Val Tyr Leu Ala 180 Thr Ser Pro Gin Phe Ile His Tyr Val Asp Giu Gly Asp 165 Asp Pro Val Ser Glu Val Val Asp Tyr Gly 55 Ala Ala 70 Gly Giu Pr.o Leu Ala Arg Pro Ala 135 Ala-Val 150 Thr Gly Arg Ile Giu Gly Leu Asn 215 Thr Asp 230 Ala His Ala Cy s Lys Leu Ser 120 Val1 Giu G hi Leu Gly 200 Glu *Phe 25 Ser Phe Giu Cys Leu 105 Ile Thr Trp Thr Thr 185 Tyr Ile His Tyz Sez G ly Ala Leu 90 Ala Val1 Tyr Ile Val1 170 Val1 Gly Thr Ile His *Tyr *Asp Pro 75 Phe Ala Ala Asn Phe 155 Asn Ser Leu Asn Ala 235 Gil Arg Asn Leu Leu Lys Ile Asn 140 Pro Val1 Gin H1is G ly 220 Ser ITyi ArS Gir Val Ala Tyr His 12 Leu Thr Leu Gly Glu 205 Ile His *Arg Pro Phe Leu Asn Arg Asn Gly T rp Lys Tyr 190 Leu Asp Tyr Al~ Gl Arc Pro Asp Pro Ile Leu Ala G ly 175 Ser Leu V/al Ser i Gly Thr Phe Leu s0 Cys Tyr Ala Pro Arg 160 Ala Trp Ser Asn Ile 240 Asn Asp Leu Ser Pro Pro Gly Lys Val Gin Cys Lys Thr Asp Leu Gln 62 Pro Asp Cys 265 Lys Giu Leu Gly Leu Pro Ile Arg 260 Pro Leu Ile Gly Ph 270 Ile Gly Arg Leu Asp Tyr Glii Ile Pro Glu Leu Met Gly 305 Lys Arg Pro Ile Pro 385 Pro Thr Met 290 Giu Asp Ile Cys Val1 370 Tyr Leu Tyr Gly Lys Lys Thr Gly 355 His Ala Thr Thr ALrg Gin Phe Ala 340 Leu Ser Gin Ser Giu 420 Asp Tyr Ar g 325 Gly Asn Thr Glu Glu 405 Hlis Gin Giu 310 Ala Cys Gin Gly Gly 390 Ly s Asn 295 Asp Trp Asp Leu Gly 375 Ly s Leu Lys Gly 280 Asp Vai Trp Met Val Gly Ile Leu 345 Tyr Ala 360 Leu Arg Gly Giu Phe Asp Val Gin Ile Ile 285 Val Met 300 Leu Leu Ser G ly Arg His Thr 315 Phe Asn Val 330 Leu Met Pro Met Arg Tyr Asp Thr Vai 380 Gly Thr Gly 395 Thr Leu Lys 410 Glu Gly Leu Glu Pro S er Gly 365 Ly s Trp Leu Met *Asn Val1 Arg 350 Thr Asp Thr Al a Lys 430 Leu Ser 335 Phe Ile Phe Phe Ile 415 Arg P he 320 His Giu Pro Asn Ser 400 Arg Gly Lys Ser Ser Trp 425 ryr Ser Trp Giu Asn Ala Ala Ile Gin Tyr Giu Gin 440 445 435 Vai Phe Thr Trp Ala Phe Ile Asp Pro Pro Tyr 450 455 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1926 base pairs ART: nucleotide STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to rnRNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanui tuberosum STRAIN: cv. Beroiina TISSUE TYPE: tuber tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in pBiuescriptSK+ (ix) FEATURE: NA1KE/FEATURE: CDS LOCATION:2. .1675 OTHER INFORMATION:/function= "Polymerization of starch" /product= "Starch synthase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: C CCC ACG AGC AAA ACT TTA GTA GAT GTT CCT GGA AAC AAG ATC CAG Gly Thr Ser Lys Ser Leu Val Asp Val Pro Gly Lys Lys Ile Gin TCT TAT ATG Ser Tyr Met CAG AGG ALAT Gin Arg Asn ACT GAA GAT Thr Giu Asp CCT TCA TTA CGT Pro Ser Leu Arg AAA GAA TCC TCA GCA TCC CAT GTG CAA Lys Glu SerSer Aia 25 Ser His Vai Ciu AAT CTT GAA GGA Asn. Leu Giu Cly TCA AGT CCT GAG GCA AAC GAA GAG 142 Ser Ser Ala Glu Ala Asn Giu Glu.

CCT GTG ART ATA Pro Val Asn Ile

GAT

Asp GAG AMA CCC CCT Giu Lys Pro Pro TTG GCA GGA 190 Leu Ala Gly ACA ART Thr Asn CTT ATG ARC Val Met Asn ATT ATT Ile Ile CTT- OCA LeU Cly 85 TTC GTC GCT TCA Leu Val Ala Ser TCC CCT CCA TGG Cys Ala Pro Trp

TCT

Ser AAA ACA CCT CCC Lys Thr Gly Cly CAT CTT GCT Asp Val Ala

CCA

Cly 90 GCA TTA CCC AAA Ala Leu. Pro Lys C CT Ala TTG GCT CGA CGT Leu. Ala Arg Arg

GGC

Gly 100 CAC AGA GTT ATG His Arg Val Met GTG GCA CCT CCT Val Ala Pro Arg TAT- CAC Tyr Asp 110 AC TAT CCT Asn Tyr Pro CAT GGT CAG Asp Gly Gin 130

CAR

Ciu 115 CCT CAR CAT TCT Pro Gin Asp Ser GTA AGA AAA ATT Val Arg Lys Ile TAT AAA GTT Tyr Lys Val.

125 ATT CAT CGT Ile Asp Cly GAT GTG GAA CTG Asp Val Giu Val

ACT

Thr 135 TAC TTC CAR CCT Tyr Phe Gin Ala

TTT

P he 140 430 GTG CAT Val Asp 145 TTT OTT TTC ATT Phe Val Phe Ile

GAC

Asp

ISO

ACT CAT ATG TTT AGA CAC ATT CGG ARAC Ser His Met Phe Arg His Ile Gly Asn 155 ARC ATT TAC GCA CCC ARC CCT CTC CAT ATT TTA AAA CCC ATC CTT TTA Asn 160 Ile Tyr Gly Gly 'Asn 165 Arg Val Asp Ile Lys Arg Met Val GTT CCA TOT GOT TTT TGC AAA GCA Phe Cys Lys Ala ATT GAG GTT CCT Ile Glu Val Pro TGG CAT Trp His 185

GGG

Giy Val Pro Cys Gly 190 GTC TGC TAT Val Cys Tyr ACT OCT TTA Thr Ala Leu 210

OGA

G ly 195 OAT GGA AAT TTA Asp Gly Asn Leu

GTG

Val 200 TTC ATT GOT AAT Phe Ilie Ala Asn GAT TGG CAT Asp Trp His 205 GAC AAT GGA Asp Asn Gly TTG CCA GTA TAT Leu Pro Val Tyr

OTG

Leu 215 AAA GOT TAT TAT Lys Ala Tyr Tyr ATT ATG Ile Met 225 AAC TAT ACA AGA Asn Tyr Thr Arg GTC OTG GTG ATT Val Leu Val Ile

CAT

His 235 AAC ATC GOT CAT Asn Ile Ala His

CAG

Gin 240 CGT CGT OCT OCT Oly Arg Cly Pro

TTC

Leu 245 GAG GAT TTT TCA Giu Asp Phe Ser GTA OAT OTT OCA Val Asp Leu Pro

CCA

Pro 255 766 CAC TAT ATG GAO His Tyr Met Asp TTC A.AG TTG TAT GAO Phe Lys Leu Tyr Asp .265 OCA GTA GGA GOT Pro Vai Gly Gly GAG CAT Glu His 270 TIC AAC AT Phe Asn Ile OTT ACT CAT Val Ser His 290

TTT

Phe 275 GCC GOT GOT OTA Ala Ala Gly Leu

AAG

Lys 280 ACA OCA GAT COT Thr Ala Asp Arg CIA OTT ACA Val Vai Thr 285 GOT GOT TG Gly Gly Trp OGA TAT TCA TG Gly Tyr Ser Trp CIA AAGiACT TOO Leu Lys Thr Ser

CAA

Gin 300 910 OGA TO Oly Leu 305 OAT CGO ATA His Gin Ilie ATT AAT Ile Asn 310 GAG A OAT TOO AAA TTA CAG GOT ATT Giu Asn Asp Trp Lys Leu Gin Gly Ile 315 AAT COO ATT CAT Asn Giy Ile Asp AAA GAO TOG AAC Lys Oiu Trp Asn

CT

Pro 330 GAG TTC GAO OTT Giu Leu Asp Vai

CAC

His 335 1006 1054 TTA CAG TCh OAT Leu Gin Ser Asp

GT

Oly 340 TAO ATO AAO TAO Tyr Met Asn Tyr TTG GAO ACG CIA Leu Asp Thr Leu CAG ACT Gin Thr 350 GGC AAG CCT CAA TGT AAA OCT OCA TTG kGGACTGTTACA AAG GAA CTT GGT TTA CCA 1102 Gly Lys Pro OTT CGT GAT Val Arg Asp 370 Cys Lys Ala Ala Lys Glu Leu Gly Leu Pro 365 CTT GAC CCA Leu Asp Pro GAT GTC CCA CTO Asp Val Pro Leu

ATC

Ile 375 GGT TTC ATT GG Gly Phe Ile Gly

AGO

Arg 380 1150 CAA AAG OGln Lys 385 GOGT GTT GAT OTO Gly Val Asp Leu GCT GAG 0CC AGT Ala Glu Ala Ser TOO ATO ATO GOT Trp Met Met Oly 1198 1246

CAG

Gin 400 OAT OTA CA.A CTG A 'sp Val Gin Leu

OTC

Val1 405 ATO TTG 000 ACO Met Leu Gly Thr 000 Gly 410 AGO COT GAO OTT A-rg Arg Asp Leu CAG ATO OTA AGO Gin Met Leu Arg

CAA

Gin 420 TTT GAO TOT CAA Phe Giu Cys Gin AAT OAT AAA ATT Asn Asp Lys Ile AGA OGA Arg Oly 430 1294 TOG OTT GOT Trp Val Oly

TTC

Phe 435 TOT OTG A.AO ACT Ser Val Lys Thr

TOT

Ser 440 CAT COT ATA ACT His Arg Ile Thr OCT OOT OCA Ala Gly Aia 445 OTO AAC CAG Leu Asn Gin 1342 GAO ATT Asp Ile CTT TAT Leu Tyr 46S OTC ATO COT TOT Leu Met Pro Ser

AGA

Arg 455 TTT GAG GCC TTG Phe Glu Ala Leu

OGA

Arg 460 OCA ATO AAA TAT Ala Met Lys Tyr 000 Gly 470 ACT ATT COT OTT Thr Ile Pro Val

