AU3225099A - Novel exo-(1-ge4)-beta-D galactanase - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGFNAL

COMPLETE

SPECIFICATION

STANDARD

PATENT

TITLE OF INVENTION NOVEL

GALACTANASE

Name and Address of Applicant: UNILEVER PLC of Unilever House, Blackfriars, London EC4P 4BQ, England The following statement is a full description of this invention, including the best method of performing it known to me:- WO 95/23228 PCT/GB95/00372 Title: Novel Exo-(l-4-B-D Galactanase Field of the Invention This invention relates to novel nucleotide sequences and to vectors and hosts comprising said sequences. The invention also relates to a method of altering the characteristics of plants.

Background of the Invention Pectin is a major matrix polysaccharide found in the cell wall of plants. Pectins are composed of two distinct regions. The smooth region comprises of long stretches of homogalacturonan interrupted by rhamnose, and is relatively unbranched. The hairy region is rich in galacturonic and rhamnose residues and is highly branched. The side branches contain different sugars but primarily comprise the neutral sugar side chains, arabinan. B-(l-4)-galactan and arabinogalactan. The function of these neutral sugar polysaccharide side chains has not been fully established. It is speculated that thev may function in modulating the pore size of the cell wall and therefore the mobility of proteins. possibly restricting access of various enzymes to their substrates. Moreover.

the interaction of the side chains between themselves and with other cell wall polymers could contribute to the structure of the cell wall and the rheological properties of products derived from them. In vitro studies carried out on a solution of apple pectin with different neutral sugar contents demonstrate that increase in branching of pectin results in higher zero-shear viscosity. It was concluded that this was due to pectin side S..chain interactions. In addition, more branched pectin gives.higher elastic or storage moduli than less branched pectin, suggesting that side chain of pectins contribute to elastic properties (Hwang er al.. (1993) Food Hydrocolloids 7. 39-53).

The hydrolysis of l-I-4)-linked galactose from polymeric galactan side chains of pectin has been demonstrated in different plants and in various physiological states (de Vetten and Huber (1990) Physiol. Plant. 78. 447-454: Fischer and Bennett (1991) Ann.

Rev. Plant Physiol Plant Mol. Bioi. 42. 675-703). During the process of fruit WO 95/23228 PCT/GB95/00372 2 rpening. the loss of the neutral sugar, galactose, is the single most extensive change in the cell walls of many fruits (Fischer and Bennett 1991). Galactose mobilization during fruit ripening has been demonstrated in several fruits including tomato, hot pepper, strawberry, apple, coffee, muskmelon, kiwi fruit, and nectarines. During the senescence of carnation petals, the decrease in. cell wall yield is due largely to a loss of the neutral sugar galactose (de Vetten and Huber 1990). In germinating lupin cotyledons, up to 80% of galactose is mobilized primarily from the B-(1-4)-linked galactan side chains of the rhamnogalacturonan backbone from secondary cell walls adapted to a storage function. A 3-galactosidase (exo-(1-4)-B-D-galactanase) would be the enzyme activity predicted to be responsible for galactose mobilization from the galactan side chains of pectin.

B-Galactosidase enzyme activities (including exo-galactanase activities) in plants have been described in the prior art (Dick et al.. (1990) Physiol. Plant. 89, 369-375: Bums (1990) Phytochemistry 29, 2425-2429: Singh and Knox. (1985) Phytochemistry 24, 1639-1643; Kundu et al., (1980) Phytochemistry 29, 2079-2082). The purification of some 3-galactosidase enzymes has been described, though in many instances synthetic substrates rather than endogenous substrates have been used for enzyme S::characterization (Ogawa et al.. (1990) Nippon Shokuin Kogyo Gakkaishi 37, 298-305: Giannakouros et al.. (1991) Physiol. Plant. 82. 413-418). There is evidence that plant 13-,-galactosidases may be associated with developmental processes requiring ceil wall turnover, like tissue elongation in Cicer arierinum epicotyl segments. In this tissue. 3galactosidase has been demonstrated to be responsible for autolysis, and the natural :substrate of the autolytic reaction is the pectic fraction of the cell wall (Dopico er al..

(1989) Physiol. Plant. 75, 458-464: Valero and Labrador (1993) Physiol. Plant. 89.

199-203). A 3-galactosidase has been highly purified from the buffer-soluble fraction of carrot cell culture homogenate (Konno et al.. (1986) Physiol. Plant. 68. 46-52).

The enzyme was active on B-(1--4)-linked galactan prepared from citrus pectin in an exo fashion. The loss of galactose in cell walls during softening has been widely documented (Bartley (1974) Phytochemistry 13. 2107-2111: Redgewell e: al.. (1992) Plant Physiol. 98. 71-81: Wegrzvn and MacRae (1992) Hort. Sci. 27, 900-902). In tomato, it has been found that the increase in monomeric galactose during fruit -I I WO 95/23228 PCT/GB95/00372 3 ripening is due to an increase in the rate of galactose solubilization from the cell wall rather than changes in the rate of metabolic utilization of the solubilized galactose (Kim et al., (1991) Postharvest Biol. Technol. 1. 67-80). This suggests the action of 13galactosidases in vivo during fruit ripening. There have been several reports of increased B-galactosidase activity during the process of fruit ripening (Bartley 1974; Pressey (1983) Plant Physiol. 71, 132-235; Ross er (1993) Planta 1889, 499-506).

The B-galactosidase purified from kiwifruit was active in cleaving terminal galactose attached at either the 2, 3, 4 or 6 position (Ross et al.. 1993). In tomato fruits, Gross and Wallner (1979, Plant Physiol. 63, 117-120) have shown that decline in wall galactans precedes or accompanies increase in soluble polyuronide. Pressey (1983) has characterized three B-galactosidase activities in ripening tomato fruits of which one (3galactosidase II), increases 3-fold during ripening. This enzyme was also able to degrade galactan extracted from the cell walls of green tomato and the author suggests a possible role for it in tomato softening. A B-galactosidase has been purified from ripe coffee beans which increases four fold during the transition from immature to ripe fruits (Golden et al., (1993) Phytochemistry 34. 355-360). The enzyme displaved activity against galactan and arabinogalctan. however pectin yielded galactose only in conjunction with an endopolygalacturonase activity.

Solubilization of pectin during fruit ripening is a well-documented phenomenon (Fischer and Bennett, 1991). The action of endopolygalacturonase was considered to be the most likely cause of pectin solubilization, which was thought to be the cause of softening of fruits. Recent studies in transgenic tomato fruits argue against PG being the sole causal agent in the process of fruit softening (Giovannoni et al., (1989) Plant Cell 1. 53-63: Smith er al., (1990) Plant Mol. Biol. 14. 369-379). Recent evidence in fruits with no apparent endo or exo polygalacturonase activity suggests a role for 8galactosidases active on the gaiactan side chains for the solubilization of pectin (Cutillas-Iturralde er al., (1993) Physiol. Plant. 89, 369-375). Ranawala e al., ((1992) Plant Physiol. 100. 1318-1325) have described a NaCl-released B-galactosidase activity from cell walls of ripe muskmelon (Cucumis melo) fruits. that has the ability to degrade (in vitro) pectin extracted from pre-ripe fruits to smaller sizes of pectin.

similar to those observed in ripe fruits. Moreover, there is no detectable PG activity at WO 95/23228 PCT/GB95/00372 4 any stage of muskmelon fruit development and ripening. De Veau et al., (1993 Physiol. Plant. 87, 279-285) have demonstrated increased pectin solubility and decreased apparent molecular weight of pectin extracted from mature green tomato fruits when digested in vitro with B-galactosidases isolated from avocado fruits.

Though tomato pectin has been shown to contain at least 10% galactose. only 0.2% was mobilized using avocado derived B-galactosidases. However, this minor change in pectin galactose composition was sufficient to change the solubility of polymeric pectin.

These results suggest that an exo-galactanase might play an important role in the pectin solubilization during the process of fruit ripening.

For all the B-galactosidase activities so far described, there are only some indications as to which of the macromoiecular components of the cell walls are the actual in vivo substrates. An exception is the B-galactosidase isolated from germinating nasturtium (Tropaeolum majus) cotyledons. The enzyme activity is coincident with xyloglucan mobilization, and the purified enzyme has the unique capability of hydrolysing the terminal B-1,2-linked galactose from the galactoxylosyl sidechain of the xylogiucan polymer (Edwards et al.. (1988) J. Biol. Chem. 263. 4333- 4337). Buckeridge and Reid recently described (in the printed abstracts of disclosures made at the 6 t Cell Wall Meeting, Nijmegen. August 25-28. 1992. and at the Scottish Cell Wall Group Meeting, April 1993) the purification of a 3 -galactosidase (an exo-(1-*4)-B-Dgalactanase) that metabolises the linear B-(l--4)-galactan component of the lupin S. cotyledonary cell wall. This enzyme is thought to play a key role in the post germinative mobilization of galactan. The enzyme activity is detectable only when galactan mobilization begins, increases during the period of galactan mobilization, and subsequently declines. The changes in exo-galactanase enzyme activity have been shown to correlate with changes in the level of the exo-galactanase enzyme, as i.: determined by immunoblotting. This enzyme is highly specific to B-(1-*4)-galactan and does not hydrolyse other plant cell wall polysaccharides known to have terminal nonreducing galactose residues, like nasturtium xyloglucan (terminal (1-2)-B-linked galactose) and larch arabinogalactan (terminal non-reducing and linked galactose residues.

WO 95/23228 PCT/GB95/00372 The enzyme, exo-(l-'4)-3-D-galactanase. (which catalyses- the hydrolysis of terminal galactose residues from (l-*4)-f-linked galactan side chains) appears to have an important role during several physiological processes. The present inventors have achieved the partial protein sequencing, cloning and sequence analysis of a full length cDNA coding for the exo-(1-4)-5-D-galactanase from germinating agricultural lupins (Lupus angustifolius).

Summary of the Invention In a first aspect the invention provides a nucieotide sequence comprising substantially nucleotides 229 to 2319 of the sequence (Seq ID No. 1) shown in Figure 1 encoding an enzyme having exo-(1-- 4 )--D-galactanase activity, or a precursor or derivative of such an enzyme, or the functional equivalent of such a nucleotide sequence.

For the purposes of the present specification, a precursor shall be understood to mean a polypeptide (active or inactive), which is longer than that encoded by nucleotides 229 to 2319 of the sequence shown in Figure 1. which can be processed (e.g by proteolysis) in vitro or. more preferably, in vivo. to yield an active enzyme.

A

derivative shall be understood to mean a polypeptide, obtained by processig in virro or in vivo. having enzyme activity, which is shorter than that encoded by nucleotides 229 .to 2319 of the sequence shown in Figure 1. Particularly preferred derivatives are those having molecular weights of about 60 kDa and 45 kDa (as determined bv SDS-PAGE).

which are formed either by intracellular C-terminal cleavage of the polypeptide encoded by nucleotides 229 to 2319 of Figure 1. or which arise during purification of the enzyme.

SPreferably the nucieotide sequence of the invention comprises a 5' ATG start signal. It is also preferred that the sequence furthe omprises a suitable 5' untranslated region, including a promoter, to enable expression in appropriate host cells. It is also preferred that the sequence comprises-signals to optimise expression in appropriate host cells, such as 3" polyadenylation signal to optimise expression in eukarvotes. The sequence of the invention may also comprise a sequence encodine a signal peptide.

Particularly preferred embodiments are those sequences which comprise substantially WO 95/23228 PCT/GB95/00372 6 the nucieotide sequence corresponding to nucleotides 163 to 228, or 151 to 228. or 130 to 228 of the sequence shown in Figure 1.

The term "functional equivalent" as used herein, is intended to refer to those sequences which differ from the precise nucleotide sequence in Figure 1. In particular, the term refers to: those nucleotide sequences which encode the same amino acid sequence as that encoded by the sequence shown in Figure 1 but which, by virtue of the degeneracy of the genetic code. possess a different nucleotide sequence; sequences which encode substantially the same polypepride but wherein there may be one or more conserved amino acid substitutions the substitution of one amino acid for another with similar properties). A functionally equivalent sequence will generally encode a polypeptide exhibiting at least 70% amino acid.homology, preferably at least 75%, and more preferably at least 85% amino acid homology with the amino acid sequence encoded by nucleotides 229 to 2319 shown in Figure 1. Accordingly, preferred functionally equivalent sequences will be able to hybridise with the complement of the sequence shown in Figure 1 under standard hybridisation conditions such as described by Sambrook et al.. 1989). but preferably under more stringent conditions.

