CA2416347C - Improvements in or relating to plant starch composition - Google Patents

Abstract

Disclosed is a nucleotide sequence encoding an effective portion of a class A
starch branching enzyme (SBE) obtainable from potato plants, or a functional equivalent thereof, together with, inter alia, a corresponding polypeptide, a method of altering the characteristics of a plant, a plant having altered characteristics; and starch, particularly starch obtained from a potato plant, having novel properties.

Description

IMPROVEMENT IN OR RELATING TO PLANT STARCH COMPOSITION
This application is a division ofI'CT Inteniational Application No.

bearing Canadian Application Serial No. 2,217,878 with the international filing date of May 3, 1996.

Field of the Invention This invention relates to novel nucleoride seauences. polypeptides encoded thereby, vectors and host cells and host orQa.nisms comprisiria one or more of the novel sequences, and to a method of alterin(2 one or more characteristics of an oraanism. The invention al;so relates to starch having novel properties and to uses thereof.

Background of the Invention Starch is the major form of carbon reserve in plants, constitutina 50% or more of the dry weiqht of many storaQe organs - e.z. tubers, seeds of cereals. Starch is used in numerous food and industrial applications. In many cases, however, it is necessary to modify the native starches, via chemical or physical means, in order to produce distinct properties to suit particular applications. It would be highly desirable to be able to produce starches with the required properties directly in the plant, thereby removing the need for additional modification. To achieve this via genetic engineering requires knowledge of the metabolic pathwav of starch biosvnthesi.s. This includes characterisation of genes and encoded gene products which catalyse the synthesis of starch. Knowledae about the regulation of starch biosvnthesis raises the possibility of "re-proarammina" biosynthetic pathways to create starches with novel properties that could have new corr.imercial applications.

The commercially useful properties of starch derive frorn the ability of the native granular form to swell and absorb water upon suitable treatment. Usuallv heat is required to cause granules to swell in a process known as aelatirisation., which has been defined (W A
Atwell et al, Cereal Foods World 33. 306-311, 1988) as "... the coliapse (disruption) of molecular orders within the starch granule manifested in irreversible changes in properties such as granular swelling, narive crystallite melting, loss of birefringence, and starch solubilisation. The point of initial gelatinisation and the range over which it occurs is governed bv srarch concentrarron. method of observation, granule type, and heterogeneities within the granule population under obsen%ation". A number of techniques are available L

for the determination of aelatinisation as induced by heating, a convenient and accurate method beinQ differential scanninQ caiorimetrs: . which detects the temperature ranae and enthalpy associated with the collapse of molecular orders within the aranule_ To obtain accurate and meaninaful results, the peak and/or onset temueran:re of the endotherm observed bv differential scanninQ calorimetn- is usually determined.

The consequence of the collapse of molecular orders within starch granules is that the aranules are capable of taking up water in a process known as pasting, which has been defined (W A Atwell et al, Cereal Foods World 33, 306-311, 1988) as "... the phenomenon following gelatinzsation in the dissolution of starch. It involves granular swelling, exudation of molecular comoonents from the granule, and eventualiv, total disruption of the granules". The best method of evaluating pastina properties is considered to be the viscoamyloaraph (Atwell et al, 1988 cited above) in which the viscosity of a stirred starch suspension is monitored under a defined time/temperature regime. A typical viscoamylograph profile for potato starch shows an initial rise in viscosity, which is considered to be due to aranule swelling. In addition to the overall shape of the viscosity response in a viscoamylograph, a convenient quantitative measure is the temperature of initial visc;ositv development (onset). Fip-ure 1 shows such a typical viscositv profile for potato starch, during and after cooking, and includes stages A-D
which correspond to viscositv onset (A). maximum viscosi.tv (B), complete dispersion (C) and reassociation of molecules (or retroaradation, D). In the fi-aure, the dotted line represents viscosity (in stirring number units) of a 10% w/w starch suspension and the unbroken line shows the temperature in deorees centiQrade. At a certain point, defined by the viscositv peak, aranule swelling is so extensive that the resulting highly expanded structures are susceptible to mechanicallv-induced fragmentation under the stirring conditions used. With increased heatina and holding at 95 C, further reduction in viscositv is observed due to increased fraamentation of swollen aranules. This veneral profile has previously always been found for native potato starch.

After heatinQ starches in water to 95 C and holdina at that temperature (for typically 15 minutes). subseauent coolins to 50 C results in an increase in viscositv due to the process of retroQradation or set-back. RetroLyradation (or set-back) is defined (Atweil et al.. 1988 cited above) as ".. . a process which occur s:::':en tite molecules comorising Qelarinised starch begin to reassociate in an ordered strucrure... ". At 50 C. it is primarilv the amvlose comaonent =hich reassociates. as indicated b%- the increase in viscoamvlograph viscositv for starch from normal maize (21.6 amvlose) compared with starch from waxy maize (l.? % amvlose) as shown in Figure 2. Fioure 2 is a viscoamvlograph of l0%wiw starch suspensions from waxv maize (,solid line), conventional maize (dots and dashes), high amylose varietv (hylon 5. dotted line) and a verv high amylose varietv (hvion 7, crosses). The temperatur,, protile is also shown by a solid line, as in Figure 1.
The extent of viscositv increase in the viscoamvlograph on cooling and holding at depends on the amount of amvlose which is able to reassociate due to its exudation from starch granules during the gelatinisation and pastinQ processes. A
characteristic of amvlose-rich starches from maize plants is that very little amylose is exuded from granules by gelatinisation and pasting up to 95 C, probably due to the restricted swellina of the granules. This is illustrated in Figure 2 which shows low viscosities for a high amvlose (44.9%) starch (Hvlon 5) from maize during gelatinisation and pasting at 95 C
and little increase in viscositv on cooling and holding at 50 C. This effect is more extreme for a higher amylose content (58%, as in Hylon 7), which shows even lower viscosities in the viscoamvlo4raph test (Figure 2). For commercially-available high amylose starches (currently available from maize plants. such as those described above), processing at greater than 100 C is usuallv necessarv in order to generate the benefits of high amylose contents with respect to increased rates and strengths of reassociation, but use of such high temperatures is energeticallv un.favourable and c ostiv. Accordingly, there is an unmet need for starches of high amvlose content which can be processed below 100 C
and still show enhanced levels of reassociation, as indicated for example bv viscoarnvlograph measurements.

The properties of potato starch are useful in a variety of both food and non-food (paper.
textiles. adhesives etc.) appiications. However. for many applications.
properties are not optimum and various chemical and physical modifn.ations well Lnown in the art are undertaken in order to improve useful properties. Two types of propertv manipulation which would be of use are: the controlled alteration of gelatinisation and pastina temperatures: and starches which suffer less granular fragmentation during pasting than conventional starches.

Currentlv the only wavs of manipulating the Qelatinisation and pastinQ
temperatures of potato starch are bv the inclusion of additives such as suaars. polvhvdroxv compounds of salts (Evans & Haisman. Starke 34. 21-4-231. 1982) or by extensive physical or chemical pre-treatments (e.Q. Stute, Starke 44. 205-214. 1992). The reduction of aranule fraQmentation durinQ pasting can be achieved either bv extensive phvsical pretreatments (Stute, Starke 44, 205-214, 1992) or bv chemical cross-linking. Such processes are inconvenient and inefficient. It is therefore desirable to obtain plants which produce starch which intrinsicallv possesses such advantageous properties.

Starch consists of two main polysaccharides, amvlose and amylopectin. Amvlose is a generallv linear polymer containing a-1.4 linked alucose units, while amylopectin is a highly branched polymer consistins; of a a-1.4 linked glucan backbone with a-1,6 linked zlucan branches. In most plant storage reserves amylopectin constitutes about 75 % of the starch content. Amylopectin is synthesized by the concetted action of soluble starch synthase and starch branching enzyme [a-1,4 glucan: a-1,4 glucan 6-Qlycosyltransferase, EC 2.4.1.18]. Starch branching enzyme (SBE) hvdrolvses a-1,4 linkages and rejoins the cleaved glucan, via an a-1,6 linkage, to an acceptor chain to produce a branched structure.
The physical properties of starch are strongly affected by the relative abundance of amylose and amvlopeetin, and SBE is therefore a crucial enzvme in determininQ
both the quantity and quality of starches produced in plant systems.

In most plants studied to date e.g. maize (Bover & Preiss, 1978 Biochem.
Biophys. Res.
Comm. 80, 169-175), rice (Smvth. 1988 Plant Sci. 57, 1-8) and pea (Smith.
Planta 175, 270-279), two forms of SBE have been identified, each encoded by a separate 2ene. A
recent review bv Burton et al.. (1995 The Plant 7ournal 7.. 3-15) has demonstrated that the two forms of SBE constitute distinct classes of the enzvme such that, in oeneral, enzymes of the same class frotn different plants may exhibit greater similarity than enzvmes of different classes from the same plant. In their review. Burton et al, termed the vvo respective enzvme families class "A" and class "B". and the reader is referred thereto (and to the references cited therein) for a detailed discussion of the distinctions between the two classes. One aPneral distinction of note would appear to be the presence, in class A SBE molecules, of a flexible ti-terminal domain. '~rtich is not found in class B molecules. The distinctions noted bv Burton er a'. are relied on herein to define class A and class B SBE molecules. which terms are to be interpreted accordinQiv.

However in potato, only one isoform ot the SEE molecule (belonging to class B) has thus far been reported and only one gene cloned (Blennow & Johansson, 1991 Phytochem. 30.
437-444, and Kof3mann er al., 1991 Liol. Gen. Genet. 230, 39-44). Further, published attempts to modify the properties of starch in potato plants (by preventing expression of the single known SBE) have aenerally not succeeded (e.2. Muller-Rober &
KoBmann 1994 Plant Cell and Environment 17, 601-613)_ Summary of the Invention In a first aspect the invention provides a nucleotide sequence encoding an effective portion of a class A starch branchinQ, enzyme (SBE) obtainable from potato plants.

Preferably the nucleotide sequence encodes a polypeptide comprising an effective portion of the amino acid sequence shown in Figure 5(excluding the sequence MNKRIDL, which does not represent part of the SBE molecule), or a functional equivalent thereof (which term is discussed below). The amino acid sequence shown in Fi-zttre 5 (Seq ID
No. 15) includes a leader sequence which directs the polypeptide. when svnthesised in potato cells, to the amvloplast. Those skilled in the art will recognise that the leader sequence is removed to produce a mature enzyme and that the leader sequence is therefore not essential for enzyme activity. Accordingly. ar. "effective portion" of the polypeptide is one which possesses sufficient SB:E activitv to complement the brarichin?
enzyme mutation in E. coli KV 832 cells (described below) and which is active when expressed in E. coli in the. phosphorvlation stimulation assay. An example of an incomplete polvpeptide which nevertheless constitutes an "effective portion" is the mature enzvme lacking the leader sequence. Bv analoav with the pea class A SBE sequence. the potato class A
sequence shown in Figure ~ probably possesses a leader sequence of about 48 amino acid residues, such that the N terminal amino acid sequence is thouaht to commence around the fflutamic n the art will appreciate acid residue (E) at position 49 (EKSSYN.. . etc_). Those skilled 1 ~

that an effective portion of the enzvme may well omit other parts of the sequence shown in the fiQure without substantial detrimental effe-_t. For example. the C-terminal slutamic acid-rich region could be reduced in lenath, or possiblv deleted entirelv, without abolishinQ class A SBE activitv. A comparison with other known SBE sequences, especiallv other class A SBE sequences (see for example. Burton et al. 1995 cited above), should indicate those portions which are hi hlv conserved (and thus likely to be essential for activity) and those portions which are less well conserved (and thus are more likely ~ to tolerate sequence changes without substantial loss of enzyme activity).

Conveniently the nucleotide sequence will comprise substantially nucleotides 289 to 2790 of the DNA sequence (Seq ID No. 14) shown in Figure 5 (which nucleotides encode the mature enzyme) or a functional equivalent thereof, and may also include further nucleotides at the 5' or 3' end. For exampie, for ease of expression, the sequence will desirably also comprise an in-frame ATG start codon, and may also encode a leader sequence. Thus, in one embodinnzent, the sequence further comprises nucleotides 145 to 288 of the sequence shown in Figure 5. Other embodiment.s are nucleotides 228 to 2855 of the sequence labelled "psbe2con.seq" in Fieure 8, and nucleotides 57 to 2564 of the sequence shown in Figure 12 (preferably comprising an in-frame ATG start codon, such as the sequence of nucleotides 24 to 56 in the same Figure), or functional equivalents of the aforesaid sequences.

The term "functional equivalent" as applied herein to nucleotide sequences is intended to encompass those sequences which differ in their nucleotide composition to that shown in Figure 5 but which, bv virtue of the de2eneracv of the aenetic code. encode polypeptides havinz identical or substantially identical amino acid sequences. It is intended that the term should also apply to sequences which are sufficientlv homologous to the sequence of the invention that they can hvbridise to the complement thereof under stringent hvbridisation conditions - such equivalents will preferably possess at least 85 %, more preferablv at least 90 %, and most preferablv at least 95% seauence homolo y with the sequence of the invention as exemplified by nucleotides 289 to 2790 of the DNA
sequence shown in FiQure 5. It will be apparent to those skilled in the an that the nucleotide sequence of the invention may also find useful application when present as an "antisense"

sequence. Accordingly, functionally equivalent sequences will also include those sequences which can hvbridise. under strinQent hvbridisation conditions. to the sequence of the invention (rather than the complement thereof). Such "antisense"
equivalents will preferably possess at least 85%. more preferably at least 90 17c, and most prer"erabiv 95%
sequence homology with the complement of the sequence of the invention as exemplifed bv nucleotides 289 to 2790 of the DNA sequence shown in Figure 5. Particular functional equivalents are shown, for example. in Figures 8 and 10 (if one disregards the various frameshift mutations twted therein).

The invention also provides vectors, particularlv expression vectors, comprising the nucleotide sequence of the invention. The vector will typically comprise a promoter and one or more reaulatory signals of the type well known to those skilled in the art. The invention also includes provision of cells transformed (which term encompasses transduction and transfection) with a vector comprising the nucleotide sequence of the invention.

The invention further provides a class A SBE polypeptide, obtainable from potato plants.
In particular the invention provides the polypeptide in substantially pure form, especially in a form free from other plant-derived (especially pctato plant-derived) components, which can be readily accomplished by expression of the relevant nucleotide sequence in a suitable non-plant host (such as anv one of the veast strairrs routinely used for expression purposes, e. a. Pichia spp. or Sacchar ornvices spp). Typically the enzyme will substantially comprise the sequence of amino acid residues 49 to 882 shown in Figure 5 (disregarding the sequence MNYRIDL., which is not part of the enzvrrie), or a functional equivalent thereof. The polypeptide of the invention may be used in a method of modifying starch in vitro. comprisine treatinQ starch under suitable conditions (e.g.
appropriate temperature, pH. etc) with an effective amount of the polvpeptide accordina to the invention.

