AU5601499A - 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 a a a a.

a. a a a.

The following statement is a full description of this invention, including the best method of performing it known to me/us: A DNA molecules encoding enzymes involved in starch synthesis, vectors, bacteria, transgenic plant cells and plants containing these molecules 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 granule-bound 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 important 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 homogeneous 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 polymerization and in the degree of branching of the glucose chains. Therefore, starch is not a homogeneous raw material. One pm/msofficelwinword/transiations/01356pct differentiates particularly between amylose-starch, a basically non-branched polymer made up of a-l,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 Srecombinant 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 lace 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 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 S 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).

n 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 S (Tyynela 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 S: 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 No. 9, preferably molecules comprising the coding region indicated under Seq ID No. 9.

Another subject matter of the invention are DNA molecules encoding an SSSB 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

SSSB

and hybridizing to any of the above-described DNA molecules. An exception are the DNA molecules from rice. The SSSB proteins encoded by the DNA molecules according to the invention preferably have a molecular weight of 78±5 kD.

The enzymatic properties of the SSSB proteins are described in the examples.

The invention furthermore relates to DNA molecules encoding proteins with the biological activity of a soluble starch synthase of the isotype A (SSSA). Such proteins can, for example, be characterized in that they are recognized by an antibody that is directed to the peptide having the amino acid sequence NH2-GTGGLRDTVENC-COOH (Seq ID No. 13).

The enzymatic properties of the SSSA proteins are described in the examples.

An example of a DNA molecule encoding such a protein is a DNA molecule having the coding region depicted under Seq ID No. 11.

*oe This DNA molecule may be used to isolate from other organisms, in particular plants, DNA molecules encoding the SSSA proteins.

Thus, the present invention also relates to DNA molecules encoding proteins with the biological activity of a soluble starch synthase of the isotype A (SSSA) 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. 12. The invention particularly relates to DNA molecules having the nucleotide sequence indicated under Seq ID No. 11, preferably molecules comprising the coding region indicated under Seq ID No. 11.

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

W

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 S 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-l,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, S 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 o 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.

SFurthermore, the proteins encoded by the DNA molecules of the 0 0 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 a protein and whereby the protein is then isolated from the to. cultivated cells and/or the culture medium.

0* 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 M 0 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 S 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 S: 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 o f 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 .14 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 S 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 S with homologies between 95 and 100%.

S 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 nono o foodstuffs.

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: 1. 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 all, the paper and cardboard industry. In this field, the starch is mainly used for retention (holding back solids), "for 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, S.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.

S 2.6 Use of starch in plant protectives and fertilizers o 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 S. 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.

*r 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 B starches.

B 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 S. 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 S* synthetic polymer are combined in a ratio of 1 1 by means of coexpression to form a 'master batch', from which various 9: 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 0 M 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.

0

M

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, 0-allyl ether, hydroxylalkyl ether, 0- S: carboxylmethyl 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.

r 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.

*o 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.

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 W092/148 27 S 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 (KoBmann 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 S. 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 0 1 1 1 1 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., S Willmitzer, 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise Rehm, G. Reed, A.

Puhler, 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 i 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 .e S 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 NaC1 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 a a.

a a a. a.

a a.

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 inN 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 inN Tris-HCl pH mM NaCi 2 mM EDTA 14.7 mM B-inercaptoethanol mM PMSF 50 mM sodium phosphate buffer pH 7.2 10 mM EDTA 0.5 mM PMSF 14.7 mM B-mercaptoethanol Fig. 1 shows plasinid 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 S..soluble starch synthase; SSSA isotype; 00

X

ba 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: S* 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 31 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 andsoluble 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 pg 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: 0, 1. Cloning methods o 0 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

S

For the Bluescript vector and for the pBinAR Hyg constructs the E. coli strain DH5 (Bethesda Research Laboratories, Gaithersburg, USA) was used. For the in vivo excision the E. coli S" strain XL1-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 H6fgen 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.

33 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 ul 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 250C and 3,000 lux for one week the leaves were placed for shoot induction on MS medium containing 1.6% glucose, 1.4 mg/l 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.

5. 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 14C glucose from ADP C 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

M

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 0 C for 4 hours (Nielsen et al., Plant Physiol. 105 .(1994), 111-117). After neutralization with 0.7 M KOH, 50 ~l 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 eoe* imidazole/HCl; 10 mM MgCl 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 PA1; 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/l.

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 50°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 S* 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 95 0 C at 120/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 0

_M

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 S"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.

37 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 'o'e 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 Sto 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 S: 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°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 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 NaHPO 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 NaHPO 4 pH 7.2, 0.25 M NaCI, 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. Desirde (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. Desirde (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 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

A sequence comparison between the sequences encoding soluble and Sgranule-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 NaCl 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 i 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 ,aa 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 pg 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 pg protein of the factions eluted from the AR column and 3 pg protein of the fractions eluted from the MonoQ column. The proteins were transferred onto a nitrocellulose membrane using the semidry electroblot method.

I

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 "o 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. D6sirde (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 :I 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 e 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

C]

Maximum viscosity [cP] 4044 3720 3756 Final viscosity at 50 0 C 3312 2904 2400 [cP] Mean granule size [gm] 29 24 27 0** 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): 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 S. 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" 1 Pastification 64.1 62.55 63.25 63.55 temperature [oC] Maximum 4057 2831 2453 2587 viscosity [cP] Final 2849 2816 2597 2587 viscosity at 0 C [cP] Mean granule 37 32 31 32 size [Am] 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 S. 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 pg 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.

e 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 Sin 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:/function= "Polymerization of starch" /product= "Starch synthase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GG CAC GAG GTC AAA AAG CTT GTT AAA TCT GAG AGA ATA GAT GGT GAT His Glu Val Lys Lys Leu Val Lys Ser Glu Arg Ile Asp Gly Asp 1 5 10 TGG TGG TAT ACA GAG GTT GTT ATT CCT GAT CAG GCA CTT TTC TTG GAT Trp Trp Tyr Thr Glu Val Val Ile Pro Asp Gin Ala Leu Phe Leu Asp c

CCC.

C

p C

CCC.

Cr

C

CCC.

CC

C C CC C SC C TGG GTT TTT GCT Trp Val Phe Ala AAT CAC CGC CAA Asn His Arg Gin 50 GAT GGT CCA CCC Asp Gly Pro Pro AAG CAT GCC ATT GCT TAT Lys His Ala Ile Ala Tvr GAT AAC Asp Asn GAC TTC CAT Asp Phe His

GCC

Ala 55 ATT GTC CCC AAC Ile Val Pro Asn

CAC

His ATT CCG GAG Ile Pro Glu GAA TTA Glu Leu 65 TAT TGG GTT GAG Tyr Trp Val Glu GAA CAT CAG ATC Glu His Gin Ile

TTT

Phe AAG ACA CTT CAG Lys Thr Leu Gin

GAG

Glu 80 GAG AGA AGG CTT Glu Arg Arg Leu

AGA

Arg 85 GAA GCG GCT ATG Glu Ala Ala Met

CGT

Arg 90 GCT AAG GTT GAA Ala Lys Val Glu

AAA

Lys 287 ACA GCA CTT CTG Thr Ala Leu Leu

AAA

Lys 100 ACT GAA ACA AAG Thr Glu Thr Lys AGA ACT ATG AAA Arg Thr Met Lys TCA TTT Ser Phe 110 CC I

C.

p.

C* S C C

CC

TTA CTG TCT Leu Leu Ser GCT GGA AGC Ala Gly Ser 130

CAG

Gin 115 AAG CAT GTA GTA Lys His-Val Val

TAT

Tyr 120 ACT GAG CCT CTT Thr Glu Pro Leu GAT ATC CAA Asp Ile Gin 125 ACA GTA CTT Thr Val Leu AGC GTC ACA GTT Ser Val Thr Val

TAC

Tyr 135 TAT AAT CCC GCC Tyr Asn Pro Ala

AAT

Asn 140 AAT GGT Asn Gly 145 CAC CGC His Arg 160 AAA CCT GAA Lys Pro Glu CTG GGT CCA Leu Gly Pro ATT TGG Ile Trp 150 TTC AGA TGT TCA Phe Arg Cys Ser

TTT

Phe 155 AAT CGC TGG ACT Asn Arg Trp Thr

TTG

Leu 165 CCA CCT CAG AAA Pro Pro Gin Lys

ATG

Met 170 TCG CCT GCT Ser Pro Ala GAA AAT Glu Asn 175 TAT ATG Tyr Met 190 GGC ACC CAT GTC Gly Thr His Val

AGA

Arg 180 GCA ACT GTG AAG Ala Thr Val Lys

GTT

Val 185 CCA TTG GAT GCA Pro Leu Asp Ala ATG GAT TTT GTA TTT TCC GAG AGA GAA GAT GGT GGG ATT TTT GAC AAT Met Asp Phe Val Phe Ser Glu Arg Glu Asp Gly Gly Ile Phe Asp Asn AAG AGC GGA ATG GAC TAT CAC ATA CCT GTG TTT GGA GGA GTC Lys Ser Gly 210 Met Asp Tyr His Ile 215 Pro Val Phe Gly Gly Val 220 GCT AAA Ala Lys GAA CCT Glu Pro 225 CCA ATG CAT ATT Pro Met His Ile

GTC

Val 230 CAT ATT GCT GTC His Ile Ala Val

GAA

Glu 235 ATG GCA CCA ATT Met Ala Pro Ile

GCA

Ala 240 AAG GTG GGA GGC Lys Val Gly Gly

CTT

Leu 245 GGT GAT GTT GTT Gly Asp Val Val

ACT

Thr 250 AGT CTT TCC CGT Ser Leu Ser Arg

GCT

Ala 255 GTT CAA GAT TTA Val Gin Asp Leu

AAC

Asn 260 CAT AAT GTG GAT His Asn Val Asp

ATT

Ile 265 ATC TTA CCT AAG Ile Leu Pro Lys TAT GAC Tyr Asp 270 815 oooo o TGT TTG AAG Cys Leu Lys TTT TGG GGT Phe Trp Gly 290

ATG

Met 275 AAT AAT GTG AAG Asn Asn Val Lys

GAC

Asp 280 TTT CGG TTT CAC Phe Arg Phe His AAA AAC TAC Lys Asn Tyr 285 GTG GAA GGT Val Glu Gly GGG ACT GAA ATA Gly Thr Glu Ile

AAA

Lys 295 GTA TGG TTT GGA Val Trp Phe Gly

AAG

Lys 300 863 911 959 CTC TCG Leu Ser 305 GTC TAT TTT TTG Val Tyr Phe Leu

GAG

Glu 310 CCT CAA AAC GGG Pro Gin Asn Gly

TTA

Leu 315 TTT TCG AAA GGG Phe Ser Lys Gly

TGC

Cys 320 GTC TAT GGT TGT Val Tyr Gly Cys AGC AAT Ser Asn 325 GAT GGT GAA Asp Gly Glu

CGA

Arg 330 TTT GGT TTC TTC Phe Gly Phe Phe

TGT

Cys 335 1007 CAC GCG GCT TTG His Ala Ala Leu TTT CTT CTG CAA Phe-Leu Leu Gin GGA TTT AGT CCG Gly Phe Ser Pro GAT ATC Asp Ile 350 1055 ATT CAT TGC Ile His Cys GAA CAA TAT Glu Gin Tyr 370

CAT

His 355 GAT TGG TCT AGT Asp Trp Ser Ser

GCT

Ala 360 CCT GTT GCT TGG Pro Val Ala Trp CTC TTT AAG Leu Phe Lys 365 GTC TTC ACG Val Phe Thr 1103 1151 ACA CAC TAT GGT Thr His Tyr Gly

CTA

Leu 375 AGC AAA TCT CGT Ser Lys Ser Arg

ATA

Ile 380 ATA CAT Ile His 385 AAC GCA Asn Ala 400 AAT CTT GAA TTT Asn Leu Glu Phe GAC AAA GCT ACA Asp Lys Ala Thr 405

GGG

Gly 390 GCA GAT CTC ATT Ala Asp Leu Ile

GGG

Gly 395 AGA GCA ATG ACT Arg Ala Met Thr 1199 ACA GTT TCA CCA ACT TAC TCA CAG GAG GTG Thr Val Ser Pro Thr Tyr Ser Gin Glu Val 410 415 1247 TCT GGA AAC CCT GTA ATT GCG CCT CAC CTT CAC AAG TTC CAT GGT ATA Ser Gly Asn Pro Val Ile Ala Pro His Leu His Lys Phe His Gly Ile 1295 GTG AAT GGG Val Asn Gly ATT CCG ATT Ile Pro Ile 450

ATT

Ile 435 GAC CCA GAT ATT Asp Pro Asp Ile

TGG

Trp 440 CCT TTA AAC GAT AAG TTC Pro Leu Asn Asp Lys Phe 445 GTT GAA GGC AAA ACA GCA Val Glu Gly Lys Thr Ala CCG TAC ACC TCA GAA AAC GTT Pro Tyr Thr Ser Glu Asn Val 1343 1391 1439 1487 GCC AAG Ala Lys 465 GAA GCT TTG CAG Glu Ala Leu Gin

CGA

Arg 470 AAA CTT GGA CTG Lys Leu Gly Leu

AAA

Lys 475 CAG GCT GAC CTT Gin Ala Asp Leu

CCT

Pro 480 TTG GTA GGA ATT Leu Val Gly Ile

ATC

Ile 485 ACC CGC TTA ACT Thr Arg Leu Thr

CAC

His 490 CAG AAA GGA ATC Gin Lys Gly Ile

CAC

His 495 CTC ATT AAA CAT Leu Ile Lys His

GCT

Ala 500 ATT TGG CGC ACC Ile Trp Arg Thr GAA CGG AAC Glu Arg Asn GTC TTG CTT Val Leu Leu AAT TTG GCA Asn Leu Ala 530

GGT

Gly 515 TCT GCT CCT GAT Ser Ala Pro Asp CCT AGG GTA CAA Pro Arg Val Gin 520 AAA TAT AAT GAC Lys Tyr Asn Asp

AAC

Asn

CGC

Arg 540 GGA CAG GTA Gly Gin Val 510 GAT TTT GTT Asp Phe Val 525 GCA CGA CTC Ala Arg Leu 1535 1583 1631 1679 AAT CAA TTG CAC Asn Gin Leu His

TCC

Ser 535 TGT CTA Cys Leu 545 ACA TAT GAC Thr Tyr Asp GAG CCA Glu Pro 550 CCT TCA Pro-Ser 565 CTT TCT CAC CTG ATA TAT GCT GGT GCT Leu Ser His Leu Ile Tyr Ala Gly Ala 555

GAT

Asp 560 TTT ATT CTA GTT Phe Ile Leu Val ATA TTT GAG Ile Phe Glu

CCA

Pro 570 TGT GGA CTA ACA Cys Gly Leu Thr

CAA

Gin 575 1727 CTT ACC GCT ATG Leu Thr Ala Met

AGA

Arg 580 TAT GGT TCA ATT Tyr Gly Ser Ile GTC GTG CGT AAA Val Val Arg Lys ACT GGA Thr Gly 590 1775 GGA CTT TAT Gly Leu Tyr CAA CAG TGT Gin Gin Cys 610

GAT

Asp 595 ACT GTA TTT GAT Thr Val Phe Asp

GTT

Val 600 GAC CAT GAC AAA Asp His Asp Lys GAG AGA GCA Glu Arg Ala 605 GGA GCA GAT Gly Ala Asp 1823 1871 GGT CTT GAA CCA Gly Leu Glu Pro AAT GGA Asn Gly 615 TTC AGC TTT Phe Ser Phe

GAT

Asp 620 GCT GGC Ala Gly 625 GGA GTT GAT TAT Gly Val Asp Tyr

GCT

Ala 630 CTG AAT AGA GCT CTC TCT GCT TGG TAC Leu Asn Arg Ala Leu Ser Ala Trp Tyr 635 1919

GAT

Asp 640 GGT CGG GAT TGG Gly Arg Asp Trp

TTC

Phe 645 AAC TCT TTA TGC Asn Ser Leu Cys CAG GTC ATG GAA Gin Val Met Glu

CAA

Gin 655 1967 GAT TGG TCT TGG Asp Trp Ser Trp CGA CCT GCT CTT Arg Pro Ala Leu TAT TTG GAG CTT Tyr Leu Glu Leu TAC CAT Tyr His 670 2015 GCT GCT AGA AAG TTA GAA Ala Ala Arg Lys Leu Glu 675 TAGTTAGTTT GTGAGATGCT AGCAGAAAAA 2063

S

0: 0. 0.