A particular example of functional equivalents are those sequences which are substantially the antisense equivalent of nucleotides 229 to 2319 of the seuence of Figure 1. Such sequences are therefore able to hybridise with the sequence shown in Figure 1. Preferably such antisense equivalents are able to interfere with the expression of the sense sequence, at the DNA and/or mRNA level.

One functional equivalent to the nucleotide sequence of the invention is the nucleotide sequence comprising substantially nucleotides 117 to 2555 of the sequence (Sea ID No.

18) shown in Figure 5. which encodes a mature exo-galactanase obtainable from tomato fruit. Also included within the scope of the invention are those nucleotide sequences encoding precursors of the mature enzyme, such as nucleotide sequences comprising substantially nucleotides 42 to 2555 of the sequence shown in Figure 5. It will be apparent to those skilled in the art that other nucleotide sequences may exist which encode substantially the same amino acid sequence as that of the polypeptide WO 95/23228 PCT/GB95/00372 7 shown in Figure 5 but which, by virtue of the degeneracy of the genetic code, possess a different nucleotide sequence to that shown in Figure 5. Such obvious variants are to be considered as functional equivalents falling within the scope of the invention.

In a second aspect the invention provides a polypeptide having exo-(1-4)-fl-Dgalactanase activity and comprising substantially the amino acid sequence encoded by nucleotides 229 to 2319 of the sequence shown in Figure 1. or a precursor or derivative of such a polypeptide. or a functional equivalent thereof.

SA particular functionally equivalent polypeptide is that encoded by nucleotides 117 to 2555 of the sequence shown in Figure 5. or a precursor or derivative thereof. One such precursor comprises, for example, the polypeptide encoded by nucleotides 42 to 2555 of the sequence shown in Figure In a third aspect the invention provides a vector comprising substantially the sequence of nucleotides 229 to 2319 of the sequence shown in Figure 1. or a functional equivalent thereof. Preferably the vector is capable of directing the expression of a polypeptide having exo-(l-- 4 )-fl-D-galactanase activity in an appropriate host. or is .capable (directly or indirectly) of interfering with the expression of such a polypeptide.

Transformation techniques for introducing the sequence of the invention into various hosts are well-known to those skilled in the art. Accordingly, in a fourth aspect the invention provides a host or host cell into which the sequence of the invention (or a functional equivalent thereof) has been artificially introduced. Preferabiv the host or host cell is a plant or plant cell, although other hosts could be employed. For example, if one wished to express large quantities of the enzyme. one could introduce the sequence of the invention into a yeast cell. Possible uses of such a purified, recombinant enzyme include the following: modification, degradation or liquefaction of plant materials in order to affect mechanical properties relating to eating texture; (b) particle sizes of. for example. fruit or vegetable juices (affecting haze): or (c) extractability of colours, flavours or vitamins.

WO 95/23228 PCT/GB95/00372 8 As will be clear to those skilled in the art, because of the role galactanase plays in breaking down polymers present in the plant cell wall, altering (increasing or decreasing) the levels, or altering the pattern, of expression of this enzyme in a plant might have an effect on certain characteristics of the plant. In particular, one might expect to be able to alter:-growth, texture or ripening of the plant or part thereof.

In a fifth aspect, the invention provides a method of altering the characteristics of a plant or part thereof, comprising introducing into the plant the sequence of the invention or a functional equivalent thereof, so as to alter the level or pattern of exo- (l--4)-/-D-galactanase activity in the plant.

If the sequence introduced into the plant is in the sense orientation relative to the promoter, it may result in increased levels of expression. Conversely, introduction into a plant of a sequence in the antisense orientation relative to the promoter may result in a reduction of levels of expression. The plant into which the sequence is introduced is preferably a commercially significant plant in which molecules comprising (l-4)-/-linked D-galactan residues perform a structural role. Examples of such plants include: alfalfa, apple, broccoli, cabbage, carrot, cauliflower. celery, cranberry, cucumber, eggplant. flax. grape. horseradish, kiwi. lettuce. mangoes.

melon. oilseed rape. papaya. pea. peaches. pears. peppers, plum. potato, raspberry.

soybean, strawberry, sugarbeat. sweet potato. tobacco, tomato and walnut.

With the knowledge of the sequence data disclosed herein, those skilled in the art will appreciate that it should prove possible to clone functionally equivalent sequences (as defined above) from other plants. Accordingly, in a further aspect the invention provides a method of isolating a functionaily equivalent sequence, comprising isolating mRNA from a plant of interest and screening (by means of hybridisation or PCR) the cDNA obtained therefrom using a probe nucleic acid sequence which is substantially complementary to at least part of the sequence shown in Figure 1.

The invention will now be described by way of example and with reference to the WO 95/23228 PCT/GB95/00372 9 drawings, of which: Figure 1 shows the nucleotide sequence (Seq ID No. 1) of the invention together with the amino acid sequence (Seq ID No. 2) of the polypeptide encoded by the nucleotide sequence: Figure 2 shows the N-terminal amino acid sequence data obtained by peptide sequencing of the 60, 45 and 15kDa ]upin polypeptides that co-purify during enzyme purification (Seq ID No.s 3-5 respectively); Figure 3 shows the N-terminal amino acid sequence data for the 60kDa lupin exogalactanase together with the nucleotide sequence of EXO1 (Seq ID No. the probe used to screen the cDNA library; Figure 4 shows a comparison between the cDNA-encoded polypeptide sequences of lupin exo-galactanase and that encoding a protein of unknown function from carnations (which sequence is already found in publicly available databases); Figure 5 shows the cDNA sequence (Seq ID No. 18) of a functionally equivalent enzyme irom tomato, together with the amino acid sequence (Seq ID No. 19) of the polypeptide encoded thereby; and Figure 6 shows a comparison of the amino acid sequences of the lupin and tomato enzymes: the tomato polypeptide sequence is shown boxed and those portions of the lupin polypeptide which possess an identical sequence are also boxed.

Examples SDS-PAGE and electroblotting for N-terminal sequence analysis: The lupin exo-galactanase enzyme was purified from 18-days after planting (dap) lupin cotyledons as described (Buckeridge and Reid 1993. abstracts described previously), WO 95/23228 PCT/GB95/00372 and supplied by Dr. J. S. Grant Reid (University of Stirling). The protein preparation was composed of a major protein (60 kDa), and two minor proteins (45 kDa and kDa) when analyzed on SDS gels. SDS-PAGE was performed by the method of Laemmli (1970. Nature 227, 680-685) on linear 10% acrylamide slab gels on a BioRad electrophoresis kit. Proteins were electroblotted onto the PROBLOTTT membrane (Applied Biosystems, Warrington, as described by Matsudaira (1987) with the following adaptations. Gels were pre-run with 50uM glutathione (Sigma) added to the cathode electrode buffer. Sodium thioglycolate (0.1mM. Sigma) was added to fresh cathode buffer for sample electrophoresis. Protein was stained with Coomassie brilliant blue following Applied Biosystem's recommendations.

Endoproteinase Lys-C digestion: A preparation containing approximately 50ug purified enzyme 8 00pmoles intact exogalactanase) was brought to pH 8.5. measured with indicator paper. using iM Tris/HCI pH 8.5. The sample was boiled for 5 min to denature the protein. Endoproteinase Lys-C (Sequencing grade: Boehringer Mannheim) was added at a ratio of 1:50 w/w.

The incubation was left at 37°C overnight and stored at -20°C prior to reversed-phase chromatography.

HPLC Separation of peptides: :Reversed-phase chromatography was performed at 30 0 C on Applied Biosytems model 130A HPLC separation system. Samples were loaded, via a 500ul looo onto a o. Brownlee RP 300-C8 microbore column (250 x 1mm id: 7u) pre-equilibrated in 0.1% TFA. Flow rate was 0. ml min Peptides were eluted with a gradient of increasing buffer B (90% acetonitrile: 0.085% TFA) 0-70% over 70 minutes.

Absorbance was monitored at 214nm and peaks were collected manually into Eppendorf tubes with a time delay to allow for the dead space between the detector and outlet. Fractions were stored at -20 0 C. Prior to loading sample onto the HPLC.

acetonitrile gradients were performed until a reproducible low baseline was obtained.

Protein sequencing: This was performed on the Applied Biosystems model 475 protein sequencer.

WO 95/23228 PCT/GB95/00372 11 At the stage of protein purification (18 the exo-galactanase enzyme was purified as a 60 kDa protein, along with a 45 kDa and a 15 kDa protein that co-purifies with it.

The N-terminal amino acid sequence of these three polypeptides is illustrated in Figure 2 (Seq ID No.s The Figure shows that the 60 and 45 kDa proteins have an identical N-terminal sequence suggesting that they are derived by C-terminal cleavage of the same parent molecule. Antiserum raised against and affinity purified on the kDa protein recognises larger proteins (-80 kDa) at earlier stages of seed germination.

It is possible that the purified enzyme (60 kDa) is proteolytically derived from a larger precursor. This is evident from the observation that the deduced protein coded for by the purified cDNA. has a molecular weight of -77 kDa. The C-terminal cleavage could occur in vivo or during the process of protein purification. The 60 kDa polypeptide clearly retains activity and is regarded as a functionally equivalent derivative of the sequence of the invention. The 45 kDa polypeptide may possess activity (this has not yet been investigated) and. if so. would also be regarded as a functionally equivalent derivative of the sequence of the invention.

mRNA isolation and cDNA library synthesis: 12 d.a.p lupin cotyledons were supplied by Dr. Reid (Univ of Stirling). Total RNA was extracted using Qiagen columns according to manufacturers recommendations, with some modifications. Total RNA was extracted in two batches. In each batch, g of 12 d.a.p lupin cotyledons was ground to a fine powder under liquid nitrogen and S. distributed to six tubes, each containing 3mls of cold extraction buffer (4M Guanidine thiocyanate, 100mM Tris HC1 pH7.5, and 25mM EDTA). To this was added 3ul of Bmercaptoethanol and 2401l of 25% Triton X-100 and the mix was incubated on ice for 15 min. 3mls of cold 3 M Sodium acetate pH6 was added and incubation on ice continued for a further 15 min. The homogenate was centrifuged at 15.000 xg for min at 4 0 C. 5mls of cold iso-propanol was added to the supernatant and incubated on ice for 5 min. The precipitate was concentrated by centrifugation at 15,000 xg for min at 4°C. The pellet was resuspended in 8ml of cold TE (20mM Tris HCI pH8, ImM EDTA). and undissolved particles were removed by an additional centrifugation at 20.000 xg for 15 min at 4~C. 2ml of S1 (2M NaCI and 250mM MOPS pH7) was added to the supernatant. which was then applied to a Qiagen-tip 100 column pre- WO 95/23228 PCT/GB95/00372 12 equilibrated with 3ml of buffer QAT (0.4M NaCl. 50mM MOPS pH7.0. 15% ethanol and 0.15% Triton X-100). Each column was then washed with 15ml of buffer QA (0.4M NaC. 50mM MOPS pH7.0. and 15% ethanol). Total RNA was eluted with 7 .5ml of buffer QRU (0.9M NaCl. 50mM MOPS pH7.0, 15% ethanol and 6M Urea), precipitated with an equal volume of iso-propanol for 10 min on ice and centrifuged at 15,000xg for 30 min at 4°C. The pellet was washed once in 80% ethanol. air dried and dissolved in a total of 1.5ml. All the fractions were pooled at this stage and the yield of total RNA quantified spectrophotometrically at O.D Messenger RNA was fractionated from 1.2mgs of lupin total RNA by affinity chromatography on oligo(dT) cellulose, using Poly Quik columns from Stratagene according to manufacturers instructions. 5ug of poly RNA was used to synthesize a directional cDNA library using a ZAP cDNA synthesis kit (Stratagene) and packaged using Gigapack II Gold T packaging extract (Stratagene) according to manufacturers instructions. The packaged library had a titre of approx 1x10 6 pfu of which 4 .8x10 4 pfu were screened in duplicate.