The term "functional equivalent". as applied herein to amino acid sequences.
is intended to encompass amino acid sequerices substantiallv similar to that shown in Figure 5. such that the polvpeptide possesses sufficient activitv to complement the branching enzvme rnutation in E. coli KV 832 cells (described below) and which is active in E.
coii in the phosphorvlation stimuiation assay. Typically such functionally equivalent amino acid sequences will preferably possess at least 85 ~, more preferably at least 90 %, and most preferably at least 95 ',,a sequence identity with the amino acid sequence of the mature enzyme (i.e. minus leader sequence) shown in Figure 5. Those skilled in the art will appreciate that conservative substitutions mav be made Qenerally throughout the molecule without substantiallv affectina the activity of the enzvme. Moreover, some non-conservative substitutions mav be tolerated, especially in the less highly conserved regions of the molecule. S~:ch substitutions may be made, for example, to modify slightly the activity of the enzyme. The polypeptide may, if desired, include a leader sequence, such as that exemplified by residues 1 to 48 of the amino acid sequence shown in Fi-aure 5, althouah other leader sequences and siQnal peptides and the like are known and may be included.

A portion of the nucleotide sequence of the invention has been introduced into a plant and found to affect the characteristics of the plant. In particular, introduction of the sequence of the invention, operably linked in the antisense orientation to a suitable promoter, was found to reduce the amount of branched starch molecules in the plant.
Additionally, it has recently been demonstrated in other experimental systems that "sense suppression" can aiso occur (i.e. expression of an introduced sequence operably linked in the sense orientation can interfere, by some unknown mechanism, with the expression of the native Qene), as described by Matzke & Matzke (1995 Plant Physiol. 107, 679-685). Anv one of the methods mentioned by Matzke & Matzke could, in theory, be used to affect the expression in a host of a homolog-ous SBE gene.

It is believed that antisense methods are mainly operable by the production of antisense mRNA which hvbridises to the sense mRh1A, preventing its translation into functional polvpeptide, possibly bv causina the hvbrid R\A to be degraded (e.a. Sheehy er al., 1988 PNAS 85. 8805-8809: %,an der Krol er al.. Mol. Gen. Genet. 220, 204-212).
Sense suppression also requires homology between the introduced seauence and the tarQet 'Zene, but the exact mechanism is unciear. It is apparent however that, in relation to both antisense and sense suppression. neither a full length nucleotide sequence.
nor a"native"
sequence is essential. Preferably the "effective portion" used in the method will comprise at least one third of the full len2th sequence, but by simple trial and error other fraaments (smaller or larger) may be found which are rsnctional in alterinQ the characteristics of the plant.

Thus. in a further aspect the invention orovides a method of altering the characteristics of a plant. comprising introducing into the plant an effective portion of the sequence of the invention operably linked to a suitabie promoter active in the plant.
Conveniently the sequence will be linked in the anti-sense orientation to the promoter.
Preferably the plant is a potato plant. Conveniently, the characteristic altered relates to the starch content and/or starch composition of the plant (i.e. amount and/or type of starch present in the plant). Preferabty the method of altering the characteristics of the plant will also comprise the introduction of one or more further sequences, in addition to an effective portion of the sequence of the invention. qhe introduced sequence of the invention and the one or more further sequences (which rnay be sense or antisense sequences) may be operably linked to a singie promoter (which would ensure both sequences were transcribed at essentially the same time), or may be operably linked to separate promoters (which may be necessary for optimal expression). Where separate promoters are employed they may be identical to each other or different. Suitable promoters are well known to those skilled in the art and include both constitutive and inducible types. Examples include the CaMV
35S promoter (e.a. single or tandem repeat) and the patatin promoter.
Advantaizeously the promoter will be tissue-specific. Desirably the promoter will cause expression of the operably linked sequence at substantial levels only in the tissue of the plant where starch svnthesis and/or starch storaae mainlv occurs. Thus, for example, where the sequence is introduced into a potato plant. the operably linked promoter may be tuber-specific, such as the patatin promoter.

Desirably, for example, the method will also comprise the introduction of an effective portlon of a sequence encoding a class B SBE. operably linked in the antisense orientation to a suitable promoter active in the plant. Desirably the further sequence will comprise an effective portion of the sequence encodina the potato class B SBE molecule.
Convenientiv the fi2rther sequence %vill comprise an effective portion of the sequence described by Blennow ~ Johansson (1991 Phytochem. 30, 437-444) or that disclosed in W092/11375. More preferablv. the further sequence will comprise at least an effe;.tive portion of the sequence disclosed in Internatiorial Patent Application No. WO
95/26407.
Use of antisense sequences against both class A and class B SBE in combination has now been found bv the present inventors to result in the production of starch havinLy very greatly altered properties (see below). Those skilled in the art will appreciate the possibility that, if the plant already comprises a sense or antisense sequence which efficiently inhibits the class B SBE activitv, introduction of a sense or antisense sequence to inhibit class A SBE activitv (therebv producing a plant with inhibition of both class A
and class B activity) miQht alter ?reatly the properties of the starch in the plant, without the need for introduction of one or more further sequences. Thus the sequence of the invention is convenientlv introduced into plants already having low levels of class A
and/or class B SBE activity, such that the inhibition resultinLy from the introduction of the sequence of the invention is likely io have a more pronounced effect.

The sequence of the invention, and the one or more further sequences if desired, can be introduced into the plant by any one of a number of well-known techniques (e.g.
Agrobacterium-mediated transformation. or by "biolistic" m.ethods). The sequences are likely to be most effective in inhibiting SBE activity in potato plants, but theoretically could be introduced into any plant. Desirable examples include pea, tomato, maize, wheat, rice, barley, sweet potato and cassava plants. Preferably the plant will comprise a natural gene encodine an SBE molecule which exhibits reasonable homology with the introduced nucleic acid sequence of the invention.

In another aspect, the invention provides a plant cell. or a plant or the progenv thereof, which has been altered by the method defined above. The progeny of the altered plant mav be obtained. for example, bv veaetative propagation. or by crossing the altered plant and reserving the seed so obtained. The inv,,mtion also provides parts of the altered plant, such as storase orQans. Convenientlv, for example, the invention provides tubers comprisina altered starch. said tubers being obtained from an altered plant or the proLyeny thereof. Potato tubers obtained from altered plants (or the progeny thereof) will be particularly useful materials in certain industrial applications and for the preparation andior processing of foodstuffs and may be used. for example. to prepare low-fat waffles and chips (amvlose L- nerallv being used as a coatina to prevent fat uptake). and to prepare mashed potato (especiallv "instant" mashed potato) havin~ particular characteristics.

In particular relation to potato plants. the invention provides a potato plant or pan thereof which, in its wild type possesses an effective SBE A aene, but which plant has been altered such that there is no effective expression of an SBE A polypeptide within the cells of at least part of the plant. The plant may have been altered by the method defined above, or may have beeri selected by conventional breeding to be deleted for the class A
SBE gene, presence or absence oF which can be readily determined by screening samples of the plants with a nucleic acid probe or antibody specific for Ihe potato class A gene or sene product respectively.

The invention also provides starch extracted from a plant altered bv the method defined above, or the proQeny of such a plant. the starch having altered properties compared to starch extracted from equivalent, but unaltered, plants. The invention further provides a method of making altered starch, comprising altering a plant by the method defined above and extracting therefrom starch having altered properties compared to starch extracted from equivalent, but unaltered, plants. Use of nucleotide sequences in accordance with the invention has allowed the present inventors to produce potato starches having a wide variery of novel properties.

In particular the invention provides the followinJ: a plant (especially a potato plant) altered bv the method defined above, containins! starch which. when extracted from the plant, has an elevated endotherm peak temperature as judged by DSC, compared to starch extracted from a similar, but unaltered, plant: a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has an elevated viscosity onset temperature (converuentlv elevated by 10 - 25 C) as judaed bv viscoamvloaraph compared to starch extracted from a similar, but unaltered, plant: a plant (especiallv a potato plant) altered by the method defined above, curuainina starch which, when extracted from the plant. has a decreased peak viscosity (conveniently decreased by 240 - 700SNUs) as iud2ed bv viscoamvlozraph compared to starch extracted from a similar, but unaltered. plant: a plant (especiallv a potato plant) altered by the method defined above. containinQ starch which, when extracted from the plant, has an increased pastinv viscosirv (conveniently increased bv 3 7-?60SNlis) as }udged bv viscoamylograph compared to starch extracted from a similar. but unaltered. plant: a plant (especially a potato plant) altered bv the method defined above, containinQ starch which, when extracted from the plant, has an increased set-back viscosity (convenientlv increased by SNUs) as judged bv viscoamvloaraph compared to starch extracted from a similar, but unaltered, plant: a plant (especially a potato plant) altered by the method defined above, containing starch which, when extracted from the plant, has a decreased set-back viscosity as judged by viscoamyloaraph compared to starch extracted from a similar, but un2ltered, plant; and a plant (especially a potato plant) altered by the method defined above, containino starch which, when extracted from the plant, has an elevated amvlose content as judged bv iodometric assav (i.e. bv the method of Morrison & Laignelet 1983. cited above) compared to starch extracted from a similar, but unaltered, plant. The invention also provides for starch obtainable or obtained from such plants as aforesaid.

In particular the invention provides for starch which, as extracted from a potato plant by wet milling at ambient temperature, has one or more of the following properties, as judged by viscoamylograph analysis performed according to the conditions defined below:
viscosity onset temperature in the range 70-95 C (preferably 75-95 C); peak viscosity in the range 500 - 12 stirring number units; pasting viscosity in the range 214 -434 stirring number units: set-back viscosity in the ranee 450 - 618 or 14 - 192 stirring number units;
or displavs no sienificant increase in viscosity during viscoamylograph. Peak, pasting and set-back viscosities are defined below. Viscosity onset temperature is the temperature at which there is a sudden, marked increase in viscosity from baseline levels during viscoamylograph, and is a term well-known to those skilled in the art.

In other particular embodiments. the invention provides starch which as extracted from a potato plant bv wet milling at ambient temperature has a peak viscositv in the range 200 -500 SNUs and a set-back viscositv in the range 275-618 SNUs as judaed bv viscoamvlograph accordino to the protocol defined below: and starch which as extracted from a potato plant bv wet miliinQ at ambient temperature l:ias a viscositv which does not decrease between the start of the heating phase (step 2) and the start of the final holding phase (step 5) and has a set-back viscosity of 303 SNUs or less as judged by viscoamvloQraph accordinsi to the protocoI defined below.

For the purposes of the present invention, viscoarnvlovraph conditions are undcrstood to pertain to analysis of a 10% (w/w) aqueous suspension of starch at atmospheric pressure, using a Newport Scientific Rapid Visco Analyser with a heating profile of:
holding at 50 C
for 2 minutes (step 1), heating from 50 to 95 C at a rate of 1.5 C per minute (step 2), holding at 95 C for 15 minutes (step 3),-coolina from 95 to 50 C at a rate of 1.5 C per minute (step 4), and then holding at 50 C for 15 minutes (step 5). Peak viscosiry may be defined for present purposes as the maximum viscositv attained during the heating phase (step 2) or the holding phase (step 3) of the viscoamvioaraph. Pasting viscosity may be defined as the viscosity attained by the starch suspensions at the end of the holding phase (step 3) of the viscoamylograph. Set-back viscosiry may be defined as the viscosity of the starch suspension at the end of step 5 of the viscoamvloaraph.

In yet another aspect the invention provides starch from a potato plant having an apparent ainylose content (% w/w) of at least 35 %, as judged by iodometric assay according to the method described by Morrison & Laignelet (1983 J. Cereal Science 1, 9-20).
Preferably the starch will have an amylose content of at least 40%, more preferably at least 50%, and most preferably at least 66 %. Starch obtained directly from a potato plant and having such properties has not hitherto been produced. Indeed, as a result of the present invention, it is now possible to generate in vivo potato starch which has some properties analogous to the very hiah amvlose starches (e.a. Hylon 7) obtainable from maize.

Starches with hiRh (at least 35 %) arnvlose contents find commercial application as, amonast other reasons. the amylose component of starch reassociates more strongly and rapidlv than the amylopectin component during retroaradation processes. This may result, for example, in pastes with higher viscosities, aels of greater cohesion, or films of areater strensth for starches with high (at least 35%) compared with normal (less than 35%) amvlose contents. Alternativelv, starches mav be obtained with verv hieh amvlose contents, such that the Qranule structure is substantially preserved during heatins, resulting in starch suspensions which demonstrate substantiallv no increase in viscosity during = ,~

cooking (i.e. there is no significant viscosity increase during viscoamvlooraph conditions defined above). Such starches typically exhibit a viscosity increase of less than 10%
(preferably less than 5 %) during viscoamvloQraph under the conditiDns defined above.
In commerce, these valuable properties are currently obtained from starches of high amylose content derived from maize plants. It would be of commercial value to have an altetnative source of high amylose starches from potato as other characteristics such as granule size, organoleptic properties and textural qualities may distinguish application performances of high amylose starches from maize and potato plants.

Thus high amylose starch obtained by the method of the present invention may find application in manv different technological fields. which may be broadly categorised into two groups: food products and processing; and "Industrial" applications. Under the heading of food products, the novel starches of the present invention may find application as, for example, films, barriers, coatings or gelling agents. In general, high amylose content starches absorb less fat during frying than starches with low amylose content, thus the high amylose content starches of the invention may be advantageously used in preparing low fat fried products (e.g. potato chips, crisps and the like). The novel starches may also be employed with advantage in preparing confectionery and in granular and retrograded "resistant" starches. "Resistant" starch is starch which is resistant to digestion by a-amylase. As such. resistant starch is not digested by a-amylases present in the human small intestine, but passes into the colon where it exhibits properties similar to soluble and insoluble dietarv fibre. Resistant starch is thus of great benefit in foodstuffs due to its low calorific value and its hiQh dietarv fibre content. Resistant starch is formed by the retrogradation (akin to recrvstallization) of amviose from starch gels.
Such retrogradation is inhibited bv amvlopectin. Accordinalv, the high amvlose starches of the present invention are excellent starting materials for the preparation of resistant starch.
Suitable methods for the preparation of resistant starch are well-known to those skilled in the art and include, for example, those described in US 5.051.1-71 and US
5.281.276.
Convenientiv the resistant starches provided by the present invention comprise at least 5 %
total dietarv fibre, as judged bv the method of Prosky et al.. (1985 J. Assoc.
Off. Anal.
Chem. 68. 677), mentioned in US 5.281. 276.

. # . .

~ ~ -Under the headina of "Industrial" appiications. the novel starches of the invention mav be advantageouslv emploved, for example. in corrugating adhesives. in biodeQradable products such as loose fill packaaina and foamed shapes, and in the production of glass fibers and textiles.