TTCACGAGAT CTGCAATCTG TACAGGTTCA GTGTTTGCGT CTGGACAGCT TTTTATTTCC TATATCAAAG TATAAATCAA GTCTACACTG AGATCAATAG CAGACAGTCC TCAGTTCATT TCATTTTTTG TGCAACATAT GAAAGAGCTT AGCCTCTAAT AATGTAGTCA TTGATGATTA TTTGTTTTGG GAAGAAATGA GAAATCAAAG GATGCAAAAT ACTCTGAAAA AAAAAAAAAA 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: 2123 2183 2243 2303 His 1 Glu Val Lys Lys Leu Val Lys Ser Glu Arg Ile Asp Gly Asp Trp Trp Tyr Thr Glu Val Val Ile Pro Asp Gin Ala Leu Phe Leu Asp Trp 25 Val Phe Ala Asp Gly Pro Pro Lys His Ala Ile Ala Tyr Asp Asn Asn 40 His Arg Gin Asp Phe His Ala Ile Val Pro Asn His Ile Pro Glu Glu 55 Leu Tyr Trp Val Glu Glu Glu His Gin Ile Phe Lys Thr Leu Gin Glu 70 75 Glu Arg Arg Leu Arg Glu Ala Ala Met Arg Ala Lys Val Glu Lys Thr 90 Ala Leu Leu Lys Thr Glu Thr Lys Glu Arg C C

C

C..

C a C *9 C

.*C

C

SO

a CC C. C S C Leu Ser Gly ser 130 Gly Lys 145 Arg Leu Thr His Asp Phe Ser Gly 210 Pro Pro 225 Lys Val Gin Asp Leu Lys Trp Gly 290 Ser Val 305 Vai Tyr Ala Ala His Cys 100 Gin Lys 115 Ser Val Pro Giu Gly Pro Val Arg 180 Val Phe 195 Met Asp Met His Gly Gly Leu Asn 260 Met Asn 275 Gly Thr Tyr Phe Gly Cys Leu Glu 340 His Asp 355 His Thr Ile Leu 165 Ala Ser Tyr Ile Leu 245 His Asn Glu Leu Ser 325 Phe Trp Val1 Val1 Trp 150 Pro Thr Giu His Val 230 Gly Asn Val1 Ile Giu 310 Asn Leu Ser Val Tyr 135 Phe Pro Val Arg Ile 215 His Asp Val Lys Lys 295 Pro Asp Leu Ser Tyr 120 Tyr Arg Gin Lys Giu 200 Pro Ile Val Asp Asp 280 Vai Gin Gly Gin Ala 360 105 Thr Asn Cys Lys Val 185 Asp Val Ala Val Ile 265 Phe Trp Asn Giu Gly 345 Pro Giu Pro Ser Met 170 Pro Gly Phe Val Thr 250 Ile Arg Phe Gly Arg 330 Gly Val1 Thr Pro Ala Phe 155 Ser Leu Gly Gly Giu 235 Ser Leu Phe Gly Leu 315 Phe Phe Ala Met Leu As n 140 Asn Pro Asp Ile Gly 220 Met Leu Pro His Lys 300 Phe Gly Ser Trp Lys Asp 125 Thr Arg Ala Ala Phe 205 Val Ala Ser Lys Lys 285 Val Ser Phe Pro Leu 365 Ser 110 Ile Val Trp Giu Tyr 190 Asp Ala Pro Arg Tyr 270 Asn Glu Lys Phe Asp 350 Phe Phe Gin Leu Thr Asn 175 Met Asn Lys Ile Ala 255 Asp Tyr Gly Gly Cys 335 Ile Ly s Leu Ala As n His 160 Gly Met Lys Giu Ala 240 Vai Cys Phe Leu Cys 320 His Ile Glu Gin Tyr Thr His Tyr Gly Leu Ser Lys Ser Arg Ile Val Phe Thr Ile 370 375 380 57 His Asn Leu Glu Phe Gly Ala Asp Leu Ile Gly Arg Ala Met Thr Asn 385 390 395 400 Ala Asp Lys Ala Thr Thr Val Ser Pro Thr Tyr Ser Gin Glu Val Ser 405 410 415 Gly Asn Pro Val Ile Ala Pro His Leu His Lys Phe His Gly Ile Val 420 425 430 Asn Gly Ile Asp Pro Asp Ile Trp Asp Pro Leu Asn Asp Lys Phe Ile 435 440 445 Pro Ile Pro Tyr Thr Ser Glu Asn Val Val Glu Gly Lys Thr Ala Ala 450 455 460 Lys Glu Ala Leu Gin Arg Lys Leu Gly Leu Lys Gin Ala Asp Leu Pro 465 470 475 480 Leu Val Gly Ile Ile Thr Arg Leu Thr His Gin Lys Gly Ile His Leu 485 490 495 Ile Lys His Ala Ile Trp Arg Thr Leu Glu Arg Asn Gly Gin Val Val 500 505 510 Leu Leu Gly Ser Ala Pro Asp Pro Arg Val Gin Asn Asp Phe Val Asn 515 520 525 Leu Ala Asn Gin Leu His Ser Lys Tyr Asn Asp Arg Ala Arg Leu Cys 530 535 540 Leu Thr Tyr Asp Glu Pro Leu Ser His Leu Ile Tyr Ala Gly Ala Asp 545 550 555 560 Phe Ile Leu Val Pro Ser Ile Phe Glu Pro Cys Gly Leu Thr Gin Leu 565 570 575 Thr Ala Met Arg Tyr Gly Ser Ile Pro Val Val Arg Lys Thr Gly Gly 580 585 590 Leu Tyr Asp Thr Val Phe Asp Val Asp His Asp Lys Glu Arg Ala Gin 595 600 605 Gin Cys Gly Leu Glu Pro Asn Gly Phe Ser Phe Asp Gly Ala Asp Ala 610 615 620 Gly Gly Val Asp Tyr Ala Leu Asn Arg Ala Leu Ser Ala Trp Tyr Asp 625 630 635 640 Gly Arg Asp Trp Phe Asn Ser Leu Cys Lys Gin Val Met Glu Gin Asp 645 650 655 Trp Ser Trp Asn Arg Pro Ala Leu Asp Tyr Leu Glu Leu Tyr His Ala 660 665 670 Ala Arg Lys Leu Glu 675 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1758 base pairs ART: nucleotide STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (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:1..1377 OTHER INFORMATION:/function= "Polymerization of starch" /product= "Starch synthase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: a. a a a a.

GGC

Gly 1 ACG AGC AAT GCT GTT-GAC CTT GAT Thr Ser Asn Ala Val Asp Leu Asp 5 GTG CGG GCC ACT GTC CAT TGC Val Arg Ala Thr Val His Cys 10 TTT GGT GAT Phe Gly Asp GTT GAT TGG Val Asp Trp CAG GAA GTA GCC Gln Glu Val Ala TAC CAT GAA TAC Tyr His Glu Tyr AGG GCA GGT Arg Ala Gly CCT GGA ACG Pro Gly Thr GTA TTT GTG GAC Val Phe Val Asp

CAC

His 40 TCT TCT TAC CGC Ser Ser Tyr Arg

AGA

Arg CCA TAT Pro Tyr GGT GAT ATT TAT Gly Asp Ile Tyr

GGT

Gly 55 GCA TTT GGT GAT Ala Phe Gly Asp

AAT

Asn CAG TTT CGC TTC Gln Phe Arg Phe

ACT

Thr TTG CTT TCT CAC Leu Leu Ser His GCA TGT GAA GCG Ala Cys Glu Ala TTG GTT CTT CCA Leu Val Leu Pro 240 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 Leu Ala Asn Asp Cys 90 AAO GCT GC Asn Ala Ala GTT OCT TTA CTT Val Pro Leu Leu TTA GCG Leu Ala 105 GCC AAG TAT Ala Lys Tyr CGT OCT TAT Arg Pro Tyr 110 AAC ATT GCA Asn Ile Ala 336 GOT GTT TAC AAG GAT GCT CGT AGT GTC GCA ATA CAC Gly Val Tyr 115 Lys Asp Ala Arg Ser 120 Val Ala Ile His 125 CAT CAG His Gin 130 GGA GTG GAG OCT Gly Val Giu Pro

GCA

Ala 13 -1 GTA ACC TAO A-AT Val Thr Tyr Asn

AAT

Asn 140 TTG GGT TTG OCT Leu Gly Leu Pro

CA

Pro 145 CAA TGG TAT GGA Gin Trp Tyr Gly

GCA

Ala 150 GTT GAA TGG ATA Val Giu Trp Ile

TTT

Phe 155 CCC ACA TGG GCA Pro Thr Trp Ala

AGG

Arg 160 p

S

*5S* S S S. 5.5.

S S 5* GOG CAT GOG OTT Ala His Ala Leu ACT GGT GAA ACA Thr Gly Olu Thr AAO GTT TTG AAA Asn Val Leu Lys GOG GOA Gly Ala 175 528 ATA GCA GTT Ile Ala Val GAA ATA ACA Glu Ile Thr 195

GOT

Ala 180 GAT COG ATA OTO Asp Arg Ile Leu

ACA

Thr 185 OTT AGO CAG GGA Val Ser Gin Gly TAO TCA TG Tyr Ser Trp 190 OTG TTG AGO Leu Leu Ser 576 624 ACT OCT GAA GGG Thr Pro Giu Gly

GGA

Gly 200 TAT 000 OTA OAT Tyr Gly Leu His

GAG

Giu 205 AGT AGA Ser Arg 210 CAG TOT OTT OTT Gin Ser Val Leu

AAT

Asn 215 OGA ATT ACT AAT Oly Ile Thr Asn

OGA

Gly 220 ATA OAT OTT AAT Ile Asp Val Asn S. *5 *5 S

S.

OAT

Asp 225 TOG AAC COG TOO Trp Asn Pro Ser ACA -OAT Thr Asp 230 GAG OAT ATO Giu His Ile

GOT

Ala 235 TOG OAT TAO TOO Ser His Tyr Ser

ATO

Ile 240 720 AAT GAO OTO TOO Asn Asp Leu Ser

CCC

Pro 245 OCT OGA AAO OTT Pro Oly Lys Val

CAG

Gin 250 TOO AAG ACT OAT Cys Lys Thr Asp OTO CA.A Leu Gin 255 AAG OAA OTO Lys Oiu Leu ATT OGA AGO Ile Oly Arg 275

GC

Gly 260 OTT OCA ATT OGA Leu Pro Ile Arg OAT TOT OCA OTO Asp Cys Pro Leu ATT OGA TTT Ile Cly Phe 270 CTG TCA OCA Leu Ser Ala 816 864 OTO GAO TAO CAG Leu Asp Tyr Gin

AAA

Lys 280 GOT OTT GAO ATA Gly Val Asp Ile

ATO

Ile 285 ATT OCA Ile Pro 290 OAA OTT ATO CAG Giu Leu Met Gin

AAT

Asn 295 OAT OTO CAA OTT Asp Val Gin Val ATO OTT OGA TOT Met Leu Oly Ser 912 GOT GAG AAA CAA TAT GAA GAO TOO ATO AGA CAT ACA GAA AAT OTT TTT 960 Gly Giu Lys Gin Tyr Giu Asp Trp Met Arg His Thr Giu Asn Leu Phe 305 310 315in AAA GAC AAA TTT CGT GOT TGG GTT Lys Asp Lys Phe Arg Ala Trp Val 325 GGA TTT AAT GTT CCA GTT TCT CAT Gly Phe Asn Val Pro Val Ser His 1008 AGG ATA ACA Arg Ile Thr CCG TGT GGC Pro Cys Gly 355

GCA

Ala 340 GGA TGC GAC ATA Gly Cys Asp Ile

CTA

Leu 345 TTG ATG CCC TCA Leu Met Pro Ser AGA TTC GAA Arg Phe Glu 350 ACC ATA OCT Thr Ile Pro 1056 1104 TTA AAC CAA TTG Leu Asn Gin Leu

TAT

Tyr 360 GOA ATG AGA TAT Ala Met Arg Tyr

GGO

Gly 365 ATT GTT Ile Vai 370 OAT AGO AOG GGG His Ser Thr Gly

GGO

Gly 375 OTA AGA GAO ACA Leu Arg Asp Thr

GTG

Vai 380 AAG GAT TTT A.AT Lys Asp Phe Asn a

OCA

Pro 385 TAT GOT CAA GAA Tyr Ala Gin Giu

GGA

Gly 390 AAA GGT GAA GGT Lys Gly Giu Gly

ACC

Thr 395 GGG TGG ACA TTT Gly Trp Thr Phe

TOT

Ser 400 1152 1200 1248 OCT OTA AOG AGT Pro Leu Thr Ser

GAA

Giu 405 AAG TTG TTT GAT Lys Leu Phe Asp

ACA

Thr 410 CTG AAG OTG GOG Leu Lys Leu Ala ATO AGG Ile Arg 415 ACT TAT ACA Thr Tyr Thr ATG GGA AGG Met Gly Arg 435

GAA

Glu 420 OAT AAG TOA TOT His Lys Ser Ser

TGG

Trp 425 GAG GGA TTG ATG Glu Gly Leu Met AAG AGA GGT Lys Arg Gly 430 TAT GAG CAA Tyr Giu Gin GAO TAT TOO TGG Asp Tyr Ser Trp