Design of oligonucleotide probe: A stretch of 8 amino acids (shown underlined and in bold type in Figure 3a) within the N-terminal peptide sequence of the mature lupin exo-galactanase enzyme was used to design an oligonucleotide probe (EXOl. shown in Figure 3b. Seq ID No 6) for the i screening of the cDNA library. The oligonucleotide was 23 nucleotides long with a degeneracy of 48 and incorporated one inosine at a wobble base position. The oligonucleotide probe was designed to be complementary to the mRNA coding for the lupin exo-galactanase.

cDNA library screening: Screening of the lupin cDNA library was essentially as described in the ZAP cDNA synthesis and cloning protocol (Stratagene). A total of 4.8x10 4 plaque forming units (pfu) were plated on four LB (Luria-Bertani) agar plates. using the Sure M strain of Escherichia coli bacteria (Stratagene). and incubated for 16 h at 39 0 C. The plates were then chilled for 2 hr at 4=C. and plaque lifts were made in duplicate onto 150 WO 95/23228 PCT/GB95/00372 13 mm nitrocellulose filters (SS) as described (Sambrook et al., (1989) Molecular Cloning, A Laboratory Manual. 2 nd edition, Cold Spring Harbor Laboratory Press).

Following denaturation and neutralization, the filters were baked at 80°C for 2 hrs.

The filters were incubated in 30mls of pre-hybridization solution (6xSSC. Denhardts, 100 ug/ml salmon testis DNA, and 0.5% SDS) at 42 0 C for 4 hrs with gentle agitation. 15pmol of oligonucleotide EXO1 was end labelled using gamma- 32

-P

ATP with polynucleotide kinase as described (Sambrook et al. 1989). Labelled oligonucleotides were separated from the unincorporated radioactive nucleotides by passage through a P-50 column, and added to the pre-hybridization solution at a concentration of 2xlO 5 cpm/ml. Hybridization was carried out for 18 hrs with gentle shaking at 42"C. following which the filters were washed briefly (1-2 mins) in two changes of 6xSSC. wrapped in saran-wrap, and exposed to x-ray film (KodakTM

LS).

Autoradiographv was carried out for 16 hrs at -80°C in cassettes with intensifying screens. Positive plaques were identified by autoradiography and the plaques picked into SM. 12 positive clones were taken through an additional round of screening to plaque purity. An estimate of the abundance of the mRNA coding for the lupin exogalactanase cannot be made based on these results as the probe used (designed to the N-terminal peptide sequence), would be expected to detect only full length cDNAs.

Following the second round of screening. 6 plaque pure positives were isolated, and in vivo excised.

In vivo excision: Positive clones were in vivo excised with the Bluescript T phagemid from the Uni-Zap XRTM vector as described in the manufacturers protocol (Stratagene), plasmid preparations of the isolated clones were made using Qiagen P-100 tip columns as recommended by the manufacturer. Clones were further investigated by PCR (polymerase chain reaction) and by restriction analysis. 5 of the 6 isolated clones were successfully amplified using EXO 1 and a vector-based primer for amplification in the PCR. The 5 clones all possessed an insert of similar size (-2.600 bp) which was released upon digestion of the purified Bluescript plasmid with the restriction enzymes Eco RI and Xho I. as determined by agarose gel electrophoresis.

WO 95/23228 PCT/GB95/00372 14 Sequence determination: The cDNA isolated by screening the lupin cDNA library using the oligonucleotide EXO 1 was analyzed by sequencing the double stranded plasmid using appropriate primers by Taq Dye-Deoxy'M terminator chemistry and analyzed on the automated

ABI

373A DNA sequencer.

Sequence analysis: The isolated cDNA clone (Seq ID No. 1, shown in Figure 1) is 2628 bp long and includes a 30 bp poly A tail. The longest open reading frame (2190bp. upper case letters) codes for a 730 amino acid polypeptide (Seq ID No. 2, 81.6 kDa, shown above the nucleotide sequence in single letter code) and includes a 33 amino acid putative signal peptide. However, there are three additional possible start sites between the first start (ATG) codon and the N-terminal of the mature protein which would reduce the signal peptide to either 26, 22 or 15 amino acids. The first three start sites all precede the hydrophobic core that is located within the signal peptide. The mature enzyme is coded for by 697 amino acids with a molecular weight of -77 kDa. As previously mentioned, the antibody raised against the -60 kDa protein cross reacts with a larger protein (-80 kDa), and this might serve as a precursor to the 60 kDa protein by cleavage at the C-terminal end. as the N-terminal sequence of the 60 kDa band and the mature enzyme (deduced from the cDNA) are identical. The serine (underlined) at residue 34 marks the start of the amino acid sequence of the mature protein. the deduced sequence immediately C-terminal of this corresponding to the amino acid sequence actually determined by protein sequencing. The N-terminal of the small kDa) protein has been located within the deduced amino acid of the cDNA. and cleavage at this point would release a 12.5 kDa protein, confirming that this molecule is derived by the cleavage of the C-terminal of the synthesized protein. All the peotide sequences derived by protease digestion and sequencing of the enzyme (peptide sequence data obtained include the following: VAKKQPLAWYKTT,

FSAPAGNDPL.

GEVWVNGQSIG, and GNCGNCNYAGTYTDTK. Seq ID No.s 7-10 respectively) have been located within the deduced amino acid sequence of the cDNA. further confirming the identity of the cDNA as the one coding.for the lupin exo-galactanase.

Whether the synthesized enzyme is specifically cleaved in vivo or whether cleavage WO 95/23228 PCT/GB95/00372 occurs during the purification process is open to speculation.

Homology to other sequences: The lupin exo-galactanase shows high homology to at least one other sequence. At the amino acid level, it has a 66.5% identity over a 717 amino acid overlap with the deduced amino acid sequence of a highly expressed ethylene regulated gene with unknown function isolated from senescing carnation petals (Raghothama et al., (1991) Plant Mol. Biol. 17. 61-71). This level of amino acid.homologv is insufficient for the sequence to be considered as a functional equivalent of the lupin exo-galactanase.

A comparison between the two polypeptide sequences is shown in Figure 4, the carnation sequence (CARS12.p.ro) above the lupin sequence (LEG11CON.pro).

Comparison of these two sequences reveals a number of peptides (shown in boxes) which might well be conserved among enzymes of this type. It is suggested that functional equivalents of lupin exo-galactanase which exhibit a higher degree of homology than that shown by the carnation polypeptide may also comprise these or similar peptide sequences.

Isolation of exo-galactanase cDNAs from other plants With knowledge of the sequence of the exo-galactanse from lupins, it should prove possible for those skilled in the art to isolate functionally equivalent cDNAs from other plants. Described below is a method which could potentially be used for this purpose.

The cDNA coding for the lupin exo-gaactanase will be used to isolate functional homologues from other plants tomato). The lupin cDNA-will be radiolabelled and used as a probe to screen a cDNA library constructed from mRNA isolated from the plant of interest. About 3x10 4 pfu will be plated on each LB-agarose plate (as i.described previously) and filters will be probed with the radiolabelled full-length lupin exo-galactanse cDNA. The hybridisation solution will be as described above, but the temperature of hybridisation will be reduced to about 50 0 C. The lower temperature is necessary because a heterologous probe lupin cDNA) will be used to screen the cDNA library. The hybridised filters will be washed in 2x SSC at room temperature for 20 minutes and exposed to x-ray sensitive films. Putative positive clones will be WO 95/23228 PCT/GB95/00372 16 plaque purified and sequence-analysed for homology with the lupin exo-galactanase cDNA. The example below illustrates how this procedure was used to isolate cDNA clones encoding an exo-galactanase from tomato fruits which is functionally equivalent to that obtained from lupin.

Purification of a polypeptide from tomato with exo-galactanase activity Step 1 Assay for activity of exogalactanase Exo-galactanase activity in various tomato fruit extracts was measured by the release of free galactose from galactan isolated from lupin seed. Each assay contained the following components: 3 0,ul of a 1% galactan solution. 15.l 1M ammonium acetate pH containing 0.1% sodium azide and up to 3 0ul tomato extract depending on enzyme activity. Assays were incubated at 30'C for 17-24 hours and terminated by boiling the solution for 2 min. Aliquots (64ul) were used for determination of galactose using p- D-galactose dehydrogenase essentially as described by Kurz and Wallenfels (1974. In "Methods of enzymatic analysis". pp1279-1282. Verlag, Chemie. Weinheim).

Step 2: Extraction of exoealactanase from tomato nericarp S::It was found that exo-galactanase activity could be efficiently extracted from tomato fruit pericarp by 0.2M sodium phosphate buffer. pH 7.2.

i Step 3: Choice of starting material for nurification Crude extracts were made (in 0.2M sodium phosphate buffer. pH 7.2) from tomato pericarp (Lycopersicum esculentum. var. Moneymaker) tissues taken from various stages of growth and ripening. It was found that pink and red fruit had the highest exogalactanase activity, whether expressed as total or specific activity. Therefore the pericarp of red fruit was used as the starting material for purification.

Sten 4: Amnmonium sulphate preciitation Pericarp tissue (500g) from red fruit was harvested, frozen in liquid nitrogen, and stored at -20'C.The tissue was homogenised in g: 1.5vol 0.2M sodium phosphate. pH WO 95/23228 PCT/GB95/00372 17 7.2 with 1.0% insoluble PVP (polyvinyl pyrrolidone) in a blender. The homogenate was stirred for 1 hour at 4'C to allow diffusion of cell wall enzymes into the extraction buffer. Insoluble material was removed by centrifugation at 20,000 x g, min. 4"C in the SS34 rotor of the Sorvall RC5B centrifuge. The supernatant was passed through glasswool, and brought to 30% ammonium sulphate saturation by addition of solid ammonium sulphate 16 4 g/100ml). The mixture was stirred for min at 4 C, and the precipitated proteins were removed by centrifugation (30,000 x g min. The supernatant was brought to 70% ammonium sulphate saturation by addition of solid ammonium sulphate 2 4 .9g!100ml). The mixture was stirred for min at 4"C, and the precipitated proteins collected by centrifugation (30.000 x g, min, The ammonium sulphate precipitated proteins were stored at without resuspension.

Step 5: DE52 chromatographv 30-70% ammonium sulphate precipitated proteins were resuspended in 48mi of Tris/HC1, pH 7.8 (Buffer A) and dialysed overnight against 4.5L of Buffer A. The sample was centrifuged at 30,000 x g for 10 min. 4'C and loaded onto a 40ml DE52 column (Whatman). equilibrated in Buffer A. at 0.5mlimin. The column was washed with Buffer A (flow rate 1.0ml/min) until all unbound proteins were removed. Bound proteins were eluted with a 0-100% gradient of Buffer B (as Buffer A. but with 1M NaC) over 6 column volumes. Fractions were collected and assayed for exogalactanase activity. Fractions containing high exo-galactanase activity were either dialysed immediately as detailed below or stored at -20 0

C.

Sten 6: Mono P chromatopraohv Fractions pooled from DE52 chromatography were dialysed overnight against 4.5L of 0.025M triethanolamine/iminodiacetic acid pH 8.3 (Buffer The dialvsed sample was centrifuged and the supernatant loaded via a 50ml superloop onto a Mono P column (Pharmacia HR 5/20), equilibrated in Buffer C, at a flow rate of Imlimin.

The column was washed in Buffer C until all unbound proteins were removed. Bound proteins were eluted from the column with 50ml 10% polybuffer 7/4. pH 4.8 /iminodiace:ic acid, at a flow rate of Imlimin. Fractions (0.5ml) were collected and WO 95/23228 PCT/GB95/00372 18 assayed for exo-galactanase activity. Fractions containing activity which eluted early in the pH gradient (approx pH 8.0) were pooled and dialysed overnight against ammonium acetate pH 5.0 (Buffer D).

Step 7: Lactose agarose chromatographv Dialysed sample was loaded onto a 5ml lactose agarose (Sigma) column. equilibrated in Buffer D, at a flow rate of 0.13mlimin. The column was washed with Buffer D to remove unbound proteins. Bound proteins were eluted from the column with 0.1M Tris/HC1 pH 8.6/0.2M NaCI. Fractions (0.5ml) were collected and assayed for exogalactanase activity.