Those skilled in the art will appreciate that the novel starches of the -invention may, if desired, be subjected in vitro to conventional enzvmatic, physical andlor chemical modificatiori, such as cross-linking, introduction of hydrophobic aroups (e.g.
octenyl succinic acid, dodecyl succinic acid), or derivatization (e.g. by n:eans of esterification or etherification).

In yet another aspect the invention provides high (35% or more) amylose starches which Qenerate paste viscosities areater than those obtained from hiQh amylose starches from maize plants after processing at temperatures below 100 C. This provides the advantage of more economical starch gelatinisation and pasting treat?nents through the use of lower processing temperatures than are currently required for high amylose starches from maize plants.

The invention will now be further described by way of illustrative example and with reference to the drawinas, of which:

Figure 1 shows a typical viscoamylograph for a 10% w/w suspension of potato starch;
Figure 2 shows vsicoamvlographs for 10% suspensions of starch from various maize varieties;

Figure 3 is a schematic representation of the clonina strategy used by the present inventors;

Figure 4a shows the amino acid alignment of the C-tetminal portion of starch branching enzvme isoforms from various sources; amino acid residues matching the consensus sequence are shaded;

Fiaure 4b shows the ali2nment of DNA sequences of various starch branchina enzvme isoforms which encode a conserved amino acid sequence:

FiQure 5 shows the DNA sequence (Seq ID No. 14) and predicted amino acid sequence (Seq ID No. 15) of a full lenEith potato class A SBE cDNA clone obtained bv PCR;
Figure 6 shows a comparison of the most hiahly conserved part of the amino acid sequences of potato class A (uppermost sequence) and class B(lowermost sequence) SBE
molecules:

FiQure 7 shows a comparison of the amino acid sequence of the full length potato class A
(uppermost sequence) and pea (lowermost sequence) class A SBE molecules:

FiQure 8 shows a DNA alignment of various full length potato class A SBE
clones obtained by the inventors;

Figure 9 shows the DNA sequence of a potato class A SBE clone determined by direct sequencin; of PCR products. together with the predicted amino acid sequence;

Figure 10 is a multiple DNA alignment of various full length potato SBE A
clones obtained by the inventors;

Figure 11 is a schematic illustration of the plasmid pSJ64;

Fia-ure 12 shows the DNA sequence and predicted amino acid sequence of the full length potato class A SBE clone as present in the plasmid pSJ90: and Fiaure 13 shows viscoamvioaraphs for 10% w/w suspensions of starch from various transgenic potato plants made by the relevant method aspect of the invention.

,.=

Examples Example 1 Cloning of Potato class A SBE
The stratesy for cloning the second form of starch branchin(2 enzyme from potato is shown in Figure 3. The small arrowheads represent primers used by the inventors in PCR and RACE protocols. The approximate size of the fragments isolated is indicated by the numeraIs on the riaht of the Figure. By way of explanation, a comparison of the amino acid sequences of several cloned plant starch branching enzymes (SBE) from maize (class A), pea (class A), maize (class B), rice (class B) and potato (class B), as well as human glvcoeen branching enzyme, allowed the inventors to identify a region in the carboxy-terminal one third of the protein which is almost completely conserved (GYLNFMGNEFGHPEWIDFPR) (Figure 4a). A multiple alignment of the DNA
sequences (human, pea class A, potato class B, maize class B, maize class A
and rice class B, respectivelv) corresponding to this reoion is shown in Figure 4b and was used to design an oligo which would potentially hybridize to all known plant starch branching enzymes:
AATTT(C/T)ATGGGIAA(C/T)GA(A/G)TT(C/T)GG (Seq ID No. 20).

Librarv PCR
The initial isolation of a partial potato class A SBE cDNA clone was from an amplified potato tuber cDNA librarv in the XZap vector (Stratagene). One half gL of a potato cDNA library (titre 2.3 x 109pfu/mL) was used as template in a 50 uL reaction containing 100 pmol of a 16 fold degenerate POTSBE primer and 25 pmol of a T7 primer (present in the XZap vector 3' to the cDNA sequences - see Figure 3), 100 gM dNTPs, 2.5 U Taq polymerase and the buffer supplied with the Taq polvmerase (StratageneTm). All components except the enzyme were added to a 0.5 mL microcentrifuze tube. covered with mineral oil and incubated at 94 C for 7 minutes and then held at C. while the Taq polvmerase was added and mixed bv pipettinz. PCR was then performed by incubatinQ for 1 min at 94 C. 1 min at 58 C and 3 minutes at 72 C. for 35 cycles. The PCR products were extracted with phenol/chloroform. ethanol precipitated and resuspended in TE
pH 8.0 before clonino into the T/A cloninQ vector pT7BlueR (Invitrogen).

Several fraQments between 600 and 1300 bp were amplified. These were isolated from an agarose Qei and cloned into the pT7BlueR T-A cloning vector. Restriction mappina of 24 randomlv selected clones showed that thev belonged to several different Qroups (based on size and presencerabsence of restriction sites). Initiallv four clones were chosen for sequencing. Of these four. two were found to correspond to the known potato class B
SBE sequence, however the other two, althoush homologous, differed significantiv and were more similar to the pea class A SBE sequence, suggesting that thev belonged to the class A family of branching enzymes (Burton er al., 1995 The Plant Journal, cited Pbove).
The latter two clones (- 800bp) were sequenced fully. They both contained at the 5' end the sequence correspondins to the degenerate oligonucleotide used in the PCR
anL' had a predicted open reading frame of 192 amino acids. The deduced amino acid sequence was hiahly homologous to that of the pea class A SBE.

The - 800 bp PCR derived cDNA fraament (corresponding to nucleotides 2281 to of the psbe2 con.seq sequence shown in Figure 8) was used as a probe to screen the potato tuber cDNA library. From one hundred and eighty thousand plaques, seven positives were obtained in the primary screen. PCR analysis showed that five of these clones were smaller than the original 800 bp cDNA clone, so these were not analysed further. The two other clones (designated 3.2.1 and 3.1.1) were approximately 1200 and 1500 bp in length respectiveltr. These were sequenced from their 5' ends and the combined consensus sequence aligned with the sequence from the PCR generated clones. The cDNA
clone 3.2.1 was excised from the phage vector and plasmid DNA was prepared and the insert fullv sequenced. Several attempts to obtain longer clones from the library were unsuccessful, therefore clones containing the 5' end of the full length gene were obtained usinQ RACE (rapid amplification of cDNA ends).

Rapid Amplification of cDNA ends (RACE) and PCR conditions RA_C.E was performed essentiallv according to Frohman (1992 Amplifications 11-15).
Two ,cQ of total R.hTA from mature potato tubers was heated to 65 C for 5 min and quick cooled on ice. The RNA was then reverse transcribed in a 20 L reaction for 1 hour at 37 C using BRL's M-hILV reverse transcriptase and buffer with 1 mM DTT, 1 mN1 dNTPs. I U/uL RNAsin (Promeaa) and 500 pmol random hexamers (Pharmacia) as primer. Excess primers were removed on a Centricon 100TM_'oiun-in and cDNA was recovered and precipitated with isopropanol. cDNA was A-tailed in a volume of usina 10 units terminal transferase (BRL), 200 ,yl dATP for 10 min at 37 C.
followed by 5 min at 65 C. The reaction was then diluted to 0.5 ml with TE pH 8 and stored at 4 C as the cDNA pool. cDNA clones were isolated bv PCR amplification using the primers RR,dTj7, R and POTSBE24. The PCR was performed in 50 L using a hot start technique: 10 L of the cDNA pool was heated to 94 C in water for 5 min with 25 pmol POTSBE24, 25 pmol R, and 2.5 pmol of R R,dTt, and cooled to 75 C. Five L of x PCR buffer (Stratagene), 200 M dNTPs and 1.25 units of Taq polymerase were added, the mixture heated at 45 C for 2 min and 72 C for 40 min follow--d by 35 cycles of 94 C
for 45 sec, 50 C for 25 sec. 72 C for I.5 min and a final incubation at 72 C
for 10 min.
PCR products were separated by electrophoresis on I% low melting aoarose gels and the smear covering the ranae 600-800 bp fragments was excised and used in a second PCR
amplification with 25 pmol of R, and POTSBE25 primers in a 50 uL reaction (28 cycles of 94 C for 1 min. 50 C 1 min, 72 C 2 min). Products were purified by chloroform extraction and cloned into pT7 Blue. PCR was used to screen the colonies and the longest clones were sequenced.

The first round of RACE only extended the lenitth of the SBE sequence approximately 100 bases, therefore a new A-tailed cDNA library was constructed using the class A
SBE
specific oliao POTSBE24 (10 pmol) in an attempt to recover longer RACE
products. The first and second round PCR reactions were performed using new class A SBE
primers (POTSBE 28 and 29 respectively) derived from the new sequence data. Conditions were as before except that the eloneation step in the first PCR was for 3 min and the second PCR consisted of 28 cycles at 94 C for 45 seconds, 55 C for 25 sec and 72 C
for 1 min 45 sec.

Clones raneine in size from 400 bp to 1.4 kb were isolated and sequenced. The combined sequence of the longest RACE products and cDNA clones predicted a full length aene of about 3150 nucleotides. excludina the polv(A) tail (psbe ?con.seq in Fig. 8).

As the sequence of the 5' half of the Qene was compiled from the sequence of severai 2n 'v RACE products senerated usinm Taq polvmerase. it was possible that the compiled sequence did not represent that of a single mRNA species and/or had nucleotide sequence chanQes. The 5' 1600 bases of the 2ene was therefore re-isolated by PCR using liltrna, a thermostable DNA polvmerase which. because it possesses a 3*-.5' exonuclease activitv, has a lower error rate compared to Taq polymerase. Several PCR products were cloned and restriction mapped and found to differ in the number of Hind III. Ssp 1, and EcoR I
sites. These differences do not represent PCR artefacts as thev were observed in clones obtained from independent PCR reactions (data not shown) and indicate that there are several forms of the class A SBE aene transcribed in potato tubers.

In order to ensure that the sequence of the full length cDNA clone was derived from a sinale mRNA species it was therefore necessarv to PCR the entire Qene in one piece.
cDNA was prepared accordiniy to the RACE protocol except that the adaptor oligo R RIdTõ (5 pmol) was used as a primer and after synthesis the reaction was diluted to 200 AL with TE pH 8 and stored at 4 C. Two L of the cDNA was used in a PCR
reaction of 50 l.cL using 25 pmol of class A SBE specific primers PBERI and PBERT (see below), and thirty cycles of 94 for 1 min, 60 C for 1 min and 72 C for 3 min. If Taq polymerase was used the PCR products were cloned into pT7Blue whereas if Ultma polymerase was used the PCR products were purified bv chloroform extraction, ethanol precipitation and kinased in a volume of 20 L (and then cloned into pBSSK IIP
which had been cut with EcoRV and dephosphorylated). At least four classes of cDNA
were isolated, which aQain differed in the presence or absence of Hind III. Ssp I
and EcoR I
sites. Three of these clones were sequenced fully, however one clone could not be isolated in sufficient quantiry to sequence.

The sequence of one of the clones (number 19) is shown in FiQure 5. The first methionine (initiation) codon starts a short open readine frame (ORF) of 7 amino acids which is out of frame with the next predicted ORF of 882 amino acids which has a molecular mass (Mr) of approximatelv 100 Kd. Nucleotides 6-2996 correspond to SBE sequence -the rest of the sequence shown is vector derived. Fizure 6 shows a comparison of the most hi2h1y conserved part cf the amino acid sequence of potato class A SBE (residues 180-871, top, row) and potato class B SBE (bottom row, residues 98-792): the middle row indicates the

2 l de2ree of similarity, identical re~idues being denoted by the common letter.
conservative changes by two dots and neutral changes bv a sir.ale dot. Dashes indicate gaps introduced to optimise the aliQnment. The class A SBE protein has 44% identity over the entire lenzth with potato class B SBE. and 56% identity therewith in the centra! --onserved domain (Fiaure 6), as judQed bv the ":vleLyaliEn" proaram (DNASTAR). However.
Fiaure 7 shows a comparison between potato class A SBE (top row, residues 1-873) and pea class A SBE (bottom row, residues 1-861). from which it can be observed that cloned potato gene is more homologous to the class A pea enzyrne, where the identity is 70 %
over nearlv the entire length, and this increases to 83 % over the central conserved region (starting at IPPP at position -170). It is clear from this analysis that this cloned potato SBE gene belongs to the class A family of SBE genes.

An E. coli culture, containinQ the piasmid pSJ78 (which directs the expression of a full length potato SBE Class A gene), has been deposited (on 3rd January 1996) under the terms of the Budapest Treaty at The National Collections of Industrial and Marine Bacteria Limited (23 St Machar Drive, Aberdeen, AB2 IRY, United Kingdom), under accession number NCIMB 40781. Plasmid pSJ78 is equivalent to clone 19 described above.
It represents a full length SBE A cDNA blunt-end ligated into the vector pBSSK.IIP.

Polymorphism of class A SBE genes Sequence analysis of the other two full length class A SBE genes showed that they contain frameshift mutations and are therefore unable to encode full length proteins and indeed they were unable to complement the branching enzyme deficiency in the KV832 mutant (described below). An alignment of the full lenath DNA sequences is shown in Fieure 8: "lOcon.seq" (Seq ID No. 12), "19con.seq" (Seq ID No. 14) and "llcon.seq"
(Seq ID
No. 13) represent the sequence of full lenath clones 10. 19 and 11 obtained by PCR using the PBER1 and PBERT primers (see below). whilst "psbe2con.seq" (Seq ID No. 18) represents the consensus sequence of the RACE clones and cDNA clone 3.2.1.
Those nucleotides which differ from the overall consensus sequence (not shown) are shaded.
Dashes indicate gaps introduced to optimise the alignment. Apart from the frameshift mutations these ciones are hiahIv homologous. It should be noted that the 5' sequence of psbe2con is longer because this is the lonaest RACE product and it also contains several G ~.

chan(zes compared tD the other clones. The upstream methionine codon is still present in this clone but the upstream ORF is shortened to just 3 amino acids and in addition there is a 10 base deletion in the 5' untranslated leader.

The other siL7nificant area of variation is in the carboxy terminal re!yzion of the protein codinLy region. Closer examination of this area reveals a GAA trinucleotide repeat structure which varies in lenath between the four clones. These are typical characteristics of a microsatellite repeat region. The most diverLrent clone is #11 which has only one GAA triplet whereas clone 19 has eleven perfect repeats and the other two clones have five and seven GAA repeats. All of these deletions maintain the ORF but char.ye the number of glutamic acid residues at the carboxy terminus of the protein.