GAA

Glu 440 AAT GOA GCC ATT Asn Ala Ala Ile COT OCA TAT GTC Pro Pro Tyr

CAA

Gin 445 1296 1344 1397 GTT TTO ACC TGG GC Val Phe Thr Trp Ala 450 TTT-ATA GAT Phe Ile Asp 455 k.GATGAT TTATOAAGAA AGATTGCAAA OGGGATAOAT CATTAAAOTA TACGOAGAGC TTTTGGTGOT ATTAGOTACT GTOATTGGGC GOGGAATGTT TGTGGTTOTT TCTGATTCAG AGAGATCAAG, TTAGTTCCAA AGAOATGTAG CCTGOOOCTG TCTGTGATGA AGTAAAAOTA CAAAGGOAAT TAGAAACOOA CCAACAACTG OOTOOTTTGG GAGAAGAGTG GAAATATGTA AAAAAGAATT TTGAGTTTAA TGTCA1ATTGA ATTAATTATT OTOATTTTTA AAAAAAAOAT OTOATOTOAT AOAATATATA AAATTGATOA TGATTGATGO CCOCTAAAAA A~AAAAAA AAA- AAAAAN

A

1457 1517 1577 1637 1697 1757 1758 INFORMATION 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 a 4* 4. 4 4*44 4 4 4**44* 4

S*

.4 U 4 Phe Val Pro Thr Gly Asn Gly His Pro 145 Al a Ile Glu Ser Asp 225 Gly Asp Tyr Leu Gly Ala Val Gin 130 Gin His Ala Ile Arg 210 Trp *Asp TrF Gly Leu Phe Ala Tyr 115 Gly Trp Ala Val Thr 195 Gin Asn Ala Val Asp Ser Thr Leu 100 Lys Val Tyr Leu Ala 180 Thr Ser Pro Gir Phe Ile His Tyr 85 Val Asp Glu Giy Asp 165 Asp Pro ,7al 3er 1Giu Val Vai Asp Tyr Gly 55 Aia Ala 70 Giy Glu Pro Leu Aia Arg Pro Ala 1.35 Ala-Val 150 Thr Gly Arg Ile Giu Gly Leu Asn 215 Thr Asp 230 Alz His 40 Ala Cys Lys Leu Ser 120 Val Giu Glu Leu 200 ily ;lu Phe 25 Ser Phe Giu Cys Leu 105 Ile Thr Trp Thr Thr 185 Tyr Ile His Tyx Ser Gly Ala Leu 90 Al a Val Tyr Ile Val 170 Val Gly rhr Ile His *Tyr *Asp Pro 75 Phe Ala Ala Asn Phe 155 Asn Ser Leu Asn Ala 235 Git Arg Asn Leu Leu Lys Ile Asn 140 Pro Val1 Gln H~is ;ly 220 er ITyr Arg Gin Val1 Ala Tyr His 125 Leu Thr Leu Gly Glu 205 Ile2 His Arc Pro Phe Leu Asn Arg 110 Asn Gly Trp Lys Tyr 190 ELeu ksp ryr Ala Gly Arg Pro Asp Pro Ile Leu Ala Gly 175 Ser Leu Val2 Ser Gly Thr Phe Leu Cys Tyr Ala Pro Arg 160 Ala i'rp Ser ksn Ile Asn Asp Leu Ser Pro 245 Pro Gly Lys Val Gin 250 Cys Lys Thr Asp Leu Gin 255 Lys Giu Leu Gly Leu Pro Ile Arg Pro Asp Cys Pro Leu Ile 260

S

S C

S.

C S S.

*SSS*~

S. 5* S. S S Ile Gly Ile Pro 290 Gly Giu.

305 Lys Asp Arg Ile Pro Cys Ile Vai 370 Pro Tyr 385 Pro Leu.

Thr Tyr Met Giy Val Phe 450 Arg Leu 275 Giu Leu Lys Gin Lys Phe Thr Ala 340 Gly Leu 355 His Ser Ala Gin Thr Ser Thr Giu.

420 Arg Asp 435 Thr Trp Asp Met Tyr Arg 325 Giy Asn Thr Giu Giu 405 His Tyr Ala Tyr Gin Gin Asn 295 Giu Asp 310 Aia Trp Cys Asp Gin Leu Gly Giy 375 Gly Lys 390 Lys Leu Lys Ser Ser Trp Phe Ile 455 Lys 280 Asp Trp Vai Ile Tyr 360 Leu Gly Phe Ser Giu.

440 265 Gly Vai Met Giy Leu 345 Aia Arg G iu Asp Trp 425 Asn Vai Gin Arg Phe 330 Leu Met Asp Giy Thr 410 Giu.

Ala Asp Vai His 315 Asn Met Arg Thr Thr 395 Leu Gly Ala Ile Vai 300 Thr Vai Pro Tyr Vai 380 Gly Lys Leu Ile Ile 285 Met Giu Pro Ser Giy 365 Lys Trp Leu Met Gin 445 270 Leu Leu Asn Val1 Arg 350 Thr Asp Thr Aia Lys 430 Tyr Gly Ser Gly Leu Ser 335 Phe Ile Phe P he Ile 415 Arg Giu.

Phe Aia Ser Phe 320 His Giu Pro Asn Ser 400 Arg Gly Gln Asp Pro Pro Tyr INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1926 base pairs ART: nucleotide STRANDEDNESS: unknown TOPOLOGY: iinear (ii) MOLECULE TYPE: cDNA to rnRNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Soianum tuberosum STRAIN: cv. Beroiina TISSUE TYPE: tuber tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in pBluescriptSK+ (ix) FEATURE: NAME/FEATURE: CDS LOCATION:2..1675 OTHER INFORMATION:/function= "Polymerization of starch" /product= "Starch synthase" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: C GGC ACG AGC AAA AGT TTA GTA GAT GTT CCT GGA AAG AAG ATC CAG Gly Thr Ser Lys Ser Leu Val Asp Val Pro Gly Lys Lys Ile Gin 9 9r 9* 9 *9 9 TCT TAT ATG CCT TCA TTA CGT AAA Ser Tyr Met Pro Ser Leu Arg Lys 20 CAG AGG AAT GAA AAT CTT GAA GGA Gin Arg Asn Glu Asn Leu Glu Gly GAA TCC TCA Glu Ser Ser 25 GCA TCC CAT Ala Ser His GTG GAA Val Glu AGT GCT GAG GCA Ser Ala Glu Ala AAC GAA GAG Asn Glu Glu TTG GCA GGA Leu Ala Gly ACT GAA GAT Thr Glu Asp CCT GTG AAT ATA Pro Val Asn Ile

GAT

Asp 55 GAG AAA CCC CCT Glu Lys Pro Pro

CCA

Pro ACA AAT Thr Asn GTT ATG AAC Val Met Asn ATT ATT Ile Ile 70

CTT-GGA

Leu Gly TTG GTG GCT TCA Leu Val Ala Ser

GAA

Glu TGC GCT CCA TGG Cys Ala Pro Trp *9 9*

S.

.9 9 9* *9

TCT

Ser 80 AAA ACA GGT GGG Lys Thr Gly Gly GAT GTT GCT Asp Val Ala GCA TTA CCC AAA Ala Leu Pro Lys 286 334 TTG GCT CGA CGT Leu Ala Arg Arg

GGC

Gly 100 CAC AGA GTT ATG His Arg Val Met

GTT

Val 105 GTG GCA CCT CGT Val Ala Pro Arg TAT GAC Tyr Asp 110 AAC TAT CCT Asn Tyr Pro

GAA

Glu 115 CCT CAA GAT TCT Pro Gin Asp Ser GTA AGA AAA ATT Val Arg Lys Ile TAT AAA GTT Tyr Lys Val 125 ATT GAT GGT Ile Asp Gly GAT GGT CAG GAT Asp Gly Gin Asp 130 GTG GAA GTG Val Glu Val

ACT

Thr 135 TAC TTC CAA GCT Tyr Phe Gin Ala

TTT

Phe 140 GTG GAT Val Asp 145 TTT GTT TTC ATT Phe Val Phe Ile

GAC

Asp 150 AGT CAT ATG TTT Ser His Met Phe

AGA

Arg 155 CAC ATT GGG AAC 478 His Ile Gly Asn AAC ATT TAC GGA GGG AAC CGT GTG GAT ATT TTA AAA CGC ATG GTT TTA 526 Asn 160 Ile Tyr Gly Gly Asn Arg Val Asp 165 Ile Leu 170 TGG CAT Trp His 185 Lys Arg Met Val TTT TGC AAA GCA Phe Cys Lys Ala ATT GAG GTT CCT Ile Giu Val Pro GTT CCA TGT Val Pro Cys GGT GGG Gly Gly 190 GTC TGO TAT Val Cys Tyr ACT GCT TTA Thr Ala Leu 210

GGA

Gly 195 GAT GGA AAT TTA Asp Gly Asn Leu

GTG

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

OTG

Leu 215 AAA GOT TAT TAT Lys Ala Tyr Tyr

OGT

Arg 220 670 ATT ATG Ile Met 225 AAO TAT ACA AGA Asn Tyr Thr Arg

TOT

Ser 230 GTO OTG GTG ATT Val Leu Val Ile

OAT

His 235 AAO ATO GOT OAT Asn Ile Ala His a..

a a a a *a a. a a GGT OGT GOT COT Gly Arg Gly Pro

TTG

Leu 245 GAG GAT TTT TOA Giu Asp Phe Ser

TAT

Tyr 250 GTA GAT OTT OCA Val Asp Leu Pro CAC TAT ATG GAO His Tyr Met Asp

OCT

Pro 260 TTO AAG TTG Phe Lys Leu TAT GAO Tyr Asp 265 OCA GTA OGA GOT Pro Val Gly Gly GAG CAT Giu His 270 814 TTC AAO ATT Phe Asn Ile GTT AGT OAT Val Ser His 290

TTT

Phe 275 000 GOT GOT OTA Ala Ala Gly Leu

AAG

Lys 280 ACA GOA OAT CGT Thr Ala Asp Arg OTA GTT ACA Val Val Thr 285 GGT GOT TGG Gly Gly Trp OGA TAT TOA TG Oly Tyr Ser Trp

GAA

Giu 295 OTA AAG ACT TOO Leu Lys Thr Ser

CAA

Gin 300 GGA TTG Gly Leu 305 OAT CAG ATA His Gin Ile

ATT-AAT

Ile Asn 310 ACA AAA Thr Lys 325 GAG AAO GAT TGO Oiu Asn Asp Trp

AAA

Lys 315 TTA CAG GGT ATT 958 Leu Gin Gly Ile

GTG

Val1 320 AAT GGG ATT GAT Asn Gly Ile Asp GAG TGG AAO OCT GAG TTG GAO OTT Glu Trp Asn Pro Oiu Leu Asp Val 330

CAC

His 335 1006 TTA CAG TOA GAT Leu Gin Ser Asp TAO ATG AAO TAO Tyr Met Asn Tyr TTG GAO ACO OTA Leu Asp Thr Leu CAG ACT Gin Thr 350 1054 OGC AAG CCT CAA TGT AAA OCT OCA TTG CAG AAG GAA CTT GGT TTA CCA 1102 Gly Lys Pro Gin 355 Cys Lys Ala Ala Leu 360 Gin Lys Glu Leu Gly Leu Pro 365 CTT GAC CCA Leu Asp Pro OTT COT OAT OAT OTC CCA CTO Val Arg Asp Asp Val Pro Leu 370 GGT TTC ATT GG Oly Phe Ile Gly

AGO

Arg 380 CAA AAO Gin Lys 385 OCT OTT OAT CTG Oly Val Asp Leu

ATT

Ile 390 OCT GAG 0CC AGT Ala Giu Ala Ser

OCT

Ala 395 TOO ATO ATO GOT Trp Met Met Oly

CAG

Gin 400 OAT OTA CAA CTO Asp Val Gin Leu

OTC

Val1 405 ATO TTO 000 ACO Met Leu Oly Thr 000 Oly 410 AGO COT GAC CTT Arg Arg Asp Leu

OAA

Glu 415 CAG ATO CTA AGO Gin Met Leu Arg

CAA

Gin 420 TTT GAG TOT CAA Phe Oiu Cys Gin

CAC

His 425 AAT OAT AAA ATT Asn Asp Lys Ile AGA GGA Arg Oly 430 1150 1198 1246 1294 1342 1390 1438 1486 a

C.

a be..

TOO OTT OCT Trp Val Oly GAC ATT CTO Asp Ile Leu 450

TTC

Phe 435 TCT GTO A.AO ACT Ser Val Lys Thr CAT COT ATA ACT His Arg Ile Thr OCT OOT OCA Ala Oly Ala 445 CTO AAC CAG Leu Asn Oin CTC ATO CCT TCT Leu Met Pro Ser

AGA

Arg 455 TTT GAG 0CC TTO Phe Oiu Ala Leu

CGA

Arg 460 CTT TAT Leu Tyr 465 OCA ATO AAA TAT Ala Met Lys Tyr 000 Gly 470 ACT ATT CCT OTT Thr Ile Pro Val

OTT

Val 475 CAT OCA OTA OGA His Ala Val Oly

OOA

Gly 480 CTC AGA OAT ACT Leu Arg Asp Thr

GTO

Val 485 CAG CCC TTT OAT Gin Pro Phe Asp

CCT

Pro 490 TTT AAT GAG TCA Phe Asn Oiu Ser

OGA

Oly 495 C. C C

C.

a.

C.

CTO 000 TOO ACC Leu Oly Trp Thr AOT AGO OCT OAA Ser Arg Ala Oiu AOC CAO CTO ATC Ser Gin Leu Ile CAC OCA His Ala 510 1534 TTA OOA AAT Leu Oly Asn 000 ATT CAG Oly Ile Gin 530 OCT CAG AAC Ala Gin Asn 545

TOC

Cys 515 TTA CTO ACT TAT Leu Leu Thr Tyr

COT

Arg 520 GAO TAC AAA AAO Oiu Tyr Lys Lys AOT TOO GAG Ser Trp, Olu 525 OAT AAT OCT Asp Asn Ala ACA COT TOT ATO Thr Arg Cys Met TAT OAA OAA OTT Tyr Oiu Glu Val 550

ACA

Thr 535 CAA GAC TTA AOT Gin Asp Leu Ser

TOO

Trp 540 1582 1630 1675 CTC ATC OCT OCT AAG Leu Ile Ala Ala Lys 555 TAT CAG TG Tyr Gin Trp TOAGOTTCAT TACTTOTAoA TATTTOOOOA TTTTOOCCAT TOTATCAAGT TCTAATOATO OCATTTCAOA OACATOTTTC TOOTATCGAC ACOAOAOGAT OCATOCAACA AGTTOOCTAA 1735 179S 66 CTATCATACT ACTACCACGT CAGGAATGAT TGCCGCACTT GATCATGTAA TCATGTATAT ACTCTATTTT GTTTGCAA.AA TGTAGTTACA TGTTGCAATT TCTAAAAAAA AAAAA.