OTT

Val1 475 CAT OCA OTA OGA His Ala Val Oly 1390 1438 1486

GGA

Oly 480 CTC AGA OAT ACT Leu Arg Asp Thr CAG COO TTT OAT Oln Pro Phe Asp

CT

Pro 490 TTT AAT GAO TOA Phe Asn Oiu Ser

GOA

Gly 495 CTG 000 TOO ACO Leu Gly Trp Thr AOT AGO OCT GAA Ser Arg Ala Olu AGO OAO OTO ATO Ser Gln Leu Ile CAO OCA His Ala 510 1534 TTA GGA AAT Leu Gly Asn 000 ATT CAG Gly Ile Gin 530 TTA OTO ACT TAT Leu Leu Thr Tyr

COT

Arg 520 GAO TAO AAA AAO Oiu Tyr Lys Lys AGT TOG GAG Ser Trp Glu 525 1582 ACA COT TOT ATO Thr Arg Cys Met

ACA

Thr 535 CAA GAO TTA AGT TOG OAT AAT OCT Gin Asp Leu Ser Trp Asp Asn Ala 540 1630 GOT CG Ala Gin 545 AAC TAT OA.A 07A Asn Tyr Oiu Oiu CTO ATC OCT OCT Leu Ile Ala Ala TAT GAG TG Tyr Gin Trp 1675 TOAGGTTCAT TAOTTGTAOA TATTTGOOOA TTTTGGCCAT TOTATOA.AGT TCTAATGATO GGATTTCAGA GAOATOTTTC TOOTATOGAC ACOAGAGGAT GCATOCAACA AGTTGGCTAA 1735 1795 66 CTATCATACT ACTACCACGT CAGGAATGAT TGCCGCACTT GATCATGTAA TCATGTATAT ACTCTATTTT GTTTGCAAAA TGTACTTACA TGTTGCAATT TCTAAAAAAA A~A-kAA AAAAAAAA A INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 558 amino acids ART: amino acid TOPOLOGY: linear (1i) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gly Thr Ser Lys Sex- Leu Val Asp Val Pro Gly Lys Lys Ile Gin Ser 1855 1915 1926 1.

Tyr Arg GI u Asn Lys Ala Tyr Gly Asp 1.45 Ile Met Asn 'Asp Val Thr Arg Pro Gin 130 Phe Tyr Pro Giu Pro Met Gly Arg G lu 115 Asp Val Gly Ser Asn Val1 Asn Gly Gly 100 Pro Val Phe Giy Leu Leu Asn Ile Leu His Gin G iu Ile Asn 165 Arg Glu Ile Ile 70 Gly Arg Asp Val1 Asp 150 Arg Lys Gly Asp Leu Asp Val1 Ser Thr 135 Ser Val Glu Ser 40 Glu Val Val Met Gly 120 Tyr His Asp *Ser 25 Ser Lys Ala Ala Val1 105 Val Phe Met Ile 10 Ser Ala Pro Ser Gly 90 Val Arg Gin P he Leu 170 Ala C iu Pro Glu 75 Ala Ala Lys Ala Arg Lys Ser Ala Pro Cys Leu Pro Ile Phe 140 His Arg His -Asn Leu Ala Pro Arg Tyr 125 Ile Ile Met Val1 Glu Ala Pro Lys Tyr 110 Lys Asp Gly Val1 1s Glu Glu Gly Trp Al a Asp Val Gly Asn Leu 175 Gin Thr Thr S er Leu Asn Asp Val Asn 160 Phe Cys Lys Ala Ala 180 Ile Glu Val Pro Trp 185 His Val Pro Cys Gly Gly Val 190 67 Cys Tyr Gly Asp Gly Asn Leu Val Phe Ile Ala 195 200 His Thr Asn Asp Trp 205 Ala Met 225 Gl Tyr Asn Ser Leu 305 Asn Gin Lys Arg Lye 385 Asp Met Val 3, Le' 21 Asi Ar Met IlE His 290 His Gly Ser Pro Asp 370 Gly Val Leu Gly u Leu 0 n Tyr I Gly Asp Phe 275 Gly Gin Ile Asp 4 Gin 355 Asp Val Gin I Arg C 4 Phe S 435 Prc Th Prc Pro 260 Ala Tyr Ile Asp Gly 340 :ys lal ksp ,eu ;in L20 ;er o Val Arg Leu 245 Phe Ala Ser Ile.

Thr 325 Tyr Lye Pro Leu Val 2 405 Phe Val I Tyr Leu Lys AL a Se: 23( GIt LyE Gly Trp Asn 310 Lys Met Ala Leu Ile 390 iet ;lu -ys ;er r Val a Asp Leu Leu Glu 295 Glu Glu Asn Ala Ile 375 Ala Leu Cys Thr Arg I Let Phe Tyr Lye 280 Leu Asn Trp Tyr Leu 360 Gly Glu Gly GIn Ser 440 ?he i Val Ser Asp 265 Thr Lye Asp Asn Ser 345 Gin Phe Ala Thr His P 425 His P Glu A Ty Ill Tyz 25C Pro Ala Thr Trp Pro 330 Leu Lys Ile Ser ;ly 110 ~sn ~rg la r Ty 6 Hi 23! Va Va Asj Ser Lye 315 Glu Asp Glu Gly Ala 395 Arg Asp Ile Leu r Arg 220 s Asn 5 L Asp 1 Gly Arg Gln 300 Leu Leu Thr Lea Arg I 380 Trp Arg Lys I Thr A 4 Arg L Asp Ile Leu Gly Val 285 Gly Gin Asp Leu Gly 365 ,eu let 2 ~sp I le A 4 la G eu A Asi Al Pr Clu 270 Val Gly Gly Val Gln 350 Leu %sp 4et ,eu rg ly .sn Gly His Pro 255 His Thr Trp Ile His 335 Thr C Pro Pro C Gly G 4 Glu C 415 Gly T Ala A Gin L Ile Gin 240 His Phe Val Gly Va1 320 Leu Giy lal In ;in ,00 In rp ep eu Ile Leu Leu Met Pro 450 Tyr Ala Met Lys Tyr 465

S

455 460 His Ala Val Gly Gly 480 Gly Thr Ile Pro Val Vai 470 475 Leu Arg Asp Thr Gly Trp Thr Phe 500 Gly Asn Cys Leu 515 Ile Gin Thr Arg Gin Pro Phe Arg Ala Glu Asp Pro Phe Asn Glu 490 Ala Ser Gin Leu Ile Ser Gly Leu 495 His Ala Leu 510 Trp Glu Gly Asn Ala Ala Leu Thr Tyr Cys Met Thr 535 Tyr Lys Lys Asp Leu Ser Trp Asp 530 540 Glr 545 (2) Asn Tyr Glu Glu Val Leu Ile Ala Ala Lys Tyr Gir 550 -555 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 2793 base pairs ART: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINA-L SOURCE: ORGANISM: Solanum tuberosum STRAIN: cv DUsir~e TISSUE TYPE: l eaf tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in Lambda ZAPII (ix) FEATURE: NAME/FEATURE: 005 LOCATION:242. .2542 Trp (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCGCCCATTT TTCACCAAAC GTTTTTGACA TTGACCTCCA TTGTCGTTAC TTCTTGGTTT CTCTTTCAAT ATTGCTTCAC AATCCCTAAT TCTCTGTACT AGTCTCTATC TCAATTGGGT TTTCTTTACT TGT CAATTAT CTCTACTGGG TCGGCTTCTA TTTCCACTAG GTCACTCTGG TTCTTGAAAT CTTGGATTCC TATTATCCCT GTGAACTTCA TCTTTTGTGA TTTCTACTGT A ATG GAG AAT TCC ATT CTT CTT CAT AGT GGA ART CAd TTC CAC CCC Met Glu Asn Ser Ile Leu Leu His Ser Gly Asn Gin Phe His Pro 1 5 10 ARC TTA CCC CTT TTA GCA CTT AGG CCC AAA AAA TTA TCT CTA ATT CAT Asn Leu Pro Leu Leu Ala Leu Arg Pro Lys Lys Leu Ser Leu Ile His 25 120 180 240 286 334 GGC TCC ACT ACA GAG CAA ATG TGG AGG ATC AAO CGC OTT AAA GC. ACA Ile Lys Arg Val Lys Ala Thr Gly Ser Ser Arg Giu Gin Met GGT CAA AAT TCT GGG GA.A OCT Gly Ciu Asn Ser Gly Ciu Ala Trp Arg GCA AGT GCT GAT GAA TCG RAT GAT Ala Ser Ala Asp Glu Ser Asn Asp 55 0CC Ala TTA CAG Leu Gin GTT ACA ATT GAA Val Thr Ile Giu AGC AAA AAG GTT Ser Lys Lys Val GCC ATO CAA CAG Ala Met Gin Gin

GAC

Asp CTA CTT CAA CAG Leu Leu Gin Gin OCA OAA AGA AGA Ala Glu Arg Arg

AAA

Ly s 90 OTA CTC TCT TCA Val Val Ser Ser

ATA

Ile AAA AGC AGT CTT Lys Ser Ser Leu

GCC

Ala 100 AAT GCC AAA GGT Asn Ala Lys Gly

ACT

Thr 105 TAT GAT GGT GG Tyr Asp Gly Giy AOT OCT Ser Cly 110 AGC TTA TCA OAT OTT CAT ATC CCT Ser Leu Ser Asp Val Asp Ile Pro OTO CAT AAA OAT Val Asp Lys Asp TAT AAT CTT Tyr Asn Val 125 OAT AAA AAT Asp Lys Asn ACT OTA CCT Thr Val Pro 130 AOT ACT OCT OCT ACT CCA ATT ACT CAT Ser Thr Ala Ala Thr Pro Ile Thr Asp

CTC

Val1 140 ACA CCC Thr Pro 145 CCT CCT ATA AC Pro Ala Ile Ser GAT TTT GTT OA.A Asp Phe Val Oiu AAA AGA OAA ATC Lys Arg Olu Ile

AAA

Ly s 160 AGO GAO CTC GCC Arg Asp Leu Ala

CAT

Asp 165 OAA AGO OCA CCC Olu Arg Ala Pro

CCA

Pro 170 CTG TCG AGA TCA Leu Ser Arg Ser ATC ACA GCC ACT Ile Thr Ala Ser

AGC

Ser 180 CAG ATT TCC TCT Gin Ile Ser Ser OTA AGT TCC AAA Val Ser Ser Lys AGA ACO Arg Thr 190 TTO AAT OTC Leu Asn Val

CCT

Pro 195 CCA, CAA ACT CCC Pro Ciu Thr Pro

AAC

Lys 200 TCC ACT CAA GAO Ser Ser Gin Clu GAT OTO Asp Val TCA COO AAA AGT Ser Arg Lys Ser CTA OAT OTT CCT Val Asp Val Pro ACA OTT TTC Thr Leu Leu 205 AAG AAG ATC Lys Lys Ile TCC CAT GTC Ser His Val 910 958 CG TCT TAT Gin Ser Tyr 225 ATO CCT TCA Met Pro Ser TTA COT Leu Arg 230 AAA GALA TC Lys Glu Ser TCA OCA Ser Ala 235