Most of the other differences between the clones are single base changes. It is quite possible that some of these are PCR errors. To address this question direct sequencing of PCR fragments amplified from first strand cDNA was performed. Figure 9 shows the DNA sequence, and predicted amino acid sequence, obtained by such direct sequencing.
Certain restriction sites are also marked. Nucleotides which could not be unambiguously assigned are indicated using standard IUPAC notation and, where this uncertainty affects the predicted amino acid sequence, a question mark is used. Sequence at the extreme 5' and 3' ends of the gene could not be determined because ot the heteroQeneity observed in the different cloned Qenes in these regions (see previous paragraph). However this can be taken as direct evidence that these differences are real and are not PCR or cloning artefacts.

There is absolutely no evidence for the frameshift mutations in the PCR
derived sequence and it would appear that these mutations are an artefact of the cloning process. resulting from negative selection pressure in E. coli. This is supported by the fact that it proved extremely difficult to clone the full length PCR products intact as many large deletions were seen and the full lenath clones obtained were all cloned in one orientation (away from the LacZ promoter), perhaps suggesting that expression of the gene is toxic to the cells. Difficulties of this nature may have been responsible. at least in pan.
for the previous failure of other researchers to obtain the present invention.

A comparison of all the full lenoth sequences is shown in Fiaure 10. In addition to clones 10. 11 and 19 are shown the sequences of a Bgl II - Xho I product cloned directly into the QE32 expression vector ("86CON.SEQ", Seq ID 'lo. 16) and the consensus sequence of the directly sequenced PCR products ("pcrsbe2con.seq". Seq ID No. l;). Those nucleotides which differ from the consensus sequence (not shown) are shaded, Dashes indicate gaps introduced to optimise the alignment. There are 11 nucleotide differences predicted to be present in the mRNA population. which are indicated by asterisks above and below the sequence The other differences are probably PCR artefacts or possihlv sequencincy errors.

Complementation of a branching enzvme deficient E. coli mutant To determine if the isolated SBE 4ene encodes an active protein i.e. one that has branching enzvme activitv, a complementation test was performed in the E. coli strain KV832. This strain is unable to make bacterial alvcoaen as the Qene for the glycoeen branching enzyme has been deleted (Keil et al., 1987 Mol. Gen. Genet. 207, 294-301).
When wild type cells are grown in the presence of glucose they svnthesise glyco;en (a highlv branched glucose polvmer) which stains a brown colour with iodine, whereas the KV832 cells make only a linear chain alueose polymer which stains blueish green with iodine. To determine if the cloned SBE aene could restore the ability of the KV832 cells to make a branched polymer, the clone pSJ90 (Seq ID No. 19) was used and constructed as below. The construct is a PCR-derived. substantiallv full length fragment (made using primers PBE 2B and PBE 2X. detailed below), which was cut with Bgl II and K'ho I and cloned into the BamH I Sal I sites of the His-tag expression vector pQE32 (Qiagen).
This clone, pSJ86. was sequenced and found to have a frameshift mutation of two bases in the 5' half of the Qene. This frameshift was removed by digestion with Nsi I and SnaB
I and replaced with the correspondinQ fra8ment from a Taq-generated PCR clone to produce the plasmid pSJ90 (sequence shown in Fi-aure 12; the first 10 amino acids are derived from the expression vector). The polypeptide encoded by pSJ90 would be predicted to correspond to amino acids 46-882 of the full SBE codinsz sequence. The construct pSJ90 was transformed into the branchinQ enzyme deficient KV832 cells and transformants were arowr, on solid PYG medium (0.85 ','o KH_PO4, 1.19o K,HPO;, 0.6 ','o veast extract) containinQ 1.0% alucose. To test for compiementation. a loop of cells was scraped off and resuspended in 150 l of water. to which was added 15kcl Lugol's solution (2Q KI and 19 I, per 300m1 water). It was found that the potato SBE fraszment-transformed KV832 cells now stained a yellow-brown colour with iodine whereas control cells containina only the pQE32 vector continued to stain blue-green.

Expression of potato class A SBE in E. coli Sinsle colonies of KV832. containins! one of the plasmids pQE32. pAGCRl or pSJ90, were picked into 50m1 of 2xYT medium contairunz carbenicillin, kanamycin and streptomycin as appropriate (100, 50 and 25 mg/L. respectively) in a 250m1 flask and grown for 5 hours, with shaking., at 37 C. IPTG was then added to a final concentration of ImM to induce expression and the flasks were further incubated overnight at 25 C.
The cells were harvested bvi centrifusation and resuspended in 50 mM sodium phosphate buffer (pH 8.0), containing 300mM NaCI, Ima/ml lysozvme and ImM PMSF and left on ice for 1 hour. The cell lysates were then sonicated (3 pulses of 10 seconds at 40% power using a microprobe) and cleared bvi centrifuzation at 12,000Q for 10 minutes at 4 C.
Cleared lysates were concentrated approximately 10 fold in a CentriconT'' 30 filtration unit. Duplicate 10 1 samples of the resultin; extract were assayed for SBE
activity by the phosphorylation stimulation method, as described in International Patent Application No.
WO 95/26407. In brief, the standard assay reaction mixture (0.2m1) was 200mM 2-(N-morpholino) ethanesulphonic acid (MES) buffer pH6.5, containing 100nCi of alucose-l-phosphate at 50mM. 0.05 ms rabbit phosphorylase A. and E. coli lysate. The reaction mixture was incubated for 60 minutes at 30 C and the reaction terminated and szlucan polymer precipitated by the addition of lml of 75 % (v/v) methanol, 1%(w/v) potassium hvdroxide, and then 0. lml Qlycogen f 10mg/ml). The results are presented below:

Construct + SBE Activitv (cpm) pQE32 (control) 1,829 pSJ90 (potato class A SBE) 14,327 pAGCRI (pea class A SBE) 29,707 The potato class A SBE activity is 7-8 fold above background levels. It was concluded therefore that the potato class A SBE aene was able to complement the BE
mutation in the phosphorylation stimulation assav and that the cloned aene does indeed code for a protein with branching enzyme activitv.

Oligonucleotides The following synthetic oligonucleocides (Seq ID No.s 1-I1 respectively) were used:
RaR1dT17 AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGA(T)17 Ro AAGGATCCGTCGACATC
RI GACATCGATAATACGAC

POTSBE28 ATGTTCAGTCCATCT.aAAGT

PBERT CGTCCCAGCATTCGACATAA

Exampie 2 Production of Transgenic Plants Construction of plant transformation vectors with antisense starch branching enzyme genes A 1200 bp Sac I - Xho I fraament, encoding approximately the -COOTti half of the potato class A SBE (isolated from the rescued XZap clone 3.2.1), was cloned into the Sac I- Sal I sites of the plant transformation vector pSJ29 to create plasmid pSJ64, which is illustrated schematicallv in Figure 11. In the fiQttre, the black line represents the DNA
sequence. The broken line represents the bacterial plasmid backbone (containina the oriQin of replication and bacterial selection marker), which is not shown in, full. The filled trianQles on the line denote the T-DNA borders (RB = right border, LB = left border).
Relevant restriction sites are shown above the black line, with the approximate distances (in kilobases) between the sites (marked bv an asterisk) -aiven bv the numerals below the line. The thinnest arrows indicate polvadenvlation signals (pAnos = nopaline synthase, pAg7 = Aarobacterium Lyene 7), the arrows intermediate in thickness denote protein coding reaions (SBE II = potato class A SBE. HYG = hvaromvcin resist.ance aene) and the thickest arrows represent promoter regions (P-?x35 = double Ca.VIV 35S
promoter, Pnos = nopaline svnthase promoter). Thus pSJ64 contained the class A SBE aene fra2rnent in an antisense orientation between the 2X 35S CaMV promoter and the nopaline svnthase polyadenylation siLynal.

For information, pSJ29 is a derivative of the binary vector pGPTV-HYG (Becker et 31., 1992 Plant Molecular Biolozy 20, 1195-1197) modified as follows: an approximately 750 bp (Sac I, T4 DNA polvmerase blunted - Sal I) fragment of pJIT60 (Guerineau et al., 1992 Plant Mol. Biol. 18, 815-818) containing the duplicated cauliflower mosaic virus (CaMV) 35S promoter (Cabb-JI strain, equivalent to nucleotides 7040 to 7376 duplicated upstream of 7040 to 7433 . Frank et al., 1980 Cell 21, 285-294) was cloned into the Hind III (Klenow polvmerase repaired) - Sal I sites of pGPTV-HYG to create pSJ29.

Plant transformation Transformation was conducted on two types of potato plant explants; either wild type untransformed minitubers (in order to give single transformants containing the class A
antisense construct alone) or minitubers from three tissue culture lines (which gave rise to plants #12, #15, #17 and #18 indicated in Table 1) which had already been successfully transformed with the class B (SBE I) antisensz construct containinQ the tandem promoter (so as to obtain double transformant plants, containing antisense sequences for both the class A and class B enzymes).

Details of the method of Aarobacterium transformation. and of the Qrowth of transformed plants. are described in International Patent Application No. WO 95/26407, except that the medium used contained 3% sucrose (not 1%) until the final transfer and that the initial incubation with Aarobacterium (strain 3850) was performed in darkness.
Transformants containinQ the class A antisense sequence were selected bv arowth in medium containina I5maiL hvaromvcin (the class A antisense construct comprisinQ the HYG gene, i.e.
hvaromvcin phosphotransferase) .

Transformation was confirmed in all cases by production of a DNA fragment from the antisense sene after PCR in the presence of appropriate primers and a crude extract of aenomic DNA from each regenerated shoot.

Characterisation of starch from potato plants Starch was extracted from plarits as follows: potato tubers were homoaenised in water for 2 minutes in a WaringTM blender operating at high speed. The homogenate was washed and filtered (initiallv throucrr 2mm, then through lmm filters) using about 4 litres of water per, llOOgms of tubers (6 eYtractions). Washed starch (zranules were finallv extracted with acetone and air dried.

Starch extracted from singly transformed potato plants (class A/SBE II
antisense. or class B/SBE I antisense), or from double transformants (class A/SBE II and class B/SBE I
antisense), or from untransformed control plants, was partially characterised.
The results are shown in Table 1. The table shows the amount of SBE activity (units/gram tissue) in tubers from each transformed plant. The endotherm peak temperature ( C) of starch extracted from several plants was determined by DSC, and the onset temperature ( C) of pastin; was determined by reference to a viscoamylograph ("RVA"), as described in WO
95/26407. The viscoamylograph profile was as follows: step 1- 50 C for 2 minutes; step 2 - increase in temperature from 50 C to 95 C at a rate of 1.5 C per minute;
step 3 -holding at 95 C for 15 minutes: step 4 - cooling from 95 C to 50 C at a rate of 1.5 C per minute; and finallv, step 5 - holding at 50 C for 15 minutes. Table 1 shows the peak, pasting and set-back viscosities in stirrinc, number units (SNUs), which is a measure of the amount of torque required to stir the suspensions. Peak viscosity may be defined for present purposes as the maximun viscositv attained durins the heating phase (step 2)= or the holding phase (step 3) of the viscoamylograph. Pasting viscosity may be defined as the viscosity attained by the starch suspensions at the end of the holding phase (step 3) of the viscoamylograph. Set-back viscosity may be defined as the viscosity of the starch suspension at the end of step 5 of the viscoamylograph.

A determination of apparent amylose content (7c w/w) was also performed. using the iodometric assav method of Morrison & Laipnelet (1983 J. Cereal Sci. 1, 9-20).
The results (percentaae apparent amylose) are shown in Table 1. The untransformed and transformed control plants gave rise to starches having apparent amvlose contents in the ranQe 29(+/-3)%.

Generally similar values for amylose content were obtained for starch extracted from most of the sin(Ylv transformed plants containina the class A (SBE II) antisense sequence.
However, some plants (#152, 249) Qave rise to starch having an apparent amylose content of 37-38%, notably higher than the control value. Starch extracted from these plants had markedly elevated pastini, onset temperatures, and starch from plant 152 also exhibiteL- an elevated endotherm peak temperature (starch from plant 249 was not tested bv DSC).

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m a Q a d d ~ ~ ~n z SUBSTITUTE SHEET (RULE 26) 4 ~ O O
r d aa ' v .- as 0 ul cp c n (N
f''N If1 ~D t0 <O ~A 0 t~~t 9 N mv VD YJ
r r r- N fy to ts.
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SUBSTITUTE SHEET (RULE 26) ry It should be noted that. even if other single transfotmants were not to provide starch with an altered amvloseiamvlopectin ratio. the starch from such plants might still have different properties relative to starch from conventional plants (e. a. different average molecular weiQht or different amvlopectin branchin(z patterns). which misht be useful.

Double transforrnant plants. containing antisense sequences for both the class A and class B enzymes, had greatly reduced SBE activity (units/gm) compared to untransformed plants or single anti-sense class A transformants. (as shown in Table 1). Moreover, certain of the double transformant plants contained starch having very significantly a'tered properties. For exa.mple, starch extracted from plants #201, 202. 208, 208a, 2'_~6 and 236a had drastically altered amylose/amylopectin ratios, to the extent that amylose was the main constituent of starch from these plants. The pasting onset temperatures of starch from these plants were also the most areatly increased (by about 25-30 C).
Starch from plants such as #150, 161, 212. 220 and 230a represented a range of intermediates, in that such starch displayed a more modest rise in both amvlose content and pasting onset temperature. The results would tend to suggest that there is generally a correlation between % amylose content and pasting onset temperature, which is in agreement with the known behaviour of starches from other sources, notably maize.

The marked increase in amylose content obtained by inhibition of class A SBE
alone, compared to inhibition of class B SBE alone (see WO 95/26407) might suggest that it would be advantageous to transform plants first with a construct to suppress class A SBE
expression (probably, in practice, an antisense construct), select those plants giving rise to starch with the most altered properties, and then to re-transform with a construct to suppress class B SBE expression (again. in practice. probably an antisense construct), so as to maximise the deQree of starch modification.

In addition to pastina onset temperatures. other features of the viscoamylograph profile e.g. for starches from plants #149, 150, 152. 161, 201. 236 and 236a showed sisnificant differences to starches from control plants. as illustrated in Figure 13-3.
Referring to Fiaure 13. a number of viscoamylograph traces are shown. The legend is as follows:
shaded box - normal potato starch control (29.8 % amylose content): shaded circle -starch from plant 149 (35.6% amv?ose)-. shaded triangle, pointina upwards - plant 152 (37.5%):
shaded trianzle, pointina downwards - plant 161 (40.9 shaded diamond - plant 150 (53.1 %);
unshaded box - plant 236a (56.7 7,c); unshaded circle - piant 236 t,60.1 ',C);
unshaded trianQle, pointing upwards - plant 201 (66.4~); unshaded trianzle. pointing downwards -Hvlon V starch. from maize (44.9 % amylose). The thin line denotes the heatina profile.
With increasing amylose content. peak viscosities during processing to 95 C
decrease, and the drop in viscosity from the peak until the end of the holding period at 95 C also generally decreases (indeed. for some of the starch samples there is an increase in viscosity during this period). Both of these results are indicative of reduced granule fraQmentation. and hence increased granule stabilitv during pasting. This property has not previously been available in potato starch without extensive prior chemical or physical modification. For applications where a maximal viscosity after processinQ to 95 C is desirable (i.e. correspondina to the viscosity after 47 minutes in the viscoamylograph test), starch from piant #152 would be selected as starches with both lower (Controls, #149) and higher (#161, #150) amylose contents have lower viscosities following this gelatinisation and pasting regime (Figure 13 and Table 1). It is believed that the viscosity at this stage is determined by a combination of the extent of granule swelling and the resistance of swollen granules to mechanical fragmentation. For any desired viscosity behaviour, one skilled in the art would select a potato starch from a range containing different amylose contents produced according to the invention by performing suitable standard viscosiry tests.