AAAAAA A INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 558 amino acids ART: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gly Thr Ser Lys Ser Leu Vai Asp Val Pro Gly Lys Lys Ile Gin Ser 1 5 10 1855 1915 1926 00 0 0e.

0000 00 toI 00 Tyr Met Arg Asn Giu Asp Asn Val Lys Thr Ala Arg Tyr Pro Gly Gin 130 Asp Phe 145 Ile Tyr Pro Giu 35 Pro Met Giy Arg Giu 115 Asp Vali Giy Ser Asn Val Asn Gly Giy 100 Pro Val Phe Giy Leu Leu As n Ile Leu His Gin Giu Ile Asn 165 Arg Lys Giu Giu Gly Ser 40 Ile Asp Giu 55 Ile Leu Vai 70 Giy Asp Vai Arg-Vai Met Asp Ser Giy 120 Val Thr Tyr 135 Asp Ser His 150 Arg Vai Asp Ser 25 Ser Lys Ala Aia Val 105 Vali Phe Met Ile Ser Ala Pro Ser Giy 90 Val Arg Gin Phe Leu 170 Aia Giu Pro Giu 75 Ala Ala Lys Ala Arg 155 Lys Ser His Ala Asn Pro Leu Cys Ala Leu Pro Pro Arg Ile Tyr 125 Phe Ile 140 His Ile Arg _Met Val Giu Giu Giu Ala Gly Pro Trp Lys Ala Tyr Asp 110 Lys Val Asp Giy Gly Asn Val Leu 175 Gin Thr Thr Ser Leu Asn Asp Val Asn 160 Phe Cys Lys Ala Ala Ile Glu Vai Pro Trp His Val Pro Cys Gly Gly Val 180 185 190 Cys Tyr Gly Asp Gly Asn Leu Vai Phe Ile 195 200 a a a a a a a. a a 0a** a. a a a. a a a.

Al a Met 225 Gly Tyr Asn Ser Leu 305 Asn Gin Lys Arg Lys 385 Asp Met Val1 Ile Leu 210 As n Arg Met Ile His 290 His Gly Ser Pro Asp 370 Gly Val Leu Gly Leu 450 Leu Tyr Gly Asp Phe 275 Gly Gin Ile Asp Gin 355 Asp Val Gin Arg Phe 435 Leu Pro Thr Pro Pro 260 Aia Tyr Ile Asp Gly 340 Cys Val Asp Leu Gin 420 Ser Met Vai Arg Leu 245 Phe Aia Ser Ile Thr 325 Tyr Lys Pro Leu Val 405 Phe Vali Pro Tyr Ser 230 Giu Lys Giy Trp Asn 310 Lys Met Ala Leu Ile 390 Met Giu Lys Ser Leu 215 Val Asp Leu Leu Giu 295 Giu Giu Asn Ala Ile 375 Aia Leu Cys Thr Arg 455 Lys Leu Phe Tyr Lys 280 Leu Asn Trp Tyr Leu 360 Gly Giu Giy Gin Ser 440 Phe Ala Val Ser Asp 265 Thr Lys Asp Asn Ser 345 Gin Phe Ala Thr His 425 His Glu Tyr Ile Tyr 250 Pro Ala Thr Trp Pro 330 Leu Lys Ile Ser Giy 410 Asn Arg Ala Ala Tyr His 235 Vai Vai Asp Ser Lys 315 Glu Asp Glu Giy Ala 395 Arg Asp Ile Leu Asn Arg 220 Asn Asp Gly Arg Gin 300 Leu Leu Thr Leu Arg 380 Trp Arg Lys Thr Arg 460 Asp 205 Asp Ile Leu Giy Vai 285 Gly Gin Asp Leu Giy 365 Leu Met Asp Ile Ala 445 Leu Trp Asn Ala Pro Glu 270 Val Gly Gly Val Gin 350 Leu Asp Met Leu Arg 430 Gly Asn His Gly His Pro 255 His Thr Trp Ile His 335 Thr Pro Pro Gly Glu 415 Gly Ala Gin Thr Ile Gin 240 His Phe Val Gly Val1 320 Leu Gly Vali Gin Gin 400 Gin Trp Asp Leu Tyr Aia Met Lys Tyr Gly Thr Ile Pro Vai Val His Ala Val Gly Gly 465 470 475 480 Gin Pro Phe Asp Leu Arg Asp Thr Pro 490 Ser Phe Asn Glu Gin Leu Ile Gly Trp Thr Gly Asn Cys 515 Ile Gln Thr 530 Set Gly Leu 495 His Ala Leu 510 Trp Glu Gly Phe 500 Leu Arg Ala Glu Leu Thr Tyr Arg 520 Gin Tyr Lys Lys Ser 525 Arg Cys Met Asp Leu Ser Trp Asp Asn 540 Ala Ala Gin 545 (2) .9 9 9 9* 9 9* *9 9 6 Asn Tyr Glu Glu Val Leu Ile Ala Ala Lys Tyr Gin 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) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum STRAIN: cv Desir6e TISSUE TYPE: leaf tissue (vii) IMMEDIATE SOURCE: LIBRARY: cDNA-library in Lambda ZAPII (ix) FEATURE: NAME/FEATURE: CDS LOCATION:242..2542 Trp 9.« 99* 99* 9.

o o ee° o e o (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCGCCCATTT TTCACCAAAC GTTTTTGACA TTGACCTCCA TTGTCGTTAC TTCTTGGTTT CTCTTTCAAT ATTGCTTCAC AATCCCTAAT TCTCTGTACT AGTCTCTATC TCAATTGGGT TTTCTTTACT TGTCAATTAT CTCTACTGGG TCGGCTTCTA TTTCCACTAG GTCACTCTGG TTCTTGAAAT CTTGGATTCC TATTATCCCT GTGAACTTCA TCTTTTGTGA TTTCTACTGT A ATG GAG AAT TCC ATT CTT CTT CAT AGT GGA AAT CAG TTC CAC CCC Met Glu Asn Ser Ile Leu Leu His Ser Gly Asn Gin Phe His Pro 1 5 10 AAC 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

N-

GGC TOO AGT Gly Ser Ser GGT GAA AAT Gly Git' Asn

AGA

Arg GAG CAA ATG TGG Giu Gin Met Trp ATC AAG OGO GTT Ile Lys Arg Val AAA GCA ACA Lys Ala Thr AAT GAT GCC Asn Asp Ala TCT GGG GAA GCT Ser Gly Giu Ala

GCA

Ala 55 AGT GCT GAT GA Ser Ala Asp Giu

TCG

Ser 430 TTA CAG Let' Gin GTT ACA ATT GAA Val Thr Ile Giu AGC AAA AAG GTT Ser Lys Lys Val GOC ATG CAA CAG Ala Met Gin Gin 478 OTA CTT CAA CAG Leu Let' Gin Gin

ATT

Ile GCA GAA AGA AGA Ala Git' Arg Arg

AAA

Lys GTA GTC TOT TCA Val Val Ser Ser 526 AAA AGC AGT CTT Lys Ser Ser Let' AAT GCC AAA GGT Asn Ala Lys Gly TAT GAT GOT GGG Tyr Asp Gly Gly AGT GGT Ser Gly 110 574

S

S

S

S

*5

S

AGC TTA TCA Ser Leu Ser ACT GTA COT Thr Val Pro 130

GAT

Asp 115 GTT GAT ATO OCT Val Asp Ile Pro

GAO

Asp 120 GTG GAT AAA GAT Val Asp Lys Asp TAT AAT OTT Tyr Asn Val 125 GAT AAA AAT Asp Lys Asn AGT ACT GOT GOT Ser Thr Ala Ala

ACT

Thr 135 OCA ATT ACT GAT Pro Ile Thr Asp

GTO

Val1 140 670 ACA COG Thr Pro 145 COT GOT ATA AGO CAA GAT TTT GTT GAA Pro Ala Ile Ser Gin Asp Phe Val Git' 150 AAA AGA GAA ATO Lys Arg Oiu Ile AGG GAO OTG GC Arg Asp Let' Ala

GAT

Asp 165 GAA AGG GCA COO Giu Arg Ala Pro

OCA

Pro 170 OTG TOG AGA TOA Let' Ser Arg Ser

TOT

Ser 17S 766 *5 5 S S

SS

S. S

OS

ATO ACA GOC AGT Ilie Thr Ala Ser

AGO

Ser 180 CAG ATT TOO TOT Gin Ile Ser Ser GTA AGT TOO AAA Val Ser Ser Lys AGA ACO Arg Thr 190 814 TTG AAT GTO Let' Asn Val GAT GTG AAT Asp Val Asn 210

COT

Pro 195 OCA GAA ACT COG Pro Git' Thr Pro

AAG

Lys 200 TOO AGT CAA GAG Ser Ser Gin Git' ACA OTT TTG Thr Let' Let' 205 AAG AAG ATO Lys Lys Ile 862 TOA OGO AA.A AGT Ser Arg Lys Ser

TTA

Let' 215 GTA GAT GTT OCT Val Asp Val Pro

GGA

Gly 220 910 CAG TOT Gin Ser 225 TAT ATG COT TCA Tyr Met Pro Ser CT AAA GAA TOO Arg Lys Git' Ser GOA TOO CAT GTG Ala Ser His Val 958

GAA

Giu 240 CAG AGG AAT GAA Gin Arg Asn Glu OTT GAA GGA TCA Leu Giu Gly Ser AGT GCT GAG GCA Ser Ala Glu Ala 250 AAA CCC CCT CCA Lys Pro Pro Pro

AAC

Asn

GAA

Giu 255 1006 1054 GAG ACT GAA GAT Giu Thr Glu Asp

CCT

Pro 260 GTG AAT ATA GAT Vai An Ile Asp

GAG

Glu 265 TTG GCA Leu Ala 270 GGA ACA AAT Gly Thr Asn TGG TOT AAA Trp Ser Lys 290

GTT

Vai 275 ATG AAC ATT ATT Met Asn Ile Ile GTG GOT TOA GAA Vai Ala Ser Glu TGO GOT OCA Cys Ala Pro 285 TTA CCC AAA Leu Pro Lys 1102 1150 ACA GGT GGG OTT Thr Gly Gly Leu

GGA

Gly 295 GAT GTT GOT GGA Asp Val Ala Gly

GCA

Ala 300 GOT TTG Ala Leu 305 GOT OGA CGT GGC Ala Arg Arg Gly AGA GTT ATG GTT Arg Val Met Val GOA OCT COT TAT Ala Pro Arg Tyr 1198 1246 0 0S0**@ 0 0*0e

S

Soot

*S

S

0*5O So 5 5 *550 5* 6* S S. S

S

S

S5

S*

*0 0O *5

GAO

Asp 320 AAO TAT COT GAA Asn Tyr Pro Giu

COT

Pro 325 CAA GAT TOT GGT Gin Asp Ser Gly

GTA

Val 330 AGA AAA ATT TAT Arg Lys Ile Tyr OTT GAT GGT CAG Val Asp Gly Gin GTG GAA GTO ACT Va'i Giu Val Thr TTC CAA GOT TTT Phe Gin Ala Phe ATT OAT Ile Asp 350 GGT GTG GAT Gly Val Asp AAO AAC ATT Asn Asn Ile 370

TTT

Phe 355 GTT TTC ATT GAO Val Phe Ile Asp

AGT

Ser 360 CAT ATG TTT AGA His Met Phe Arg CAC ATT GGG His Ile Gly 365 OGO ATG GTT Arg Met Vai 1294 1342 1390 TAO GGA GGG AAC Tyr Gly Gly Asn GTG OAT ATT TTA Val Asp Ile Leu

AAA

Lys 380 TTA TTT Leu Phe 385 TGO AAA GCA GOG Cys Lys Ala Ala

ATT

Ile 390 GAG GTT OCT TGG Glu Val Pro Trp

CAT

His 395 GTT OCA TGT GGT Val Pro Cys Giy

GG

Gly 400 GTO TOO TAT GGA Val Cys Tyr Gly GGA AAT TTA GTG Giy Asn Leu Val

TTO

Phe 410 ATT GOT AAT GAT Ile Ala Asn Asp

TGG

Trp 415 1438 1486 1534 OAT ACT GOT TTA His Thr Ala Leu

TTG

Leu 420 OCA GTA TAT OTG Pro Val Tyr Leu

AAA

Lys 425 GOT TAT TAT OGT Ala Tyr Tyr Arg GAO AAT Asp Asn 430 GGA ATT ATO Gly Ile Met CAT CAG GOT His Gin Gly 450

A.AC

Asn 435 TAT ACA AGA TOT Tyr Thr Arg Ser OTG GTG ATT OAT Leu Val Ile His AAC ATO GOT Asn Ile Ala 445 GAT OTT OCA Asp Leu Pro 1582 1630 COT GGT COT TTG Arg Gly Pro Leu

GAG

Giu 455 GAT TTT TOA TAT Asp Phe Ser Tyr

GTA

Val 460 CCA CAC Pro His 465 TAT ATG GAO CCT Tyr Met Asp Pro TTC AAG Phe Lys 470 TTG TAT GAO CCA GTA GGA GGT Leu Tyr Asp Pro Val Gly Gly 475

GAG

Giu

GTT

Val1 495 1678

CAT

His 480 TTC AAC ATT TTT Phe Asn Ile Phe

GCG

Ala 485 GOT GGT OTA AAG Ala Gly Leu Lys

ACA

Thr 490 GCA GAT CGT GTA Ala Asp Arg Val 1726 1774 ACA GTT AGT CAT Thr Val Ser His

GGA

Gly 500 TAT TOA TGG GAA Tyr Ser Trp Giu

OTA

Leu 505 AAG ACT TOO CAA Lys Thr Ser Gin GGT GGT Gly diy 510 TGG GGA TTG Trp Giy Leu ATT GTG AAT Ile Val Asn 530

OAT

His 515 CAd ATA ATT AAT Gin Ile Ile Asn

GAG

Giu 520 AAC dAT TGG AA Asn Asp Trp Lys TTA CAG GOT Leu Gin Gly 525 TTG GAO GTT Leu Asp Val GGG ATT OAT ACA Gly Ile Asp Thr

AAA

Lys 535 GAG TGG AAO COT Giu Trp Asn Pro

GAG

diu 540

S.

S

*9S S 9.