Stet 8: N-terminal amino acid seouencino Fractions from the lactose agarose column with high exo-galactanase activity were pooled. concentrated 16-fold using a centricon 10 (Amicon). and separated according to apparent molecular weight on a 12.5% SDS polyacrylamide gel. After electrophoresis, proteins were transferred onto a ProBlott membrane (transfer buffer 10mM CAPS pH 11 in 10% methanol). The ProBlott membrane was stained in 0.1% Coomassie Brilliant Blue R250 in 1% acetic acid, 40% methanol and destained in 50% methanol.

Several protein bands were visible. Two bands, of approximate molecular weight 40kDa and SOkDa. in fractions coincident with exo-galactanase activity, were excised and subjected to N-terminal amino acid sequencing on an ABI model 475 protein sequencer.

The 80kDa tomato polypeptide was identified as an exogalactanase by its homoiogy to the lupin enzyme.

Tomato polypeptide: SVSYDDRA" NG*R (Seq ID No. 11) Lupin Exo-gaiactanase: SVTYDHKAIMINGQR...etc unassigned amino acid) The identity of the 40kDa protein is unknown: WO 95/23228 PCT/GB95/00372 19 Tomato polypeptide: FSNNNFVATDGTHFALNGKS (Seq ID No. 12) The retention of exo-galactanase activity is highly dependent on ionic strength, and activity can be lost if the ionic strength of chromatography buffers etc. is less than 100mM. An improved purification procedure would be to include 100mM NaCI (minimum) in all chromatography buffers. The above protocol would need reevaluation and optimisation to realise the same purification but yield of exo-galactanase protein and activity should be enhanced.

Isolation of partial cDNA clones encoding tomato exo-galactanase Using a heterologous cDNA probe (2628 bp) coding for lupin exo-galactanase and a hybridization temperature of 55 0 C. 2 partial cDNA clones. TEG13 (1082 bp) and TEG6 (415 bp), were isolated from a commercial tomato fruit (breaker stage) cDNA library obtained from Clontech Laboratories Inc. [prepared using mRNA from ripening (breaker stage) fruit (Ailsa craig cultivar VFN8). primed with oligo-(dT) and random primers and cloned into lambda gtl Approximately 300,000 cDNA clones from this library were screened using a 3 2 P-dCTP (Amersham International plc) radiolabelled probe prepared using a kit supplied by United States Biochemicals Inc. (Sequenase v2.0). Unincorporated nucleotides were removed by chromatography through a 9Sephadex G-50 column. Both clones TEG13 and TEG6 were PCR amplified using TAQ polymerase and lambda gtll specific primers (GTI1 ACTCCTGGATCCCGTCAGTAT. Seq ID No. 13; and GT11 3'K: T T A Se.ID N TAATGGTACCGACCGGCG CT. Seq ID No. 14). cloned into pT7 Blue PCR cloning vector (AMS Biotechnology Limited) and transformed into competent E. coli, according to the manufacturer's instructions. Colonies containing recombinant plasmids were used to inoculate 30 ml Lennox Broth containing 100.ug/l Carbenicillin: after overnight growth at 37 plasmid DNA was purified using a Qiagen plasmid DNA extraction kit, PEG precipitated and washed thoroughly with 70 EtOH.

DNA sequence data were obtained using an automated ABI sequencer, and analysed using an Apple Mac computer and DNAstar software. TEG13 exhibited 64.2 homology with nucleotides 678 to i760 of the lupin exo-galactanase sequence. TEG6 WO 95/23228 PCT/GB95/0037 2 exhibited 62.7% homology with nucleotides 2014 to 2428 of the lupin exo-galactanase sequence.

Isolation or an overlapping cDNA fragment Because the cDNA clones TEG13 and TEG6 aligned with discreet regions of the lupin exo-galactanase cDNA, a RACE procedure was performed (according to Frohman

M,

Rapid amplification of cDNA ends (RACE): User friendly cDNA cloning, Amplifications: A forum for PCR users, pp 11-14) in order to obtain an overlapping cDNA fragment extension of TEG6). A 5' cDNA pool was prepared by reverse transcription of tomato fruit (breaker stage) total RNA with MuMLV-RT and random hexanucleotide primers, and subsequent tailing with terminal deoxy-transferase

(BRL)

in the presence of dATP. In a first round PCR amplification of the 5' cDNA pool, the primer 6A1 (position 84-64 in TEG6 and 1844-1824 in fig. 5) was used in combination with an outer adaptor primer (Ro: AAGGATCCGTCGACATC, Seq ID No. 15) and

AAGGATCCGTCGACATCGATAATACGACTCA

CTATAGGGAI 1 i 1 1 1 IT T, Seq ID No. 16) which annealed to the 5' tail of the cDNA. In a second round of PCR amplification. the primer 6A3 (position 42-22 in TEG6 and 1802-1782 in fig. 5) was used in combination with a nested inner adaptor primer GACATCGATAATACGAC. Seq. ID No. 17). again annealing to the of the cDNA. A RACE product of 511 bp was obtained, extending 469 bp beyond the 5 end of TEG6. This fragment was cloned into pT7 and sequenced. The sequence data revealed that the 5' 209 bp of the RACE product was 100% homologous to the 3' end of TEG13. suggesting that TEG13 and TEG6 were 2 partial cDNA clones representing the same gene (TEGi). A homology of 59.2 was found between this overlapping RACE product (TEG7) and nucleotides 15--2054 of lupin exo-galactanase.

All DNA alignments were calculated using a DNAStar Megalign program (multiple alignment using the Clustal method. Gap penalty: 10. Gap length penalty: By overlapping the sequences of TEG13. TEG7 and TEG6. a hybrid cDNA molecule Sof 1757 bp length, containing a continuous open reading frame, is formed. The encoded polypeptide exhibits 68.17 identity to amino acids 135 to 717 of lupin exo- WO 95/23228 PCT/GB95/00372 21 galactanase.

In order to obtain the sequence information at the 5' end of this clone. 3 subsequent RACE experiments were performed on the 5' tomato cDNA pool (5 ul).

1st 5' RACE In the first round of PCR amplification, the primer 13A2 (position 106-86 in TEG13 and 524-504 in fig. 5) was used in combination with the outer adaptor primer (Ro) to amplify sequences from the 5' cDNA pool. In the second round of PCR. the primer 13A5 (position 62-42 in TEG13 and 480-460 in fig. 5) was used in combination with the inner adaptor primer (R3. A specific fragment of 214 bp was recovered from a 0.8 agarose gel using DEAE paper. cloned into plasmid pT7 and transformed into competent E. coli The resultant clone was called 5"TEG1.1 and had a 62 bp overlap with the hybrid cDNA molecule, extending the sequence to 1909 bp.

2nd 5' RACE 2 new primers, 1A1 (position 28-8 in 5'TEG1.1 and 294-274 in fig. 5) and 1A2 (position 63-43 in 5'TEG1.1 and 329-309 in fig. 5) were designed for use in a second RACE experiment. Primer 1A2 was used in the first round of amplification of the cDNA pool and primer 1A1 in the second round of amplification. in combination with S:

R

0 and R. respectively. A fragment of 206 bp. was obtained. purified, cloned and sequenced as described above. The clone was called 5'TEG1.2 and had a 26 bp overlan with the extended hybrid cDNA molecule, further extending the sequence to 2089 bp.

The 5' end of the extended sequence encoded the C terminal 10 amino acids of a signal peptide followed by the mature N-terminus of tomato exo-galactanase (11/12 amino acids conserved with the N-terminus of purified tomato exogalactanase described above).

3r. d 5' RACE In order to obtain sequence encoding a complete signal peptide, 2 further primers, 1A6 (position 52-32 in 5'TEG1.2 and 138-118 in fig. 5) and 1A7 (position 32-12 in 5'TEG1.2 and 118-98 in fig. 5) were synthezised. Primer 1A6 was used in a first WO 95/23228 PCT/GB95/00372 22 round of amplification and primer 1A7 in the second round, as described above. A cDNA product of 117 bp was obtained, purified, cloned and sequenced as above. The clone was called 5'TEG1.3 and had a 32 bp overlap with the extended hybrid cDNA molecule, further extending the sequence to 2 175bp. The extended sequence encoded a complete signal peptide of 25 aa.

3' RACE In order to obtain a cDNA molecule corresponding to the 3' end of the TEG1 gene, a RACE experiment was carried out on a 3'cDNA pool derived from reverse transcription of tomato fruit (breaker stage) total RNA with MuMLV-RT and the primer. The primer 6S2 (position 340-360 in TEG6 and 2100-2120 in fig was used in the first PCR reaction, in combination with R, and the primer 6S3 (position 373-393 in TEG6 and 2133-2153 in fig. 5) in the second (nested)

PCR

reaction, in combination with The resultant RACE product was 812 bp long and had a 43bp overlap with the extended hybrid cDNA molecule, further extending the sequence to 2944bp, including 23 "A"s at the 3' end.

The complete TEGI cDNA sequence (Sea ID No. 18) is shown in Figure 5 and was found to contain a 2514 bp long open reading frame (nucleotide 42 to 2555) encoding a polypeptide (Seq ID No. 19) of 838 amino acids. The signal sequence cleavage site is marked with an arrow. The mature exo-galactanase protein encoded by TEG1 (minus a 25 amino acid signal peptide) is 813 amino acids in length, has a molecular weight of 90.623 daltons and exhibits 70% homology (Lipman-Pearson algorithum. gap penalty: 4. K tuple: 2. length: 12) with the mature exo-galactanase protein encoded by the luoin cDNA and may therefore be regarded as functionally equivalent to the lutin enzyme.

In Figure 5. nucleotide 1445 is shown as a Y (IUPAC code) whilst in Sea. ID No. 18 in the sequence listing the nucleotide is shown as T. This is purely for convenience: 0Sthe precise identity of the nucleotide is uncertain. It will be appreciated that, as the nucleotide is at position 3 of a codon, its precise identity is not critical. It will further be noted that the deduced amino acid sequence of the polypeptide encoded by TEG1 differs slightly from the N-terminal sequence of the purified polypeptide as determined WO 95/23228 PCT/GB95/00372 23 by peptide sequencing. Presumably there are a number (at least two) of related genes allelic variants) in tomato plants which encode functionally equivalent enzymes having slightly different amino acid sequences.

Figure 6 shows a comparison of the full length amino acid sequences of the lupin enzyme (LEG11CON.pro) and the tomato enzyme (CONTIG.TEG1.PRO). The tomato sequence is shown boxed, and the boxed regions include those portions of the lupin enzyme where the sequence is identical to that of the tomato enzyme. It will be observed that there are several portions of extensive homology tomato residues 120-138 lupin residues 128-146; tomato residues 396-408 lupin residues 404-416) which may be important to the catalytic function of the molecules.

onstruction of lan transformation vectors for antisense exoeriments.

In order to assess the phenotypic consequences of down regulating the expression of TEG1 in transgenic plants, plant transformation vectors were constructed for antisense experiments. The vectors were transferred into Agrobacterium tumefaciens LBA4404.

Tomato cotyledons (variety "Moneymaker") were infected with A. tumefaciens carrying the vectors.

The vector p35STEG1A was constructed by inserting a 1082 bp Sstl/XbaI fragment of TEG1 (419-1500) in the antisense orientation between the constitutive 35S promoter and the nos polvadenylation signal of The vector p35S/TEG1B was constructed by inserting a 511 bp SstI/XbaI fragment of TEG1 (1292-1802) in the antisense orientation between the constitutive 35S promoter and the nos polyadenylation site of The vector pKan35S was constructed by inserting a 0.8kb HindIII-Xba fragment, contaning a 35S cauliflower mosaic virus constitutive promoter (from the vector pcTAK. which vector is described by Toepfer e al., 1987 NAR 15. 5890 er seq.), behind the nos poly-adenylation site of pGPTV-kan (Becker et al., 1992 Plant Molecular Biology 20. 1195-1197).

WO 95/23228 PCT/GB95I00372 24 The vec-tor pPG/TEG1A was constructed by inserting a 1082 bp PstI/Blunt/SstI fragment of TEGI (419-1500) in the antisense orientation between a fruit-specific (-942 to +33) PG promoter (Bird et al., 1988 Plant Molecular Biology 11, 651-662) and the nos polyadenylation site of pGrPTV-kan.