Upon cooling pastes from 95 C to 50 C, potato starches from most plants transformed in accordance with the invention showed an increase in viscoamvlograph viscosity as expected for partial reassociation of amviose. Starches from plants #149, 152 and 161 all show viscosities at 50 C significantlv in excess of those for starches from control plants (Fiaure 13 and Table 1). This contrasts with the effect of elevated amvlose contents in starches from maize plants (Figure 2) which show verv low viscosities throuQhout the viscoamvloQraph test. Of particular note is the fact that, for similar amvlose contents, starch from potato plant 150 (53 % amvlose) shows markedly increased viscosity compared with Hvlon 5 starch (44.9% amvlose) as illustrated in Figur.e 13. This demonstrates that useful properties which require elevated (35~ or ~reater) amviose ievels can be obtained bv processing starches from potato plants below 100 C. whereas more enerL7v-intensive processing is required in order to Qenerate similarly useful properties rrom hiLyh amylose starches derived from maize plants.

Final viscositv in the viscoamylograph test (set-back viscositv after 92 minutes) is Qreatest for starch from plant #161 (40.9% amylose) amongst those tested (Figure 13 and Table 1). Decreasing final viscosities are obtained for starches from plant #152 (37.5%
amylose), #149 (35.6% amylose) and #150 (53.1% amylose). Set-back viscositv o~,curs where amylose molecules, exuded from the starch granule durin7 pastinlg, start r; re-associate outside the 2ranule and form a viscous gel-like substance. It is believed that the set-back viscositv values of starches from transaenic potato plants represent a balance between the inherent amvlose content of the starches and the ability of the amviose fraction to be exuded from the aranule durinlg pasting and therefore be available for the reassociation process which results in viscosity increase. For starches with low amylose content, increasinz the amylose content tends to make more amylose available for re-association, thus increasin' the set-back viscosity. However, above a threshold value, increased amylose content is thought to inhibit granule swelling, thus preventin-2 exudation of amvlose from the starch Qranule and reducing the amount of amylose available for re-association. This is supported bv the RVA results obtained for the very high amylose content potato starches seen in the viscoamvlograph profiles in Figure 13. For any desired viscositv behaviour following set-back or retro2radation to any desired temperature over anv desired timescale, one skilled in the art would select a potato starch from a ranae containina different amylose contents produced according to the invention by performing standard viscositv tests.

Further experiments witll starch from plants #201 and 208 showed that this had an apparent amvlose content of over 6? r7c (see Table 1). Viscoamylogyraph studies showed that starch from these plants had radically altered properties and behaved in a manner similar to hvlon 5 starch from maize plants (FiQure 13). Under the conditions emploved in the viscoamvloaraph. this starch exhibited extremely limited (nearly undetectable) aranule swelling. Thus, for example, unlike starch from control plants, starch from plants .J 3 201. 208 and 208a did not displav a clearly dPfined pasting viscositv peak during the heatinp phase. Microscopic analvsis confirmed that the starch granule structure underwent only minor swelling during the experimental heatinQ process. This property may well be particularly useful in certain appiications, as will be apparent to those skilled in the art.
Some re-arown plants have so far been found to increase still further the apparent amylose content of starch extracted therefrom. Such increases may be due to:-i) Growth and development of the first aeneration transformed plants may have been affected to some degree by the exogenous growth hormones present in the tissue culture svstem, which exoaenoous hormones were not present durin- growth of the second 2eneration plants; and ii) Subsequent aenerations were arown under field conditions, which may allow for attainment of greater maturity than growth under laboratory conditions, it being generally held that amylose content of potato starch increases with maturitti of the potato tuber.
Accordingly, it should be possible to obtain potato plants Qiving rise to tubers with starch having an amvlose content in excess of the 66% level so far attained, simply by analysing a greater number of transformed plants andlor by re-growing transgenic plants through one or more generations under field conditions.

Table 1 shows that another characteristic of starch which is affected by the presence of anti-sense sequences to SBE is the phosphorus content. Starch from untransformed control plants had a phosphorus content of about 60-710mg/100aram drv weight (as determined according to the AOAC Official Methods of Analysis, 15th Edition, Method 948.09 "Phosphorus in Flour"). Introduction into the plant of an anti-sense SBE B
sequence was found to cause a modest increase (about two-fold) in phosphorus content, which is in agreement with the previous findings reported at scientific meetings.
Similarly, anti-sense to SBE A alone causes only a small rise in phosphorus content relative to untransformed controls. However, use of anti-sense to both SBE A and B in combination results in up to a four-fold increase in phosphorus content. which is far areater than any in planta phosphorus content previously demonstrated for potato starch.

This is useful in that, for certain applications. starch must be phosphorylated in virro by 34 _ chemical modification. The ability to obtain potato starch which, as extracted from the plant, alreadv has a high phosphorus content will reduce the amount of in vitro phosphorylation required suitabiv to modify the starch. Thus, in another aspect the invention provides potato starch which. as extracted from the plant. has a phosphorus content in excess of 200mg/ 100Qram drv weiaht starch. Tvpicallv the starch will have a phosphorus content in the range 200 - 240ma/ I00gram dr,v weight starch.

SEQUENCE LISTING
(1) GENERAL INFORMATION:

('t) APPLICANT:
(A) NAME: National Starch and Chemical (nvestment Holding Corporation (B) STREET: 501 Silverside Road, Suite 27 (C) CITY: Wilmington (D) STATE: Delaware (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP): 19809 (ii) TITLE OF iNVENTIQN: Improvements in or Relating to Plant Starch Composition (iii) NUMBER OF SEQUENCES: 20 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disK
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentfn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AAGGATCCGT CGACATCGAT AATACGACTC ACTATAGGGA Ti TTI-TTTTT TTTTTTT 57 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:

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(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear . "a , .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATÃC1'~', FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucieic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTÃOfj: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3003 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GATGGGGCCT TGAACTCAGC AATTTGACAC TCAGTTAGTT ACACTGCCAT CACTTATCAG

ATCTCTATTT TTTCTCTTAA TTCCAACCAA GGAATGAATA AAAAGATAGA TTTGTAAAAA

CCCTAAGGAG AGAAGAAGAA AGATGGTGTA TACACTCTCT GGAGTTCGTT TTCCTACTGT

TCCATCAGTG TACAAATCTA ATGGATTCAG CAGTAATGGT GATCGGAGGA ATGCTAATAT

TTCTGTATTC TTGAAAAAAC ACTCTCTTTC ACGGAAGATC TTGGCTGAAA AGTCTTCTTA

CAATTCCGAA TCCCGACCTT-CTACAATTGC AGCATCGGGG AAAGTCCTTG TGCCTGGAAT

CCAGAGTGAT AGCTCCTCAT CCTCAACAGA TCAATTTGAG TTCGCTGAGA CATCTCCAGA

AAATTCCCCA GCATCAACTG ATGTAGATAG TTCAACAATG GAACACGCTA GCCAGATTAA

AACTGAGAAC GATGACGTTG AGCCGTCAAG TGATCTTACA GGAAGTGTTG AAGAGCTGGA

TTTTGCTTCA TCACTACAAC TACAAGAAGG TGGTAAACTC GAGGAGTCTA AAACATTAAA

TACTTCTGAA GAGACAATTA TTGATGAATC TGATAGGATC AGAGAGAGGG GCATCCCTCC

ACCTGGACTT GGTCAGAAGA TTTATGAAAT AGACCCCCTT TTGACAAACT ATCGTCAACA

CCTTGATTAC AGGTATTCAC AGTACAAGAA ACTGAGGGAG GCAATTGACA AGTATGAGGG

TGGTTTGGAA GCTTTTTCTC GTGGTTATGA AAGAATGGGT TTCACTCGTA GTGCTACAGG

TATCACTTAC CGTGAGTGGG CTCCTGGTGC CCAGTCAGCT GCCCTCATTG GGGATTTCAA

CAATTGGGAC GCAAATGCTG ACTTTATGAC TCGGAATGAA TTTGGTGTCT GAGAGATTTT

TCTGCCAAAT AATGTGGATG GTTCTCCTGC AATTCCTCAT GGGTCCAGAG TGAAGATACG

TATGGACACT CCATCAGGTG TTAAGGATTC CATTCCTGCT TGGATCAACT ACTCTTTACA

GCTTCCTGAT GAAATTCCAT ATAATGGAAT ATATTATGAT CCACCCGAAG-AGGAGAGGTA

TATCTTCCAA CACCCACGGC CAAAGAAACC AAAGTCGGTG AGAATATATG AATCTCATAT

TGGAATGAGT AGTCCGGAGC CTAAAATTAA CTCATACGTG AATTTTAGAG ATGAAGTTCT

TCCTCGCATA AAAAAAGCTT GGGTACAATG CGGTGCAAAT TATGGCTATT CAAGAGCATT

GAACGCCCGA CGACCTTAAG TCTTTGATTG ATAAAGCTCA TGAGCTAGGA ATTGTTGTTC

TCATGGACAT TGTTCACAGC CATGCATCAA ATAATACTTT AGATGGACTG AACATGTTTG

ACGGCACAGA TAGTTGTTAC TTTCACTCTG GAGCTCGTGG TTATCATTGG ATGTGGGATT

TCCGCCTCTT TAACTATGGA AACTGGGAGG TACTTAGGTA TCTTCTCTCA AATGCGAGAT

GGTGGTTGGA TGAGTTCAAA TTTGATGGAT TTAGATTTGA TGGTGTGACA TCAATGATGT

GTACTCACCA CGGATTATCG GTGGGATTCA CTGGGAACTA CGAGGAATAC TTTGGACTCG

CAACTGATGT GGATGCTGTT GTGTATCTGA TGCTGGTCAA CGATCTTATT CATGGGCTTT

TCCCAGATGC AATTACCATT GGTGAAGATG TTAGCGGAAT GCCGACATTT TGTGTTCCCG

TTCAAGATGG GGGTGTTGGC TTTGACTATC GGCTGCATAT GGCAATTGCT GATAAATGGA

TTGAGTTGCT CAAGAAACGG GATGAGGATT GGAGAGTGGG TGATATTGTT CATACACTGA

CAAATAGAAG ATGGTCGGAA AAGTGTGTTT CATACGCTGA AAGTCATGAT CAAGCTCTAG

TCGGTGATAA AACTATAGCA TTCTGGCTGA TGGACAAGGA TATGTATGAT TTTATGGCTC

TGGATAGACC GTCAACATCA TTAATAGATC GTGGGATAGC ATTACACAAG ATGATTAGGC

TTGTAACTAT GGGATTAGGA GGAGAAGGGT ACCTAAATTT CATGGGAAAT GAATTCGGCC

ACCCTGAGTG GATTGATTTC CCTAGGGCTG AACAACACCT CTCTGATGGC TCAGTAATTC

CCAGAAACCA ATTCAGTTAT GATAAATGCA GACGGAGATT TGACCTGGGA GATGCAGAAT

ATTTAAGATA CCGTGGGTTG CAAGAATTTG ACCGGGCTAT GCAGTATCTT GAAGATAAAT

ATGAGTTTAT GACTTCAGAA CACCAGTTCA TATCACGAAA GGATGAAGGA GATAGGATGA

TTGTATTTGA AAAAGGAAAC CTAGTTTTTG TCTTTAATTT TCACTGGACA AAAGGCTATT

, ~ a Il CAGACTATCG CATAGGCTGC CTGAAGCCTG GAAAATACAA GGTTGCCTTG GACTCAGATG

ATCCACTTTT TGGTGGCTTC GGGAGAATTG ATCATAATGC CGAATATTTC ACCTTTGAAG

GATGGTATGA TGATCGTCCT CGTTCAATTA TGGTGTATGC ACCTAGTAGA ACAGCAGTGG

TCTATGCACT AGTAGACAAA GAAGAAGAAG AAGAAGAAGA AGTAGCAGTA GTAGAAGAAG

TAGTAGTAGA AGAAGAATGA ACGAACTTGT GATCGCGTTG AAAGATTTGA ACGCCACATA

GAGCTTCTTG ACGTATCTGG CAATATTGCA TTAGTCTTGG CGGAATTTCA TGTGACAACA

GGTTTGCAAT TCTTTCCACT ATTAGTAGTG CAACGATATA CGCAGAGATG AAGTGCTGAA

CAAAAACATA TGTAAAATCG ATGAATTTAT GTCGAATGCT GGGACGATCG AATTCCTGCA

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2975 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTiON: SEQ ID NO: 13:

GATCTCTATT TTTTCTCTTA ATTCCAACCA GGGGAATGAA TAAAAGGATA GATTTGTAAA

AACCCTAAGG AGAGAAGAAG AAAGATGGTG TATATACTCT CTGGAGTTCG TTTTCCTACT

GTTCCATCAG TGTACAAATC TAATGGATTC AGCAGTAATG GTGATCGGAG GAATGCTAAT

GTTTCTGTAT TCTTGAAAAA GCACTCTCTT TCACGGAAGA TCTTGGCTGA AAAGTCTTCT

TACAATTCCG AATTCCGACC TTCTACAGTT GCAGCATCGG GGAAAGTCCT TGTGCCTGGA

ACCCAGAGTG ATAGCTCCTC ATCCTCAACA GACCAATTTG AGTTCACTGA GACATCTCCA

GAAAATTCCC CAGCATCAAC TGATGTAGAT AGTi'CAACAA TGGAACACGC TAGCCAGATT

AAAACTGAGA ACGATGACGT TGAGCCGTCA AGTGATCTTA CAGGAAGTGT TGAAGAGCTG

GATTTTGCTT CATCACTACA ACTACAAGAA GGTGGTAAAC TGGAGGAGTC TAAAACATTA

AATACTTCTG AAGAGACAAT TATTGATGAA TCTGATAGGA TCAGAGAGAG GGGCATCCCT

CCACCTGGAC TTGGTCAGAA GATTTATGAA ATAGACCCCC TTTTGACAAA CTATCGTCAA

CACCTTGATT ACAGGTATTC ACAGTACAAG AAACTGAGGG AGGCAATTGA CAAGTATGAG

GGTGGTTTGG AAGCTTTTCT CGTGGTTATG AAAAAATGGG TTTCACTCGT AGTGCTACAG

GTATCACTTA CCGTGAGTGG GCTCCTGGTG CCCAGTCAGC TGCCCTCATT GGAGATTTCA

ACAATTGGGA CGCAAATGCT GACATTATGA CTCGGAATGA ATTTGGTGTC TGGGAGATTT

TTCTGCCAAA TAATGTGGAT GGTTCTCCTG CAATTCCTCA TGGGTCCAGA GTGAAGATAC

GTATGGACAC TCCATCAGGT GTTAAGGATT CCATTCCTGC TTGGATCAAC TACTCTTTAC

AGCTTCCTGA TGAAATTCCA TATAATGGAA TATATTATGA TCCACCCGAA GAGGAGAGGT

~=.