S

9* 9

S

o CAC TTA His Leu 545 ACT GGC Thr Gly 560 CAd TCA GAT GGT Gin Ser Asp Gly

TAO

Tyr 550 ATG AAO TAO TOO Met Asn Tyr Ser GAO ACG OTA CAG Asp Thr Leu Gin 1822 1870 1918 1966 2014 AAG COT CAA Lys Pro Gin AAA GOT GOA TTG Lys Ala Ala Leu

OAG

Gin 570 AAG GAA OTT GGT Lys Giu Leu Gly

TTA

Leu 575 OCA GTT COT GAT Pro Val Arg Asp

GAT

Asp 580 OTO OCA CTG ATO Val Pro Leu Ile

GT

Gly 585 TTO ATT COG AGG Phe Ile Gly Arg OTT GAO Leu Asp 590 OCA CAA AAG Pro Gin Lys GOT CAd OAT Oly Gin Asp 610

OCT

Gly 595 GTT GAT OTG ATT Val Asp Leu Ile GAG GOC ACT GOT Giu Ala Ser Ala TGG ATG ATG Trp Met Met 605 OGT GAO OTT Arg Asp Leu 2062 2110 GTA CAA OTO OTO Val Gin Leu Val

ATG

Met 615 TTG dOG AOG dG Leu Oly Thr Gly

AGG

Arg 620 GAA CAG Oiu Gin 625 ATG OTA AGd CAA Met Leu Arg Gin

TTT

Phe 630 GAG TGT CAA CAC Oiu Cys Gin His

AAT

Asn 635 GAT AAA ATT AGA Asp Lys le.Arg ATA ACT GOT GGT Ile Thr Ala Gly 655

GGA

Gly 640 TGG GTT GGT TTO Trp Val Gly Phe

TOT

Ser 645 GTG AAG ACT TOT Val Lys Thr Ser CAT COT His Arg 650 2158 2206 2254 GOA GAO ATT OTG OTO Ala Asp Ile Leu Leu 660 ATG OCT TOT AdA Met Pro Ser Arg

TTT

Phe 665 GAG OCT TGC OGA Giu Pro Cys Giy OTO AAO Leu Asn 670 CAG CTT TAT GCA ATG AAA TAT GGG ACT ATT CCT GTT GTT CAT GCA GTA Gin Leu Tyr GGA GGA CTC Gly Gly Leu 690 Ala 675 Met Lys Tyr Gly Ile Pro Val Val His Ala Val 685 AAT GAG TCA Asn Glu Ser 2302 2350 AGA GAT ACT GTG Arg Asp Thr Val

CAG

Gin 695 CCC TTT GAT CCT Pro Phe Asp Pro

TTT

Phe 700 GGA CTG Gly Leu 705 GGG TGG ACC TTC Gly Trp Thr Phe

AGT

Ser 710 AGG GCT GAA GCT AGC CAG CTG ATC CAC Arg Ala Glu Ala Ser Gin Leu Ile His 715 2398

GCA

Ala 720 TTA GGA AAT TGC Leu Gly Asn Cys

TTA

Leu 725 CTG ACT TAT CGT Leu Thr Tyr Arg

GAG

Glu 730 TAC AAA AAG AGT Tyr Lys Lys Ser

TGG

Trp 735 GAG GGG ATT Glu Gly Ile GCT GCT CAG Ala Ala Gin CAG ACA Gin Thr 740 AAC TAT Asn Tyr 755 CGT TGT ATG ACA CAA GAC Arg Cys Met GAA GAA GTT Glu Glu Val Thr Gin Asp 745 TTA AGT TGG Leu Ser Trp GAT AAT Asp Asn 750 CTC ATC Leu Ile 760 GCT GCT AAG Ala Ala Lys TAT CAG TGG Tyr Gin Trp 765 2446 2494 2542 2602 2662 2722 2782 2793 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 Gin Phe His Pro Asn Leu Pro Leu Leu Ala Leu Arg Pro Lys Lys Leu Ser Leu Ile His Gly 25 Ser Ser Arg Glu Gin Met Trp Arg Ile Lys Arg Val Lys Ala Thr Gly 40 73 Glu Asn Ser Gly Giu Ala Ala Ser Ala Asp Glu sr Ser Asn Asp Ala .r 9 9* 9.55

S

9* 9* 9 *9 Gl 6! Let Se Let Val Pro 145 Arg Thr Asn Val Ser 225 Gin Thr Thr Ser Leu 305 n Val 5 u Leu Ser Ser Pro 130 Pro Asp Ala Val Asn 210 Tyr Arg Glu Asn Lys 1 290 Ala A Th G1i Le Asi 115 Ser Ala Leu Ser Pro 195 Ser Met ksn ksp Tal 'hr Lrg r Ile Gin i Ala 100 a Val Thr Ile Ala Ser 180 Pro Arg Pro Glu Pro 260 Met Gly G Arg G Gli Il 8! Asr AsI Ala Ser Asp 165 Gin Glu Lys Ser ksn 245 lal ~sn ;ly ;ly u Lys 70 a Ala 5 i Ala SIle Ala Gin 150 Glu Ile Thr Ser Leu 230 Leu Asn Ile I Leu G 2 His A 310 Sei G1 Lyr Prc Thr 135 Asp Arg Ser Pro Leu 215 Arg 3lu Ile le ;ly 195 ~rg r Lys i Arg 3 Gly Asp 120 Pro Phe Ala Ser Lys 200 Vai Lys Gly Asp C Leu 1 280 Asp I.

Val M Ly Ar Th Val Ile Val Pro Thr 185 Ser Asp Glu Ser ;lu !65 Tal Tal [et s Va.

3 Ly 91 7 Tya As1 Thi Glu Pro 170 Val Ser Val Ser Ser 250 Lys Ala Ala Val 1 Leu 75 S Val 0 Asp Lys Asp I Ser 155 I Leu Ser Gin Pro Ser 235 Ala Pro I Ser C Gly m Val A Al Va.

Glt As1 Val 140 Lys Ser Ser Glu Gly 220 Ala flu ?ro ;lu la 100 la a Met 1 Ser r Gly a Tyr 125 Asp Arg Arg Lys Thr 205 Lys Ser I Ala I Pro I 2 Cys P 285 Leu.P Pro A Gl Se Sel Asr Lys Glu Ser Arg 190 Leu Lys iis ksn 4 eu ila ro rg n Gi.

9! G1 SVa Asr IlE Ser 175 Thr Leu lie Val Glu 255 Ala Pro Lys Tyr n Asp Lys y Ser 1 Thr Thr Lys 160 Ile Leu Asp Gin Glu 240 Glu Gly Trp Ala Asp 320 Asn Tyr Pro Glu Pro 325 Gin Asp Ser Gly Va1 330 Arg Lys Ile Tyr Lys Val 335 Asp Gly Gin Asp Val Giu Vai Thr Tyr Phe GIi ft...

ft ft ft.

ft ft...

*ft ft.

ft ft...

at ft ft.

ft.

ft ft ft ft at Val Asn Phe 385 Val1 Thr Ile Gin His 465 Phe Val Gly Val1 Leu 545 Gly Val1 Gin Asp Ile 370 Cys Cys Aia Met Gly 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 Giy Leu 420 Tyr Gly Asp Phe G ly 500 Gin Ile Asp Gin Asp 580 Vali Phe Giy Ala Asp 405 Pro Thr Pro Pro Ala 485 Tyr Ile Asp Gly Cys 565 Val Asp Ile As r Ile 390 Gly Val Arg Leu Phe 470 Ala Ser Ile Thr Tyr 550 Lys Pro Leu Asp 1Arg 375 Giu Asn Tyr Ser Giu 455 Lys Gly Trp Asn Lys 535 Met Ala Leu Ile Sel 3 60 ValI Val1 Leu Leu Val 440 Asp Leu Leu Giu Giu 520 Giu Asn Al1a I le kl a 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 Phc Lei.

His 395 Ile Tyr Ile Tyr Pro 475 Ala Thr Trp Pro Leu 555 Lys Ile Ser iAla Arg Lys 380 Val Ala Tyr His Val 460 Val Asp Ser Lys Glu 540 Asp Giu Gly Ala Ph~ HiE 365 Arc Pro Asn Arg Asn 445 Asp Gly Arg Gin Leu 525 Leu l'hr Leu Arg rrp 605 Ile 350 Ile Met Cys Asp Asp 430 Ile Leu Gly Vali Gly 5:10 Gin Asp Leu Gly Leu 590 Met Asl Gi Val Gly Trp 415 Asn Ala Pro Giu Val1 495 Gly Gly Val Gln Leu 575 Asp -let Giy Asn *Leu *Gly 400 His Gly His Pro His 480 Thr Trp Ile His Thr 560 Pro Pro Gly Gin Asp Val Gin Leu Val Met Leu Gly Thr 610 615 Gly Arg Arg Asp Leu Giu Gin Met Leu Arg Gin Phe Glu Cys Gin His Asn Asp Lys Ile Arg Gly 625 630 635 640 Trp Val Gly Phe Ser Val Lys Thr Ser His Arg Ile Thr Ala Gly Ala 645 650 655 Asp Ile Leu Leu Met Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gin 660 665 670 Leu Tyr Ala Met Lys Tyr Gly Thr Ile Pro Val Val His Ala Val Gly 675 680 685 Gly Leu Arg Asp Thr Val Gin Pro Phe Asp Pro Phe Asn Glu Ser Gly 690 695 700 Leu Gly Trp Thr Phe Ser Arg Ala Glu Ala Ser Gin Leu Ile His Ala 705 710 715 720 Leu Gly Asn Cys Leu Leu Thr Tyr Arg Glu Tyr Lys Lys Ser Trp Glu 725 730 735 Gly Ile Gin Thr Arg Cys Met Thr Gin Asp Leu Ser Trp Asp Asn Ala 740 745 750 Ala Gin Asn Tyr Glu Glu Val Leu Ile Ala Ala Lys Tyr Gin Trp 755 760 765 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 2360 base pairs ART: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:-cDNA (vi) ORIGINAL SOURCE: :o ORGANISM: Solanum tuberosum STRAIN: cv. D6sirde 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: AGATTTTCTA TATTGAAAGA TTTTGTCTTT ACATGATTCT TGATTTTACA GCAGGTGTCA ATACOAA ATG GGG TOT CTG CAA ACA COO ACA AAT Met Gly Ser Leu Gin Thr Pro Thr Asn OTT AGC Leu Ser AAT AAG TCA Asn Lys Ser TGT TTA TGT GTG TCA Cys Leu Cys Val Ser AGA GTT GTG AAG Arg Val Val Lys

GGT

Gly TTG AGG GTA GAA Leu Arg Val Giu CAA GTG GGG TTG Gin Val Gly Leu

GGG

Gly TTT TCT TGG TTG Phe Ser Trp Leu AAG GGA CGA AGA Lys Gly Arg Arg AAC AGA Asn Arg AAA GTT CAA Lys Vai Gin ATT GOT GAA Ile Ala Giu

TCT

Ser TTG TGT GTT ACA Leu Cys Val Thr

AGT

Ser 55 AGT GTT TCA GAT Ser Val Ser Asp GGT TCA TCA Gly Ser Ser GGT GCT GAG Giy Ala Giu AAT AAG AAT GTG Asn Lys Asn Vai

TCA

Ser 70 GAA GGG OTT CTT Giu Gly Leu Leu

TTG

Leu a *a AGA GAT Arg Asp GTT GOA Val Ala 95' GGT TOT GGO TOT Giy Ser Gly Ser GTT GGT TTT CAA Val Gly Phe Gin ATT OOA OAT TOT Ile Pro His Ser GGA GAT GOA Gly Asp Ala

ACA

Thr 100 ATG GTA GAA TOT Met Val Giu Ser

OAT

His 105 GAT ATT GTA GOO Asp Ile Val Ala

AAT

Asn 110 GAT AGA GAT GAO Asp Arq Asp Asp AGT GAG GAT ACT Ser Giu Asp Thr

GAG

Glu 120 GAG ATG GAG GAA Giu Met Glu Giu ACC OOA Thr Pro 125 ATO AAA TTA Ile Lys Leu TAT TOT AAG Tyr Ser Lys 145

ACT

Thr 130 TTO AAT ATO ATT Phe Asn Ile Ile GTT ACT GOT GAA Val Thr Ala Glu GOA GOT OOA Ala Ala Pro 140 TTG OCA ATG Leu Pro Met 493 a. a 9* aa .9 a a a ACT GGT GGA TTA Thr Gly Cly Leu

GGA

Gly 150 GAT GTT TGT GGT Asp Vai Cys Gly

TOT

Ser 155 GOA OTA Ala Leu 160 GOT GOT OGG GGT Ala Ala Arg Gly

OAT

His 165 OGT GTA ATG GTO Arg Val Met Val

GTT

Val 170 TOA OOT AGG TAT Ser Pro Arg Tyr

TTG

Leu 175 AAT GGA GGT OOT Asn Gly Giy Pro

TOA

Ser 180 GAT GAA AAG TAO Asp Giu Lys Tyr

GOO

Ala 185 AAT GOT GTT GAO Asn Ala Val Asp

OTT

Leu 190 CAT GTG OGG CO Asp Val Arg Ala

ACT

Thr 195 GTO OAT TGO TTT Val His Cys Phe

GGT

Giy 200 GAT GOA OAG GAA Asp Ala Gin Giu GTA GC Val Ala 205 TTO TAO OAT Phe Tyr His GAA TAO Ciu Tyr 210 AGG GOA GGT Arg Ala Cly CAT TGG GTA Asp Trp Val TTT GTG GAO CAC Phe Val Asp His 220 TCT TCT TAC TGC AGA CCT Ser Ser Tyr 225 Cys Arg Pro GGA ACG Gly Thr 230 CCA TAT GGT GAT ATT TAT GCT GCA Pro Tyr Giy Asp Ile Tyr Gly Ala 235 TTT GGT Phe Giy 240 CAT AAT CAG TTT Asp Asn Gin Phe

CC

Arg 245 TTC ACT TTC CTT Phe Thr Leu Leu CAC GCA GCA TGT His Ala Ala Cys

GAA

Giu 255 GCG CCA TTG GTT Ala Pro Leu Val

CTT

Leu 260 CCA CTG GGA CCC Pro Leu Gly Gly

TTC

Phe 265 ACT TAT GGA GAG Thr Tyr Gly Glu

AAG

Lys 270 TGC TTG TTT CTC Cys Leu Phe Leu

GCT

Ala 275 AAT CAT TCG CAT Asn Asp Trp His

GCT

Ala 280 CCC CTG GTT CCT Ala Leu Val Pro TTA CTT Leu Leu 285

S

*5*S

C.

a.