WO 95/23228 PCT/GB95/00372 SEQUENCE

LISTING

GENERAL INFORMATION:

APPLICANT:

NAME: Unilever PLC STREET: Unilever House. Blackfriars CITY: London COUNTRY: United Kinadom POSTAL CODE-(ZIP): EC4P 4BQ NAME: Unilever NV STREET: 455 Weena CITY: Rotterdam COUNTRY: Netherlands POSTAL CODE (ZIP): 3013 AL (ii) TITLE OF INVENTION: Novel Exo-(1-4)-Beta-D galactanase (iii) NUMBER OF SEQUENCES: 19 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2628 base pairs TYPE: nucleic acid STRANDEDNESS: singae TOPOLOGY: linear S(ii) MOLECULE TYPE: cDNA (ii) HYPOTHETICAL: NO S'0: (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION:130..2322 (xi) SEQUENCE DESCRIPTION: SEO ID NO:1: S- CAACACTCTT ATAC.ATAAG AGACTCTCAA AAAGTAGCAA AATAAAAAGA CACTATATAC AAAACAGAAA ATATTTCTTC TTCTATAGAA AGACAACATT GCTTATATAG AAACATAGCA 120 WO 95/23228 PCIGB9S/00372 TTTTTTGTT ATG TTT GGT TCA AGA ATT Met Phe Gly Sen Ag Ilie GTG ATG GAG AGT Val Met Giu Sen TTA ATG TCT Leu Met Ser AGG~ AGA Ara Aro

AD

MAT TlT CAT ATG Asn Phe His Met

GTG

Val1 20 TTG CTGTTA

HTG

Leu Leu Leu Leu mT Phe TTT TGG GTT TGT Phe Trp Vai Cys

TAT

Ty r GTC ACA GCC TCT Val Thr Ala Sen ACT TAT GAT CAT Thr Tyn Asp His

A

Lys GCC ATT ATG ATT Ala Ilie Met Ilie

MAT

As n 168 216 264 312 360 GGG CAG AGA AGA Gly Gin Ang Ang Ail Ile HiG ATC TCT GGT Leu Ile Sen Gly AYE CAC TAT CCA Ilie His Tyr Pro AGA AGO Ang Sen ACA CCT CAG Thr Pro Gin TGG CCA GAC OHT AYE CMA MG Trp Pro Asp Leu Ilie Gin Lys GCC AAA Ala Lys GAT GGA GGG Asp Gly Giy GMA COT TCT Giu Pro Ser C7E GAT Leu Asp CCT GGA Pro G11 G7 Val1 ATA GAG ACT TAT Ilie Giu Thr Tyr

GTG

Val1 85 HTC TGG MAT GGA Phe Trp Asn Giy

CAT

His AAA TAT TAT7 Lys Tyr Tyr Phe GAT AGG TTT GAO Asp Ang Phe Asp C7 Leu 105 GHT GGG HiC ATA Val Gly Phe Ilie iiG GYE CAG CAA Leu Val Gin Gin

GOT

Ala 115 GGT OTA TT GYE Gly Leu Phe Val

CAT

His 120 CTC AGG AT GGT Leu Arg Ilie Giv

CT

Pro 125 408 456 504 552 600 S S

S

S S S. S

*SS.

S S

S

*5

S

*5S

*SS*

S TTC ?TA TGT GCT Phe l!e Cys Ala TAT GTE COT GGT Tyr val Pro Gly 145 GCA ?TG CMA A Ala Met Gin Lys 160

GMA

Gi u 130 TGG MAC mE GGA Trp Asn Phe Gly

GGA

Gi y 1A5 TVT CCT GTT TGG Phe Pro Val Trp CTC A Leu Lys 140- AT GOT TO AGA lie Ala Phe Arg

ACA

Th r 150 GAC MAT GAG Asp Asn Giu TC ACT GAG Phe Thr Glu AT GTA MAT ATA Ie Val Asn lie COT TO MAG GAG Pro Phe Lys Glu ATG AAA GOA GAG Met Lys Al a Giu 170 TCT CAG ATA GAG Sen Gin lie Giu MAG Lys Leu iii CMA TOO LAG Phe Gin Sen Gin

GGA

Gly 180

GGT

Giv OCA ATA AT Pro lie ile

CTG

L eu 185 696 701 GAG TT GGA COA Giu Tvr Gly Pro

GTG

Val1 115 GMA TGG GMA AT Glu Trp Giu ie

GGT

Gi y 200 GOT OCT GGA A Ala Pro Gly Lys

GCT

Ala 205 TAT ACO MAA TGG Tyr.Thr Lys Trp

GCT

Ala 210 GOT CMA ATG GCT Ala Gin met Ala GGT CTA GAT ACT Gly Leu Asp Thr GGT GTC Gly Val 220 792 WO 95123228 WO 9523228PCTIGB951oo372 CCA TGGS GTT Pro Trp Val ATG TGC MAG CMA GMA GAT Met Cys Lys Gin Giu As[) 4225 230 GCA CTT7 GAT Ala Leu Asp CCT All Pro Ile 235 AllF GAT Ilie Asp ACC TGC Thr Cys AMA rCC Lys Pro 255

MAT

As n 240 GGA TTT TAC TGT Giy Phe Tyr Cys

GMA

Giu 245 MCL llC ACT CCA Asn Phe Thr Pro

MAC

As n 250 MAG MAC TAC Lys Asn Tyr AAA 77lG TGG ACA ILys Leu Trp Thr

GAA

Gi u 260 MAT TGG ACT GGC Asn Trp Th rGly

TGG

TrD 265 TAC ACT GCT Tyr Thr Ala Phe 936 GGT G^A ACC CCT G!lY Ala Thr Pro

TAT

Ty r 275 AGA CCA GCA GMA Arg Pro Ala Giu

GT

Aso 280 ATA GCA i77 TCA le Ala Phe Ser Va 1 285 984 GCC 7 TTC All A la Aro CPhe le

CAG

Gin 290 MAT CGCrr- GGC T CA1 Asn Arc Giy Ser

CTC

Leu 295 T 77 MAC TAC TAT Phe Asn Tyr Tyr ATG TATJ Met yr 300 1032 CAT GGA GG A His Gilv Giv ACA AGTTA Thr Ser Tyr 320

ACT

Th r 305 MAC TTT1 GGC C"GG Asn Phe Gly Arg AC A Th r 310 TCG MAT USGC CTC Ser Asn Gly Leu llIC GllT G C Phe Vai Ala 315 Cll7 CTA MAT Leu Leu Asn 1080 GAC TAT GAT GCT CC Asp Tyr Asp Ala Pro 325 A"l GAT GMA TAT lie Asp Glu Tyr

GGA

Gi y '33 0 1128 a a a a. a a GMA C CA AM4 TGG GGG CAT CTG Glu Pro Lys Trp Gly His Leu 335 340 AGA GMA llA CAT Arg Giu Leu His

AGA

Arg 345 GCA ATA AMA CMA Ala lie Lys Gin 1176

TGC

Cys 350 GAG 7CG GCT 7 A G3i Ser Ala Leu

GTG

Val1 355 TGGTSz GAT CCC Ser Val Aso Pro

ACA

Thr 360 GTG TCA TGG CCT Val Ser Trp Pro

GGA

Giv 365 1224 AA MCAL C77 GAG Lys Asn Leu Giu

GTA

Val1 370 CAT llG TAC" MAG His Leu Tyr Lys

ACA

Thr 375 GAG TCT GCC TGT Giu S-r Ala Cys GCT GCA Ala Ala 380 llC GSA Phe Giv 1272 llT Il G CA Phe Leu Ala

MAT

As n 385 TAT MAC ACC G AC Tyr Asn Thr Asp Ty r TCA ACG CMA Gll7 Ser Thr Gin Val

AMA

Ly s 395- 1320 MAT GGA Asn Giv TGC M Cys Lys 415

CM-%

Gin 400 TAT GAT CTA CCA CCT T1GGU" TCT AT AG Al Tyr Asp Leu Pro Pro Trp Ser Ilie Ser Ilie 405 410 ACT GMA G7l 7C Thr Giu Val Phe

MAC

As n 420 ACT

GCAMGGT

Thr Ala Lys Val

MAT

As n 425

TCC

Ser Cll CCT SAC Leu Pro ASD CCG AGA 7 A Pro Arg Leu 1 368- 1416 WO 95/23228 PTG9/o7 PCIVGB95MO372

CAT

His 430 AGG AAA ATG ACT CCA Arg Lys Met Thr Pro 435 GTA MAC AGT GCA Val Asn Sen Ala mT Phe 440 GCT TGG CAG TCA Ala Trp Gin Sen

TAC

Ty r 445

TAT

Tyrn MAT GMA GMA CCT Asn Giu Glu Pro

GCA

Ala 450 TCA TCA AGC GMA Ser Ser Sen GiU

MAT

As n 455 GAT CCC GTC ACA Asp Pro Val Thn

GGA

Gly 460 1464 1512 1560 GCA CTA TGG Ala Leu Trp TGG TAC CTG Trp Tyr Leu 480 CAG GTT GGC GTG Gin Val Giy Val

ACC

Th r 470

GGT

Gi y CGC GAT TCT TCC GAT TAT 1TG Ang Asp Sen Sen Asp Tyr Leu 475 ACA GAT GTC MAC Thr Asp Val Asn

AT

Ile 485 COT MAT GAT Pro Asn Asp

ATA

Ilie 490 MAG GAT GGG Lys Asp Giy AAA TGG Lys Trp 495 OCT GmT CTG ACA Pro Val Leu Thr

GCA

Ala 500 ATG TCA GOA GGT Met Sen Ala Gly

OAT

His 505 GTT CTG MAT Q7 Val .Leu Asn Val 1608 1656 1704 1752

TO

Phe 510 ATC MAT GGT CMA le Asn Gly Gin GOA GGA ACT GCA Ala Giy Thr Ala

TAT

Ty n 520 GGG AGT CTA GAT Gly Sen Leu Asp

GAT

AspD 525 OCT AGA iTA ACA Pro Ang Leu Thr MAG ATT TOT TTA Lys Ilie Sen Leu 545 CAC iTT GAG ACA His Phe Glu Thr 560 Phe 530 AGT CMA AGT GTG Sen Gin Sen Val

MAT

As n 535 CTG AGA GT7 GGC Leu Ang Val Gly MAT MOC Asn Asn 540 C0T AGT GIT TOO Leu Sen Val Sen GTi Val1 550 GGT CTC GCG MAT Gly Leu Ala Asn 00.

N GmT GGT ACT Val Gly Thn ACA CTG ACA Thr Leu Tnn TGG MAT ?CT TnD Asn Thnn

GGA

Gily GTG C-F GGT OCA Val Leu Giy Pro

GTC

ValI 570 GGT CTA Gly Leu 575 AGT AGO GGA ACA Sen Sen Gly Thr GAT C7T TOG MAG Asp Leu Sen Lys

CMA

Gin 585 AAA TGG TOT TAO Lys Trp Sen Tyr 1800 1848 1896 0092 2040

MAG

Lys 590 AiT GGT GIG A Ilie Giy Leu Lys

GGT

Gi y 595 GAA AGC TG AGO Giu Sen Leu Sen 0TT Leu 600 CAT ACT GMA GOT His Thr Giu Ala

GGG

Gly 605 4**e

S.

AGT MOC TOT GTi Sen Asn Sen Val OCT TTG GCA TGG Pro Leu Ala Tro 625 TGG GTA CMA GGA Tnp Vai Gin Giv -r Sen 615 TTA GTG GOT A Leu Val Aia Lys AAA CMA Lys Gin 620 MAC GAT Asn Asp TAT MAG ACA ACT wT Tyr Lys Thn Thr Phe 630 AGO GCA OCA GC Sen Ala Pro Ala

CCC-

Gi y 635 WO 95/23228 PCT/GB95/0037 2 CCG TTG GCT Pro Leu Ala 640 CTG GAT 17A GG-T Leu Asp Leu Gly

AGC

Ser 645

CAT

H-is ATG GGT MAG GGT Met Gly Lys Gly

GMA

Gi u GTA TGG GTA Val Trp, Val 2088 AAT GGT Asn Gly 0655 CMA AGC ATT GGA Gin Ser Ile Gly C GC Arg D60 TGG CCT GGA Trp, Pro Gly

MAT

As n AAA GCT CGT GGT Lys Ala Arg Gly

AAT

As n 67 D TGC GGC MAT TGT Cys Gly Asn Cys

MAT

As n 675 TAC GCT1 GGA ACT Tyr Ala Giy Thr

TAT

Ty r 680 ACC GAT ACA A Thr Asp Thr Lys

TGC

Cys 685 1!TA GCA MAC TGT Leu A la Asn Cys

GGA

Gly 690 CMA CCC T'CC CMA Gin Pro Sen Gin 2136 2184 2232 2280 2 32 2

AGA

Ara 695 TGG TAT CAT GTT1 CCT C'GG Trp Tyr His Val Pro Arg 700 TCA TUSG CTG Ser TrD Leu GGA GGT GAT G*'y Gl iAsp .720 TCG GU^T G-GT MCr Ser Gly Gly Asn

TAC

Ty r 710 T7h GTGT GTG CTj'A Leu Val Val Leu

GMA

G-I u GMA TGG Glu Trp, CCT MAT GGA ATF Pro Asn Gly lie

GCT

Al a TG GT1G GMA AGA ACA TMA Leu Val Giu Arg Thr* 730 S S S *5 *5 S

S.

A GT GT-1ATT CA TGTGAT ACCA MATGT1ACATG T TATGT-1ACAT TAT7T UTTC CA TATACTACAT TACAGGGSTT7

GTGTCACMAT

ITGGT-ATAGA AGGGAMAGAG TTGMATACCC

MAAATGGG'TC

AAATAGA-iTT C -TFICAT -1 CTATATCMC^

TATTATGTMA

T.AATA"AATAG,- TG"TGCATTT GGL' AAAA .AMkAAA kAAMA AGT1GMAACTA

TTIATGCTGAA-

GMCATTGAG TCC7, AAACA MAAATACTAC ATT GTCCTAG GMACAMTTG -5MAGTMTAC 2382 2442 2502 2 56 2 20522 2 62 8 IN'FORMATION FOR SEQ ID NO:2: SEDUENCE

CHARACTERISTICS:

LENGTH: 731 amino acids (8BYTYPE: ami no acid TOPOLOGY: linear Ii)MOLECULE TYPE: Drotel n (xl) SEQOUENCE: DESCRIPTION: SEQO ID NO:2: Me: Phe Gly Ser Arg Ilie Val Met Glu Ser Leu 10 Phe His Met Val Leu Leu Leu Leu Rne Phe Trp 25 Met Ser Arg Arg Asn Val Cys Tyr Val Thr WO 95/23228 PCT/GB95/00372 Ala Arg Met Ile Ty r Gin Al a Gly 145 Lys Gl n Gly Trp Met 225 Gi y L eu Th r Ile Th r 305 SOr Tyr Gi n Giu IPno T I Phe 290 -f AP fl Val1 Lea.u Pro Th r Phe Ala 115 Trp Ala Th r Gin Val1 195 L-ys y r Thr Tyr As n Phe Th r Ie Asp Tyrn Gi u 100 Gly As n Phe Gi u Gly 180 Gi u Gin Gin Cy s G11u 260 Arg Ara Gly Ty r Sen Leu Val1 Asp Leu .Phe Ang Ly s 165 Gly Trnp Met G1 u Gi u 245' Asn Pro Glv Arg Pro Asp Gly Ile 70 Phe Arg Phe Gly Th n 150 Ile Pro Gl u Ala Asp 230 As n Trp Ala Sen Th r 310 His Sen Gin Trp Phe Val1 Gi y 135 Asp Val1 ile ile Val1 Ai a Phe nhr G1iu Leu 2905 Ser Ly s 40 Ile Ly s As n As p His 120 Phe As n As n Ie Gly 200 Gly Leu T~h r Gly As o 280 ?he As n Ala His Ala Gly Leu 105 Leu Pro Giu Ile Leu 185 Ala Leu As o Pro Tro As n G~ 1,l I Ie Ty r Ly s His 90 Val1 Ara Val1 Pro Met 170 Sen Pro As p Pro As n 250 Ty r Al a Tyr Leu Gly 330 Met Pro Asp 75 Gi u Gly Ile Trp Phe 155 Ly s Gin Gly Th r ile 235 Lys Th n Phe Tyr Phe 315 I Ie Arg Gly Pro Phe Gly Leu 140 Lys Al a Ile Lys Gly 2'20 Ile As n Ala Sen Met 300 Val1 As n Sen Gly Sen Ie Pro 125 Lys Gi u Gi u Gi u Ala 205 Val1 Asp Ty r Phe Va 1 285 T yr Ala Gly Th n Leu Pro Lys 110 Phe Ty r Al a Lys As n 190 Ty r Prno Th r Lys Gly 270 Al1a His Th n Gin Pro As p Gly Leu Ie Va l Met Leu Gi1 u Th n cvs Gi v Sen Ang Gin Val1 Ly s Val1 Cys Pro Gin 160 Phe yr Ly s Va, As n 240 Lys Al a Phe Gly Ty r 320 0 *0 0 0* 0000 90 0 0000 0* 0 0.

0*0* 0**0 0* 0 00 00 Aso Tyr Aso Ala le Aso Giu Tvn Leu.Leu Asn Glu Pro Lys 13Ed WO 95/23228 PC.TIGB95,'00372 Tr Ala Gil As r 385 TyrF Glu Met Pro 61 u 465 Tb r Val1 Gly Tbr Leu 545 Th r Ser Leu Val1 Trp 625 Gly Leu IVal 370 As ID Val1 Thr 450 Gin As p Leu Gi1n Phe 530 Leu T np G,1y Lys Gi1u 610 His Val1 355 His As n Leu Phe Pr o 4 35 Sea.r Val1 Val1 Th yr Ser Sen As n Tb r Gly 595S Leu 340 Sen Leu Th r Pro As n 420 Val1 Sen Gly As n Al a 500 Al a G-1n Va 1 Thn Tnp 580 Glu Val Arg Val1 Ty r Asp Pro 405 Tb r As n Sen Val1 Ile 485 Met Gly Sen Sen Gly 565 4k5 p Ser Gin 61 u Asp Lys Tyrn 390 Trp Al a Ser 61 u Th n 470 Gly Sen Th n Val1 Val1 550 Val1 Leu Leu G1 y Leu Pro Th n 375 Sen Sen Lys Ala As n 455 Ang Pro Al a Ala As n 535 Gly Leu Ser Ser Sen 615 His Th n 360 61 u Tb r ie Val1 Ph e ,440 Asp Asp As n Gly Tv r Leu Leu Gly Lys L eu 600 Leu Ang 345 Val1 Sen 61 n Sen Asn 425 Al a Pro Sen Asp His 505 Gly Arg Al a Pro Gin 585 His Val1 Ala Sen Ala Val1 Ile 410 Sen Val1 Sen Ilie 490 Val1 Sen Val1 As n Vali 570 Ly s Tb n Al a Ile Tr Cys Lys 395 Pro GI n Th n Asp) Ly s Leu Leu Gly Val Th r nrp Giu Lys 61IV 635 Lys Pro Ala 38 0 Phe Pro Ang Sen GI y 460 Ty r Asp As n As o As n 540 61 y Leu Sen.

Al a Lys 620 Gln Gly 365 Al a 61 y As p Leu Tyrn 445 Ty r Leu Gly Val1 As p 525 As n Th r T1hr Tyr Gly 605 Gin Cys 350 Lys Phe Asn Cys His 430 As n Al a Trp Lys Phe 510 Pro Lys His Gly Lys 590 Sen Pro Gil As r LeL Gly Lys 415 Arg 61 u Leu Tyrn TrD Ile Ang lie Phe Leu 575 Ilie As n Leu Sen Leu IAla Gin 400 Tb n Lys 61 u Tnrp Leu 480 Pro As n Leu Sen 61 u 5)60 Sen G1v Sen Al a Al a 640 a a a. a a a a a a a a. a a a. a. a Tyr Lys Thr Thn Phe Sen Ala Pro Ala 630 Asn Asp Pro Leu

M

WO 95123228 PCT/GB95/00372 Leu Asp Leu Gly Met Gly Lys Gly Glu Val 650.

Trp Val Asn Gly Gin 655, Sen Ile Gly Asn Cys Asn 675 Ara 660 His TroD Pro Gly As n 665 Lys Ala Arg Gly Asn Cys Gly 670 Leu Ala Asn Tyr Ala Gly Thr Ty r 680 Thr Asp Thr Lys Cy s 685 Cys Glyv 690 Gin Pro Sen Gin Arg 695 Trp Tyr His Val Ara- Sen Trp Leu Arg 705 Sen Gly Gly Asn Leu Val Val Leu Glu Trp Gly Gly Asp 720 Pro Asn Gly Ilie Ala 725 Leu Val Glu Arg Th r 730 a a.

a. a a a.

a a a. a 0* INFORMATION 'FOR SEQ ID NO:3: Mi SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (1iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-terminal (xi) SEQ_,_UENCE DESCRIPTION: SEQ ID NO: Ser VIl Thr Tyr Asp Hi s Lys Al a T Ie Met Leu -Ie Ser INFORMATION FOR SE ID NO: i)SEQUENCE CHARACTERISTICS: ()LENGTH: 12 amino acids k3) TYPE: amino acid STRANDEDNESS: sinaie TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ii) HYPOTHETICA"L.-

NO

A'NTi-SE'NSE: NO Ile Asn Gly Gin Arg Arg WO 95/23228 PCT/GB95/00372 33 FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ser Val Thr Tyr Asp His Lys Ala ile Met Ile Asn 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID Val Ala Lys Lys Gin Pro Leu Ala Trp Tyr 1 5 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATCATDATNG CYTTRTGRTC RTA 23 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

WO 95/23228 WO 95/23228 PCT/GB95/00372 34 FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Val Ala Lys Lys Gin Pro Leu Ala Trp Tyr Lys Thr Thr 1 5 INFORMATION FOR SEQ.ID NO:8: SEQUENCE CHARACTERISTICS- LENGTH: 10 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEO-ID NO:8: Phe Ser Ala Pro Ala Gly Asn Asp Pro Leu 1 5 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS- LENGTH: 11 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear MOLECULE TYPE: peptide (ii) HYPOTHETICAL:

NO

v) ANTI-SENSE:

NO

FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Gly Glu Val Trp Val Asn Gly Gin Ser Ile Gly i 5

S.

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids WO 95/23228 PCT/GB95/00372 TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Asn Cys Gly Asn Cys Asn Tyr Ala Gly Thr Tyr Thr Asp Thr Lys INFORMATION FOR SEQ ID NO:-l: SEQUENCE

CHARACTERISTICS-

LENGTH: 9 amino acids TYPE: amino acid

STRANDEDNESS-

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Ser Val Ser Tyr Asp Asp Arg Ala Ile 1" INFORMATION FOR SEQ ID NO:12: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 amino acids TYPE: amino acid to*

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 1(iii) HYPOTHETICAL:

NO'

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-terminal WO 95/23228 PCT/GB95/00372 36 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Phe Ser Asn Asn Asn Phe Val Ala Thr Asp Gly Thr His Phe Ala Leu 1 5 10 Asn Gly Lys Ser INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic oligonucieotide" (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ACTCCTGGAT CCCGTCAGTA T 21 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single S- TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic oilgonucieozide" iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEO ID NO:14: TAATGGTACC GACCGGCGCT CT 22 INFORMATION FOR SEO ID SEQUENCE

CHARACTERISTICS:

LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: sinale TOPOLOGY: linear WO 95/23228 PCT/GB95/00372 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic oligonucieotide" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID AAGGATCCGT CGACATC 17 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS- LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic oligonucleotide" (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AAGGATCCGT CGACATCGAT AATACGACTC ACTATAGGGA 1liilliii liTTTTll 57 INFORMATION FOR SEQ ID NO:17: SEOUENCE CHARACTERISTICS- LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single S(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic oligonucleotide" (iii) HYPOTHETICAL:

NO

ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GACATCGATA ATACGAC I7 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: WO 95/23228 PCT/GB9S/oo372 LENGTH: 2944 base pairs TYPE: nucleic acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA 00i) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(ix) FEATURE: NAME/KEY: COS LOCATION:42..2555 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TTAAAAAGGC ACAATCTEGA TAGAAAAGGA GATMFFFETA

C

ATG GGT TGT ACG Met Gly Cys Thr 1 ATA CTA ATG TTG Ilie Leu met Leu MAT GTG TTG TTG GTG Asn Val Leu Leu Val TCT GT7 TCA TAT GAC Ser Val Sen Tyr Asp TTG TTG GGT TCA TGG Leu Leu Gly Ser Trp Val1 TTT TCT GGA ACA Phe Sen Gly Thr

GCT

Al a CAT AGG GCT ATT His Arg Ala Ile Ile Val CCA AGA Pro Arg

S

5

S.

S. 55** *5 S

S

S.

*SS 5 *5 5*

S

5* MAT GGA CAA Asn Gly Gin AGC ACT OCT Ser Thn Pro 55 AGA ATA CT! AilT Arg ile Leu le

TT

Sen GGT TCT GiT CAT Gly Ser Val His GAG ATG TGG CCA Glu Met Trp Pro

GGT

Glv 60 ATE ATE CMA MG lie Ile Gln Lys Ala AAA GMA GGA- Lys Giu Gly GGT GIG Gly Val GAT GTG ATE CAG ACT TAT Asp Val Ile Gin-Thr Tvr 75 GTE TEC TGG Val Phe Trp GGA CAT GAG CCT Gly His Giu Pro

CMA

Gin CA GGG AAA TAT Gin Gly Lys Tyn

TAT

Tyrn 90 TE GMA GGG AGA Phe Glu Gly Ang GAT TEA GTG MAG Asp Leu Val Lys ATE QG CTG GTG lie Lys Leu Val

CAC

His 105 CMA GCA GGA CTE Gin Ala Gly Leu

TAT

Tyrn 110 GTC CAT CF AGA Val His Leu Ang GTE GGA Val Giv 115 341 389 437 OCT TAT GCT Pro Tyr Ala

TGT

Cys 120 GCT-GAA TGG MAT Ala Giu Trp Asn TFE GGG Phe Gly 125 GGC T15 COT GTE TGG CYG Gly Phe Pro Val Tnp Leu 130 WO 95/23228 PCTIGB95/O372 AAA TAT GTT Lys Tvr Val 135 CCA GGT ATC AGT Pro Gly Ile Ser

TTC

Phe 140 AGA ACA GAT AAT Arg Thr Asp Asn

GGA

Gly 145 CCT TTC AAG Pro Phe Lys

GCT

Ala

GAA

Glu 165 GCA ATG CAA AAA TTT Ala Met Gln Lys Phe 150 CGT TTG TAT GAA ACT Arg Leu Tyr Glu Thr 170

ACT

Thr 155 GCC AAG ATT GTC Ala Lys lie Val AAT ATG ATG AAA GCG Asn Met Met Lys Ala 160 ATT TTA TCT CAG ATT Ile Leu Ser Gin Ile CAA GGG GGG CCA ATA Gin Gly Gly Pro Ile 175 533 581 629 677 GAG AAT GAA TAT Glu Asn Glu Tyr

GGA

Gly 185 CCC ATG GAA TGG Pro Met Glu Trp

GAA

Glu 190 CTG GGA GCA CCA Leu Gly Ala Pro GGT AAA Gly Lys 195 ACT GGT Thr Gly TCT TAC GCA Ser Tvr Ala GTC CCA TGG Val Pro Trp 215

CAG

Gin 200 TGG GCC GCC AAA Trp Ala Ala Lys

ATG

Met 205 GCT GTG GGT CTT Ala Val Gly Leu GTT ATG TGC AAG Val Met Cys Lys CAA GAC GAT GCC CCT GAT Gin Asp Asp Ala Pro AsD 220 225 CCT ATT ATA Pro lie Ile AAT GCT Asn Ala 230 TGC AAT GGC TTC Cys Asn Gly Phe

TAC

Tyr 235 TGT GAC TAC TTT TCT Cys Asp Tyr Phe Ser 240 CCA AAC AAG GCT Pro Asn Lys Ala 773 821

TAT

Tvr 245 AAA CCA AAG ATA Lys Pro Lys Ile

TGG

Trp 250 ACT GAA GCC TGG Thr Glu Ala Trp

ACT

Thr 255 GCA TGG TTT ACT Ala Trp Phe Thr

GGT

Gly 260

C

C. TTT GGA AAT CCA Phe Gly Asn Pro

GTT

Val 265 CCT TAC CGT CCT Pro Tyr Arg Pro

GCT

Ala 270 GAG GAC TTG GCA Glu Asp Leu Ala TT TCT Phe Ser 275 GTT GCA AAA Val Ala Lys TAT CAT GGA Tyr His Gly 295 Phe 280 ATA CAG AAG GGA Ile Gin Lys Gly

GGT

Gly 285 TCC TTC ATC AAT Ser Phe Ile Asn TAT TAC ATG Tyr Tyr Met 290 CCA TTT ATT Pro Phe Ile GGA ACA AAC TTT Gly Thr Asn Phe GGA CGG Gly Arg 300 ACT GCT.GGT Thr Ala Gly

GGT

Gly 305 869 917 965 1013 1061 1109 GCT ACT Al.a Thr 310 AGT TAT GAC TAT Ser Tyr Asp Tyr

GAT

AsD 315 GCA CCA CTT Ala Pro Leu GAT GAA TAT GGA TTA TTG Asp Glu Tyr Gly Leu Leu 320 CTG CAT AGA GCA ATA AAG Leu His Arg Ala lie Lys 335 340

CGA

Arg 325 CAA CCA AAA TGG Gin Pro. Lys Trp

GGT

Gly 330 CAC CTG AAA GAT His Leu Lys Asp C.

C

C CTT TGT GAA CCA Leu Cys Glu Pro

GCT

Ala 345.

TTA GTC TCT GGA Leu Val Ser Gly

GAT

Asp 350 CCA GCT GTG ACA Pro Ala Val Thr

GCA

Ala 355 WO 95/23228 WO 9523228PCTIGB95/00372 GGA CAC CAG CAG Gly His Gin Gin 360 GAG GCC CAT GTT Glu Ala His Val TTT AGG Phe Ang 365 TCG MAG GCT Sen Lys Ala

GGC

Gly 370 TCT TGT Sen Cys GCT GC"A Ala Al a TT-1~i ^CA Phe Ala 300 -I-iC Phe 375 CTT GCT MAC TAC Leu Ala Asn Tyr

GAC

Asp 380 CMA CAC TCT 7T Gin His Sen Phe

G.CT

Al a 385 ACT GTG TCA Thn Val Sen MAC AGG CAT TAG Asn Arg His Tyr

MAC

As n 395 TTG CCA CCA TGG Leu Pro Pro Trp

TCA

Sen 400 ATC AGC ATT CTT Ilie Sen Ie Leu ATC GGT GCT CMA Ilie Gly Ala Gin 420 1157 1205 1253 1301 1349 CCC GAC TGC MAG MAC ACT Pro Asp Cys Lys Asn Thn 405 410 GTA TT M AT ACA Vai Phe Asn Thr GCA CGG Ala Ang 415 AGT GCT CAG ATG Sen A la Gin Met ATG ACT CCA GTC Met Thn Pro Val

AGC

Sen 430 AGA GGA TTG C CC TGG CAG Arg Gly Leu Pro Trp Gin 135 TCA F7C MvT Sen Phe Asn

GMA

Gi u 440 GAG ACA TCA TCT Giu Thr Sen Sen

TAT

Tyrn 445 GMA GAC AGT AGT Giu Asp Sen Sen 7-7 ACA GT7 Phe Thn V a, 450 TCT GAT TAT Sen' Asp Tyr 1397 GT7 GGG Val _G'iy Leu Tr p 470

CTA

Leu 455 TTG GMA CAG ATA Leu Giu Gin lie AAT ACA ACA AGA Asn Thn Thn Ang 460 MAG ATT1 GAT TCA Lys Ilie Asp Sen

GAC

Asp

AGA

Ana 480

GTG

Val 465 TAT TCA ACA GAT Tyr Sen Thn Asp

GTC

Val1 475 GMA MG T TTG Glu Lys Phe Leu 1445 1493 1541l I 589

AGA

Aro 485S G G:C GU-A, AAA TGG GlyI Gly Lys Tnp a a a a a C CT Prno /0 7,33G CFT ACG ATC Trp Leu Thn Ie

ATG

Met 4905 TCA GOT GGG CAT Sen Ala G'iv Hi1s Ala 500 17G C AT G7 T-i Leu His Val Phe GTG Val1 505 MAT GGT CMk 7A Asn Giy Gin Leu GCA GGA Ala Gly 510 ACT G CA >T7 Thn Ala 7v r GGAI AGT Gly Sen AiM .AA Leu CGl1u Lys Gly Val Asn 535

W

Prno 520 MAA CTA ACT FC- Lys Leu Thn Phe AGT A Sen Lys 525 GCC GTA MAT Ala Val Asn CGAGAI' GCA i eu Arg Ala CF, CCG AAT Leu Pro Asn MAG ATl TCT CTA, Lys Ile Sen Leu Leu 540

TGG

Tnp AGO AT GCT G'F Sen lie Ala Val

G'C

Gly 545 JTC GGC 11ie Giv CCA CAT iii GAG Pro His Phe Giu AnCA Th n MAT GOT GGT As n Ala Gl1Y GF7 Val1 560 C7 GGE C-A, GTC Leu Gly Pro Val 1685 1733 _181

TCA

Sen 56c)5 CTA ACT GGT CF7 Leu Thn Gly Leu GAG GGG MAA AGA Giu Gly Lys Ang

GAT

Asp 575 TA ACA TGG CAG Leu Thn Tro Giln

AAA

Ly s 580 WO 95/23228 1PCT/GB95/00372 TGG NTC TAC MAG Trp Phe Tyr Lys

GTT

Val1 585 GGT CTA AMA GGA Gly Leu Lys Gly

GMA

590 GCC CTG AGT CTT Ala Leu Ser Leu CAT TCA His Ser 595 GTG GCA Val Ala 1829 1877 CTC AGT GGT Leu Ser mly CCA TCC GTG GAG TGG Pro Ser Val Giu Trp 605 GTG GMA CGC TCT Val Ciu Gly Ser

NTA

Leu GAG MAG Gin Lys GGA MAT Gly Asn 630

CAC

Gin 615 CCA CTC AGT TGG Pro Leu Ser Trp

TAT

Ty r 620 MAG ACT ACA NC Lys Thr Thr Phe

MAT

Asn 625 GCT GCA GAT Ala Pro Asp GMA CCT NTG GCT Gilu Pro Leu Ala

NTA

Leu 6 35 GAT ATG MAT ACC Asp Met Asn Thr

ATG

Met 640 CCC AMA GGT GMA Cly Lys Gly Gin 1925 1973 2021 2 06 9

GTA

Val1 -645D TGG ATA MAT GGT Trp lie Asn Giy AC GTG GGA CC Ser Leu Gly Ara TGG CCT GCA TAT r p Pro Ala Tyr

AMA

Ly s TCA TCT GCUA ACT Ser Sen Cly Sen AA MG CTA Lys Lys Cys Leu 680 AGT GTC TGT MAC Ser Val Cys Asn

TAT

Ty r 670 ACT GGC TGG T Thr Gly Trp Phe CAT GAG Asp Giu ACT MAC TGT CGT Thr Asn Gys Cly

GAG

Gi u 685 G GC( TCA CMA AGA Gly Senr Gin Ara TGG TAG GAGC Trp Tyr His 690 GTT GTA N-C Val Val Phe 2117 GTA CCC Val Pro GAG GMA mlu Glu 710L

CGG

Ara 695 TCT TGG CTG TAT Sen Trp Leu Tyr

CCT

Pro 700 ACT GGA MATTNG Thr Gly Asn Leu

NA

Leu 705 4. 4* .4 S. TGG CGA GLA CAT Trnc Cly Giy Asp CCT TAT GGA ATC ACT NA Pro -Iyr Gly Ilie Thr leu I~Z' 20 GTG AMA AGA GMA Val Lys Arg m'u

ATA

Ile 725 GGG ALT GN, TG,-T Giy Sen Val Gys

GCT

Al a 730 CAT ATA TAT*GAG Asp TIea Tyr Glu

TGG

Trp 735b CMA CGIA GAG NA Gin Pro Gin Leu Leu 2165 22 13 2261 2309 2357 MAT TGG GAG AGUK *Asn Trp Gin Arg AMA GCC L CAT C7 :Lys Ala Hs Leu 760 Leu 7405 GTA TGT G^GT.AAG Val Sen Gly Lys 77 Phe 750 GAG AGA CGT CTG.

Asp Ara Pro Leu AGA CCT Arc Pro 755 ATC AM4 Ilie Lys MAG TGT GCA Lys Cys Al a CT GGurT GAG MCG AN TCT TCA Pro Gly Gin Lys "ie-Sen Sen 7 65 770 54 5444 .4 NTT GCA Phel Ala G GA AGC Sen TTT GCA ACA CCA Phe C-1v Thr Pro

GAG

'l u 780 G GA G7 TGT GGG Cly Val Cys Gly

MAC

As n 785 7NC GAG GAG Phe Gin Gin 210 2453 TGC CAT GCT CCC Cys His Ala Pro

CC

Ara -795; TCA TAT CAT GCT Sen Tyr Asp Ala 7NC Phe 800 AA MG MAT TGT1 Lys Lys Asn Cys .WO095/23228 PCTIGB95/00372 G7l Val1 805 GGG AAA GAG TCT Gly Lys Giu Ser TCA GTA CAG GTA Ser Val Gin Val

ACA

Th r 815 CCA GAG MAT 77F Pro Giu Asn Phe

GGA

Giy 820 2501 2549 GGT GAT CCA TG-T Giy Asp Pro Cys

CGA

Arg 825 MAC Gil1 CTA MAG Asn Val Leu Lys

A

Lys 830 CTC TCA GTG GMA Leu Sen Val Giu GCC Ail Ala Ile TGT AGT TGATMATTCT GAGTATACMA GTGAAVAAAT ACTTGMACCA

CTCATATAAA

Cys Ser CA-7TTCMA ACGAGCTACT AGACATCCAT TAACCCACAC TACCATTTTTI

TGGUCTTTGCT

GGGG ilGMG 7uGT ACAG T MAGGAACACA CCTCTFGAT CAAAGCTCAC CTGA17ATGA AGATGATTGA CGAAAGATTC TGTACATGTA AGGTFCGTC TAATTACACA

TACAGATATG

ATTCF GAT'G MT,-CG-ATGTG CAAAYFT1GT TTiGTGTTAGG' GTIGAGAGAGA

CTTGAAAAGC

ATTTTGCTTT CATGATGTTC TACATTATAC MATCATMTG TAAGTAAGCA

AGCAATMTT,

CATGCTiiG CACATFGAAA MAAAAAAMA

AAAAAAAAA

INFORMATION FOR SEQ ID NO:109: SEOUENCE CHARACTERISTICS: LENGTH: 838 amino acids TYPE: amino acid TOPOLOGY: linear 0ii) MOLECULE TYPE: orotein SEQUENCE DESCRIPTION: SEQ ID NO:19: 2605 2665 2725 2785 2845 2905 2944 C 6* a

C

C

C a

C

C C C. C

C

C. C.

Cy n Ilie Leu Met Leu As n Val Leu Leu Val L eu Gly Sen Trp Al a T' "I i 1 3 5 Va I 20 Phe Sen Gly Thr Ala 25 Sen Val Sen Tyr As p H, :s Arc Giy Sen Val Val Asn Gly Gin Arm Ang Ile Leu lie Ser 415 lHis 7Tvyr 00 Pro Arc Ser Thn Giu Met Trp Pro lie lie G-In Lys

A.

LYS GluGly Gly Asp Val T'ie Gin Th n Tyr Val Phe Tr:) G;ly H's Gi-u Pro CC C a. C C

C.

Gi n 85 Gin Gly Lys Tyn Ty r .90 Phe Giu Gly Ang Tyr Asp 95 Leu Val Lys Ilie Lys Leu Val Hi s 105 Giln Ala Gly Leu Tyr Vai His *110 WO 95/23228 PCTIGB95/00372 Leu Ara Val 115 Gly Pro Tyr Ala Cys 120 Ala Glu Trp Asn Phe Gly Gly Phe 125 Pro Val .130 Gly Pro 145 Met Met L eu Sen Ala Pro Lemu As A-so Pro 225 P ro As n Trp Phe Leu Ala Asn Tvr 290 U-IV Pro 305 Tyr 31y Val Thr/ Ala Gly 370 .Ala Thr 385 Tr~ Phf Lys~ Gi r 31 y 195 Tb r Ile Lys Th r Phe 275 Tyr Phe Leu er al1 )Leu Lys Ala IIle 180 Lys Gly Al a Gly 260 Sen Met !]ie Leu Lys 340 Leu Cys Sn Ly~ Al 31IL Ser Val1 As n Ty r 245 Phe Val1 7 yr Ala Ara 3 25 Leu G1v Phe sTyr Ala 150 Arg Asn Tvr Pro Al a 230 Lys Gly Ala His Th r 310 Gin Cys His Ala Ala 390 Val1 135 Met Leu Glu Al a I no 215 Cy s Pro As n Lys Gly 295 Ser Pro 31 u 31 n Phe 375 As n Pro Gin Tyr Ty r Gin 200 Val1 As n Lys Pro Phe 280 Gly Tyr Ly's Pro Gin 360 Leu Ang I Gl Lys Gly 1 85 Trp met Gly lie Val1 265 Ile Th r As o Trp Ala 345 31 u Mla .ns I Ie Phe Thr 170 Pro Ala Cys Phe Trp 250 Pro Gin As n Ty r Gly 3330 Leu Ala Asn Tyr Ser Thr Met Al a Lys Ty r 235 Thr Ty r Lys Phe As p 315 His Val1 Hi's Fy r \s n 395 Phe 140 Ala Gly Glu Lys Gin 220 Cys 31 u Arg Gly Gly 300 Al a Leu Sen Val Asp 380 Leu Arc Lys~ Met 205 As pD Asp Al a Pro 3 2865 Arg Pro Lys 31 y Phe 365 31 n P ro Thi 11 Prc Gil 190 Al a As p Ty r Trp Al a 270 Ser Th r Leu As p Asp 350 Ara His Pro rAs~ Val Ile Leu Val Ala Phe Tb r 255 Giu Phe Ala Asp Leu 335 Pro Se r Ser Trp Asn Asn 160 Ile Gly Gly Pro Ser 240 Al a Asp Ile Gly Giu 320 His Al a Lys Phe Sen 400 9* 9 9 .9 9.

9* 9* 9 9* 9 9.

9 *9 .9 9 9S .9 lie Sen 1lie Leu. Pro Asp Cys Lys Asn Tb r 410 Val Phe Asn Thr Al a Arg 415 7 WO 95/23228 PCTIGB95/OO372 Ie Gly Ala Gin Sen Ala Gin Met Lys Met Thn Pro Va] Sen Ang Gly 420 425 430 Leu Pro Trp Gin Sen Phe Asn Giu Giu Thr Sen Sen Tyn Giu Asp Sen 435 440 445 Sen Phe Thn Va] Val Gly Leu Leu Giu Gin lie Asn Thn Thn Ang Asp 450 455 460 Val Sen Asp Tyr Leu Trp Tyr Sen Thn Asp Vai Lys Ilie Asp Sen Arg 465 470 475 480 Giu Lys Phe Lou Arg Gly Gly Lys Tnp Pro Tnp Lou Th Ilie Met Ser 485 490 495 Ala Gly His Ala Lou His Val Phe Vai Asn Gly Gin Leu Ala Gly Thn 500 505 510 Ala Tyn Gly Ser Leu Giu Lys Pro ILys Lou Thr Phe Sen Lys Ala Val 515 520 525 Asn Leu Arg Ala Gly Val Asn Lys Ile Senr Lou Leu Sen Ilie Ala Val 530 535 540 Gly Lou Pno Asn Ilie Gly Pro His Phe Glu Thn Trp Asn Ala GlIy Val 545 550 55560 Leu Gly Pno Vai Sen Leu Thn Gly Leu Asp Giu Gly Lys Ang Asp Leu 565 570 575 Thn Trp Gin Lys Trp Phe Tyr Lys Val Gly Lou Lys Gly Glu Ala Leu 580 585 590 Sen Leu His Sen Leu Sen Gly Sen P ro Ser 'Ia] Glu Tro Val Glu Gly 595600 605 Sen L eu Val Al a Gin Lys Gin Pro Leu Sen Trp Tyr Lys Thn 7h, h *610 6,15 620 Asn Ala Pro Asp o.ly As n Giu Pro L eu Ala Lou Asp Met AsnThMe *625 630 635 Gly Lys Giy Gin Val Tnp Ie Asn Gly Gin Sen Leu Gly Arg Hiro 64565 Pro Ala Tyr Lys Sen Sen Gly Ser Cys Sen Val Cys Asn Tyn Thr Gly -060 6 65 670 *goo Trp Phe Aspo Giu Lys Lys Cys Leu Thn Asn Cys Gly Giu Gly Sen Gin 675 6,80 685 *Ang Tro Tvr His Val Pro'-Arg Sen Tnp Lou Tyr Pro Thn Gly Asn Leu *:690 695 700 Leu Val Val Phe Giu Giu Tnp Gly Gly AsoD Pro Tyr Gly Ile Thn Leu 705- 7.10 715 7120 WO 95/23228 WO 9523228PCTIGB95/oO372 Val1 Pro Pro Sen As n 785 Lys Gi u Val1 Lys Ang Gin Leu Leu Ang 755 E Sen li e 770 Phe Gin Lys As n Asn Phe Gl u A' ,a 835 Gi u Leu 740 Pro Lys Gin Cys Giy 820 i Ie Ilie Gly 725 Asn Trp Lys Al a Phe Al a G1ly Ser 790 Val Gi 805 Gly As P Cys Sen Sen Gin His Sen 775 Cys Lys Pro Val1 Arg Leu 760 Phe His Gi u Cys Cy s Leu 745 Lys Gly Ala Senr Ang 825 Ala 730 Val1 Cys Th r Pro Cys 810 As n Asp Sen Ala Prno Ang 7905 Sen Vali I Ie Gly Pro G1 u 780 Sen Val1 Tyr Glu Lys Phe 750 Gly Gin 765 Gly Val Tyn Asp Gin Val Lys Lys 830 Tnp 735 Asp Lys Cys Al a Th n 815 Leu Gl n Ang Ile Gly Phe 800 Pro Sen p.

p p p. p. p p.

p. p.

p p pp a. p a p p. p p p p p p p.

p.

Claims (9)

1. A nucleotide sequence comprising substantially nucleotides 419 to 1500 of the sequence shown in Figure 5, or the functional equivalent of such a nucleotide sequence wherein the functional equivalent encodes a polypeptide exhibiting at least 70% amino acid homology with the polypeptide encoded by the sequence of nucleotides 419 to 1500 of the sequence shown in Figure 5, excluding the sequence disclosed as Seq. ID No. 2 in W095/10622.

2. A sequence according to claim 1, comprising an ATG start signal.

3. A nucleotide sequence which is an antisense functional equivalent of, and capable of hybridising under standard hybridisation conditions with the complement of nucleotides 419 to 1500 of the sequence shown in Figure

4. A nucleotide sequence comprising substantially nucleotides 1292 to 1802 of the sequence shown in Figure 5, or the functional equivalent of such a nucleotide sequence wherein the functional equivalent encodes a polypeptide exhibiting at least 70% amino acid homology with the polypeptide encoded by the sequence of nucleotides 1292 to 1802 of the sequence shown in Figure 5, excluding the sequence disclosed as Seq. ID No. 2 in W095/10622.

5. A nucleotide sequence which is an antisense functional equivalent of, and capable of Shybridising under standard hybridisation conditions with the complement of, nucleotides

1292-1802 of the sequence shown in Figure

6. A vector comprising a sequence according to any one of claims 1 to

7. A vector according to claim 6 capable, when introduced into a suitable host, of giving rise to an RNA transcript of a sequence according to any one of claims 1 to

8. A host cell into which has been introduced a sequence according to any one of claims 1 to 47

9. A host plant or part thereof, into which has been introduced a sequence according to any.one of claims 1 to A plant or part thereof according to claim 9, having altered physical characteristics as a result of the introduction of a sequence according to any one of claims 1 to 11. A method of altering the physical characteristics of a plant or part thereof, comprising introducing into the plant or part thereof a sequence according to any one of claims 1 to DATED y\ 1°) Signed for and on behalf of UNILEVER PLC b liev, ustralia Limited B. F.JONES, C Secretary. a