ATATCTTCCA ACACCCACGG CCAAAGAAAC CAAAGTCGCT GAGAATATAT GAATCTCATA

TTGGAATGAG TAGTCCGGAG CCTAAAATTA ACTCATACGT GAATTTTAGA GATGAAGTTC

TTCCTCGCAT AAAAAAGCTT GGGTACAATG CGCTGCGAAT TATGGCTATT CAAGAGCATT

CAACGCCCGA CGACCTTAAG TCTTCGATTG ATAAAGCTCA TGAGCTAGGA ATTGTTGTTC

TCATGGACAT CGTTCACAGC CATGCATCAA ATAATACTTT AGATGGACTG AACATGTTTG

ACGGCACCGA TAGTTGTTAC TTTCACTCTG GAGCTCGTGG TTATCATTGG ATGTGGGATT

CCGCCTCTTT AACTATGGAA ACTGGGAGGT ACTTAGGTAT CTTCTCTCAA ATGCGAGATG

GTGGTTGGAT GAGTTCAAAT TTGATGGATT TAGATTCGAT GGTGTGACAT CAATGATGTA

TACTCACCAC GGATTATCGG TGGGATTCAC TGGGAACTAC GAGGAATACT TTGGACTCGC

AACTGATGTG GATGCTGTTG TGTATCTGAT GCTGGTCAAC GATCTTATTC ATAGGCTTTT

CCCAGATGCA ATTACCATTG GTGAAGATGT TAGCGGAATG CCGACATTTT GTATTC(-,CGT

TGAGTTGCTC AAGAAACGGG ATGAGGATTG GAGAGTGGGT GATATTGTTC ATACACTGAC

AAATAGAAGA TGGTCGGAAA AGTGTGTTTC ATACGCTGAA AGTCATGATC AAGCTCTAGT

CGGTGATAAA ACTATAGCAT TCT.GGCTGAT GGACAAGGAT ATGTATGATT TTATGGCTCT
2iu0 GGATAGACCG CCAACATCAT TAATAGATCG TGGGATAGCA~TTGCACAAGA TGATTAGGCT

TGTAACTATG GGATTAGGAG GAGAAGGGTA CCTAAATTTC ATGGGAAATG AATTCGGCCA

CCCTGAGTGG ATTGATTTCC CTAGGGCTGA GCCACACCTT TCTGATGGCT CAGTAATTCC

CGGAAACCAA TTCAGTTATG ATAAATGCAG ACGGAGATTT GACCTGGGAG ATGCAGAATA

TTTAAGATAC CATGGGTTAC AAGAATTTGA CTGGGCTATG CAGTAT G, e; G HAGNTAAATA

TGAGTTTATG ACTTCAGAAC ACCAGTTCAT ATCACGAAAG GATGAAGGAG ATAGGATGAT

TGTATTTGAA AGAGGAAACC TAGTTTTCGT CTTTAATTTT CACTGGACAA ATAGCTATTC

AGACTATCGC ATAGGCTGCC TGAAGCCTGG AAAATACAAG GTTGTCTTGG ACTCAGATGA

TCCACTTTTT GGTGGCTTCG GGAGAATTGA TCATAATGCC GAATATTTCA CCTCTGAAGG

ATCGTATGAT GATCGTCCTT GTTCAATTAT GGTGTATGCA CCTAGTAGAA CAGCAGTGGT

CTATGCACTA GTAGACAAAC TAGAAGTAGC AGTAGTAGAA GAACCCATTG AAGAATGAAC

GAACTTGTGA TCGCGTTGAA AGATTTGAAC GTTACTTGGT CATCCACATA GAGCTTCTTG

ACATCAGTCT TGGCGGAATT GCATGTGACA ACAAGGTTTG CAGTTCTTTC CACTATTAGT

AGTCCACCGA TATACGCAGA GATGAAGTGC TGAACAAACA TATGTAAAAT CGATGAATTT

(2) iNFORMAT(ON FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3033 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: singie (D) TOPOLOGY: linear (iX) FEATURE:
(A) NAME(KEY: CDS
(B) LOCATION:145..2790 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

AGATCTCTAT TTZTTCTCTT AATTCCAACC AAGGAATGAA TAAAAGGATA GATTTGTAAA

Met Val Tyr Thr Leu Ser GIy Val Arg Phe Pro Thr Val Pro Ser Val Tyr Lys Ser Asn Gly Phe Ser Ser Asn Gly Asp Arg Arg Asn Ala Asn Val Ser Val Phe Leu Lys Lys His Ser Leu Ser Arg Lys lle Leu Ala Glu Lys Ser Ser Tyr Asn Ser Glu Phe Arg Pro Ser Thr Val Ala Ala Ser Gly Lys Val Leu Val Pro Gly Thr Gin Ser Asp Ser Ser Ser Ser Ser Thr Asp Gin Phe Glu Phe Thr Glu Thr Ser Pro Glu Asn Ser Pro Ala Ser Thr Asp Val Asp Ser Ser Thr Met Glu His Ala Ser Gln lie Lys Thr Glu Asn Asp Asp Val Glu Pro Ser Ser Asp Leu Thr Gly Ser Val Glu GIu Leu Asp Phe Ala Ser Ser . ' ~

Leu Gin Leu Gin Glu Gly Gly Lys Leu Glu Glu Ser Lys Thr Leu Asn ACT TCT GAA GAG ACA AT T A i- Gr",T Cr,A TCT GAT AGG ATC AGA GAG AGG 651 Thr Ser Glu Giu Thr Ile Ile Asp Glu Ser Asp Arg Ile Arg Glu Arg Giy Ile Pro Pro Pro Gly Leu Giy G1n Lys Ile Tyr Glu Ile Asp Pro Leu Leu Thr Asn Tyr Arg Gin His Leu Asp Tyr Arg Tyr Ser Gln Tyr -Lys Lys Leu Arg Glu Ala Ile Asp Lys Tyr Giu Gly Gly Leu Giu Ala Phe Ser Arg Gly Tyr Glu Lys Met Gly Phe Thr Arg Ser Ala Thr Gly Ile Thr Tyr Arg Glu Trp Ala Leu Gly Ala Gin Ser Ala Ala Leu Ile Gly Asp Phe Asn Asn Trp Asp Ala Asn Ala Asp Ile Met Thr Arg Asn GAA TTT GGT GTC TGG GAG ATT T'rT CTG CCA AAT AAT GTG GAT GGT TCT 987 Glu Phe Gly Val Trp Giu Ile Phe Leu Pro Asn Asn Val Asp GIy Ser Pro Ala Ile Pro His Gly Ser Arg Va) Lys lie Arg Met Asp Thr Pro Ser Gly Vai Lys Asp Ser Ile Pro Ala Trp lie Asn Tyr Ser Leu Gin Leu Pro Asp Giu Ile Pro Tyr Asn Gly Ile His Tyr Asp Pro Pro Glu Glu Glu Arg Tyr Ile Phe Gin His Pra Arg Pro Lys Lys Pro Lys Ser Leu Arg Ile Tyr Glu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys Ile Asn Ser Tyr Val Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Leu Gin Ile Met Ala lle Gin Giu His Ser Tyr Tyr Ala Ser Phe GIV Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro Asp Asp Leu Lys Ser Leu lie Asp Lys Ala His Glu Leu GIV lie Va) Val Leu Met Asp Ile Val His Ser His Ala TCA AAT AAT ACT TTA GAT GGA CTG AAC ATG T-fT GAC TGC ACC GAT AGT 1515 Ser Asn Asn Thr Leu Asp Gly Leu Asn Met Phe Asp Cys Thr Asp Ser Cys Tyr Phe His Ser GIV Ala Arg Gly Tyr His Trp Met Trp Asp Ser 460 465 "- 470 Arg Leu Phe Asn Tyr Gly Asn Trp Glu Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp Leu Asp Ala Phe Lys Phe Asp GIV Phe Arg Phe Asp GIV Val Thr Ser Met Met Tyr lle His His Giy Leu Ser Val Gly Phe Thr Gly Asn Tyr Glu Glu Tyr Phe Gly Leu Ala Thr Asp Val Asp GCT GTT r,TG TAT CTC .qTe rTr GTC AAC GAT CTT ATT CAT GGG CTT TTC 1803 Ala Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile His Gly Leu Phe Pro Asp Ala Ile Thr Iie Giy Glu Asp Vai Ser Gly Met Pro Thr Phe Cys Ile Pro Val Gin Glu Gly Gly Val Gly Phe Asp Tyr Arg Leu His Met Ala Ile Ala Asp Lys Ara Ile Glu Leu Leu Lys Lys Arg Asp Giu Asp Trp Arg Val Giy Asp Ile Val His Thr Leu Thr Asn Arg Arg Trp Ser G(u Lys Cys Val Ser Tyr Ala Glu Ser His Asp Gln Ala Leu Vai -GIy Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ser Thr Ser Leu ile Asp Arg Gly Ife Ala Leu His Lys Met Ile Arg Leu Val Thr Met Giy Leu Gly Giy Glu Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe GIy His Pro Giu Trp Ile Asp Phe Pro Arg Ala Glu Gin His Leu Ser Asp Gly Ser Vai ife ~; o . m _..

Gly Asn Gin Phe Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu GIy Asp Ala Glu Tyr Leu Arg Tyr Arg Gly Leu Gin Glu Phe Asp Arg Pro Met Gin Tyr Leu Giu Asp Lys Tyr Glu Phe Met Thr Ser Glu His Gln Phe ile Ser Arg Lys Asp Glu Giy Asp Arg Met i(e Val Phe Glu Lys GIy Asn Leu Val Phe Val P"e Asn Phe His Trp Thr Lys Ser T'yr Ser Asp Tyr Arg iie Ala Cys Leu Lys Pro GIy Lys Tyr Lys Val Ala Leu Asp Ser Asp Asp Pro Leu Phe Gly Gly Phe Giy Arg lie Asp His Asn Ala Giu Tyr Phe Thr Phe Giu Giy Trp Tyr Asp Asp Arg Pro Arg Ser lle Met Val Tyr Ala Pro Cys Lys Thr Ala Val Val Tyr Ala Leu Val Asp Lys Glu Glu Glu Ciu Glu Glu Glu Glu Glu Glu Glu Val Ala Ala Val Giu GIu Val Val Val Glu Glu Giu TTGAAAGATT TGAACGCTAC ATAGAGCTTC TTGACGTATC TGGCAATATT GCATCAGTCT

TGGCGGAATT TCATGTGACA CAAGGTTTGC AATTCTTTCC ACTATTAGTA GTGCAACGAT

ATACGCAGAG ATGAAGTGCT GAACAAACAT ATGTAAAATC GATGAATT'i'A TGTCGAATGC

(2) INFORMATION FOR SEQ ID N0:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 882 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Met Val Tyr Thr Leu Ser Gly Val Arg Phe Pro Thr Val Pro Ser Val Tyr Lys Ser Asn Giy Phe Ser Ser Asn Gly Asp Arg Arg Asn Ala Asn Val Ser Val Phe Leu Lys Lys His Ser Leu Ser Arg Lys lie Leu Ala Glu Lys Ser Ser Tyr Asn Ser Glu Phe Arg Pro Ser Thr Va1 Ala Ala Ser Gly Lys Val Leu Val Pro GIy Thr Gln Ser Asp Ser Ser Ser Ser Ser Thr Asp Gin Phe Glu Phe Thr Glu Thr Ser Pro GIU Asn Ser Pro Ala Ser Thr Asp Val Asp Ser Ser Thr Met Glu His Ala Ser Giri lle 100 105 - 11.0 Lys Thr GIu Asn Asp Asp Vai Glu Pro Ser Ser Asp Leu Thr Gly Ser Val Glu Glu Leu Asp Phe Ala Ser Ser Leu Gin Leu Gin Glu Gly Gly Lys Leu Glu Glu Ser Lys Thr Leu Asn Thr Ser Glu Glu Thr lle Ile Asp Glu Ser Asp Arg Ile Arg Glu Arg GIy lle Pro Pro Pro Gly Leu 165 170 175 _ Gly Gin Lys lie Tyr GIu lie Asp Pro Leu Leu Thr Asn Tyr Arg Gln His Leu Asp Tyr Arg Tyr Ser Gin Tyr Lys Lys Leu Arg Glu Ala lie Asp Lys Tyr Glu Gly Gly L'u Glu Ala Phe Ser Arg Gly Tyr Glu Lys Met G(y Phe Thr Arg Ser Ala Thr Gly Ile Thr Tyr Arg Glu Trp Ala Leu Gly Ala Gln Ser Ala Ala Leu Ile Gly Asp Phe Asn Asn Trp Asp Ala Asn Ala Asp lie Met Thr Arg Asn Giu Phe Gly Val Trp Glu Ile Phe Leu Pro Asn Asn Val Asp Gly Ser Pro Ala 11e Pro His Gly Ser Arg Val Lys Ile Arg Met Asp Thr Pro Ser Giy Val Lys Asp Ser lie Pro Ala Trp Ile Asn Tyr Ser Leu Gin Leu Pro Asp Glu lie Pro Tyr Asn Gly lie His Tyr Asp Pro Pro Glu Glu Glu Arg Tyr lie Phe Gin His Pro Arg Pro Lys Lys Pro Lys Ser Leu Arg Ile Tyr Giu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys lie Asn Ser Tyr Val Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Leu G1n iie Met Ala Ile Gin Glu His Ser Tyr Tyr Ala Ser Phe GIy Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe G1y Thr Pro Asp Asp Leu Lys Ser Leu (! A.cn N,is !~ His Glu Leu Gly Ile Val Val Leu Met Asp Ile Val His Ser His Ala Ser Asn Asn Thr Leu Asp Gly Leu Asn Met Phe Asp Cys Thr Asp Ser Cys Tyr Phe His Ser Gly Ala Arg GIV Tyr His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Asn Trp Glu Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp Leu Asp Ala Phe Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met Tyr lle His His GIV Leu Ser Val Gly Phe Thr GIV Asn Tyr Glu Glu Tyr Phe GIV Leu Ala Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile His GIV Leu Phe Pro Asp Ala Ile Thr Ile GIV

Glu Asp Val Ser Gly Met Pro Thr Phe Cys Ile Pro Val Gin Glu Gly Gly Val GIV Phe Asp Tyr Arg Leu His Met Ala lle Ala Asp Lys Arg ife Glu Leu Leu Lys Lys Arg Asp Giu Asp Trp Arg Val GIV Asp Ile Val His Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val Ser Tyr Ala Glu Ser His Asp G(n Ala-Leu Val Gly Asp Lys Thr {ie Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ser Thr Ser Leu Ile Asp Arg Gly lie Ala Leu His Lys Met lie Arg Leu Val Thr Met Gly Leu GIV Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn Giu Phe GIV His Pro Giu Trp Ile Asp Phe Pro Arg Ala Glu Gin His Leu Ser Asp Gly Ser Val Ile Pro Gly Asn Gin Phe Ser Tyr'Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala GIU Tyr Leu Arg Tyr Arg Giy Leu Gin G1u Phe Asp Arg Pro Met Gin Tyr Leu Glu Asp Lys Tyr Glu Phe Met Thr Ser Glu His Gin Phe Ife Ser Arg Lys Asp Glu Gly Asp Arg Met IIe Val Phe Glu Lys Gly Asn Leu Val Phe Val Phe Asn Phe His Trp Thr Lys Ser Tyr Ser Asp Tyr Arg lie Ala Cys Leu Lys Pro Giy Lys Tyr Lys Val Ala Leu Asp Ser Asp Asp Pro Leu Phe Gly Gfy Phe G1y Arg Ile Asp His Asn Ala Glu Tyr Phe Thr Phe Glu Gly Trp Tyr Asp Asp Arg Pro Arg Ser Ile Met Val Tyr Ala Pro Cys Lys Thr Ala Val Val Tyr Ala Leu Val Asp Lys Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Val Ala Ala Val Glu Glu Va1 Val Val Glu Giu Glu (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2576 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TCATTAAAGA GGAGAAATTA ACTATGAGAG GATCTCACCA TCACCATCAC CATGGGATCT

TGGCTGAAAA GTCTTCTTAC AATTCCGAAT TCCGACCTTC TACAGTTGCA GCATCGGGGA

AAGTCCTTGT GCCTGGAACC CAGAGTGATA GCTCCTCATC CTCAACAAAC CAATTTGAGT

TCACTGAGAC ATCTCCAGAA AATTCCCCAG CATCAACTGA TGTAGATAGT TCAACAATGG

AACACGCTAG CCAGATTAAA ACTGAGAACG ATGACGTTGA GCCGTCAAGT GATCTTACAG

GAAGTGTTGA ACAGCTGGAT TTTGCTTCAT CACTP.Cn,ACT ACPn,CAACGT GGTAAACTGC

AGGAGTCTAA AACATTAAAT ACTTCTGAAG AGACAATTAT TGATGAATCT GATAGGATCA

GAGAGAGGGG CATCCCTCCA CCTGGACTTG GTCAGAAGAT TTATGAAATA GACCCCCTTT

TGACAAACTA TCGTCAACAC CTTGATTACA GGTATTCACA GTACAAGAAA CTGAGGGAGG

CAATTGACAA GTATGAGGGT GGTTTGGAAG CTTTTTCTCG TGGTTATGAA AAAATGGGTT

TCACTCGTAG TGCTACAGGT ATCACTTACC GTGAGTGGGC TCCTGGTGCC CAGTCAGCTG

CCCTCATTGG AGATTTCAAC AATTGGGACG CAAATGCTGA CATTATGACT CGGAATGAAT

GGTCCAGAGT GAAGATACGT ATGGACACTC CATCAGGTGT TAAGGATTCC ATTCCTGCTT

GGATCAACTA CTCTACAGCT TCCTGATGAA ATTCCATATA ATGGAATATA TTATGATCCA

CCCGAAGAGG AGAGGTATAT CTTCCAACAC CCACGGCCAA AGAAACCAAA GTCGCTGAGA

g a a _.

ATATATGAAT CTCATATTGG AATGAGTAGi CCGGAGCCTA AAATTAACTC ATACGTGAAT

TTTAGAGATG AAGTTCTTCC TCGCATAAAA AAGCTTGGGT ACAATGCGCT GCAAATTATG

CCAAGCAGCC GTTTTGGAAC GCCCGACGAC CTTAAGTCTT TGATTGATAA AGCTCATGAG

CTAGGAATTG TTGTTCTCAT GGACATTGTT CACAGCCATG CATCAAATAA TACTTTAGAT

GGACTGAACA TGTTTGACGG CACCGATAGT TGTTACTTTC ACTCTGGAGC TCGTGGTTAT

CATTGGATGT GGGATTCCCG CCTTTTTAAC TATGGAAACT GGGAGGTACT TAGGTATCTT

CTCTCAAATG CGAGATGGTG GTTGGATGAG TTCAAATTTG ATGGATTTAG ATTTGATGGT

GTGACATCAA TGATGTATAC TCACCACGGA TTATCGGTGG GATTCACTGG GAACTACGAG

GAATACTTTG GACTCGCAAC TGATGTGGAT GCTGTTGTGT ATCTGATGCT GGTCAACGAT

CTTATTCATG GGCTTTTCCC AGATGCAATT ACCATTGGTG AAGATGTTAG CGGAATGCCG

ACATTTTGTA TTCCCGTTCA 'kGATGGGGGT GTTGGCTTTG ACTATCGGCT GCATATGGCA

ATTGCTGATA AATGGATTGA GTTGCTCAAG AAACGGGATG AGGATTGGAG AGTGGGTGAT

ATTGTTCATA CACTGACAAA TAGAAGATGG TCGGAAAAGT GTGTTTCATA CGCTGAAAGT

CATGATCAAG CTCTAGTCGG TGATAAAACT ATAGCATTCT GGCTGATGGA CAAGGATATG

TATGATTTTA TGGCTCTGGA TAGACCGCCA ACATCATTAA TAGATCGTGG GATAGCATTG

CACAAGATGA TTAGGCTTGT AACTATGGGA TTAGGAGGAG AAGGGTACCT AAATTTCATG

GGAAATGAAT TCGGCCO.CCC TGAGTGGATT GATTTCCCTA GGGCTGAACA ACACCTCTCT

GATGACTCAG TAATTCCCGG AAACCAATTC AGTTATGATA AATGCAGACG GAGATTTGAC

CTGGGAGATG CAGAATATTT AAGATACCGT GGGTTGCAAG AATTTGACCG GGCTATGCAG

TATCTTGAAG ATAAATATGA GTTTATGACT TCAGAACACC AGTTCATATC ACGAAAGGAT

GAAGGAGATA GGATGATTGT ATTTGAAAAA GGAAACCTAG TTTTTGTCTT TAATTTTCAC

TGGACAAAAA GCTATTCAGA CTATCGCATA GGCTGCCTGA AGCCTGGAAA ATACAAGGTT

GCCTTGGACT CAGATGATCC ACTTTTTGGT GGCTTCGGGA GAATTGATCA TAATGCCGAA

TATTTCACCT TTGAAGGATG GTATGATGAT CGTCCTCGTT CAATTATGGT GTATGCACCT

TGTAGAACAG CAGTGGTCTA TGCACTAGTA GACAAAGAAG AAGAAGAAGA AGAAGAAGAA

GAAGAAGTAG CAGTAGTAGA AGAAGTAGTA GTAGAAGAAG AATGAACGAA CTTGTG

(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2529 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEO ID NO: 17:

AAAGTCTTCT TACAATTCCG AA-TCCCGACC TTCTACAGTT GCAGCATCGG GGAAAGTCCT

CA 02416347 2003-02-11 .,, -S7a-TGTGCCTGGA AYCCAGAGTG ATAGCTCCTC ATCCTCAACA GACCAATTTG AGTTCACTGA

GACATCTCCA GAAAATTCCC CHGCATCAAC TGATGTAGAT AGTTCAACAA TGGAACACGC

TAGCCAGATT AAAACTGAGA ACGATGACGT TGAGCCGTCA AGTGATCTTA CAGGAAGTGT

TGAAGAGCTG GATTTTGCTT CATCACTACA ACTACAAGAA GGTGGTAAAC TGGAGGAGTC

TAAAACATTA AATACTTCTG AAGAGACAAT TATTGATGAA TCTGATAGGA TCAGAGAGAG

GGGCATCCCT CCACCTGGAC TTGGTCAGAA GATTTATGAA ATAGACCCCC TTTTGACAAA

CTATCGTCAA CACCTTGATT ACAGGTATTC ACAGTACAAG AAACTGAGGG AGGCAATTGA

CAAGTATGAG GGTGGTTTGG AAGCTTTTTC TCGTGGTTAT GAAAAAATGG GTTTCACTCG

TAGTGCTACA GGTATCACTT ACCGTGAGTG GGCTCCTGGT GCCCAGTCAG-CTGCCCTCAT

TGGAGATTTC AACAATTGGG ACGCAAATGC TGACATTATG ACTCGGAATG AATTTGGTGT

CTGGGAGATT TTTCTGCCAA ATAATGTGGA TGGTTCTCCT GCAATTCCTC ATGGGTCCAG

AGTGAAGATA CGYATGGACA CTCCATCAGG TGTTAAGGAT TCCATTCCTG CTTGGATCAA

CTACTCTTTA CAGCTTCCTG ATGAAATTCC ATATAATGGA ATATATTATG ATCCACCCGA

AGAGGAGAGG TATRTCTTCC AACACCCACG GCCAAAGAAA CCAAAGTCGC TGAGAATATA

TGAATCTCAT ATTGGAATGA GTAGTCCGGA GCCTAAAATT AACTCATACG TGAATTTTAG

AGATGAAGTT CTTCCTCGCA TAAAAAASCT TGGGTACAAT GCGGTGCAAA TTATGGCTAT

-57b-TCAAGAGCAT TCTTATTATG CTAGTTTTGG TTATCATGTC ACAAATTTTT TTGCACCAAG

CAGCCGTTTT GGAACGCCCG ACGACCTTAA GTCTTTGATT GATAAAGCTC ATGACCTAGG

AATTGTTGTT CTCATGGACA TTGTTCACAG CCATGCATCA AATAATACTT TAGATGGACT
"1260 GAACATGTTT GACGGCACAG ATAGTTGTTA CTTTCACTCT GGAGCTCGTG GTTATCATTG

GATGTGGGAT TCCCGCCTCT TTAACTATGG AAAC ~'GGGAG GTACTTAGGT ATCTTCTCTC

AAATGCGAGA TGGTGGTTGG ATGAGTTCAA ATTTGATGGA TTTAGATTTG ATGGTGTGAC

ATCAATGATG TATACTCACC ACGGATTATC GGTGGGATTC ACTGGGAACT ACGAGGAATA

CTTTGGACTC GCAACTGATG TGGATGCTGT TGTGTATCTG ATGCTGGTCA ACGATCTTAT

TCACGGGCTT TTCCCAGATG CAATTACCAT TGGTGAAGAT GTTAGCGGAA TGCCGACATT

TTGTATTCCC GTTCAAGATG GGGGTGTTGG CTTTGACTAT CGGCTGCATA TGGCAATTGC

TGATAAATGG ATTGAGTTGC TCAAGAAACG GGATGAGGAT TGGAGAGTGG GTGATATTGT

TCATACACTG ACAAATAGAA GATGGTCGGA AAAGTGTGTT TCATMCGCTG AAAGTCATGA

TCAAGCTCTA GTCGGTGATA AAACTATAGC ATYCTGGCTG ATGGACAAGG ATATGTATGA

TTTTATGGCT CTGGATAGAC CGYCAACAYC ATTAATAGAT CGTGGGATAG CATTGCACAA

GATGATTAGG CTTGTAACTA TGGGATTAGG AGGAGAAGGG TACCTAAATT TCATGGGAAA

TGAATTCGGC CACCCTGAGT GGATTGATTT CCCTAGGGCT GARCAACACC TCTCTGATGG

-57c-CTCAGTAATT CCCGGAAACC AATTCAGTTA TGATAAATGC AGACGGAGAT TTGACCTGGG

AGATGCAGAA TATTTAAGAT ACCATGGGTT GCAAGAATTT GACCGGGCTA TGCAGTATCT

TGAAGATAAA TATGAGTTTA TGACTTCAGA ACACCAGTTC ATATCACGAA AGGATGAAGG

AGATAGGATG ATTGTATTTG AAAi-\r-,CC :CCT" GTi T T T GTCTTTAATT TTCACTGGAC

AAATAGCTAT TCAGACTATC GCATAGGCTG CCTGAAGCCT GGAAAATACA AGGTTGGCTT

GGACTCAGAT GATCCACTTT TTGGTGGCTT CGGGAGAATT GATCATAATG CCGAATATTT

CACCTCTGAA GGATCGTATG ATGATCGTCC TCGTTCAATT ATGGTGTATG CACCTAGTAG

AACAGCAGTG GTCTATGCAC TAGTAGACAA ANTAGAAGNA GAAGAAGAAG AAGAANCCGN

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3231 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: iinear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GATTTAATAC GACTCACTAT AGGGATTTTT TTl?TT T TTT TTTTAAAAAC CTCCTCCACT 60 CCAAGGAATG AATTAAAAGA TTAGATTTGA AGGAGAGAAG AAGAAAGATG GTGTATACAC
240 - :

-57d-ATGGTGATCG GAGGAATGCT AATGTTTCTG TATTCTTGAA AAAGCACTCT CTTTCACGGA

AGATCTTGGC TGAAAAGTCT TCTTACGATT CCGAATCCCG ACCTTCTACA GTTGCAGCAT

CGGGGAAAGT CCTTGTACCT GGA.-~,TCCAGA GTGATAGCTC CTCATCCTCA ACAGACCAAT

TTGAGTTCAC TGAGACAGCT CCAGAAAATT CCCCAGCATC AACTGATGTG GATAGTTCAA

CAATGGAACA CGCTAGCCAG ATTAAAACTG AGAACGATGA CGTTGAGCCG TCAAGTGATC

TTACAGGAAG TGTTGAAGAG TTGGATTTTG CTTCATCACT ACAACTACAA GAAGGTGGTA

AACTGGAGGA GTCTAAAACA TTAAATACTT CTGAAGAGAC AATTATTGAT GAATCTGATA

GGATCAGAGA GAGGGGCATC CCTCCACCTG GACTTGGTCA GAAGATTTAT GAAATAGACC

CCCTTTTGAC AAACTATCGT CAACACCTTG ATTACAGGTA TTCACAGTAC AAGAAAATGA

GGGAGGCAAT TGACAAGTAT GAGGGTGGTT TGGAAGCTTT TTCTCGTGGT TATGAAAAAA

TGGGTTTCAC TCGTAGTGCT ACAGGTATCA CTTACCGTGA GTGGGCTCCT GGTGCCCAGT

CAGCTGCTCT CATTGGAGAT TTCAACAATT GGGACGCAAA TGCTGACATT ATGACTCGGA

ATGAATTTGG TGTCTGGGAG ATTTTTCTGC CAAATAATGT GGATGGTTCT CCTGCAATTC

CTCATGGGTC CAGAGTGAAG ATACGCATGG ACACTTCATC AGGTGTTAAG GATTCCATTC

CTGCTTGGAT CAACTACTCT TTACAGCTTC CTGATGAAAT TCCATATAAT GGAATATATT

ATGATCCACC CGAAGAGGAG-AGGTATGTCT TCCAACACCC ACGGCCAAAG AAACCAAAGT

-57e-CGCTGAGAAT ATATGAATCT CATATTGGAA TGAGTAGTCC GGAGCCTAAA ATTAACTCAT

ACGTGAATTT TAGAGATGAA GTTCTTCCTC GCATAAAAAA CCTTGGGTAC AATGCGGTGC

AAATTATGGC TATTCAAGAG CATTCTTATT ATGCTAGTTT TGGTTATCAT GTCACAAATT

TTTTTGCACC AAGCAGCCGT TTTGGAACGC CCGACGACCT TAAGTCTTTG ATTGATAAAG

CTCATGAGCT AGGAATTGTT GTTCTCATGG ACATTGTTCA CAGCCATGCA TCAAATAATA

CTTTAGATGG ACTGAACATG TTTGACGGCA CAGATAGTTG TTACTTTCAC TCTGGAGCTC

GTGGTTATCA TTGGATGTGG GATTCCCGCC TCTTTAACTA TGGAAACTGG GAGGTACTTA

GGTATCTTCT CTCAAATGCG AGATGGTGGT TGGATGAGTG CAAATTTGRT GGATTTAGAT

TTGATGGTGT GACATCAATG ATGTATACTC ACCACGGATT ATCGGTGGGATTCACTGGGA

ACTACGAGGA ATACTTTGGA CTCGCAACTG ATGTRGATGC TGCCGTGTAT CTGATGCTGG

CCAACGATCT TATTCATGGG CTTTTCCCAG ATGCAATTAC CATTGGTGAA GATGTTAGCG

GAATGCCGAC ATTTTGTATT..CCCGTTCAAG ATGGGGGTGT TGGCTTTGAC TATCGGCTGC
1980 ' ATATGGCAAT TGCTGATAAA TGGATTGAGT TGCTCAAGAA ACGGGATGAG GATTGGAGAG

TGGGTGATAT TGTTCATACA CTGACAAATA GAAGATGGTC GGAAAAGTGT GTTTCATACG

CTGAAAGTCA TGATCAAGCT CTAGTCGGTG ATAAAACTAT AGCATTCTGG CTGATGGACA

AGGATATGTA TGATTTTATG GCTTTGGATA GACCGTCAAC ATCATTAATA GATCGTGGGA

-57f-TAGCATTGCA CAAGATGATT AGGCTTGTAA CTATGGGATT AGGAGGAGAA GGGTACCTAA

ATTTCATGGG AAATGAATTC GGCCACCCTG AGTGGATTGA TTTCCCTAGG GCTGAACAAC

ACCTCTCTGA TGGCTCAGTA ATTCCCGGAA ACCAATTCAG TTATGATAAA TGCAGACGGA

GATTTGA.CCT ~rr,AGATGrA GA.ATATTTAA GATACCGTGG GTTGCAAGAA TTTGACCGGG

CTATGCAGTA TCTTGAAGAT AAATATGAGT TTATGACTTC AGAACACCAG TTCATATCAC

GAAAGGATGA AGGAGATAGG ATGATTGTAT TTGAAAAAGG AAACCTAGTT TTTGTCTTTA

ATTTTCACTG GACAAAAAGC TATTCAGACT ATCGCATAGG CTGGCTGAAG CCTGGAAAAT

ACAAGGTTGC CTTGGACTCA GATGATCCAC TTTTTGGTGG CTTCGGGAGA ATTGATCATA

ATGCCGAATG TTTCACCTTT GAAGGATGGT ATGATGATCG TCCTCGTTCA ATTATGGTGT

ATGCACCTAG TAGAACAGCA GTGGTCTATG CACTAGTAGA CAAAGAAGAA GAAGAAGAAG

AAGTAGCAGT AGTAGAAGAA GTAGTAGTAG AAGAAGAATG AACGAACTTG TGATCGCGTT

GAAAGATTTG AACGCTACAT AGAGCTTCTT GACGTATCTG GCAATATTGC ATCAGTCTTG

GCGGAATTTC ATGTGACAAA AGGTTTGCAA TTCTTTCCAC TATTAGTAGT GCAACGATAT

ACGCAGAGAT GAAGTGCTGA ACAAACATAT GTAAAATCGA TGAATTTATG TCGAATGCTG

r-r n nr, r-r-r1rr i=n r=rn rrTrT T/~.rTTr:Tr./1 (:TTrT(~.Td QA, TTrT('ATCTC
TTTA._ TGTA
NA

CAGCCCACTA GAAATCAATT ATGTGAGACC TAAAAAACAA TAACCATAAA ATGGAAATAG

-57g-(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2578 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

TCATTAAAGA GGAGAAATTA ACTATGAGAG GATCTCACCA TCACCATCAC CATGGGATCT

TGGCTGAAAA GTCTTCTTAC AATTCCGAAT TCCGACCTTC TACAGTTGCA GCATCGGGGA

AAGTCC T TG T GCCTGGAACC CAGAGTGATA GCTCCTCATC CTCAACAAAC CAATTTGAGT

TCACTGAGAC ATCTCCAGAA AATTCCCCAG CATCAACTGA TGTAGATAGT TCAACAATGG
240 ' AACACGCTAG CCAGATTAAA ACTGAGAACG ATGACGTTGA GCCGTCAAGT GATCTTACAG

GAAGTGTTGA AGAGCTGGAT TTTGCTTCAT CACTACAACT ACAAGAAGGT GGTAAACTGG

AGGAGTCTAA AACATTAAAT ACTTCTGAAG AGACAATTAT TGATGAATCT GATAGGATCA

GAGAGAGGGG CATCCCTCCA CCTGGACTTG GTCAGAAGAT TTATGAAATA GACCCCCTTT

TGACAAACTA TCGTCAACAC CTTGATTACA GGTATTCACA GTACAAGAAA CTGAGGGAGG

CAATTGACAA GTATGAGGGT GGTTTGGAAG CTTTTTCTCG TGGTTATGAA AAAATGGGTT

TCACTCGTAG TGCTACAGGT ATCACTTACC GTGAGTGGGC TCCTGGTGCC CAGTCAGCTG

-57h-CCCTCATTGG AGATTTCAAC AATTGGGACG CAAATGCTGA CATTATGACT CGGAATGAAT

GGTCCAGAGT GAAGATACGT ATGGACACTC CATCAGGTGT TAAGGATTCC ATTCCTGCTT

GGATCAACTA CTCTTCACAG CTTCCTGATG AAATTCCATA TAATGGAATA TATTATGATC

CACCCGAAGA GGAGAGGTAT ATCTTCCAAC ACCCACGGCC AAAGAAACCA AAGTCGCTGA

GAATATATGA ATCTCATATT GGAATGAGTA GTCCGGAGCC TAAAATTAAC TCATACGTGA

-ATTTTAGAGA TGAAGTTCTT CCTCGCATAA AAAAGCTTGG GTACAATGCG GTGCAAATTA

CACCAAGCAG CCGTTTTGGA ACGCCCGACG ACCTTAAGTC TTTGATTGAT AAAGCTCATG

AGCTAGGAAT TGTTGTTCTC ATGGACATTG TTCACAGCCA TGCATCAAAT AATACTTTAG

ATGGACTGAA CATGTTTGAC GGCACCGATA GTTGTTACTT TCACTCTGGA GCTCGTGGTT

ATCATTGGAT GTGGGATTCC CGCCTTTTTA ACTATGGAAA CT'GGGAGGTA CTTAGGTATC

TTCTCTCAAA TGCGAGATGG TGGTTGGATG AGTTCAAATT TGATGGATTT AGATTTGATG

GTGTGACATC AATGATGTAT ACTCACCACG GATTATCGGT GGGATTCACT GGGAACTACG

AGGAATACTT TGGACTCGCA ACTGATGTGG ATGCTGTTGT GTATCTGATG CTGGTCAACG

ATCTTATTCA TGGGCTTTTC CCAGATGCAA TTACCATTGG TGAAGATGTT AGCGGAATGC

CGACATTTTG TATTCCCGTT CAAGATGGGG GTGTTGGCTT TGACTATCGG CTGCATATGG

-57i-CAATTGCTGA TAAATGGATT GAGTTGCTCA AGAAACGGGA TGAGGATTGG AGAGTGGGTG

ATATTGTTCA TACACTGACA AATAGAAGAT GGTCGGAAAA GTGTGTTTCA TACGCTGAAA

GTCATGATCA AGCTCTAGTC GGTGATAAAA CTATAGCATT CTGGCTGATG GACAAGGATA

TGTATGATTT TATGGCTCTG GATAGACCGC CAACATCATT AATAGATCGT GGGATAGCAT

TGCACAAGAT GATTAGGCTT GTAACTATGG GATTAGGAGG AGAAGGGTAC CTAAATTTCA

TGGGAAATGA ATTCGGCCAC CCTGAGTGGA TTGATTTCCC TAGGGCTGAA CAAGACCTCT

CTGATGACTC AGTAATTCCC GGAAACCAAT TCAGTTATGA TAAATGCAGA CGGAGATTTG

ACCTGGGAGA TGCAGAATAT TTAAGATACC GTGGGTTGCA AGAATTTGAC CGGGCTATGC

AGTATCTTGA AGATAAATAT GAGTTTATGA CTTCAGAACA CCAGTTCATA TCACGAAAGG

ATGAAGGAGA TAGGATGATT GTATTTGAAA AAGGAAACCT AGTT?TTGTC TTTAATTTTC

ACTGGACAAA AAGCTATTCA GACTATCGCA TAGGCTGCCT GAAGCCTGGA AAATACAAGG

TTGCCTTGGA CTCAGATGAT CCACTTiTTG GTGGCTTCGG GAGAATTGAT CATAATGCCG

AATATTTCAC CTTTGAAGGA TGGTATGATG ATCGTCCTCG TTCAATTATG GTGTAi'GCAC

CTTGTAGAAC AGCAGTGGTC TATGCACTAG TAGACAAAGA AGAAGAAGAA GAAGAAGAAG

AAGAAGAAGT AGCAGTAGTA GAAGAAGTAG TAGTAGAAGA AGAATGAACG AACTTGTG

(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs -57j-(B) TYPE: nucieic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRlPT(ON: SEQ ID NO: 20:

Claims (21)

CLAIMS:

1. Starch extracted from a potato plant and having an amylose content of at least 35%
to about 66.4%, as judged by the iodometric assay method of Morrison &
Laignelet (1983 J. Cereal Science 1, 9-20).

2. Starch according to claim 1, having an amylose content of at least 37% to about 66.4%, as judged by the method defined in claim 1.

3. Starch according to claim 1, having an amylose content of at least 40% to about 66.4%, as judged by the method defined in claim 1.

4. Starch according to claim 1, having an amylose content of at least 50% to about 66.4%, as judged by the method defined in claim 1.

5. Starch according to claim 1, having an amylose content of at least 66% to about 66.4%, as judged by the method defined in claim 1.

6. Starch according to claim 1, having an amylose content of 35 - 66%, as judged by the method defined in claim 1.

7. Starch having an amylose content of at least 35% to about 66.4%, which as extracted from a potato plant by wet milling at ambient temperature has a viscosity onset temperature in the range 70 - 95°C, as judged by viscoamylograph of a 10% w/w aqueous suspension thereof, performed at atmospheric pressure using the Newport Scientific Rapid Visco Analyser 3C with a heating profile of holding at 50°C for 2 minutes (step 1), heating from 50 to 95°C at a rate of 1.5°C per minute (step 2), holding at 95°C for 15 minutes (step 3), cooling from 95 to 50°C at a rate of 1.5°C per minute (step 4), and then holding at 50°C for 15 minutes (step 5).

8. Starch according to claim 7, which as extracted from a potato plant by wet milling at ambient temperature has peak viscosity in the range 214 - 497 stirring number units (SNUs), as judged by viscoamylograph conducted according to the protocol defined in claim 7.

9. Starch according to claim 7, which as extracted from a potato plant by wet milling at ambient temperature has a pasting viscosity in the range 214 - 434 SNUs, as judged by viscoamylograph conducted according to the protocol defined in claim 7.

10. Starch according to claim 7, which as extracted from a potato plant by wet milling at ambient temperature has a set-back viscosity in the range 450 - 618 SNUs, as judged by viscoamylograph conducted according to the protocol defined in claim 7.

11. Starch according to claim 7 which as extracted from a potato plant by wet milling at ambient temperature has a set-back viscosity in the range 14 - 192 SNUs, as judged by viscoamylograph conducted according to the protocol defined in claim 7.

12. Starch according to claim 7 which as extracted from a potato plant by wet milling at ambient temperature has a peak viscosity in the range 200 - 500 SNUs and a set-back viscosity in the range 275 - 618 SNUs as judged by viscoamylograph according to the protocol defined in claim 7.

13. Starch according to claim 7 which as extracted from a potato plant by wet milling at ambient temperature has a viscosity which does not decrease between the start of the heating phase (step 2) and the start of the final holding phase (step 5) and has a set-back viscosity of 303 SNUs or less as judged by viscoamylograph according to the protocol defined in claim 7.

14. Starch according to claim 7 which as extracted from a potato plant by wet milling at ambient temperature displays no significant increase in viscosity as judged by viscoamylograph conducted according to the protocol defined in claim 7.

15. Starch according to any one of claims 7 to 14, having an amylose content in the range of 35 - 66%, as judged by the method of Morrison & Laignelet defined in claim 1.

16. Starch according to any one of claims 1 to 6, which as extracted from a potato plant, has a phosphorus content between 200 and 240 mg/100 grams dry weight starch.

17. Starch according to any one of claims 1 to 16, further being resistant starch.

18. Use of the starch according to any one of claims 1 to 17, in the preparation or processing of a foodstuff.

19. Use according to claim 18, wherein the starch is used to provide a film, barrier, coating or as a gelling agent.

20. Use according to claim 18, to prepare resistant starch compositions.

21. Use of the starch according to any one of claims 1 to 17, in the preparation or processing of corrugating adhesives, biodegradable products, packaging, glass fibers or textiles.