S

TTA GC CC Leu Ala Ala ATT CTC CCA Ile Val Ala 305

AAC

Lys 290 TAT CCT CCT TAT Tyr Arg Pro Tyr CTT TAC AAG CAT Val Tyr Lys Asp CCT CGT ACT Ala Arg Ser 300 CCT CCA CTA Pro Ala Val 973 ATA CAC AAC ATT Ile His Asn Ile

CCA

Ala 310 CAT CAC CGA GTG His Gin Cly Val ACC TAC Thr Tyr '320 AAT AAT TTC CCT Asn Asn Leu Gly

TTG

Leu 325 CCT CCA CAA TCC Pro Pro Gin Trp

TAT

Tyr 330 GCA CCA GTT GA A Cly Ala Val Giu 1021 1069 1117

TGG

Trp 335 ATA TTT CCC ACA Ile Phe Pro Thr

TCC

Trp 340 GCA ACC CC CAT Ala Arg Ala His

CC

Ala 345 CTT CAC ACT CCT Leu Asp Thr Gly ACA GTG AAC CTT Thr Val Asn Val AAA CCC GCA ATA Lys Cly Ala Ile CTT CCT CAT CCC Val Ala Asp Arg ATA CTG Ile Leu 365 1165

S

S

*9 *9 a 4* 5* ACA CTT AGO Thr Val Ser TAT CCC, CTA Tyr Gly Leu 385 ATT ACT AAT Ile Thr Asn 400

CAC

Gin 370 CCA TAC TCA TCC Cly Tyr Ser Trp

CAA

Glu 375 ATA ACA ACT COT Ile Thr Thr Pro CAA CCC GCA Ciu Cly Cly 380 CTT AAT CGA Leu Asn Cly 1213 1261 CAT GAG CTC TTG His Clu Leu Leu GGA ATA CAT GTT Gly Ile Asp Val 405 ACT AGA CAG TCT Ser Arg Gin Ser

GTT

Val 395 AAT CAT TGC AAC CCC TOG ACA CAT GAG Asn Asp Trp Asn Pro Ser Thr Asp Glu 410 1309

CAT

His 415 ATT GCT TCC CAT Ile Ala Ser His TAC TCC ATC AAT CAC Tyr Ser Ile Asn Asp 420 CTC TCC CGA Leu Ser Gly 425 AAC CTT CAC Lys Val Gin 430 1357 TGC AAG ACT Cys Lys Thr TGT CCT OTG Cys Pro Leu GAO ATA ATC Asp Ile Ile 465 CAT CTC Asp Leu 435 CAA AAG GAA CTG GGC OTT OCA ATT OGA OCT GAT Gin Lys Giu Leu Gly Leu Pro Ile Arg Pro Asp 440 445 1405 ATT GGA TTT Ile Gly Phe 450 ATT GGA AGG OTC GAO TAO OAG AAA Ile Cly Arg Leu Asp Tyr Gin Lys 455 460 GGT GTT Giy Val 1453 OTG TOA GOA ATT Leu Ser Aia Ile

OCA

Pro 470 GAA OTT ATG CAG Giu Leu Met Gin

AAT

Asn 475 GAT GTC CAA Asp Val Gin 1501 GTT GTA Vai Val 480 ATG OTT CGA TOT Met Leu Cly Ser

GGT

Giy 485 GAG AAA CAA TAT Giu Lys Gin Tyr

GAA

Giu 490 GAO TCG ATC AGA Asp Trp Met Arg 1549 1597

OAT

His 495 ACA GAA AAT OTT Thr Ciu Asn Leu

TTT

Phe 500 AAA GAO AAA TTT Lys Asp Lys Phe

CT

Arg 505 GOT TGG GTT GCA Aia Trp Vai Gly

TTT

Phe 510

S

595.

S.

S

S AAT GTT OCA GTT Asn Vai Pro Val

TOT

Ser 515 CAT AGC ATA ACA His Arg Ile Thr

GOA

Ala 520 GGA TGO GAO ATA Ciy Cys Asp Ile OTA TTG Leu Leu 525 ATC COO TOA Met Pro Ser AGA TAT CGC Arg Tyr Civ 545

AGA

Arg 530 TTO GAA COG TCT Phe Ciu Pro Cys

GC

Cly 535 TTA AAO CAA TTC Leu Asn Gin Leu TAT GCA ATC Tyr Ala Met 540 OTA AGA GAO Leu Arg Asp i1645 1693 1741 ACC ATA COT ATT Thr Ile Pro Ile OAT AGO ACG CCC His Ser Thr Gly

GC

Gly 555 ACA GTC Thr Vai 560 AAC CAT TTT AAT Lys Asp Phe Asn

OCA

Pro 565 TAT CT CAA GAA Tyr Aia Gin Ciu

GCA

Giy 570 ATA GCT GAA GGT Ile Gly Giu Ciy 1789 1837 S S

C.

S. 9 a.

*5

ACC

Thr 575 GCC TGG ACA TTT Cly Trp Thr Phe

TOT

Ser 580 OCT OTA AOG ACT Pro Leu Thr Ser

GAA

Giu 585 AAG TTGCOTT CAT Lys Leu Leu Asp

ACA

Thr 590 OTG AAG OTG GCA Leu Lys Leu Aia

ATO

Ile 595 CCC ACT TAT ACA Cly Thr Tyr Thr

CAA

Giu 600 OAT AAG TOA TOT His Lys Ser Ser TGG GAG Trp Giu 1885 GGA TTG ATG Giy Leu Met CCC ATT CAA Ala Ile Gin 625 TAT GTC AGA Tyr Val Arg 640 AGA GCT ATG GCA Arg Gly Met Giy GAO TAT TOO TGC Asp Tyr Ser Trp CAA AAT GCA Giu Asn Ala 620 CAT OCT OCA Asp Pro Pro 1933 1981 TAT GAA CAA CTT Tyr Ciu Gin Val

TTO

Phe 630 ACC TGC CCC TTT Thr Trp Ala Phe

ATA

Ile 635 TGATTTATCA AGAAAGATTG CAAACGCGAT ACATOATTAA 2030 79 ACTATACGCG GAGCTTTTGG TGCTATTAGC TACTGTCATT GGGCGCGGAA TGTTTGTGGT TCTTTCTGAT TCAGAGAGAT CAAGTTAGTT CCAAAGACAT ACGTAGCCTG TCCCTGTCTG TGAGGGAGTA AAACTACA AGGCAATTAG AAACCACCAA GAACTGGCTC CTTTGGGAGA AGAGTGGAAA TATGTAAAAA AGAATTTTGA GTTTAATGTC AATTGATTAA TTGTTCTCAT TTTTA-AAAAA AACATCT CAT CTCATACAAT ATATAAAATT GATCATGATT GATGAAAAAA AaAAAAAA AAAAAAAA AAAAAA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 641 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Gly Ser Leo Gin Thr Pro Thr Asri Leu Ser Asn Lys Ser Cys Leo 2090 2150 2210 2270 2330 2360 *9ee** a a 9*a S a a a a a.

9* 0 a.

Cys Gly Gin Glu 65 Gly Gly Asp Leu 'Val Leu Ser 50 Asn Ser Asp Asp Thr 130 Ser Gly Leu Lys Gly Ala Leo 115 Phe Gly 20 Phe Cy s Asn Ser Thr 100 Ser Asn Arg Ser Val Val Val1 Met Glu Ile Val Trp Thr Ser 70 Val Val Asp Ile Val Leu Ser 55 -Glu Gly Glu Thr Phe Lys Leu 40 Ser Gly Phe Ser Glu 120 Val1 Gly 25 Lys Val Leu Gin His 105 Glu Thr Leo Gly Ser Leo Leo 90 Asp Met Ala Arg Arg Asp Leo 75 Ile Ile Glu Glu Val Arg Gly Gly Pro Val Glu Ala 140 Glu Arg Asn Arg Ser Ser Ala Glu His Ser Aia Asn 110 Thr Pro 125 Ala Pro Gin Val Lys Val Ile Ala Arg Asp Val Ala Asp Arg Ile Lys Tyr Ser 135 Asp Val Cys Gly Lys 145 Thr Gly Gly Leo Gly 150 Ser 155 Leo Pro Met Ala Leo 160 Val Val Ser Ala Ala Arg Gly His Arg Val. Met Pro Arg Tyr Leu Asn 175 165 170

S

S.

p. *5 Gi Arg His Tyr 225 Asp Pro Phe Ala Ala 305 Asn Phe Asn Ser Leu 385 Asn Ala Gly Ala Giu 210 Cys Asn Leu Leu Lys 290 Ile Asn Pro Val Gin4 370 His Gly Ser Prc Thr 195 Tyr Argc Gin Val Al a 275 Tyr His Leu Thr Leu 355 Gly Giu Ile iis Ser 180 *Val *Arg Pro Phe Leu 260 Asn Arg Asn Gly Trp 340 Lys Tyr Leu Asp Tyr AsI His Ala Gly Arg 245 Pro Asp Pro Ile Leu 325 Ala Gly Ser Lieu Ta 1 105 3er Glu Cys *Gly *Thr 230 Phe Leu Trp Tyr Ala 310 Pro Arg Ala Trp Ser 390 Asn2 Ile Lys Phe Val1 215 Pro Thr Gly His Gly 295 His Pro Ala Ile Glu 375 Ser %.sp %.sn Tyi G13 20OC Asr Tyr Leu Gly Al a 280 Val1 Gin Gin His Ala 360 Ile Arg Trp Asp Ala 185 Asp Trp *Gly *Leu Phe 265 Aia Tyr Gly Trp Ala 345 Val Thr Gin Asn Leu Asr Ala ValI Asp Ser 250 Thr Leu Lys Val Tyr 330 Leu Ala rhr Ser Pro 410 Ser Ala Gin Phe Ile 235 His Tyr Val Asp Giu 315 Gly Asp Asp Pro Val.

395 Ser Gly I Va.

GI~

Va 22( Tyi Alz Gly Pro Ala 300 Pro Ala Thr Arg Glu 380 Leu Chr ~ys 1 Asp u Val 205 1 Asp Gly Ala Glu Leu 285 Arg Ala Val Gly Ile 365 Gly Asn Asp Val C Le.

190 Ala His Ala Cys Lys 270 Leu Ser Val Glu Giu 350 Leu Giy ;iy 1lu 1ln 1Asp Phe Ser Phe Giu 255 Cys Leu Ile Thr Trp 335 Thr Thr Tyr Ile His 415 Cys I Val.

Tyr Ser Gly 240 Ala Leu Ala Vai Tyr 320 Ile Vali Val1 Giy rhr 400 Ile -ys 420 Thr Asp Leu Gin Lys Glu Leu Gly Leu Pro Ile Arg Pro Asp Cys Pro 435 440 445 Leu Ile 450 Ile Leu 465 Met Leu Giu Asn Pro Val Ser Arg 530 Gly Thr 545 Lys Asp Trp Thr Leu Ala Met Arg 610 G).y Phe Ser Ala Gly Ser Leu Phe 500 Ser His 515 Phe Giu Ile Pro Phe Asn Phe Ser 580 Ile Gly 595 Arg Gly Ile Ile Giy 485 Lys Arg Pro Ile Pro 565 Pro Thr Met G ly Pro 470 Giu Asp Ile Cys Val 550 Tyr Leu Tyr Gly Arg 455 Giu Lys Lys Thr Gly 535 His Ala Thr Thr Arg Leu Leu Gin Phe Ala 520 Leu Ser Gin Ser Giu 600 Asp Asp Met Tyr Arg 505 G ly Asn Thr Giu Glu 585 His Tyr Tyr Gin Lys Gin Giu 490 Ala Cys Gin Gly Gly 570 Lys Ly s Ser Asn 475 Asp Trp Asp Leu Gly 555 Ile Leu Ser Trp 460 Asp Trp Val1 Ile Tyr 540 Leu Gly Leu Ser Glu 620 Gly Val Val Gin Met Arg Gly Phe 510 Leu Leu 525 Ala Met Arg Asp Glu Gly Asp Thr 590 Trp Giu 605 Asn Ala Asp Val1 His 495 Asn Met Arg Thr Thr 575 Leu Gly Ala Ile Val 480 Thr Val Pro Tyr Val 560 Gly Lys Leu Ile Vai 640

S

*5

S.

*9

S

*5 615 Gin Tyr Giu Gin Val Phe Thr Trp Ala Phe Ile Asp Pro Pro Tyr 625 Arg (2) 630 6315 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-iibrary in Lambda ZAPII (ix) FEATURE: NAME/FEATURE: CDS LOCATION:307. .3897 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTTTTTAATA GATTTTTAAA ACCCCATTAA AGCAAATACG TATATAATTG CAGCACAGAT ACAGAGAGGG AGAGAGAAAG ATAGTGTGTT GATGAAGGAG AAGAGAGATA TTTCACATGG GATGTTCTAT TTGATTCTGT GGTGAACAAG AGTTTTACAA AGARCATTCC TTTTTCTTTT TTTCTTGGTT CTTGTGTGGG TCAGCCATGG ATGTTCCATT TCCACTGCAT AGACCATTGA GTTGCACAAG TGTCTCCAAT GCAATAACCC ACCTCAAGAT CAAACCTTTT CTTGGGTTTG TCTCTC ATG GAA CCA CAA GTC TAT CAG TAC AAT CTT CTT CAT GGA GGA Met Giu Pro Gin Val Tyr Gin Tyr Asn Leu Leu His Gly Gly 120 180 240 300 348 396

A

A.

A A

A

A A AGG ATG Arg Met TCG GGA Ser Gly GAA ATG GTT Giu Met Val AGA AGA CGG Arg Arg Arg ACT GGG GTT TCA TTT CCA TTT TGT GCA ART CTC Thr Gly Val Ser Phe Pro Phe Cys Ala Asn AGA AAA GTT TCA Arg Lys Val Ser ACT AGG AGT CAR Thr Arg Ser Gin GGA TCT Gly Ser 444 TCA CCT ARG Ser Pro Lys AGA ARG GTT Arg Lys Val

GGG

Gly so TTT GTG CCA AGG Phe Val Pro Arg

AG

Lys 55 CCC TCA GGG ATG Pro Ser Gly Met AGC ACG CAR Ser Thr Gin AGT ACT TCA Ser Thr Ser A V A. p

A

CAG ARG AGC ART Gin Lys Ser Asn GAT AAA GAR AGT Asp Lys Giu Ser ACA TCT Thr Ser GTT GAR Vai Giu AAA GAR TCT GAA Lys Giu Ser Glu

ATT

Ile 85 TCC ARC CAG ARG Ser Asn Gin Lys

ACG

Thr GTT GAR GCA AGA Vai Giu Aia Arg 588 ACT AGT GAC Thr Ser Asp

GAT

Asp 100 GAC ACT AAA GTA Asp Thr Lys Vai

GTG

Val 105 GTG AGG GAC CAC Val Arg Asp His

ARG

Lys 110 TTT CTG GAG GAT Phe Leu Giu Asp

GAG

Giu 115 GAT GAR ATC ART Asp Giu Ile Asn

GGT

Gly 120 TCT ACT AAR TCA Ser Thr Lys Ser ATA AGT Ile Ser 125 GAR ACT Giu Thr 684 ATG TCA CCT GTT CGT GTA TCA TCT CAA Met Ser Pro Val Arg Val Ser Ser Gin 130 135 TTT GTT GAA AGT GAA Phe Vai Giu Ser Giu 140 GGT GGT GAT GAO AAG GAT GOT GTA AAG TTA AAO AAA TOA AAG AGA TOG Gly Gly Asp 145 Asp Lys Asp Ala Val Lys Leu Asn Lys 150 Lys Arg Ser GAA GAG Glu Giu 160 AGT GAT TTT Ser Asp Phe OTA ATT GAT TOT GTA ATA AGA GAA CAA AGT GGA 828 Leu Ile 165 Asp Ser Vai Ile Giu Gin Ser Gly

TOT

Ser 175 OAG GGG GAA ACT Gin Gly Giu Ihr

AAT

Asn 180 GOO AGT AGO AAG Ala Ser Ser Lys

GGA

Gly 185 AGO OAT GOT GTG Ser His Ala Val

GGT

Gly 190 ACA AAA OTT TAT Thr Lys Leu Tyr AlA TTG CAG GIG Ile Leu Gln Val

GAT

Asp 200 GTT GAG OOA CAA Val Giu Pro Gin CAA TTG Gin Leu 205

S

S

S.