GAA

Glu 240 CG AGO AAT GAA Gin Arg Asn Glu

AAT

Asn 245 CTT GAA GGA TCA Leu Glu Gly Ser

AGT

Ser 250 GCT GAG GCA AAC Ala Glu Ala Asn 1006 GAG ACT GAA GAT Glu Thr Giu Asp GTG A.AT ATA GAT Val Asn Ile Asp AAA CCC CCT CCA Lys Pro Pro Pro TTG GCA Leu Ala 270 1054 GOA ACA AAT Gly Thr Asn TOG TCT AA.A Trp Ser Lys 290

GTT

Val 275 ATG AAC ATT ATT Met Asn Ile Ile

TTG

Leu 280 GTG GCT TCA GAA Val Ala Ser Glu TGC OCT CCA Cys Ala Pro 285 TTA CCC AAA Leu Pro Lys 1102 1150 ACA GGT GGG CTT Thr Gly Gly Leu GAT GTT OCT GGA Asp Vai Ala Gly

GCA

Ala 300 GCT TTG Ala Leu 305 GAC A.AC Asp Asn 320 GTT GAT Val Asp GCT CGA COT GGC Ala Arg Arg Gly

CAC

His 310 AGA OTT ATO OTT Arg Val Met Val GCA CCT CGT TAT Ala Pro Arg Tyr TAT OCT GAA Tyr Pro Glu GOT CAG OAT Gly Gin Asp 340 CAA OAT TCT GOT Gin Asp Ser Oly

OTA

Val1 330 AGA AA.A ATT TAT Arg Lys Ile Tyr

AAA

Lys 335 1198 1246 1294 OTO GAA OTO ACT Va'i Oiu Val Thr

TAC

Tyr 345 TTC CAA OCT TTT Phe Gin Ala Phe ATT GAT Ile Asp 350 GGT OTO OAT Gly Val Asp AAC A.AC ATT Asn Asi Ile 370

TTT

Phe 355 OTT TTC ATT GAC Val Phe Ile Asp CAT ATO TTT AGA His Met Phe Arg CAC ATT GO His Ile Oly 365 1342 TAC OGA 000 AAC Tyr Oly Oly Asn

CGT

Arg 37S OTO OAT'ATT TTA AAA COC ATO GTT Val Asp Ile Leu Lys Arg Met Val 380 1390 TTA TTT Leu Phe 385 TOO AAA OCA GCO Cys Lys Ala Ala GAG OTT COT TOO Olu Val Pro Trp

CAT

His 395 OTT CCA TOT GOT Val Pro Cys Gly 1438 1486 GO Gly 400 OTC TOC TAT OGA Val Cys Tyr Oly

OAT

Asp 405 OGA ALAT TTA OTO Oly Asn Leu Vai

TTC

P he 410 ATT OCT AAT OAT Ile Ala Asn Asp CAT ACT OCT TTA His Thr Ala Leu CCA OTA TAT CTO Pro Vai Tyr Leu OCT TAT TAT COT Ala Tyr Tyr Arg GAC AAT Asp Asn 430 1534 OGA ATT ATO AAC TAT ACA AGA TCT OTC CTO Oly Ile Met Asn Tyr Thr Arg Ser Val Leu

OTG

Val ATT CAT ALAC ATC OCT Ile His Asn Ile Ala 445 1582 CAT CG GGT His Gin Gly 450 COT GOT CCT TTG Arg Oly Pro Leu

GAO

Giu 455 OAT TTT TCA TAT OTA OAT OTT CCA Asp Phe Ser Tyr Val Asp Leu Pro 460 1630 CCA CAC Pro His 465 CAT TTC His Phe 480 TAT ATG GAC Tyr Met Asp AAC ATT TTT Asn Ile Phe CCT TTC AAG TTG TAT GAC CCA GTA GGA GGT GAG 1678 Pro Phe 470 Lys Leu Tyr Asp Val Gly Gly Glu GOT GCT CTA AAG ACA Ala Gly Leu Lys Thr 490 GCA GAT CGT GTA OTT Ala Asp Arg Val Val 495 1726 1774 ACA GTT AGT CAT Thr Val Ser His

GOA

Cly 500 TAT TCA TGG GAA Tyr Ser Trp Glu AAG ACT TCC CAA Lys Thr Ser Gin GGT GGT Gly Gly 510 TOG GGA TTG Trp Gly Leu ATT GTG AAT Ile Val Asn 530

CAT

His 515 CAG ATA ATT AAT Gin Ile Ile Asn

GAG

Glu 520 AAC GAT TOG AAA Asn Asp Trp Lys TTA CAG GGT Leu Gin Gly 525 TTG GAO GTT Leu Asp Val 1822 1870 COG ATT GAT ACA Gly Ile Asp Thr GAG TGG AAC CCT Glu Trp Asn Pro CAC TTA His Leu 545 ACT GGC Thr Gly 560 CAG TCA GAT GGT Gin Ser Asp Gly

TAO

Tyr 550 ATG AAC TAC TCC Met Asn Tyr Ser

TTG

Leu 555 GAC ACG CTA CAG Asp Thr Leu Gln 1918 1966 AAG CCT CAA Lys Pro Gin

TGT

Cys 565 AAA OCT CCA TTG Lys Ala Ala Leu AAC GAA CTT GOT Lys Glu Leu Gly CCA GTT CCT GAT Pro Val Arg Asp GTC CCA CTG ATC Val Pro Leu Ile

GGT

Oly 585 TTC ATT GGG AGC Phe Ile Gly Arg OTT GAC Leu Asp 590 2014 *CCA CAA AAG Pro Gin Lys COT CAG GAT Gly Gin Asp 610

GOT

Gly 595 OTT CAT CTG ATT Val Asp Leu Ile GAG CCC ACT GCT Glu Ala Ser Ala TOG ATG ATG Trp Met Met 605 CGT CAC OTT Arg Asp Leu 2062 2110 GTA CAA CTG OTC Val Gin Leu Val

ATG

Met 615 TTG GGC ACG GGG Leu Gly Thr Cly OAA CAO Glu Gin 625 ATO CTA AGO CAA Met Leu Arg Gin GAG TOT CAA CAC Olu Cys Gin His OAT AAA ATT AGA Asp Lys Ile Arg 2158 2206

OGA

Cly 640 TGG OTT GOT TTC Trp Val Cly Phe

TCT

Ser 645 OTG AAG ACT TCT Val Lys Thr Ser

CAT

His 650 CGT ATA ACT GOCT Arg Ile Thr Ala

GOT

Gly 655 OCA GAO ATT CTG CTC ATG CCT TOT AGA Ala Asp Ile Leu Leu Met Pro Ser Arg GAG OCT TOC GGA Clu Pro Cys Gly CTG AAC Leu Asn 670 2254 CAG CTT TAT GCA ATG AAA TAT GGG ACT ATT CCT GTT GTT CAT GCA GTA 2302 Gin Leu Tyr Ala 675 Met Lys Tyr Gly Thr Ile Pro Val 680 Val His Ala Val 685 GGA GGA CTC AGA GAT ACT GTG CAG CCC TTT GAT CCT Gly Gly Leu Arg Asp Thr Val Gin Pro Phe Asp Pro 690 695

TTT

Phe 700 AAT GAG TCA Asn Glu Ser 2350 GGA CTG Gly Leu 705 GGG TGG ACC TTC Gly Trp Thr Phe AGG GCT GAA GCT Arg Ala Glu Ala

AGC

Ser 715 CAG CTG ATC CAC Gin Leu Ile His 2398 2446 GCA Ala 720 TTA GGA AAT TGC Leu Gly Asn Cys

TTA

Leu 725 CTG ACT TAT CGT Leu Thr Tyr Arg TAC AAA AAG AGT Tyr Lys Lys Ser

TGG

Trp 735 GAG GGG ATT CAG Glu Gly Ile Gin CGT TGT ATG ACA Arg Cys Met Thr GAC TTA AGT TGG Asp Leu Ser Trp GAT AAT Asp Asn 750 2494 GCT CCT CAG Ala Ala Gin TAT GAA GAA GTT Tyr Glu Glu Val ATC GCT GCT AAG Ile Ala Ala Lys TAT CAG TGG Tyr Gin Trp 765 2542 TGAGGTTCAT TACTTGTAGA TATTTGGGGA TTTTGGCCAT TGTATCAAGT TCTAATGATG GGATTTCAGA GACATGTTTC TGGTATCGAC ACGAGAGGAT GCATGCAACA AGTTGGCTAA CTATCATACT ACTACCACGT CAGGAATGAT TGCCGCACTT GATCATGTAA TCATGTATAT ACTCTATTTT GTTTGCAAAA TGTAGTTACA TGTTGCAATT TCTAAAAAAA AAAAAAAAAA AAAAAAAAAA A INFORMATION FOR SEQ-ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 767 amino acids ART: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Glu Asn Ser Ile Leu Leu His Ser Gly Asn Gln Phe His Pro Asn 1 5 10 Leu Pro Leu Leu Ala Leu Arg Pro Lys Lys Leu Ser Leu Ile His Gly 25 2602 2662 2722 2782 2793 Ser Ser Arg Glu Gin Met Trp Arg Ile Lys Arg Val Lys 40 Ala Thr Gly 73 Glu Asn Ser Gly Glu Ala Ala Ser Ala Asp Glu Ser Asn Asp Ala Leu so Gln Leu Ser Leu Val1 Pro 145 Arg Thr Asn Val Ser 225 Gin Thr Thr Ser Leu 305 ValI Leu Ser Ser Pro 130 Pro Asp Ala Val1 .s n 210 Tyr Arg Glu %sn ys ~la *Thi IGlr Leu Asp 115 Ser Ala Leu Ser Pro 195 Ser Met Asn Asp Val 275 Thr Arg Sle 1Gin IAla 100 Val Thr Ile Ala S er 180 Pro Axrg Pro Glu Pro 260 Met Gly Ar g Gil Ile Asn Asp Ala Ser Asp 165 Gin Giu Ly s Ser Asn 245 Val Asn ;ly ;ly Lys 70 *Ala Ala Sle Ala Gin 150 Ile Thr Ser Leu 230 Leu Asn Ile Leu C His 310 Ser Gitu Lys Pro Thr 135 Asp Arg Ser Pro Leu 215 Arg Glu Ilie Ile ;ly ~rg *Lys Arg Gly Asp 120 Pro Phe Ala Ser Lys 200 Val Lys Gly Asp Leu 1 280 Asp Val L y A-r T h 101 Val Ile Val1 Pro Thr 185 Ser Asp Glu Ser ;lu 265 Ia 1 alI e t s Va.

g L y -AsI *Thr Giiu Pro 170 Val Ser Val1 Ser Ser 250 Lys Ala Ala Val1 1 Leu 75 s Val.

2 rAsp 'Lys Asp Ser 155 Leu Ser Gln Pro Ser2 :235 AlaC Pro I Ser G Gly A 3 Val A~ 315 Al Va) 01) Asp Val 140 Lys Ser Ser Glu Gly 220 kla al ~ro iu la .00 l1a aMet L Ser Giy Tyr 125 Asp Arg Arg Lys Thr 205 Lys Ser Ala2 Pro I Cys 285 Leu.P Pro A Gl.

Sel Sei Asr Lys Glu Ser Arg 190 Leu Lys His5 kLsnf .eu lia ro ~rg n GI r Ii 9 Gi' Va Asr Ile Ser 175 Thr Leu Ile Val Glu 255 Ala Pro Lys Tyr Asp Lys y' Ser 1 Thr Thr *Lys 160 Sle Leu Asp Gin Glu 240 Glu Gly Trp Ala Asp 320 Asn Tyr Pro Giu Pro Gin Asp Ser Gly Val Arg Lys Ile Tyr Lys Val Asp Gly Gin Asp Val Giu Val Thr Tyr Phe Gin Ala Phe Ile Asp Gly Val As f P he 385 Val Thr Ile Gin His 465 Phe Val1 C ly Val Leu 545 Gly Val.

Gin Asp Ile 370 Cy 5 cy s Al a Met Giy 450 Tyr Asn Ser Leu Asn 530 Gin Lys Arg Lys Phe 355 Tyr Lys Tyr Leu Asn 435 Arg Met Ile His His 515 Gly Ser Pro Asp Gly 595 Val Gly Ala Gly Leu 420 Tyr Gly Asp Phe Giy 500 Gin Ile Asp Gin Asp 580 Val P he Gly Al a Asp 405 Pro Thr Pro Pro Al a 485 Tyr Ile Asp Gly Cy s 565 Val1 Asp Ile Asn Ile 390 Gly Val1 Arg Leu Phe 470 Ala Ser Ile T hr Tyr 550 Lys Pro Leu Asp Arg 375 C iu Asn Tyr Ser Glu 455 Ly s Gly Trp Asn Lys 535 Met Ala Leu Ile Ser 360 Val1 Val1 Leu Le u Val 440 Asp Leu Leu Glu G1 u 520 G iu Asn Ala Ile Ala 600 345 His Met Asp Ile Pro Trp Val Phe 410 Lys Ala 425 Leu Val Phe Ser Tyr Asp Lys Thr 490 Leu Lys 505 Asn Asp Trp Asn Tyr Ser Leu Gin 570 Gly Phe 585 Giu Ala 350 P he Leu His 395 Ile Tyr Ile Tyr Pro 475 Ala Thr Trp Pro Leu 555 Lays Ile Ser *Arg *Lys 380 Val1 Al a Tyr His Val 460 Val App Ser Ly s Giu 540 Asp Giu Giy Ala Hi 365 Arc Prc As n Arg Asn 445 Asp Gly Arg Gin Leu 525 Leu Thr Le u Arg rrp 605 Ile Met Cys Asp *Asp 430 Ile Leu Giy ValI Gly 510 Gin Asp Leu Gly Leu 590 Met Gl Val Gly Trp 415 Asn Ala Pro Giu Val 495 Gly Gly Val1 Gin Leu ksp 4et Asn *Leu Gly 400 *His Gly His Pro His 480 Thr Trp Ile His Thr 560 Pro Pro G iy Gin Asp Val. Gin Leu Val Met Leu Gly Thr Giy Arg Arg Asp Leu Giu 610 615 1620 Gin Met Leu Arg Gin Phe Glu Cys Gin His Asn ASp Lys Ile Arg 625 Trp Asp Leu Gly Leu 705 Leu Giy Ala 630 635 640 Val1 Ile Tyr Leu 690 Giy Gly Ile Gin Giy Leu Ala 675 A.rg Trp Asn Gin Asn 755 Phe Leu 660 Met Asp Thr Cys Thr 740 Tyr Se r 645 Met Ly s Thr Phe Leu 725 Arg Giu Vai Pro Tyr Vali Ser 710 Leu Cy s G iu Lys Ser dly Gin 695 Arg Thr Met Val1 T hr Ar g Thr 680 Pro Ala Tyr Thr Leu 760 Ser P he 665 Ile P he Giu Ar; Gin 745 Ile His *650 Glu Pro Asp Ala Glu 730 Asp Ala Arg Pro Val Pro Ser 715 Tyr Leu Aila Ile Cys Val P he 700 Gin Lys Ser Lys Thr Giy His 685 As n Leu Lys Trp Tyr 765 Ala Leu 670 Ala Giu Ile Ser Asp 750 Gin Gly 655 Asn Val Ser His Trp 735 As n Trp Ala Gin Gly Gly Al a 720 Glu Ala INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 2360 base pairs ART: nucleotide STRANDEDNESS: single AD) TOPOLOGY: linear (ii) MOLECULE TYPE: -cONA (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum STRAIN: cv. D~sir~e TISSUE TYPE: leaf tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in Lambda ZAPII (ix) FEATURE: NAME/FEATURE: CDS LOCATION:68. .1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACATTTTCTA TATTGAAAGA TTTTGTCTTT ACATGATTCT TGATTTTACA GCAGG TGTCA ATACCAA ATG GGG TCT CTG Ck.A ACA Met Gly Ser Leu Gin Thr CCC ACA AAT CTT ACC N.AT A.AG TCA Pro Thr Asn Leu Ser Asn Lys Ser

TGT

Cys TTA TGT GTG TCA Leu Cys Val Ser AGA GTT GIG AAG Arg Val Vai Lys

GGT

Giy TTG AGO GTA GAA Leu Arg Vai Glu CAA GIG GGG TTG Gin Vai Gly Leu

GGG

Gly TTT TCI TOG TTG Phe Ser Trp Leu AAG GGA CGA AGA Lys Gly Arg Arg AAC AGA Asn Arg AAA OTT CA.A Lys Val Gin ATT OCT GAA Ile Ala Giu TTG TOT GTT ACA Leu Cys Val Thr

AGT

Ser ACT GTT TCA GAT Ser Vai Ser Asp OT TCA TCA Giy Ser Ser GGT GCT GAG Giy Ala Giu A-AT AAG AAT GTG Asn Lys Asn Vai GAA OGG CTT CTT Giu Gly Leu Leu

TTG

Leu AGA GAT Arg Asp OTT OCA Vai Aia GOT TCT GGC TCT Giy Ser Oly Ser G TI Vali OTT GGT ITT CAA Val Giy Phe Gin ATT CCA CAT TCT Ile Pro His Ser GGA GAT GCA Gly Asp Ala ATG GTA GAA TCT Met Val Ciu Ser GAT ATT GTA GCC Asp Ile Val Aia

A.AT

As n 110 GAT AGA GAT GAC Asp Arg Asp Asp

TTG

Leu 115 ACT GAG GAT ACT Ser Giu Asp Thr

GAG

Giu 120 GAG ATG GAG GAA Giu Met Glu Giu ACC CCA Thr Pro 125 ATC AAA TIA Ile Lys Leu TAT TCT AAG Tyr Ser Lys 145 TTC AAT ATC ATT Phe Asn Ile Ile GTT ACT GCT GAA Val Thr Ala Glu GCA OCT CCA Ala Ala Pro 140 TO CCA ATG Leu Pro Met 493 ACT GGT GGA TTA Thr Gly Gly Leu GAT GTT IGI GGT Asp Val Cys Gly OCA CIA Ala Leu 160 GCT GCT COG GGT Ala Ala Arg Gly

CAT

His 165 CGT CIA ATCOGTC Arg Val Met Val TCA CCT AGO TAT Ser Pro Arg Tyr

TTG

Leu 175 AAT OGA GOT CCT Asn Oly Gly Pro CAT GAA AAG TAC Asp Giu Lys Tyr AAT GCT OTT GAC Asn Ala Val Asp GAT GIG COO GCC ACT GTC CAT ICC TT Asp Val Arg Ala Thr Vai His Cys Phe 195

GT

Giy 200 GAT GCA CAG GAA CIA GCC Asp Ala Gin Giu Val Ala .205 TTC TAC CAT Phe Tyr His

GAA

Glu 210 TAC AGO GCA GOT Tyr Arg Ala Gly

OTT

Val1 215 GAT TOG OTA TTT Asp Trp Vai Phe GIG GAC CAC Val Asp His 220 TCT TCT TAO Ser Ser Tyr 225 TGC AGA OCT GGA Cys Arg Pro Gly ACG CCA TAT GOT GAT ATT Thr Pro Tyr Gly Asp Ile 230 235 TAT GGT GOA Tyr Gly Ala TTT GOT Phe Gly 240 GAT AAT CAG TTT Asp Asn Gin Phe

COO

Arg 245 TTC ACT TTO OTT Phe Thr Leu Leu CAC OCA OCA TOT His Ala Ala Cys

GAA

Olu 255 GOG OCA TTO OTT Ala Pro Leu Val

OTT

Leu 260 O.CA OTO OOA G00 Pro Leu Oly Oly

TTO

P he 265 ACT TAT OGA GAG Thr Tyr Oly Olu TOO TTO TTT OTO Cys Leu Phe Leu AAT OAT TOO OAT Asn Asp Trp His OCT 000 OTO OTT OCT TTA OTT Ala Ala Leu Val Pro Leu Leu 280 285 TTA 000 000 Leu Ala Ala ATT OTO GOA Ile Val Ala 305

AAG

Lys 290 TAT COT OCT TAT Tyr Arg Pro Tyr OTT TAO AAG OAT Val Tyr Lys Asp GOT COT AOT Ala Arg Set 300 OCT OCA OTA Pro Ala Val ATA CAC AAO ATT Ile His Asn Ile

GCA

Ala 310 CAT CAG OGA OTO His Gin Gly Val 1021 ACC TAO Thr Tyr '320 AAT AAT TTG GOT Asn Asn Leu Oly COT OCA OA.A TG Pro Pro Gin Trp OGA OCA OTT GA.A Giy- Ala Val Glu 1069 1117

TG

Trp, 335 ATA TTT 000 ACA Ile Phe Pro Thr

TOO

Trp 340 OCA AGO 000 CAT Ala Arg Ala His 000 Ala 345 OTT GAO ACT GOT Leu Asp Thr Gly ACA OTO A.AC OTT Thr Val Asn Val AAA 000 OCA ATA Lys Oly Ala Ile OTT OCT OAT CG Val Ala Asp Arg ATA OTO Ile Leu 365 1165 ACA OTT AGO Thr Val Ser TAT 000 OTA Tyr Gly Leu 385 ATT ACT AAT Ile Thr Asn 400

OAO

Oln 370 OGA TAO TOA TOO Gly Tyr Ser Trp ATA ACA ACT COT Ile Thr Thr Pro GAA 000 OGA Oiu Oly Oly 380 OTT NAT GOA Leu Asn Gly 1213 1261 CAT GAG CTO TTG His Glu Leu Leu

AGO

Ser 390 AOT AGA CAG TOT Ser Arg Gin Ser GGA ATA OAT Gly Ile Asp

OTT

Val 40S AAT OAT TOO AAO 000 TOG ACA OAT GAO Asn Asp Trp Asn Pro Ser Thr Asp Olu 4 1309

CAT

His 415 ATT GOT TOG OAT Ile Ala Ser His

TAO

Tyr 420 TOO ATO AAT GAO OTO TOO OGA AAO Ser Ile Asn Asp Leu Ser Gly Lys OTT CAG Val Gin 430 1357 TGC AAG ACT GAT Cys Lys Thr Asp CTG CAA A.AG CAA Leu Gin Lvs Ciu 435 OGA T'TT ATT GGA Gly Phe Ile Giy CTG GCC Leu Gly 440 CTT CCA ATT CGA Leu Pro Ile Arg COT CAT Pro Asp 445 1405 TOT COT CTO Cys Pro Leu GAC ATA ATC Asp Ile Ilie 465

ATT

Ile 450 CTG GAC TAC CAG Leu Asp Tyr Gin AAA GGT GTT Lys Giy Val 460 GAT GTC CAA Asp Val Gin 1453 1501 CTG TCA GCA ATT Leu Ser Ala Ile

CCA

Pro 470 OAA OTT ATG CAG Giu Leu Met Gin

AAT

Asn 475 GTT GTA Val. Val 480 ATG OTT GGA TCT Met Leu Gly Ser GAG AAA CA.A TAT Giu Lys Gin Tyr GAC TOG ATO AGA Asp Trp, Met Arg 1549 1597

CAT

His 495 ACA CAA AAT CTT Thr Giu Asn Leu AAA GAO AAA TTT Lys Asp Lys Phe OCT TGG OTT GGA Ala Trp Val Giy AAT OTT CCA OTT Asn Val Pro Val

TOT

Ser 515 CAT AGG ATA ACA His Arg Ile Thr

GCA

Ala 520 GGA TOC GAO ATA Gly Cys Asp Ile OTA TTG Leu Leu 525 1645 ATC COO TOA Met Pro Ser AGA TAT GGO Arg Tyr Ciy 545 TTC GAA CCcd TOT Phe Giu Pro Cys TTA AAC CAA TTG Leu Asn Gin Leu TAT GOA ATO Tyr Ala Met 540 OTA AGA GAO Leu Arg Asp 1693 1741 ACC ATA OCT ATT Thr Ile Pro Ile OAT AGO ACO GG His Ser Thr Gly GG0 Oly 555 ACA OTC Thr Val 560 AAG CAT TTT AAT Lys Asp Phe Asn TAT CT CAA GAA Tyr Ala Gin Giu ATA OCT CAA GOT Ile Gly Giu Giy 1789 1837 GGG TOO ACA TTT Cly Trp Thr Phe

TOT

Ser 580 OCT OTA AOG AGT Pro Leu Thr Ser AAG TTG OTT CAT Lys Leu Leu Asp OTG AAG OTO GCA Leu Lys Leu Ala COG ACT TAT ACA Cly Thr Tyr Thr OAT AAG TOA TOT His Lys Ser Ser TOG GAG Trp Giu 605 1885 GGA TTG ATG Gly Leu Met 000 ATT OA-A Ala Ile Gin 625 TAT OTO AGA Ty~r Val Arg 640 AGA GOT ATG OCA Arg Cly Met Gly GAO TAT TOO TG Asp Tyr Ser Trp GAA AAT GCA Oiu Asn Ala 620 OAT OCT OCA Asp Pro Pro 1933 1981 TAT GA.A CAA OTT Tyr Oiu Gin Val

TTC

Phe 630 ACC TGG CO TTT Thr Trp Ala Phe TOATTTATCA AGAAAGATTG CAAACGGGAT ACATCATTAA 2030 ACTATACGCG GAGCTTTTGG TGCTATTAGC TACTGTCATT GGGCGCGGAA TGTTTGTGGT TCTTTCTGAT TCAGAGAGAT CAAGTTAGTT CCAAAGACAT ACGTAGCCTG TCCCTGTCTG TGAGGGAGTA AAACTACAAA AGGCAATTAG A.AACCACCAA GAACTGGCTC CTTTGGGAGA AGAGTG-GAAA TATGTAAA.AA AGAATTTTGA GTTTA.ATGTC XATTGATTAA TTGTTCTCAT TTTTAAAAAA A.ACATCTCAT CTCATACAAT ATATAAAATT GATCATGATT GATGAAAAAA ~AAAAAAA AAAAAAAA AAAAAAAAA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 641 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQU ENCE DESCRIPTION: SEQ ID NO: 2090 2150 2210 2270 2330 2360 Met Gly Ser 1 Cys Val Ser Gly Leu Gly Gin Ser Leu Leu Gin Thr Pro Thr Asn Leu Ser Asn Lys Ser Cys Leu 1s Gly Phe Val Val Lys Arg Val Giu Ser Trp Leu Leu S er Giy Arg Arg Arg Gin Val Arg Lys Val Ser Ile Ala Glu Asn Ly s Cys Val Thr Ser Asn Val Ser -Giu 70 Ser Val Val Glv Val Ser Asp Gly Leu Leu Gly Leu 75 Ile Giy Ala Giu Arg Asp Ser Giy Phe Gin Pro His Ser Val Ala Gly Asp Ala Asp Asp Leu 115 Leu Thr Phe 130 Val Giu Ser Ile Val Ala Glu Asp Thr Met Giu Giu Asn Asp Arg 110 Pro Ile Lys Pro Tyr Ser Asn Ile Ile Thr Ala Glu Lys Thr Gly Gly Leu Giy Asp Val Cys Gly Ser'Leu Pro Met Ala Leu 145 150 155 160 Ala Ala Arg Gly His Arg Val Met Val Val Ser Pro Arg Tyr Leu Asn 165 175 Gly Arg His Tyr 225 Asp Pro Phe Ala Ala 305 Asn Phe Asn Ser Leu 385 Asn Ala Gly Ala Glu 210 Cys Asn Leu Leu Lys 290 Ile Asn Pro Val Gin 370 His Gly Ser Pro Thr 195 Tyr Arg Gin Val Ala 275 Tyr His Leu Thr Leu 355 Gly Glu Ile His SSer 180 Val Arg Pro Phe Leu 260 Asn Arg Asn Gly Trp 340 Lys Tyr Leu Asp Tyr 420 Asp His Ala Gly Arg 245 Pro Asp Pro Ile Leu 325 Ala Gly Ser Leu Val 405 Ser Glu Cys Gly Thr 230 Phe Leu Trp Tyr Ala 310 Pro Arg Ala Trp Ser 390 Asn Ile Lys Phe Val 215 Pro Thr Gly His Gly 295 His Pro Ala Ile Glu 375 Ser Asp Asn Tyr Gly 200 Asp Tyr Leu Gly Ala 280 Val Gin Gin His Ala 360 Ile Arg Trp Asp Ala 185 Asp STrp Gly Leu Phe 265 Ala Tyr Gly Trp Ala 345 Val Thr Gin Asn Leu 425 Asn Ala Val Asp Ser 250 Thr Leu Lys Val Tyr 330 Leu Ala Thr Ser Pro 410 ;er Ala Gin Phe Ile 235 His Tyr Val Asp Glu 315 Gly Asp Asp Pro Val 395 Ser Gly Val Glu Val 220 Tyr Ala Gly Pro Ala 300 Pro Ala Thr Arg Glu 380 Leu Thr Lys SAsp Val 205 Asp Gly Ala Glu Leu 285 Arg Ala Val Gly Ile 365 Gly Asn Asp Val SLeu 190 SAla SHis Ala Cys Lys 270 Leu Ser Val Glu Glu 350 Leu Gly Gly Glu Gin 430 Asp Phe Ser Phe Glu 255 Cys Leu Ile Thr Trp 335 Thr Thr Tyr Ile His 415 :ys SVal Tyr Ser Gly 240 Ala Leu Ala Val Tyr 320 Ile Val Val Gly Thr 400 Ile Lys Thr Asp Leu Gin Lys Glu Leu Gly Leu Pro Ile Arg Pro Asp Cys Pro Leu Ile Gly Phe Ile Gly Arg Leu Asp Tyr Gin Lys Gly Val Asp Ile 450 455 460 Ile Leu Ser Ala Ile Pro Glu Leu Met Gin Asn Asp Val Gin Val Vai 465 470 475 480 Met Leu Giy Ser Gly Glu Lys Gin Tyr Giu Asp Trp Met Arg His Thr 485 490 495 Glu Asn Leu Phe Lys Asp Lys Phe Arg Ala Trp Val Gly Phe Asn Val 500 505 510 Pro Vai Ser His Arg Ilie Thr Ala Gly Cys Asp Ile Leu Leu Met Pro 515 520 525 Ser Arg Phe Glu Pro Cys Gly Leu Asn Gin, Leu Tyr Ala Met Arg Tyr 530 535 540 Gly Thr Ile Pro Ile Val His Ser Thr Gly Giy Leu Arg Asp Thr Vai 545 550 555 560 Lys Asp Phe Asn Pro Tyr Ala Gin Glu Gly Ile Gly Giu Gly Thr Gly 565 570 575 Trp Thr Phe Ser Pro Leu Thr Ser Glu Lys Leu Leu Asp Thr Leu Lys 580 585 590 Leu Ala Ile Gly Thr Tyr Thr Glu His Lys Ser Ser Trp Glu Gly Leu 595 600 605 Met Arg Arg Gly Met Gly Arg Asp Tyr Ser Trp Glu Asn Ala Ala Ile 610 615 620 Gin Tyr Giu Gin Val Phe Thr Trp Ala Phe Ile Asp Pro Pro Tyr Val 625 630 635 640 Arg INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 4168 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to RNA (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum STRAIN: cv. D~sir~e TISSUE TYPE: leaf tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in Lambda ZAPII (ix) FEATURE: NAME/FEATURE: CDS LOCATION:307. .3897 (xi) SEQUYENCE DESCRIPTION: SEQ ID NO: 11: TTTTTTAATA GATTTTTAAA ACCCCATTAA ACCAAATACG TATATAATTG CACCACAGAT ACAGAGAGGG AGAGAGAAAG ATAGTCTGTT GATGAAGGAG AAOAGAGATA TTTCACATGG GATGTTCTAT TTGATTCTGT GGTCAACAAG AGTTTTACAA AGAACATTCC TTTTTCTTTT TTTCTTGGTT CTTGTGTGGG TCAGCCATGG ATGTTCCATT TCCACTGCAT AGACCATTCA GTTGCACAAG TGTCTCCAAT GCAATAACCC ACCTCAACAT CAAACCTTTT CTTGGGTTTC TCTCTC ATG GAA CCA CAA GTC TAT CAG TAC ART CTT CTT CAT OGA GGA Met Ciu Pro Gin Vai Tyr Gin Tyr Asn Leu Leu His Gly Gly 120 180 240 300 348 AGG ATG Arg Met GAR ATG OTT Giu Met Val

ACT

Thr 20 COG GTT TCA TTT Gly Val Ser Phe

CCA

Pro 25 TTT TGT GCA Phe Cys Ala ART CTC Asn Leu GGA TCT Gly Ser TCG GGA AGA AGA Ser Gly Arg Arg AGA AAA GTT TCA Arg Lys Val Ser ACT AGO AGT CAR Thr Arg Ser Gin TCA CCT ARC Ser Pro Lys AGA ARC GTT Arg Lys Val

GGG

Gly so TTT GTG CCA AGG Phe Val Pro Arg

ARC

Lys 55 CCC TCA GOG ATG Pro Ser Cly Met ACC ACC CAR Ser Thr Gin CAG ARC AGC ART Gin Lys Ser Asn GAT AAR CAR ACT CAR AGT ACT TCA 540 Asp Lys Glu Ser Gin Ser Thr Ser ACA TCT Thr Ser AAA CAR TCT GARA Lys Giu Ser Giu TCC ARC CAC ARC Ser Asn Gin Lys

ACG

Thr OTT OAR GCA AGA Val Giu Ala Arg

OTT

Val1 OAR ACT ACT CAC Oiu Thr Ser Asp

GAT

Asp 100 GAC ACT AAA OTA Asp Thr Lys Val

GTO

Val 105 OTO AGO CAC CAC Val Arg Asp His TTT CTG GAO OAT Phe Leu Olu Asp

GAG

Oiu 115 CAT OAR ATC ARAT Asp Giu Ile Asn

GOT

Gly 120 TCT ACT AAA TCA Ser Thr Lys Ser ATA ACT Ile Ser 125 ATO TCA OCT Met Ser Pro

OTT

Val 130 COT GTA TCA TOT CAR TTT OTT CAR ACT Arg Val Ser Ser Gin Phe Val Oiu Ser 135 CAR OAR ACT Ciu Olu Thr 140 GOT GGT OAT Gly Gly Asp 145 GAO AAG GAT OCT Asp Lys Asp Ala AAG TTA ALAC AA.A Lys Leu Asn Lys AAG AGA TCG Lys Arg Ser GA.A GAG Oiu Giu 160 AGT OAT TTT Ser Asp Phe CTA ATT Leu Ile 165 AAT GCC Asn Ala 180 OAT TCT OTA ATA AGA OAA CAA ACT OGA 82.8 Asp Ser Val Ile Arg Oiu Gin Ser Oly 170 T CT Ser 175 CAG 000 OAA ACT Gin Oly Oiu Thr ACT AGO AAG Ser Ser Lys AOC CAT OCT OTO Ser His Ala Val

GT

Gly 190 876 ACA AAA OTT TAT Thr Lys Leu Tyr ATA TTG CG OTO Ile Leu Gin Val

GAT

Asp 200 OTT GAG CCA CA~A Val Oiu Pro Gin CAA TTG Gin Leu 205 AAA GAA AAT Lys Oiu Asn

AAT

Asn 210 OCT 000 AAT OTT Ala Gly Asn Val

GAA

Olu 215 TAC AAA OGA CCT Tyr Lys Oly Pro OTA OCA AGT Val Ala Ser 220 ACT GAA AGO Thr Oiu Ser AAG CTA Lye Leu AAT GAG Asn Olu 240

TTO

Leu 225 GAA ATT ACT AAG Oiu Ile Thr Lys AOT GAT OTO OA.A Ser Asp Val Olu

CAC

His 235 ATT OAT GAO TTA Ile Asp Asp Leu

GAO

Asp 245 ACT AAT AOT TTC Thr Asn ser Phe AAA TGA OAT TTA Lys Ser Asp Leu 1020 1068 1116

ATT

Ile 255 GAA GAG OAT GAG Glu Giu Asp Oiu

CCA

Pro 260 TTA OCT OCA OGA Leu Ala Ala Gly

ACA

Thr 265 GTO GAO ACT OGA Val. Giu Thr Gly TOT TCT OTA AAC Ser Ser Leu Asn TTA AGA Leu Arg 275 TTG GAG ATO Leu Giu Met

OAA

Oiu 280 OCA AAT CTA COT Ala Asn Leu'Arg AGO CAG Arg Gin 285 1164 OCT ATA GAA Ala Ile Giu TTT TOT TTT Phe Cys Phe 305

AGG

Arg 290 OTT 0CC GAG GA.A Leu Ala Oiu Glu TTA TTO CAA 000 Leu Leu Gin Oly ATC AGA TTA Ile Arg Leu 300 1212 OCA GAG OTT GTA Pro Olu Val Val CCT OAT GA.A OAT OTC GAG ATA TTT Pro Asp Giu Asp Val Glu Ile Phe 315 1260 OTT A.AC Leu Asn 320 AGAGOT CTT TOO ACT TTG AAG AAT GAG Arg Gly Leu Ser Thr Leu Lys Asn Glu 325 OAT OTC TTG ATT Asp Val Leu Ile 1308

ATG

Met 335 OGA OCT TTT AAT Gly Aia Phe Asn

GAG

Glu 340 TOG COO TAT AGO Trp Arg Tyr Arg TTT ACT ACA AGO Phe Thr Thr Arg 1356 ACT GAG ACT CAT Thr Giu Thr His

CTC

Leu 355 AP.T OGA GAT TOG TGG TCT TGC A.AG Asn Gly Asp Trp Trp Ser Cys Lys 360 CCC AAG GAA Pro Lys lu GTC TAT GAC Val Tyr Asp 385

GCA

Ala 370 TAC AGG GCT GAT Tyr Arg Ala Asp GTG TTT TTT AAT Val Phe Phe Asn ATO CAT GTT Ile His Val 365 GGA CAA GAT Gly Gin Asp 380 GTG AAA GGT Vai Lys Gly 1404 1452 1500 AAC AAT GAT GGA Asn Asn Asp Gly

AAT

Asn 390 GAO TTC AGT ATA Asp Phe Ser Ile

ACT

Thr 395 GGT ATG Gly Met 400 CAA ATC ATT GAC Gin Ile Ile Asp

TTT

Phe 405 GAA AAT TTC TTG Glu Asn Phe Leu GAG GAG AAA TGG Glu Glu Lys Trp 1548 1596 GAA CAG GAG AAA Glu Gin Glu Lys OCT AAA GAA CAA Ala Lys Oiu Gin

OCT

Ala 425 GAA AGA GAA AGA Glu Arg Olu Arg

CTA

Leu 430 GCG GAA GAA CAA Ala Glu Glu Gin GAC AGA OCA OAA Asp Arg Ala Gin 450 OGA GAA TTG ATG Arg Glu Leu Met 465

AGA

Arg 435 CGA ATA GAA GCA Arg Ile Glu Ala AAA OCT GAA ATT GAA OCT Lys Ala Glu Ile Giu Ala 445 1644 OCA AAG OAA GAG Ala Lys Glu Glu

OCT

Ala 455 OCA AAG AAA AAG Ala Lys Lys Lys AAA OTA TTG Lys Val Leu 460 ACO TGG TAO Thr Trp Tyr 1692 1740 OTA AAA 0CC Val Lys Ala

ACO

Thr 470 AAG ACT OGT OAT Lys Thr Arg Asp

ATO

Ile 475 ATA GAG Ile Olu 480 OCA ACT CAA TTT Pro Ser Giu Phe

AAA

Lys 485 TGC GAG GAO AAG Cys Glu Asp Lys AGO TTA TAO TAT Arg Leu Tyr Tyr 1788 1836

AAO

Asn 495 AAA AOT TCA GGT Lys Ser Ser Oly CTC TOC OAT OCT Leu Ser His Ala

AAC

Lys 505 GAO TTC TGG ATO Asp Leu Trp Ile OGA OGA TAT AAT Oly Oly Tyr Asn

AAT

Asn 515 TGG AAG GAT GGT Trp Lys Asp Gly TCT ATT GTC AAA Ser Ile Val Lys AAG CTT Lys Leu 525 1884 OTT AAA TCT Val Lys Ser ATT COT OAT Ile Pro Asp 545 AGA ATA OAT GOT OAT TOG TGG TAT ACA GAG OTT OTT Arg Ile Asp Gly Asp Trp Trp Tyr Thr Glu Vai Val 1932 CAG OCA OTT TTC Gin Ala Leu Phe

TTC

Leu 550 OAT TOO GTT TTT Asp Trp Val Phe

OCT

Ala 555 OAT GOT OCA Asp Oly Pro 1980 CCC AAG Pro Lys 560 OAT GCC ATT GCT His Ala Ile Ala

TAT

Tyr 565 OAT AAO AAT CAC Asp Asn Asn His CAA GAO TTC CAT Gin Asp Phe His 2028

GCC

Ala 575 ATT GTC CCC XAC Ile Val Pro Asn ATT CCG GAG GAA Ile Pro Giu Giu

TTA

Leu 585 TAT TGG GTT GAG Tyr Trp Val Giu 2076 2124 CXA CAT CAG ATC Giu His Gin Ile

TTT

P he 595 AAG ACA CTT CAG Lys Thr Leu Gin GAG AGA AGG CTT Giu Arg Arg Leu AGA GAA Arg Giu 605 GCG GCT ATO Ala Ala Met ACA AAG GA.A Thr Lys Glu 625 GCT AAG GTT GAA Ala Lys Vai Giu

~AA

Lys 615 ACA GCA CTT CTG Thr Ala Leu Leu AAA ACT GAA Lys Thr Giu 620 AAG CAT GTA Lys His Val 2172 2220 AGA ACT ATG AAA Arg Thr Met Lys

TCA

Ser 630 TTT TTA CTG TCT Phe Leu Leu Ser GTA TAT Val Tyr 640 TAC TAT Tyr Tyr 655 ACT GAG CCT CTT Thr Giu Pro Leu ATC CAA GCT GGA Ile Gin Ala Gly

AGC

Ser 650 AGC GTC ACA GTT Ser Val Thr Val 2268 2316 A.AT CCC GCC Asn Pro Ala ACA GTA CTT AAT Thr Val Leu Asn AAA CCT GAA ATT Lys Pro Giu Ile TTC AGA TCT TCA TTT Phe Arg Cys Ser Phe 675 AAT CGC TGG ACT Asn Arg Trp Thr

CAC

His 680 CGC CTG GGT CCA Arg Leu Gly Pro TTG CCA Leu Pro 685 2364 CCT CAG AAA Pro Gin Lys GTG AAG GTT Val Lys Val 705 TCG CCT GCT GAA Ser Pro Ala Glu GGC ACC CAT GTC Gly'Thr His Val AGA GCA ACT Arg Ala Thr 700 TTT TCC GAG Phe Ser Giu 2412 2460 CCA TTG GAT GCA Pro Leu Asp Ala

TAT

Tyr 710 ATG ATG GAT TTT Met Met Asp Phe

GTA

Vai 715 AGA GA.A Arg Glu 720 GAT GGT GOG ATT Asp Gly Gly Ile GAC AAT AAG AGC Asp Asn Lys. Ser

GGA

Gly 730 ATG GAC TAT CAC Met Asp Tyr His 2508 2556 CCT GTG TTT GGA Pro Val Phe Gly GTC GCT AAA GA-A Val Ala Lys Giu CCA ATG CAT ATT Pro Met His Ile CAT ATT GCT GTC GAA ATG CCA CCA ATT His Ile Ala Val Glu Met Ala Pro Ilie 755

GCA'

Ala 760 AAG GTG GGA GCC Lys Val Gly Gly CTT COT Leu Gly 765 2604 GAT GTT GTT Asp Val Val

ACT

Thr 7770 AGT CTT TCC CCT Ser Leu Ser Arg CTT CAA CAT TTA Val Gin Asp Leu AAC CAT N.AT Asn His Asn 780 2652 GTG OAT ATT Val Asp Ile 785 ATC TTA OCT AAG Ile Leu Pro Lys GAC TGT TTG AAG Asp Cys Leu Lys A.AT AAT GTG Asn Asn Val 2700 AAG GAC Lys Asp 800 TTT CGG TTT CAC Phe Arg Phe His

AAA

Lys 605 AAC TAC TTT TG Asn Tyr Phe Trp

GGT

Gly 810 COG ACT GAA ATA Gly Thr Giu Ile 2748 2796

AAA

Lys 815 GTA TGG TTT GGA Val Trp Phe Gly

AAG

Ly s 820 GTG GAA GGT CTC Val Glu Gly Leu

TCG

Ser 825 GTC TAT TTT TTG Val Tyr Phe Leu OCT CAA AAC G Pro Gin Asn Gly

TTA

Leu 835 TTT TOG AAA GG Phe Ser Lys Gly GTC TAT GGT TOT Val Tyr Gly Cys AGO AAT Ser Asn 845 2844 OAT GOT GAA Asp Gly Glu CTG CA.A GOT Leu Gin Gly 86S

CGA

Arg 850 TTT GOT TTO Phe Oly Phe TTC TOT Phe Cys .855 CAC CG OCT TTG His Ala Ala Leu GAG TTT OTT Glu Phe Leu 860 OAT TGG TOT Asp Trp Ser 2892 2940 OGA TTT AGT COO Oly Phe Ser Pro

OAT

Asp 870 ATO ATT OAT TOO Ile Ile His Cys

OAT

His 87S AGT GOT Ser Ala 8680 COT OTT GOT TG Pro Val Ala Trp TTT AAG GAA C~A Phe Lys Giu Gin

TAT

Tyr 890 ACA CAC TAT GOT Thr His Tyr Gly 2988 3036

CTA

Leu 895 AGC AAA TOT COT Ser Lys Ser Arg

ATA

Ile 900 OTO TTC ACG ATA Val Phe Thr Ile

OAT

His 905 AAT OTT GAA TTT Asn Leu Glu Phe OCA OAT OTO ATT Ala Asp Leu Ile 000 G-ly 915 AGA OCA ATG ACT Arg Ala Met Thr

AAO

Asn 920 OCA GAO AA.A GOT Ala Asp Lys Ala ACA ACA Thr Thr 925 3084 OTT TOA OCA Val Ser Pro TAO TOA CAG GAG Tyr Ser Gin Oiu TOT OGA AAO OCT Ser Gly Asn Pro OTA ATT 000 Val Ile Ala 940 GAO OCA OAT Asp Pro Asp 3132 OCT CAC Pro His WIT TG Ilie Trp 960

CTT

Leu 945 CAC AAd TTO CAT His Lys Phe His ATA GTG AAT 000 Ile Val Asn Giy OAT OCT TTA AAO Asp Pro Leu Asn

GAT

Asp 965 PLAG TTO ATT 000 Lys Phe Ilie Pro

ATT

Ile 970 CCG TAO ACC TOA Pro Tyr Thr Ser 3180 3228 3276

GA

Giu 975 AAO OTT OTT GAA Asn Val Val Giu AAA ACA OCA 000 Lys Thr Ala Ala

ALAG

Lys 985 GAA GOT TTG CAG Glu Ala Leu Gin

OGA

Arg 990 AAA OTT OGA OTO AA.A CAG GOT GAO OTT OCT TTG OTA OGA ATT Lys Leu Gly Leu Lys Gin Ala Asp Leu Pro Leu Val Oly Ile 995 1000 ATO ACC Ile Thr 1005 3324 CGC TIA ACT CAC CAG Arg Leu Thr His Gin 1010 AAA GGA ATC CAC CTC ATT AAA CAT GCT ATT TGG Lys Gly Ile His Leu Ile Lys His Ala Ile Trp 1015 1020 3372 CGC ACC TTG GAA Arg Thr Leu Giu 1025 GAT CCT AGG GTA Asp Pro A-rg Vai 1040 CGG AAC GGA CAG GTA Arg Asn Gly Gin Val 1030 CAA AAC GAT TTT GTT Gin Asn Asp Phe Vai 1045 GTC TTG CTT GGT TCT GCT CCT Vai Leu Leu Gly Ser Ala Pro 1035 AAT TTG GCA AAT CAA TTG CAC Agn Leu Ala Asn Gin Leu His 1050 3420 3468 3516 TCC AAA Ser Lys 1055 TAT AAT GAC Tyr Asn Asp CGC GCA Arg Ala 1060 CGA CTC TOT Arg Leu Cys CTA ACA Leu Thr 1065 TAT GAC GAG Tyr Asp Glu

CCA

Pro 1070 CTT TCT CAC CTG Leu Ser His Leu

ATA

Ile 1075 TAT GCT GGT OCT CAT TTT ATI CTA GTT CCT TCA Tyr Ala Cly Ala Asp Phe Ile Leu Val Pro Ser 3564 1080 1085 ATA TTT GAG CCA TGT GGA CTA Ile Phe GIU Pro Cys Gly Leu 1090 ACA CAA Thr Gin 1095 CTT ACC GCT ATO AGA TAT CGT Leu Thr Ala Met Arg Tyr Gly 1100 3612 TCA ATT Ser Ile CCA CTC Pro Val 1105 GTG CCT AAA Val Arg Lys ACT GGA GGA Thr Gly Gly 1110 CAT GTT GAC CAT GAC AAA GAG AGA GCA CAA Asp Val Asp His Asp Lys Giu Arg Ala Gin 1120 1125 CTT TAT CAT ACT GTA TTT Leu Tyr Asp Thr Val Phe 1115 CAG TOT GCT CTT GAA CCA Gin Cys Gly Leu Giu Pro 1130 CCC CGA GTT CAT TAT GCT Gly Gly Val Asp Tyr Ala 1145 1150 GGT CCC CAT TGG TTC AAC Gly Arq Asp Trp Phe Asn 1165 3708 3660 AAT GGA Asn Cly 1135 TTC ACC TTT Phe Ser Phe CAT GGA Asp Gly 1140 CCA CAT GCT Ala Asp Ala CTC AAT AGA GCC Leu Asn Arg Ala CTC TCT GCC ICC Leu Ser Ala Trp 1155 TAC CAT Tyr Asp 1160 3756 3804 3852 TCT TTA TG Ser Leu Cys OCT CTT CAT Ala Leu Asp 1185

A.AG

Ly s 1170 CAC GTC ATG GAA.CAA CAT TCC TCT ICC Gin Val Met Glu Gin Asp Trp Ser Irp AAC CGA CCI Asn Arg Pro 1180 1175 TAT TTG GAG CTT TAC CAT OCT OCT AGA Tyr Leu Clu Leu Tyr His Ala Ala 'Arg 1190 AAG TTA CAA Lys Leu Clu 3897 TAGTTAGTTT OTOAGATC AGCAGAAAAA TTCACGAGAT CTGCKATCTG TACACCTTCA GTGTTTGCGT CTOOACAGCT TTTTTATTTC CTATATCAAA GTATAAATCA ACICTACACT CAGATCAATA GCAGACACTC CTCAGTTCAT TTCATTTTTT GTGCAACATA TCAAACAGCT TAGCCTCTAA TA.ATGTAGTC ATTOATGATT ATTTGTTTTG GGA.AGAA.ATG ACAAATCAAA 3957 4017 4077 4137 GGATGCAAAA TACTCTGAAA AAAAAA A INFORMATION FOPR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 1197 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID'NO: 12: 4168 Met Glu Pro Gin Vai Tyr Gin Tyr Asn Leu Leu His Gly Gly Arg Met Giu Arg Lys Val1 Lys Thr Glu Pro Asp 145 Ser Gly Met Arg Gly so Gin Giu Ser Asp Val1 130 Asp Asp GJlu Val Arg 3S Phe Ly s Ser Asp Giu 115 A.rg Lys Phe Thr -Thr Arg Val Ser Giu Asp 100 Asp Val Asp Leu Asn dly Lys Pro Asn Ile Asp Giu Ser Ala Ile 165 Ala Val1 Val Arg Gly 70 S er Thr Ile Ser Val1 150 Asp Ser *Ser Phe Pro 25 Ser Thr Thr 40 Lys Pro Ser 55 Asp Lys Giu Asn Gin Lys Lys Val Val 105 Asn Gly Ser 120 Gin Phe Val 135 Lys Leu Asn Ser Val Ile Ser Lys Giy 185 1s Phe Cys Ala Asn Leu Ser Gly Arg Ser Gin dly Ser Thr 90 Va 1 Thr Glu Lys Arg 170 Ser Met Gin 75 Val Arg Lys Ser Ser 155 Glu Ser Ser Giu Asp Ser Glu 140 Ly s Gin Gly Thr Thr Ala His Ile 125 Glu Arg Ser Ser Gin Ser Arq Lys 110 Ser Thr Ser Gly *Ser Arg Thr ValI Phe Met Gly Glu Ser Pro Lys Ser Giu Leu S er Gly Giu 160 Gin 175 His Ala Val 180 Gly Thr Lys 190 Leu Lys Giu Leu Tyr Glu 195 Ile Leu Gin Val Val GIlu Pro Gin Gin 205 Asn Asn Ala Gly Asn Va 210 Leu Glu 225 Ile Asp Glu Asp Leu Asn Glu Arg 290 Phe Pro 305 Arg Gly Ala Phe Thr His Glu Ala 370 Asp Asn 385 Gin Ile Gin Glu Glu Gin Ala Gin 450 Leu Met 465 Ile Asp Glu Leu 275 Leu Glu Leu Asn Leu 355 Tyr Asn Ile Lys Arg 435 Ala fal Thr Leu Pro 260 Arg Ala Val Ser Giu 340 Asn Arg Asp Asp Leu 420 Arg Lys Lys J Lys Asp 245 Leu Leu Glu Vai Thr 325 Trp Gly Ala ly Phe 405 Ala Ile flu kla Al 23C Thi Ala Glu Glu Lys 310 Leu Arg Asp Asp Asn 390 Glu Lys Glu lu rhr 470 1 Giu Tyr Lys Gly Pro 215 i Ser Asp Val Giu His 235 Asn Ser Phe Phe Lys 250 Ala Giy Thr Vai Glu 265 Met Giu Aia Asn Leu 280 Asn Leu Leu Gin Gly 295 Pro Asp Giu Asp Vai 315 Lys Asn Glu Ser Asp 330 Tyr Arg Ser Phe Thr 345 Trp Trp Ser Cys Lys 360 Phe Val Phe Phe Asn 375 Asp Phe Ser Ile Thr 395 Asn Phe Leu Leu Giu G 410 Glu Gin Ala Glu Arg G 425 Ala Giu Lys.Ala Giu I 440 Ala Ala Lys Lys Lys L 455 4 Lys Thr Arg Asp Ile T 475 Va 22C Thi Ser Thr Arg Ile 300 Glu Vai Thr Ile fly 380 lai ;lu lu le ,ys 'hr 1 Ala Glu Asp Gly Arg 285 Arg Ile Leu Arg His 365 Gin Lys C Lys Arg I Glu 7 445 Vai I Trp T Se Se Lei As 1 27C GIr Leu Phe Ile Leu 350 Val Asp ly rrp .eu 1 a ,eu yr r Lys Asn 1 Ile 255 Ser Ala Phe.

Leu Met 335 Thr Pro Val Gly Arg 415 Ala C Asp T Arg C Ile C 4 Leu Glu 240 Glu Ser Ile Cys Asn 320 Gly Glu Lys Tyr M1et 400 ;lu flu ~rg lu lu Pro Ser Glu Phe Lys 485 Cys Glu Asp Lys Val 490 Arg Leu Tyr Tyr Asn Lys 495 Asp Leu Ser Ser Gly Pro Leu Ser His Ala 500 Tyr Ser Asp 545 His Val Gln Met Glu 625 Thr Asn Cys Lys Val 705 Asp 4 Val Ala Asn Glu 530 Gin Ala Pro Ile Arg 610 Arg Glu Pro Ser Met 690 Pro ly Phe lal Asn 515 Arg Ala Ile Asn Phe 595 Ala Thr Pro Ala Phe 675 Ser Leu Gly Gly Glu 755 Trp Ile Leu Ala His 580 Lys Lys Met Leu Asn 660 Asn Pro Asp Ile I Gly N 740 Met Lys Asp Phe Tyr 565 Ile Thr Val Lys Asp 645 rhr Arg la kia ?he 125 ial la Asj Gly Leu 550 Asp Pro Leu Glu Ser 630 Ile Val Trp Glu Tyr 710 Asp Ala Pro Gly Asp 535 Asp Asn Glu Gin Lys 615 Phe Gin Leu Thr Asn 695 Met Asn Lys c Ile Ala 775 Leu 520 Trp Trp Asn Glu Glu 600 Thr Leu Ala Asn His 580 Gly iet 'ys flu la I r60 Lys 505 Sex Trp Vai His Leu 585 Glu Ala Leu Gly Gly 665 Arg Thr Asp Ser ?ro 745 ,ys Trp Ile His Gly Gly 510 iIE Tyr Phe Arg 570 Tyr Arg Leu Ser Ser 650 Lys Leu His Phe Gly 730 Pro Val Val Thr Ala 555 Gin Trp Arg Leu Gln 635 Ser Pro Gly Val Val 715 Met 2 Met t Gly C Lys Glu 540 Asp Asp Val Leu Lys 620 Lys Val Glu Pro Arg 700 Phe ksp iis ;ly Lys 525 I Val Gly Phe Glu Arg 605 Thr His Thr Ile Leu 685 Ala Ser Tyr I Ile Leu C 765 Lei Val Pro His Glu 590 Glu Glu Val Val Trp 670 Pro Thr lu His al 750 ;ly i Val Ile Pro Ala 575 Glu Ala Thr Val Tyr 655 Phe Pro Val Arg Ile i 735 His Asp I Lys Pro Lys 560 Ile His Ala Lys Tyr 640 Tyr Arg Gin Lys lu 720 Pro Ile Ial Val Thr 77b Ser Leu Ser Arg Pal Gin Asp Leu Asn His Asn Vai Asp Ile Ile Leu Pro Lys Tyr Asp Cys Leu Lys Met 795 Asn Lys Lys 815 Phe Arg Phe Trp Phe Gly Asn Gly Leu 835 Glu Arg Phe 850 Gly Gly Phe 865 Pro Val Ala Lys Ser Arg Leu Ile Gly 915 Pro Thr Tyr 930 Leu His Lys 945 Asp Pro Leu Val Val Glu Gly Leu Lys 995 Thr His Gin 1010 Leu Glu Arg 1025 Arg Val Gin 2 His Lys 820 Phe Gly Ser Trp Ile 900 Arg Ser Phe Asn Gly 980 Gin Lys Asn Asn Lys Asn Tyr Phe Tri 805 Val Glu Gly Leu Sei 825 Ser Lys Gly Cys Val 840 Phe Phe Cys His Ala 855 Pro Asp Ile Ile His 870 Leu Phe Lys Glu Gin .885 Val Phe Thr Ile His 905 Ala Met Thr Asn Ala 920 Gin Glu Val Ser Gly 935 His Gly Ile Val Asn 950 Asp Lys Phe Ile Pro 965 Lys Thr Ala Ala Lys 985 Ala Asp Leu Pro Leu 1000 Gly Ile His Leu Ile 1015 Gly Gin Val Val Leu 1030 Asp Phe Val Asn Leu 1045 p Gly Gly Thr Glu Ile 810 Val Tyr Phe Leu Glu 830 Tyr Gly Cys Ser Asn 845 Ala Leu Glu Phe Leu 860 Cys His Asp Trp Ser 875 Tyr Thr His Tyr Gly 890 Asn Leu Glu Phe Gly 910 Asp Lys Ala Thr Thr 925 Asn Pro Val Ile Ala 940 Gly Ile Asp Pro Asp 955 Ile Pro Tyr Thr Ser 970 Glu'Ala Leu Gin Arg 990 Val Gly Ile Ile Thr J 1005 Lys His Ala Ile Trp.) 1020 Leu Gly Ser Ala Pro 1035 Ala Asn Gin Leu His S 1050 1 Pro Gin Asp Gly Leu Gin Ser Ala 880 Leu Ser 895 Ala Asp Val Ser Pro His Ile Trp 960 Glu Asn 975 Lys Leu Arg Leu Arg Thr Asp Pro 1040 ;er Lys .055 Tyr Asn Asp Arg Ala Arg Leu Cys Leu Thr Tyr Asp Glu Pro Leu Ser 1060 1065 1070 92 His Leu Ile Tyr Ala Gly Ala Asp Phe Ile Leu Val Pro Ser Ile Phe 1075 1080 1085 Glu Pro Cys Gly Leu Thr Gin Leu Thr Ala Met Arg Tyr Gly Ser Ile 1090 1095 1100 Pro Val Val Arg Lys Thr Gly Gly Leu Tyr Asp Thr Val Phe Asp Val 1105 1110 1115 1120 Asp His Asp Lys Glu Arg Ala Gin Gln Cys Gly Leu Glu Pro Asn Gly 1125 1130 1135 Phe Ser Phe Asp Gly Ala Asp Ala Gly Gly Val Asp Tyr Ala Leu Asn 1140 1145 1150 Arg Ala Leu Ser Ala Trp Tyr Asp Gly Arg Asp Trp Phe Asn Ser Leu 1155 1160 1165 Cys Lys Gin Val Met Glu Gln Asp Trp Ser Trp Asn Arg Pro Ala Leu 1170 1175 1180 Asp Tyr Leu Glu Leu Tyr His Ala Ala Arg Lys Leu Glu 1185 1190 1195 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Gly Thr Gly Gly Leu Arg Asp Thr Val Glu Asn Cys 1 5 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc.= "Oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACAGGATCCT GTGCTATGCG GCGTGTGAAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc.= "OligonucleotidE" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTGGGATCCG CAATGCCCAC AGCATTTTTT TC INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Pro Trp Ser Lys Thr Gly Gly Leu Gly Asp Val Cys 1 5 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gln Leu Tyr 1 5 This application is divided from our copending application 39279/95 and entire disclosure in the complete specification and claims of that application is by this cross-reference incorporated into the present specification.

Claims (22)

1. 2. An isolated DNA molecule according to claim 1, which has more than 90% overall sequence homology.
2. 3. A vector comprising a DNA molecule according to claim 1 or claim 2.
3. 4. A vector according to claim 3, wherein the DNA molecule is linked in sense orientation to DNA elements ensuring transcription and synthesis of a translatable RNA in prokaryotic or eukaryotic cells. A host cell comprising a vector according to claim 3 or claim 4.
4. 6. A protein or biologically active fragment thereof encoded by a DNA molecule according to claim 1.
5. 7. A method for producing a protein according to claim 6 or a biologically active fragment thereof, wherein a host cell according to claim 5 is cultivated under conditions allowing synthesis of the protein, and wherein the protein is isolated from the cultivated cells and/or the culture medium. H:\terryr\Keep\Retype\P48083 Amended claims BAYER Nov 2005.doc 24/11/05 n- 95
6. 8. A plant cell comprising a DNA molecule: O DNA molecules encoding a protein having the amino acid sequence indicated under SEQ ID NO:8; and CI 5 DNA molecules comprising the nucleotide sequence depicted under SEQ ID NO:7, wherein the DNA molecules or encode a NO 0 protein with the biological activity of a starch synthase of isotype II (GBSSII), in combination with a heterologous 10 promoter. C 9. A plant comprising plant cells according to claim 8.
7. 10. A plant according to claim 9, which is a useful plant.
8. 11. A plant according to claim 10, which is a starch-storing plant.
9. 12. A plant according to claim 11, which is a potato plant.
10. 13. Propagation material of a plant according to any one of claims 9 to 12, comprising plant cells according to claim 8.
11. 14. Starch obtained from a plant according to any one of claims 9 to 12. A transgenic plant cell, in which the activity of the protein according to claim 6 is reduced.
12. 16. A plant cell according to claim 15, wherein an antisense RNA to transcripts of a DNA molecule DNA molecules encoding a protein having the amino acid sequence indicated under SEQ ID NO:8; and H:\terryr\Keep\Retype\P48083 Amended claims BAYER Nov 2005.doc 24/11/05 96 o DNA molecules comprising the nucleotide sequence depicted under SEQ ID NO:7, O wherein the DNA molecules or encode a protein with CI 5 the biological activity of a starch synthase of isotype II (GBSSII) is expressed.
13. 17. A plant cell according to claim 15, wherein a sense RNA of a DNA molecule according to claim 1 or 2 is C 10 expressed, thereby to achieve a cosuppression effect. CI 18. A plant cell according to claim 15, wherein a ribozyme is expressed which specifically cleaves transcripts of a DNA molecule according to claim 1 or 2.
14. 19. A plant comprising plant cells according to any one of claims 15 to 18. A plant according to claim 17, which is a useful plant.
15. 21. A plant according to claim 18, which is a starch-storing plant.
16. 22. A plant according to claim 19, which is a potato plant.
17. 23. Propagation material of a plant according to any one of claims 19 to 22, comprising cells according to any one of claims 15 to 18.
18. 24. Starch obtained from a plant according to any one of claims 19 to 22.
19. 25. An isolated DNA molecule according to claim 1, substantially as herein described with reference to any one of the Examples. H:\terryr\Keep\Retype\P48083 Amended claims BAYER Nov 2005.doc 24/11/05 97
20. 26. A vector according to claim 3, substantially as 0 herein described with reference to any one of the Examples. c-
21. 27. A protein according to claim 6, substantially as herein described with reference to any one of the \O 0 Examples. C 10 28. A method according to claim 7, substantially as herein described with reference to the Examples.
22. 29. A plant cell according to claim 8 or claim substantially as herein descried with reference to the Examples. Dated this 24th day of November 2005 BAYER CROPSCIENCE GMBH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\terryr\Keep\Retype\P48083 Amended claims BAYER Nov 2005,doc 24/11/05