S

S.

S

S

S S S. AAA GAA AAI Lys Giu Asn AAG OTA TIG Lys Leu Leu 225

AAT

Asn 210 GOT GGG AAT GTT Ala Gly Asn Val

GAA

Giu 215 TAO AAA GGA 001 Tyr Lys Gly Pro GTA GCA AGT Val Ala Ser 220 ACT GAA AGO Thr Giu Ser GAA AlT ACT AAG Giu Ile Thr Lys

GOT

Ala 230 AGT GAT GTG GAA Ser Asp Val Glu

CAC

His 235 AAT GAG Asn Giu 240 AlT GAl GAO TIA Ile Asp Asp Leu

GAO

Asp 245 ACT AAT AGT TIO Thr Asn Ser Phe AAA IOA GAT TTA Lys Ser Asp Leu 972 1020 1068 1116 1164

ATT

Ile 255 GAA GAG GAT GAG Glu Giu Asp Giu

OOA

Pro 260 TTA GOT GOA GGA Leu Ala Ala Gly

ACA

lhr 265 GTG GAG ACT GGA Val Glu Thr Gly GAl Asp 270 TOT TOT OTA AAO Ser Ser Leu Asn

TTA

Leu 275 AGA TTG GAG ATG Arg Leu Giu Met

GAA

Giu 280 GOA AAT OTA OGT Ala Asn Leu Arg AGG OAG Arg Gin 285 S. 5 55

S.

S5 9 S S

S.

GOT ATA GAA Ala Ile Giu TTT IGT ITT Phe Oys Phe 305

AGG

Arg 290 OTT CO GAG GAA Leu Ala Glu Giu

AAT

Asn 295 ITA TTG CAA GG Leu Leu Gin Gly ATO AGA TIA Ile Arg Leu 300 1212 OOA GAG GTT GTA Pro Glu Val Val

AAA

Lys 310 001 GAT GAA GAT GTO GAG AlA TTT Pro Asp Glu Asp Val Giu Ile Phe 315 1260

OTT

Leu

ATG

Met 335 AAO AGA GGT OTT TOO ACT TTG AAG AAT GAG Asn Arg Gly Leu Ser Thr Leu Lys Asn Giu 320 325 GGA GOT TTT AAI GAG TGG OGO TAT AGG TOT GAT GlO TTG AT Asp Val Leu Ile 1308 1356 Gly Ala Phe Asn Giu Trp Arg Tyr Arg Ser 340 345 TIT ACT ACA AGC Phe Thr Thr Arg

OTA

Leu 350 ACT GAG ACT CAT CTC AAT GGA GAT TGG TGG TCT TGC AAG ATC CAT GTT Thr Glu Thr His Leu Asn Gly Asp Trp Trp 360 Ser Cys Lys CCC AAG GAA GCA Pro Lys Glu GTC TAT GAC Val Tyr Asp 385 Ala 370 TAC AGG GCT GAT TTT Tyr Arg Ala Asp Phe 375 GTG TTT TTT AAT Val Phe Phe Asn Ile His Val 365 GGA CAA GAT Gly Gin Asp 380 GTG AAA GGT Val Lys Gly 1404 1452 1500 AAC AAT GAT GGA Asn Asn Asp Gly

AAT

Asn 390 GAC 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

CTT

Leu 410 GAG GAG AAA TGG Glu Glu Lys Trp 1548 1596

AGA

Arg 415 GAA CAG GAG AAA Glu Gin Glu Lys

CTT

Leu 420 GCT AAA GAA CAA Ala Lys Glu Gin

GCT

Ala 425 GAA AGA GAA AGA Glu Arg Glu Arg

CTA

Leu 430 99

S

S

4 *r ,t

S

GCG GAA GAA CAA Ala Glu Glu Gin

AGA

Arg 435 CGA ATA GAA GCA Arg Ile Glu Ala AAA GCT GAA ATT Lys Ala Glu Ile GAA GCT Glu Ala 445 GAC AGA GCA Asp Arg Ala CGA GAA TTG Arg Glu Leu 465

CAA

Gin 450 GCA AAG GAA GAG Ala Lys Glu Glu

GCT

Ala 455 GCA AAG AAA AAG Ala Lys Lys Lys AAA GTA TTG Lys Val Leu 460 ACG TGG TAC Thr Trp Tyr 1644 1692 1740 ATG GTA AAA GCC Met Val Lys Ala

ACG

Thr 470 AAG ACT CGT GAT Lys Thr Arg Asp

ATC

Ile 475 ATA GAG Ile Glu 480 CCA AGT GAA TTT Pro Ser Glu Phe

AAA

Lys 485 TGC GAG GAC AAG Cys Glu Asp Lys

GTC

Val 490 AGG TTA TAC TAT Arg Leu Tyr Tyr 1788 1836

C

S S S

S.

*5 S S S S

S.

AAC

Asn 495 AAA AGT TCA GGT Lys Ser Ser Gly

CCT

Pro 500 CTC TCC CAT GCT Leu Ser His Ala GAC TTG TGG ATC Asp Leu Trp Ile

CAC

His 510 GGA GGA TAT AAT Gly Gly Tyr Asn

AAT

Asn 515 TGG AAG GAT GGT Trp Lys Asp Gly

TTG

Leu 520 TCT ATT GTC AAA Ser Ile Val Lys AAG CTT Lys Leu 525 1884 GTT AAA TCT Val Lys Ser ATT CCT GAT Ile Pro Asp 545

GAG

Glu 530 AGA ATA GAT GGT Arg Ile Asp Gly

GAT

Asp 535 TGG TGG TAT ACA Trp Trp Tyr Thr GAG GTT GTT Glu Val Val 540 GAT GGT CCA Asp Gly Pro 1932 1980 CAG GCA CTT TTC Gln Ala Leu Phe

TTG

Leu 550 GAT TGG GTT TTT Asp Trp Val Phe

GCT

Ala 555 CCC AAG Pro Lys 560 CAT GCC ATT GCT His Ala Ile Ala TAT GAT Tyr Asp 565 AAC AAT CAC Asn Asn His CAA GAC TTC CAT Gin Asp Phe His 2028 GCC ATT GTC CCC AAC CAC Ala 575 Ile Vai Pro Asn His 580 ATT CCG GAG GAA Ile Pro Giu Ciu TTA TAT TGG GTT GAG Leu Tyr Trp Val Ciu 585 GAG AGA AGG CTT AGA Ciu Arg Arg Leu Arg 605

GAA

Giu 590

GAA

Giu 2076 GAA CAT CAG ATC Giu His Gin Ile

TTT

Phe 595 AAG ACA CTT CAG Lys Thr Leu Gin

GAG

Giu 600 2124 CC GCT ATG Ala Ala Met ACA AAG CAA Thr Lys Ciu 625

CGT

Arg 610 OCT AAG GTT GAA Ala Lys Vai Giu 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

CAG

Gin 635 GTA TAT Vai Tyr 640 ACT GAG CCT CTT Thr Giu Pro Leu

GAT

Asp 645 ATC CAA GCT GGA Ile Gin Ala Giy

AGC

Ser 650 AGC GTC ACA OTT Ser Val Thr Val 2268 2316 es..

0 OS*@*0 0 0*O# 0000 0 0 000S

S.

S S 050 S *5S3 S I, S.

S

S. S 5555

S

OeSSeO @5 0* 0* S 5* 0@

TAC

Tyr 655 TAT AAT CCC GCC Tyr Asn Pro Ala

AAT

Asn 660 ACA GTA CTT AAT Thr Val Leu Asn AAA CCT GAA ATT Lys Pro Giu Ile TTC AGA TOT TCA Phe Arg Cys Ser 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 OTT Vai Lys Val 705

ATG

Met 690 TCG CCT GCT GAA Ser Pro Ala Giu

AAT

Asn 695 GGC ACC CAT GTC Gly Thr His Vai 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

Val 715 AGA GAA Arg Giu 720 GAT GGT GGG 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 COT GTG TTT GGA Pro Vai Phe Gly

GGA

Gly 740 GTC GCT AAA GAA Vai Ala Lys Giu

CCT

Pro 745 CCA ATG CAT ATT Pro Met His Ile

GTC

Val 750 CAT ATT GCT CTC CAA ATO GCA CCA ATT His Ile Aia Val Giu Met Aia Pro Ile 755

GCA

Ala 760 AAG GTG GGA GGC Lys Vai Giy Giy OTT GGT Leu Giy 765 2604 CAT GTT OTT Asp Vai Val AGT OTT TCC CGT GCT GTT CAA GAT TTA Ser Leu Ser Arg Ala Vai Gin Asp Leu AAC CAT AAT Asn His Asn 780 2652 -m -m GTG GAT ATT ATO TTA CCT AAG Val Asp Ile Ile Leu Pro Lys 785 CAC TGT TTG AAG ATG AAT AAT GTG Asp Cys Leu Lys Met Asn Asn Val 795 2700 AAG GAC Lys Asp 800 TTT CGG TTT GAG Phe Arg Phe His

AAA

Lys 805 AAC TAG TTT TGG Asn Tyr Phe Trp

GGT

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

AAA

Lys 815

CCT

Pro GTA TGG TTT GGA Val Trp Phe Giy CAA AAC GG TTA Gin Asn Gly Leu 835

AAG

Lys 820 GTG GAA GGT CTC Vai Giu Giy Leu

TG

Ser 825 GTC TAT TTT TTG Val Tyr Phe Leu

GAG

Giu 830 TTT TCG AAA GGG Phe Ser Lys Gly GTC TAT GGT TGT Vai Tyr Gly Cys AGC AAT Ser Asn 845 2844 GAT GGT GAA Asp Giy Giu CTG CAA GGT Leu Gin Gly 865

CGA

Arg 850 TTT GGT TTC TTC Phe Gly Phe Phe

TGT

Cys 855 CAC GGG GCT TTG His Ala Ala Leu GAG TTT CTT Giu Phe Leu 860 GAT TGG TCT Asp Trp Ser GGA TTT AGT CG Ciy Phe Ser Pro

CAT

Asp 870 ATC ATT CAT TGC Ile Ile His Gys

CAT

His 875 ACT CCT Ser Aia -'880 CCT GTT GCT TGG Pro Vai Aia Trp

CTC

Leu 885 TTT AAG GAA CA Phe Lys Giu Gin ACA CAC TAT CGT Thr His Tyr Giy

CTA

Leu 895 AGC AAA TCT GGT Ser Lys Ser Arg

ATA

Ile 900 GTC TTC ACG ATA Val Phe Thr Ile

CAT

His 905 AAT CTT GAA TTT Asn Leu Giu Phe

CCC

Giy 910 2892 2940 2988 3036 3084 3132 3180 CCA CAT CTC ATT Aia Asp Leu Ile

GG

Giy 915 AGA GCA ATG ACT Arg Aia Met Thr AAG GCA GAG AAA Asn Ala Asp Lys 920 TCT CGA AAG CCT Ser Gly Asn Pro CTT TCA GCA Val Ser Pro GGT GAG GTT Pro His Leu 945

ACT

Thr 930 TAG TCA GAG GAG Tyr Ser Gin Giu

CTG

Vali 935 GCT ACA ACA Ala Thr Thr 925 CTA ATT CC Val Ile Ala 940 GAG CCA GAT Asp Pro Asp GAG AAG TTG CAT His Lys Phe His ATA GTG AAT CCC Ile Val Asn Gly

ATT

Ile 955 ATT TGG Ile Trp 960 CAT CCT TTA AAG Asp Pro Leu Asn

CAT

Asp 965 AAG TTG ATT CCC Lys Phe Ile Pro

ATT

Ile 970 CCC TAG ACC TGA Pro Tyr Thr Ser 3228 3276

GAA

Giu 975 AAG GTT GTT GAA Asn Val Vai Giu

GGC

Gly 980 AAA ACA GGA CC Lys Thr Ala Ala

AAG

Lys 985 GAA GGT TTGCGAG Giu Ala Leu Gin AAA GTT GGA CTG Lys Leu Gly Leu

AAA

Lys 995 GAG GGT GAG GTT Gin Ala Asp Leu GGT TTG GTA CCA ATT Pro Leu Vai Gly Ile 1000 ATC ACC Ile Thr 1005 3324 CGC TTA ACT CAC CAG AAA GGA ATC CAC CTC Arg Leu Thr His Gin Lys Gly Ile His Leu 1010 1015 CGC ACC TTG GAA CGG AAC GGA CAG GTA GTC Arg Thr Leu Giu Arg Asn Giy Gin Val Val 1025 1030 GAT CCT AGG GTA CAA AAC GAT TTT GTT AAT Asp Pro Arg Val Gin Asn Asp Phe Val Asn 1040 1045 ATT AAA CAT GCT ATT TGG Ile Lys His Aia Ile Trp 1020 TTG CTT GGT TCT GCT CCT Leu Leu Oiy Ser Ala Pro 1035 TTG OCA AAT CAA TTG CAC Leu Ala Asn Gin Leu His 1050 3372 3420 3468 TOO AAA Ser Lys 1055 TAT A.AT GAC COC GCA Tyr Asn Asp Arg Aia 1060 OGA CTC TOT Arg Leu Cys CTA ACA Leu Thr 1065 TAT GAC GAG CCA Tyr Asp Giu Pro 1070 3516 CTT TCT CAC OTO ATA TAT OCT GOT OCT OAT TTT ATT CTA OTT OCT TCA Leu Ser His Leu Ile Tyr Ala Oly Aia Asp Phe Ile Leu Vai Pro Ser 3564 1075 1080 1085 ATA TTT GAO OCA TOT OGA OTA ACA Ile Phe Oiu Pro Cys Oly Leu Thr 1090 CAA OTT ACC Gin Leu Thr 1095 OCT ATO AGA TAT GOT Ala Met Arg Tyr Oly 1100 3612 3660 TOA ATT OCA OTO Ser Ile Pro Vai 1105 OTO COT AAA Vai Arg Lys ACT OGA Thr Oly 1110 OGA OTT TAT Oly Leu Tyr OAT ACT OTA TTT Asp Thr Val Phe 1115 OAT OTT GAO Asp Val Asp 1120 OAT GAO AAA His Asp Lys GAG AGA OCA Olu Arg Ala 1125 CAA CAG TOT GOT OTT GAA OCA Gin Gin Cys Oly Leu Oiu Pro 1130 3708 AAT OGA Asn Oly 1135 TTO AGO TTT Phe Ser Phe OAT OGA Asp Gly 1140 OCA OAT GOT 000 OGA Ala Asp Ala Gly Oly 1145 OTT OAT TAT Val Asp Tyr

GOT

Ala 1150 3756 OTO AAT AGA GOT OTO TOT GOT TOO TAO Leu Asn Arg Ala Leu Ser Ala Trp Tyr 1155 OAT GOT Asp Oly 1160 000 OAT TOO TTO AAC Arg Asp Trp Phe Asn 1165 TOT TOO AAO OGA OCT Ser Trp Asn Arg Pro 1180 3804 3852 TOT TTA TOO Ser Leu Cys AAO CAG OTO Lys Gin Val 1170 ATO OAA CAA OAT TG Met Olu Gin Asp Trp 1175 GOT OTT OAT TAT TTO GAG OTT TAO OAT GOT GOT Ala Leu Asp Tyr Leu Giu Leu Tyr His Ala Ala 1185 1190 AGA AAG TTA OAA Arg Lys Leu Oiu 1195 3897 TAOTTAOTTT OTOAGATOOT AOOAOAAAAA TTOAOGAGAT OTGOAATOTO TAOAOOTTOA GTGTTTGOOT OTGGACAOOT TTTTTATTTO OTATATOAAA OTATAAATCA AOTOTAOACT OAGATCAATA GOAOAOAGTO OTOAOTTOAT TTOATTTTTT GTGCAAOATA TGAAAGAOCT TAOOOTOTAA TAATOTAOTO ATTOATOATT ATTTGTTTTO GGAAGAAATG AGAAATCAAA 3957 4017 4077 4137

I

GGATGCAJAJAJ TACTCTGAAA AAAAAA A INFORMATION FOR 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: Met Giu Pro Gin Val Tyr Gin Tyr Asn Leu Leu His Gly Gly Arg Met 4168 a.

a a a. 0 *a a. a *aaaa.

a a.

a.

a.

Glu Arg Lys Val1 65 Lys Thr Glu Pro Asp 145 Ser Gly Met Arg Gly 50 Gin Glu Ser Asp Val 130 Asp Asp Glu Val Arg Phe Lys Ser Asp Glu 115 Arg Lys Phe Thr Thr Arg Val Ser Glu Asp 100 Asp Val Asp Leu Asn 180 Gly Lys Pro Asn Ile 85 Asp Glu Ser Ala Ile 165 Al a Val Ser Val Ser Arg Lys 55 Gly Asp 70 Ser Asn Thr Lys Ile Asn Ser Gin 135 Vai Lys 150 Asp Ser Ser Ser Phe Thr 40 Pro Lys Gin Val1 Gly 120 Phe Leu Val1 Lys Pro 25 Thr Ser Giu Lys Val 105 Ser Val Asn Ile Gly 185 Phe Arg G ly Ser Thr 90 Val Thr Giu Lys Arg 170 Ser Cys Ala Ser Gin Met Ser Gin Ser 75 Val Glu Arg Asp Lys Ser Ser Glu 140 Ser Lys 155 Giu Gin His Ala Asn Leu Gly Ser Thr Gin Thr Ser Ala Arg His Lys 110 Ile Ser 125 Glu Thr Arg Ser Ser Giy Val Gly 190 Ser Gly Ser Pro Arg Lys Thr Ser Val Glu Phe Leu Met Ser Giy Gly Giu Glu 160 Ser Gin 175 Thr Lys Leu Tyr Giu Ile Leu Gin Val Asp Val Giu Pro Gin Gin Leu Lys Giu 200 205 Asn Asn Ala Gly Asn Val Giu Tyr Lys Gly Pro Val a.

a. I I Leu 2 2 5 le Glu Leu Glu Phe 305 Arg Al a Thr Glu Asp 385 Gin Gln Giu Ala Leu 465 210 Glu Asp Asp Asn Arg 290 Pro Gly Phe His Ala 370 Asn Ile Glu Gln Gln 450 .,let Ile Asp Giu Leu 275 Leu Glu Leu Asn Leu 355 Tyr Asn Ile Lys Arg 435 Ala Val 215 Thx Leu Prc 260 Arg Ala Val Ser G lu 340 Asn Arg Asp Asp ELeu 420 Arg Lys :,ys Lys Ala 230 Asp Thr 245 Leu Ala Leu Giu Glu Giu Val Lys 310 Thr Leu 325 Trp Arg Gly Asp Ala Asp Gly Asn 390 Phe Giu 405 Ala Lys Ile Glu Giu Giu Ala Thr 470 Ser Asn Ala Met Asn 295 Pro Lys Tyr Trp Phe 375 Asp Asn Giu Ala Ala 455 Lys *Asp Ser Gly Glu 280 Leu Asp Asn Arg Trp 360 Val Phe Phe Gin Glu 440 Ala Thr Val PhE Thr 265 Ala Leu Glu Glu Ser 345 Ser Phe Ser Leu Ala 425 Lys Lys Arg *Glu Phe 250 *Val Asn Gin Asp Ser 330 Phe Cys Phe Ile Leu 410 Glu Ala Lys Asp His Lys Glu Leu Gly Val 315 Asp Thr Lys Asn Thr 395 Glu Arg Glu Lys Ile 4'75 220 Thr Ser Thr Arg Ile 300 Glu Val1 Thr Ile Gly 380 Val1 Giu Giu Ile Lys 460 Thr A12 Gitu Asp Gly Arg 285 Arg Ile Leu Arg His 365 Gin Lys Lys Arg Glu 445 Val L'rp Ser Ser Leu Asp 270 Gin Leu Phe Ile Leu 350 Val Asp Gly Trp Leu 430 Ala Leu Tyr Lys As r Ile 255 Ser Ala Phe Leu Met 335 Thr Pro Val Gly Arg 415 Ala Assp krg SIle Leu Giu 240 Glu *Ser Sle Cys Asn 320 Giy Giu Lys Tyr Met 400 Giu Giu Arg Giu Giu 480 Pro Ser Giu Phe Lys Cys Giu Asp Lys Val Arg Leu Tyr Tyr Asn Lys 485 490 495 m m Lys Asp Leu Ser Ser Gly Pro 500 Leu Ser His Ala Trp Ile His Gly Gly 510 505

*SSS

.555 S.r o 5*

S

C.

S.

Ty Se As 54! Hi Va Glr Met Glu 625 Thr Asn Cys Lys Val 705 Asp Val Ala r Asn r Glu 530 p Gin 5 s Ala L Pro 1 Ile Arg 610 Arg Glu Pro Ser I Met 690 Pro L Gly G Phe G Val G 7 As 51 Ar Al Ile Asr Phe 595 Ala Thr Pro kia ?he 575 er Aeu ly ly lu 55 n Trp 5 g Ile a Leu I Ala His 580 Lys Lys Met Leu Asn 660 Asn I Pro I Asp A Ile P 7 Gly V 740 Met A Ly As Ph Ty 56! I le Thi Val Lys Asp 645 Lhr irg la la he 25 *al la s Asp p Gly e Leu 550 Asp Pro Leu Glu Sen 630 Ile Val I Trp IJ Glu 1 6 Tyr M 710 Asp A Ala L Pro I G1 As 53! As As Gl.

Gir Lys 615 Phe Gin .eu :hr ~sn ;95 [et .sn ys le y Leu 520 p Trp 5 p Trp i Asn 1 Glu 1 Glu 600 Thr Leu Ala Asn I His 680 Gly Met I Lys Glu P 7 Ala L 760 Ser Ile Va Tr Va HiE Let 585 Glu Ala Leu Gly Gly 665 Arg rhr ksp er 'ro '45

YS

p Tyr 1 Phe Arg 570 l Tyr Arg Leu Ser Ser 650 Lys Leu His Phe 1 Gly IN 730 Pro M Val G Thr Ala 555 Gin Trp Arg Leu Gin 635 Ser Pro ;ly 7al alI 115 let I [et I .ly G 1 Ly G1 54 Asj Asj Val Leu Lys 620 Lys Val Glu Pro Arg 700 ?he ~sp is ly s Ly 52 u Va p Gi Ph Gli I Arc 605 Thr His Thr Ile Leu 685 Ala Sen Tyr Ile Leu 765 s Leu

S

1 Val y Pro e His .i Glu 590 Glu Glu Val Val Trp 670 Pro I Thr Glu J His I 7 Val H 750 Gly A Va Ii, Pre Al 57! Gli Al Thr Val ryr 555 ?he ?ro Tal Lrg le is sp 1 Lys e Pro o Lys 560 a Ile i His I Ala Lys Tyr 640 Tyr Arg Gin Lys Glu 720 Pro Ile Val Val Thr 770 Ser Leu Sen Arg Ala 775 Val Gin Asp Leu Asn His Asn Val Asp 780 91 Leu Lys Met Ile 785 Ile Leu Pro Lys Tyr Asp Cys 790 Asn Asn Val Lys Asp

S.

S a

S

9

S

S

S

59 *5 *9 Phe Arg Phi Trp Phe Gl Asn Gly Let 83! Glu Arg Phf 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 Giu Gly Leu Lys 995 Thr His Gin 1010 Leu Giu Arg 1025 Arg Val Gin Tyr Asn Asp eHis Lys Asn Tyr Phe Trp 805 yr Lys Val Glu Gly Leu Ser 820 825 .i Phe Ser Lys Gly Cys Val 840 Gly Phe Phe Cys His Ala 855 Ser Pro Asp Ile Ile His 870 Trp Leu Phe Lys Giu Gin 885 Ile Val Phe Thr Ile His 900 905 Arg Ala Met Thr Asn Ala 920 Ser Gin Giu Vai Ser Gly 935 Phe His Gly Ile Vai Asn 950 Asn Asp Lys Phe Ile Pro 965 Gly Lys Thr Ala Ala Lys 980 985 Gin Aia Asp Leu Pro Leu 1000 Lys Giy Ile His Leu Ile 1015 Asn Gly Gin Val Vai Leu I 1030 Asn Asp Phe Val Asn Leu 1045 2 Arg Ala Arg Leu Cys Leu TI 1060 1065 G13 81C Val Tyr Ala Cys Tyr 890 Asn Asp Asn Gly Ile 970 Glu lal .'ys ~eu lia .050 'hr 795 rGly Thr Giu Ile -Tyr Phe Leu Giu 830 Gly Cys Ser Asn 845 Leu Giu Phe Leu 860 His Asp Trp Ser 875 Thr His Tyr Gly Leu Glu Phe Gly 910 Lys Ala Thr Thr 925 Pro Val Ile Aia 940 Ile Asp Pro Asp 955 Pro Tyr Thr Ser Ala Leu Gin Arg 990 Gly Ile Ile Thr 1 1005 His Ala Ile Trp P 1020 Giy Ser Ala Pro A 1035 Asn Gin Leu His S 1 Tyr Asp Giu Pro L 1070 800 Lys Vai 815 Pro Gin Asp Gly Leu Gin Ser Ala 880 Leu Ser 895 Ala Asp Val Ser Pro His Ile Trp 960 ilu Asn 975 .sys Leu ~rg Leu Lrg Thr ~sp Pro 1040 er Lys 055 eu Ser 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 Gin 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 Gin 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 S: (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 32 S" 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 (18)

1. DNA molecule encoding a protein with the biological activity of a starch synthase selected from the group consisting of DNA molecules encoding a protein having the amino acid sequence indicated under Seq ID No. 8; DNA molecules comprising the nucleotide sequence depicted under Seq ID No. 7; DNA molecules the nucleotide sequence of which differs from the sequence of the DNA molecules under or (b) due to the degeneracy of the genetic code; and DNA molecules which hybridize to the DNA molecules mentioned under or wherein the DNA molecules mentioned under or encode a protein with the biological activity of a starch synthase of isotype II (GBSSII) or a biologically active fragment of such a protein; and DNA molecules encoding a protein having the amino acid sequence depicted under Seq ID No. DNA molecules comprising the nucleotide sequence depicted under Seq ID No. 9; DNA molecules the nucleotide sequence of which differs from the sequence of the DNA molecules under or (f) due to the degeneracy of the genetic code; and DNA molecules which hybridize to the DNA molecules mentioned under or except for DNA molecules from rice, wherein the DNA molecules mentioned under or encode a protein with the biological activity of a soluble starch synthase of the isotype B (SSSB) or a biologically active fragment of such a protein; and DNA molecules encoding a protein having the amino acid sequence depicted under Seq ID No. 12; DNA molecules comprising the nucleotide sequence depicted under Seq ID No. 11; DNA molecules the nucleotide sequence of which is different from the sequence of the DNA molecules under or due to the degeneracy of the genetic code; and DNA molecules which hybridize to the DNA molecules mentioned under or S. S a wherein the DNA molecules mentioned under or encode a protein with the biological activity of a soluble starch synthase of the isotype A (SSSA) or a biologically active fragment of such a protein.

2. DNA molecule encoding a protein with the biological activity of a soluble starch synthase of the isotype A (SSSA) or a biologically active fragment thereof, wherein the protein encoded by the DNA molecule is recognized by an antibody that is directed to the peptide NH 2 -GTGGLRDTVENC-COOH (Seq ID No. 13).

3. Vector containing a DNA molecule according to claim 1 or 2.

4. The 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. Host cells containing a vector according to claim 3 or 4.

6. Protein or biologically active fragment thereof encoded by a DNA molecule according to claim 1 or 2 or a vector according to claim 3 or 4.

7. 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.

8. Plant cell containing a DNA molecule according to claim 1 or 2 in combination with a heterologous promoter.

9. Plant containing plant cells according to claim 8. The plant according to claim 9, which is a useful plant.

11. The plant according to claim 10, which is a starch-storing plant. 96

12. The plant according to claim 11, which is a potato plant.

13. Propagation material of a plant according to any of claims 9 to 12 containing plant cells according to claim 8.

14. Starch obtainable from a plant according to any of- claims 9 to 12. Transgenic plant cell, characterized in that in this plant cell the activity of at least one of the proteins according to claim 6 is reduced.

16. The plant cell according to claim 15, wherein in this cell an antisense RNA to transcripts of a DNA molecule according to claim 1 or 2 is expressed.

17. Plant containing plant cells according to claim 15 or 16.

18. The plant according to claim 17, which is a useful plant.

19. The plant according to claim 18, which is a starch-storing plant.

20. The plant according to claim 19, which is a potato plant.

21. Propagation material of a plant according to any of claims 17 .to 21, containing cells according to claim 15 or 16. :22. Starch obtainable from plants according to any of claims 17 to 21. Dated this 21st day of October 1999 HOECHST SCHERING AGREVO GMBH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia