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WO1999060103A2 - Galactosyltransferase from plants involved in galactomannan biosynthrsis - Google Patents

Galactosyltransferase from plants involved in galactomannan biosynthrsis Download PDF

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Publication number
WO1999060103A2
WO1999060103A2 PCT/GB1999/001610 GB9901610W WO9960103A2 WO 1999060103 A2 WO1999060103 A2 WO 1999060103A2 GB 9901610 W GB9901610 W GB 9901610W WO 9960103 A2 WO9960103 A2 WO 9960103A2
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WIPO (PCT)
Prior art keywords
nucleic acid
sequence
plant
seq
cell
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Ceased
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PCT/GB1999/001610
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French (fr)
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WO1999060103A3 (en
Inventor
Michael John Gidley
Sumant Chengappa
John Spence Grant Reid
Mary Elizabeth Edwards
Cathryn Anne Dickson
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Hindustan Unilever Ltd
Unilever NV
Original Assignee
Hindustan Lever Ltd
Unilever NV
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Application filed by Hindustan Lever Ltd, Unilever NV filed Critical Hindustan Lever Ltd
Priority to AU40509/99A priority Critical patent/AU4050999A/en
Priority to EP99923746A priority patent/EP1086208A2/en
Publication of WO1999060103A2 publication Critical patent/WO1999060103A2/en
Publication of WO1999060103A3 publication Critical patent/WO1999060103A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans

Definitions

  • the present invention relates to nucleic acids and other materials having a role in the biosynthesis of complex non-cellulosic plant cell wall polysaccharides. It further relates to various applications for such materials.
  • Plant cell walls contain a number of non-cellulosic polysaccharides which play important roles in vivo both structurally and as energy stores. Some of these are discussed in Brett & aldron (1996) "Physiology and Biochemistry of Plant Cell Walls - 2nd Edition” Pub. Chapman & Hall, London, especially pages 4-43. Generally these occur in the cell wall matrix phase as pectins and hemicelluloses .
  • CWSPs Two principal cell wall storage polysaccharides
  • galactomannan e.g. guar gum, locust bean gum
  • xyloglucan e.g. tamarind seed polysaccharide
  • Galactomannans are found in the endosperm cells of leguminous seeds, and in the endosperms of the seeds of a small number of non-leguminous species. In general they act as storage reserves, being broken down following germination to monosaccharides which are used by the developing seedling. Their overall biological functions are more complex.
  • the galactomannan of fenugreek has been shown to be multifunctional, imbibing large amounts of water during seed hydration, deploying it as a buffer to protect the germinating embryo from post- imbibition drought, and serving as a substrate reserve following successful germination.
  • Structurally galactomannans comprise a (1-4) - ⁇ -linked D-mannan backbone which carries single-unit ⁇ -D-galactosyl substituents attached (1-6) - ⁇ to backbone mannose.
  • Mannose/Galactose [Man/Gal] ratios in galactomannans range from about 3.5 [low-galactose] to about 1.1 [high galactose] .
  • Man/Gal ratio is constant and fixed for the galactomannan of a given species [genetic control] , and Man/Gal ratios are similar within taxonomic sub-groupings of the Leguminosae .
  • Galactomannans are closely related structurally to other cell wall storage polysaccharides of seeds [mannans, glucomannans , galactoglucomannans] and to non- cellulosic polysaccharides of non-seed plant cell walls [glucomannans, galactoglucomannans] .
  • the nature of the interaction between the mannan synthase and the galactosyltransferase was also investigated using the membrane preparations . This demonstrated that the mannan synthase can operate independently of the galactosyltransferase, that the galactosyltransferase cannot operate in the absence of simultaneous mannan synthase action and that (1-4) - ⁇ -D-mannan preformed at the site of synthesis using the mannan synthase is not accessible as a substrate for the galactosyltransferase.
  • an experimental model for galactomannan biosynthesis involves stepwise chain-elongation of the mannan chain towards the non-reducing end catalysed by the mannan synthase and transfer of galactose, catalysed by the galactosyltransferase, to a hypothetical galactosyl acceptor mannose residue at or close to the [elongating] non-reducing chain-end.
  • Man/Gal ratio increases during late seed development from about 2 to 3.3, and this change is accompanied by the appearance and increase of the activity of a galactomannan-active -galactosidase .
  • Man/Gal ratio is determined only by the pathway of biosynthesis.
  • the Man/Gal ratio of the primary biosynthetic product is controlled by the biosynthetic process, and the primary biosynthetic product undergoes a post-depositional modification catalysed by a galactomannan-active ⁇ -galactosidase .
  • the labelled galactomannans can be fragmented, using a pure structure-sensitive endo- (1-4) - ⁇ -D-mannanase, to give a series of diagnostic manno-and galactomanno-oligosaccharides, the relative amounts of which can be determined accurately using quantitative digital autoradiography after separation on thin layer chromatography [TLC] plates
  • TLC thin layer chromatography
  • the (galacto) mannan substrate subsite recognition of the galactosyltransferase ⁇ from fenugreek, guar and senna must encompass at least three backbone mannosyl residues the one which is the site of reaction, and the two preceding ones, towards the reducing end of the chain Other backbone mannosyl residues may be recognised by the galactosyltransferase, but their states of substitution do not influence greatly the probability of obtaining galactosyl- substitution at the reacting mannosyl residue
  • EP 0 255 153 (Unilever NV/Unilever pic) discusses the use of recombmantly produced guar alpha-galactosidase for providing galactomannans having improved properties
  • WO 97/20937 discusses methods of m vivo modification of mannose/galactose ratios in galactomannans
  • the Examples apparently disclose the cloning of a phosphomannose isomera ⁇ e gene (involved m mannose-6 -phosphate generation) from guar, and also the use of senna alpha-galactosidase
  • the present inventors have used novel techniques to identify and isolate a membrane-bound glycosyltransferase, and encoding nucleic acid, which catalyses the biosynthesis of a complex non-cellulosic plant cell wall polysaccharide
  • the glycosyltransferase has demonstrated activity as a galactosyltransferase, involved in the biosynthesis of galactomannan.
  • the polypeptide has a single membrane-spanning ⁇ -helix near the N- terminus which appears to serve to anchor the whole polypeptide to a biological membrane. This is the first time that a plant enzyme with activity appropriate for hemicellulose or pectin synthesis has been isolated, and that a nucleic acid sequence has been positively identified as encoding the same.
  • Protein distribution within IEF gel strips was determined using two procedures In the first, the strips were cut into narrow slices, which were soaked m SDS-PAGE sample buffer and placed within individual sample wells of SDS-PAGE gels In the second, entire strips were soaked in SDS-PAGE sample buffer turned at right angles and applied as sample to SDS-PAGE gels, giving effectively a 2 -dimensional gel, the first dimension being the IEF separation carried out in the presence of the solubilismg detergent
  • sequence apparently shares limited but significant homology with yeast galactosyltransferases, plus also low homologies with yeast mannan synthases and a plant ⁇ -mannanase
  • Each nucleic acid encoding a glycosyltransferase provided by the present inventors may be used to manipulate (e.g. galactomannan) synthesis both in vi tro and in vivo thereby allowing galactomannans to be tailored for particular applications. Additionally it can be used, for instance, to alter the cell wall rheology, and hence mechanical properties (e.g. texture) of plant tissues, thereby permitting the production of improved plants and plant products for consumption or industrial use (e.g. fruits, vegetables, timber, paper etc.) .
  • the galactosyltransferase nucleic acid can also be used to prepare novel genes (variants) having altered properties with respect to the wild-type, or alternatively to facilitate the isolation of homologous genes from natural sources.
  • nucleic acid molecule encoding a polypeptide which is capable of catalysing the biosynthesis of a complex non-cellulosic plant cell wall polysaccharide .
  • the polysaccharide may be a pectin or a hemicellulose, preferably the latter.
  • hemicelluloses include xylan, glucomannan, mannan, galactomannan, glucuronoxylan, xyloglucan, callose or arabinogalactan.
  • the polypeptide is preferably a glycosyltransferase, which is to say that it catalyses, inter alia , the addition of monosaccharides (optionally from an activated precursor or donor e.g. a sugar nucleotide, such as a diphosphate precursor e.g. ADP- CDP- GDP- TDP- or UDP-sugar) to a polysaccharide chain (the 'acceptor') generally, but not exclusively, at the non-reducing end.
  • an activated precursor or donor e.g. a sugar nucleotide, such as a diphosphate precursor e.g. ADP- CDP- GDP- TDP- or UDP-sugar
  • a polysaccharide chain the 'acceptor'
  • Such enzymes are occasionally also termed polysaccharide synthases or synthetases by those skilled in the art.
  • the hemicellulose is one which contains galactose, and the glycosyltransferase is membrane-bound in vivo .
  • the activity of the encoded polypeptide may be tested, for instance, by observing the addition of radiolabelled sugar residues from exogenously supplied radioactive sugar nucleotides to saccharides, for instance oligosaccharides , or more preferably polysaccharides. Such methods are described in more detail below.
  • Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid) .
  • a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
  • Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term “isolated” encompasses all of these possibilities.
  • the nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.
  • nucleic acid encodes a galactosyltransferase, which is capable of catalysing the biosynthesis of galactomannan.
  • nucleic acid comprising the nucleotide sequence shown in Seq ID No 1 (Annex la) .
  • This sequence represents that of a cDNA molecule encoding a galactosyl transf rase gene from fenugreek.
  • the encoded polypeptide (Seq ID No 2) is also shown in Annex lb.
  • nucleic acid comprising the nucleotide sequence shown in Seq ID No 3 (Annex 2a) .
  • This sequence represents that of a cDNA molecule encoding a galactosyl transferase gene from guar.
  • the encoded polypeptide (Seq ID No 4) is also shown in Annex 2b.
  • nucleic acids which are variants of the sequences provided.
  • a variant nucleic acid molecule shares homology with, or is identical to, all or part of the coding sequence discussed above.
  • variants may encode, or be used to isolate or amplify nucleic acids which encode, polypeptides which are capable of catalysing the biosynthesis of a complex non-cellulosic plant cell wall polysaccharide by binding nucleotide sugar precursors and transfer sugar residues to polysaccharides in the golgi compartment (s) .
  • Such polypeptides may include not only galactosyltransferases, but also other (golgi located) glycosyltransferases e.g. those involved in galacto (gluco) mannan biosynthesis such as mannosyl and glucosyl transferases. Also included may be galactosyltransferases which act on pectin or xyloglucan.
  • polypeptides having the requisite characteristics may include arabinosyltrans erase, glucosyltransferase, xylosyltran ⁇ ferase, mannosyltransferase, fucosyltransferase, rhamnosyltransferase, galacturonyltransferase and glucuronyltransferase .
  • Activities may conveniently be assessed using in si tu analysis in chromatographic gels (e.g. agarose gels) containing a suitable substrate (e.g. galactomannan for galactosyltransferase activity). Such methods of assessment form one part of the present invention.
  • chromatographic gels e.g. agarose gels
  • suitable substrate e.g. galactomannan for galactosyltransferase activity
  • a typical method will comprise the steps of :
  • Suitable 'compatible' substrates may include xyloglucan, xylan, glucomannan and pectin
  • Variants of the present invention can include not only novel, naturally occurring, nucleic acids, isolatable using the sequences of the present invention, but also artificial nucleic acids having novel sequences, which can be prepared by the skilled person m the light of the present disclosure
  • a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided
  • the fragments may encode particular functional parts of the polypeptide, e g portions lacking the transmembrane ⁇ -helix near the N-termmus (e g between residues 15 to 41 of the fenugreek sequence, or as underlined in Fig 1) which may have improved properties such as solubility or activity
  • fragments may have utility m probing for, or amplifying, the sequence provided or closely related ones Suitable lengths of fragment, and conditions, for such processes are discussed m more detail below
  • nucleic acids which have been extended at the 3 ' or 5' terminus
  • sequences e g genomic sequence ⁇ having additional, non-expressed, portions ('introns')
  • Sequence variants which occur naturally may include homologous galactosyltransferases from other species, alleles (which will include polymorphisms or mutations at one or more bases) or pseudoalleles (which may occur at clo ⁇ ely linked loci to the galactosyl transferase gene from fenugreek)
  • isogenes, or other homologou ⁇ gene ⁇ which may belong to the same family as the galactosyltran ⁇ fera ⁇ e gene (e.g galactoglucomannan synthases) Although these may occur at different genomic loci to the gene, they are likely to share conserved regions with it
  • Artificial variants may be prepared by those skilled m the art, for instance by site directed or random mutagenesi ⁇ , or by direct synthe ⁇ i ⁇
  • the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence shown in Seq ID No 1 or 3.
  • it encodes a polypeptide which is capable of catalysing the biosynthe ⁇ i ⁇ of a complex non-cellulo ⁇ ic plant cell wall poly ⁇ accharide .
  • variant' nucleic acid a ⁇ u ⁇ ed herein encompasses all of these possibilities.
  • polypeptides or proteins indicates the encoded expres ⁇ ion product of the variant nucleic acid.
  • Similarity or homology may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J " . Mol . Biol . 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.
  • Homology may be at the nucleotide sequence and/or encoded amino acid sequence level.
  • the nucleic acid and/or amino acid sequence share ⁇ at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
  • Homology may be over the full-length of the relevant sequence shown herein, or may be over a part of it, preferably over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400 or more amino acids or codons, compared with Seq ID Nos 1 to 4 as appropriate.
  • a variant polypeptide in accordance with the present invention may include within the sequence shown in Seq ID No 2 or 4 , a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes.
  • a variant polypeptide may include additional amino acids at the C- terminus and/or N-termmu ⁇
  • change ⁇ to the nucleic acid which make no difference to the encoded polypeptide (1 e 'degeneratively equivalent') are included
  • a further a ⁇ pect of the invention there is disclosed a method of producing a derivative nucleic acid comprising the step of modifying the coding sequence of Seq ID No 1 or 3
  • Changes to a sequence, to produce a derivative may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more ammo acids the encoded polypeptide
  • Changes may be desirable for a number of reasons , including introducing or removing the following feature ⁇ restriction endonuclease sequences, codon usage, other site ⁇ which are required for po ⁇ t tran ⁇ lation modification, cleavage sites m the encoded polypeptide, motifs in the encoded polypeptide for glycosylation, lipoylation etc Leader or other targeting sequences (e g membrane or golgi locating sequences) may be added to the expressed protein to determine its location following expres ⁇ ion All of these may as ⁇ ist efficiently cloning and expressing an active polypeptide recombinant form (a ⁇ described below)
  • Other desirable mutations may be random or site directed mutagenesis m order to alter the activity (e g specificity) or stability of the encoded polypeptide
  • Changes may be by way of conservative variation, I e sub ⁇ titution of one hydrophobic re ⁇ idue ⁇ uch a ⁇ l ⁇ oleucme, valme, leucme or methionine for another, or the ⁇ ub ⁇ titution of one polar re ⁇ idue for another, such as argmine for lysme, glutamic for aspartic acid, or glutamine for asparagine A ⁇ l ⁇ well known to tho ⁇ e skilled in the art, altering the primary structure of a polypeptide by a conservative sub ⁇ titution may not ⁇ ignificantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which i ⁇ critical in determining the peptide' ⁇ conformation.
  • Figures 6A and 6B show the predicted secondary structures of the f
  • variants having non-conservative substitutions are also included.
  • sub ⁇ titution ⁇ to region ⁇ of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide' s three dimensional structure.
  • regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
  • a method of identifying and/or cloning a nucleic acid variant from a plant which method employs Seq ID No 1 or 3 or a derivative thereof .
  • clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequence ⁇ , probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.
  • the original DNA source e.g. a clone library, mRNA preparation etc.
  • nucleotide sequence information provided herein may be used in a data-ba ⁇ e (e.g. of expre ⁇ ed ⁇ equence tags, or sequence tagged sites) search to find homologous sequences, such as those which may become available in due course, and expres ⁇ ion products of which can be tested for activity as de ⁇ cribed below.
  • a data-ba ⁇ e e.g. of expre ⁇ ed ⁇ equence tags, or sequence tagged sites
  • a variant in accordance with the pre ⁇ ent invention i ⁇ also obtainable by means of a method which includes: (a) providing a preparation of nucleic acid, e.g. from plant cell ⁇ , (b) providing a nucleic acid molecule having a nucleotide sequence ⁇ hown in or complementary to Seq ID No 1 or 3 or a derivative thereof, (c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with ⁇ aid nucleic acid molecule.
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes . Restriction fragments may then be separated by electrophoresis on an agaro ⁇ e gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
  • Test nucleic acid may be provided from a cell a ⁇ genomic DNA, cDNA or RNA, or a mixture of any of the ⁇ e, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as is de ⁇ cribed hereinafter. Probing may optionally be done by mean ⁇ of ⁇ o-called 'nucleic acid chip ⁇ ' (see Mar ⁇ hall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review) .
  • this method may be u ⁇ ed to isolate promoters or other regulatory elements, the activity of which may be confirmed by analogy with the method ⁇ below e.g. using promoterless con ⁇ truct ⁇ in which isolated fragments are operably linked to detectable gene ⁇ .
  • Preliminary experiment ⁇ may be performed by hybridi ⁇ ing under low stringency conditions.
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisation ⁇ identified as positive which can be investigated further.
  • the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE
  • a formamide concentration of about 20% or a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE
  • hybridisation conditions will be selected (e g using higher temperatures) which allow the identification of sequences having 70% or more (e g 80%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the probe, while discriminating against sequences which have a lower level of sequence identity with respect to the probe
  • low stringency hybridisation has been used to identify several nucleic acids having a substantial degree of similarity with the probe sequence
  • this subset is then subjected to high stringency hybridisation, so as to identify tho ⁇ e clone ⁇ having a particularly high level of homology with respect to the probe sequences
  • High stringency conditions comp ⁇ e a temperature of about 42 °C or le ⁇ , a formamide concentration of le ⁇ s than about 20%, and a low salt (SSC) concentration Alternatively they may comprise a temperature of about 65°C or less, and a low salt (SSPE) concentration
  • Preferred conditions for ⁇ uch screening comprise a temperature of about 42°C, a formamide concentration of about 20%, and a
  • nucleic acid libraries e.g cDNA libraries representative of expre ⁇ ed sequences
  • suitable conditions of the desired stringency for selective hybridisation taking into account factors such as oligonucleotide length and base composition, temperature and so on
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the dispo ⁇ al of tho ⁇ e skilled m the art
  • probes may be radioactively, fluorescently or enzymatically labelled
  • Other methods not employing labelling of probe include amplification using PCR (see below), RN'ase cleavage and allele specific oligonucleotide probing
  • the identification of successive ⁇ ful hybridi ⁇ ation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host
  • hybridisation of nucleic acid molecule to a variant may be determined or identified indirectly, e g using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of glycosyltran ⁇ fera ⁇ es are employed
  • PCR polymerase chain reaction
  • a method involving use of PCR in obtaining nucleic acid according to the present invention may include
  • Nucleic acids for probing or amplification may be about 30 or fewer nucleotide ⁇ m length (e g 18, 21 or 24) Generally specific primers are upwards of 14 nucleotides length For optimum specificity and co ⁇ t effectivene ⁇ s, primers of 16-24 nucleotides m length may be preferred Those skilled in the art are well versed m the design of primers for use in processes such as PCR If required, probing can be done with entire restriction fragments of the gene disclo ⁇ ed herein which may be 100' s or even 1000' s of nucleotides m length
  • primers or probes which are distinctive for particular parts of the sequence which are likely to be associated with particular activities e g it may be desirable to avoid using sequence from the helix region as these are more likely to cross react with sequences not forming part of the present invention.
  • the nucleic acid encoding the galactosyltransferase described above is in the form of a recombinant and preferably replicable vector.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacteri um binary vector in double or single stranded linear or circular form which may or may not be self transmi ⁇ ible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organi ⁇ ms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast or fungal) cells.
  • a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell.
  • a host cell such as a microbial, e.g. bacterial, or plant cell.
  • the vector may be a bi-functional expres ⁇ ion vector which functions in multiple hosts.
  • thi ⁇ may contain it ⁇ own promoter or other regulatory element ⁇ and in the case of cDNA thi ⁇ may be under the control of an appropriate promoter or other regulatory element ⁇ for expression in the host cell
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3 ' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter i ⁇ "under transcriptional initiation regulation" of the promoter.
  • this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the fenugreek galactosyltransferase gene or a variant thereof.
  • tho ⁇ e ⁇ killed in the art are well able to con ⁇ truct vectors and design protocols for recombinant gene expression.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • thi ⁇ a ⁇ pect of the present invention provides a gene con ⁇ truct, preferably a replicable vector, compri ⁇ ing an inducible promoter operatively linked to a nucleotide sequence provided by the present invention, such as Seq ID No 1 or 3.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expres ⁇ ion under the control of an inducible promoter i ⁇ " ⁇ witched on” or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expres ⁇ ion) in the ab ⁇ ence of the appropriate stimulus. Other inducible promoters cause detectable con ⁇ titutive expre ⁇ sion in the ab ⁇ ence of the stimulus . Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus .
  • nucleic acid constructs which operate as plant vectors .
  • Suitable promoters which operate in plants include the Cauliflower Mo ' saic Virus 35S (CaMV 35S) ; the cauliflower meri 5 and the Arabidopsi s thaliana LEAFY promoter that is expressed very early in flower development.
  • Other promoters include the rice actin promoter.
  • Inducible promoters may include the GST-II-27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledons and dicotyledons. Other examples are di ⁇ clo ⁇ ed at pg 120 of Lind ⁇ ey & Jone ⁇ (1989) "Plant Biotechnology in Agriculture” Pub. OU Press, Milton Keynes, UK.
  • the promoter may be selected to include one or more sequence motif ⁇ or elements conferring developmental and/or tissue - specific regulatory control of expres ⁇ ion.
  • selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes ⁇ uch as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinone ⁇ and glypho ⁇ ate) .
  • antibiotics or herbicides e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinone ⁇ and glypho ⁇ ate
  • the present invention also provides method ⁇ compri ⁇ ing introduction of such a construct into a cell and/or induction of expression of a con ⁇ truct within a cell, by application of a ⁇ uitable stimulus e.g. an effective exogenous inducer .
  • a ⁇ uitable stimulus e.g. an effective exogenous inducer
  • a host cell containing a heterologous construct according to the present invention especially a plant or a microbial cell (e.g. yeast cell) .
  • a microbial cell e.g. yeast cell
  • heterologous is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (e.g. encoding galactosyltransferase) have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention.
  • a heterologous gene may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the in ⁇ erted sequence may be additional to the endogenous gene or other sequence.
  • Nucleic acid heterologous to a plant cell may be non- naturally occurring in cells of that type, variety or species.
  • heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or specie ⁇ or variety of plant.
  • a further po ⁇ ibility is for a nucleic acid sequence to be placed within a cell in which it or a homolog is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
  • the host cell e.g. plant cell
  • the host cell is preferably transformed by the con ⁇ truct, which i ⁇ to say that the construct becomes established within the cell, altering one or more of the cell's characteristics and hence phenotype e.g. with respect to CCWP production.
  • Nucleic acid can be transformed into plant cells using any ⁇ uitable technology, ⁇ uch as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al .
  • any ⁇ uitable technology ⁇ uch as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO
  • Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous specie ⁇ . Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (see e.g. Hiei et al . (1994) The Plant Journal 6, 271-282)). Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone i ⁇ inefficient or ineffective. Alternatively, a combination of different technique ⁇ may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co- cultivation with Agrobacterium (EP-A-486233 ) .
  • a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a construct as described above into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid according to the present invention into the genome.
  • the invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g comprising the galactosyltransferase sequence) especially a plant or a microbial cell.
  • a host cell transformed with nucleic acid or a vector according to the present invention e.g comprising the galactosyltransferase sequence
  • the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome.
  • a plant may be regenerated, e.g. from single cells, callu ⁇ tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Va ⁇ il et al . , Cell Cul ture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weis ⁇ bach and Weissbach, Methods for Plant Molecular Biology, Academic Pre ⁇ , 1989.
  • Plants which include a plant cell according to the invention are also provided.
  • a plant according to the present invention may be one which does not breed true one or more properties Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights It is noted that a plant need not be considered a "plant variety" simply because it contains stably with its genome a transgene, introduced into a cell of the plant or an ance ⁇ tor thereof
  • the present invention embraces all of the following a clone of such a plant, ⁇ eed, selfed or hybrid progeny and descendants (e g Fl and F2 descendants) and any part of any of these
  • the invention al ⁇ o provides a plant propagule from ⁇ uch a plant, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on
  • the plant is an endo ⁇ permic legume which contains galactomannan as a CWSP
  • galactomannan as a CWSP
  • a CWSP galactomannan
  • the present invention also encompas ⁇ es the expression product of any of the galactosyltransferase or variant nucleic acid sequences disclosed above, and al ⁇ o methods of making the expres ⁇ ion product by expre ⁇ ion from encoding nucleic acid therefore under ⁇ uitable conditions, which may be ⁇ uitable ho ⁇ t cells
  • truncated polypeptide lacking the transmembrane helix, which is soluble and not membrane-associated and which also has galacto ⁇ yltran ⁇ ferase activity
  • the product may be l ⁇ olated from the expre ⁇ ion ⁇ y ⁇ tem (e g microbial) and may be u ⁇ ed as desired, for instance formulation of a composition including at least one additional component
  • the product may be used to perform its function m vivo and in particular in planta as di ⁇ cussed above.
  • Purified galactosyltran ⁇ ferase protein may be used to raise antibodies employing techniques which are standard in the art.
  • Antibodies and polypeptides comprising antigen-binding fragments of antibodies may be used in identifying variants from other species as discussed further below.
  • Methods of producing antibodie ⁇ include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof .
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the a t, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al , 1992, Nature 357: 80-82) .
  • Antibodies may be polyclonal or monoclonal. Single chain antibodies e.g. from Camelidae may be preferred ( ⁇ ee WO 94/25591 of Unilever) .
  • Antibodie ⁇ may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, thi ⁇ term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or ⁇ ynthetic. Chimaeric molecule ⁇ compri ⁇ ing an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expres ⁇ ion of Chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023. It has been shown that fragments of a whole antibody can perform the function of binding antigens.
  • binding fragment ⁇ examples include (I) the Fab fragment con ⁇ i ⁇ ting of VL, VH, CL and CHI domain ⁇ ; (ii) the Fd fragment consisting of the VH and CHI domain ⁇ ; (iii) the Fv fragment consisting of the Vl and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al .
  • Diabodies are multimer ⁇ of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the as ⁇ ociation of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804) .
  • antibodie ⁇ with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • Antibodie ⁇ rai ⁇ ed to a polypeptide or peptide can be used in the identification and/or isolation of variant polypeptides, and then their encoding genes.
  • the present invention provides a method of identifying or isolating a galactosyltransferase or variant thereof (as di ⁇ cu ⁇ sed above) , comprising screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind said galactosyltransferase polypeptide or variant thereof, or preferably has binding specificity for such a polypeptide.
  • an antibody for example whole antibody or a fragment thereof
  • Specific binding members such as antibodie ⁇ and polypeptides comprising antigen binding domains of antibodies that bind and are preferably specific for a galactosyltransferase polypeptide or mutant or derivative thereof represent further aspects of the present invention, as do their use and methods which employ them.
  • Candidate polypeptide ⁇ for ⁇ creening may for instance be the products of an expres ⁇ ion library created u ⁇ ing nucleic acid derived from an plant of interest, or may be the product of a purification proce ⁇ from a natural ⁇ ource .
  • a polypeptide found to bind the antibody may be i ⁇ olated and then may be subject to amino acid sequencing. Any suitable technique may be used to sequence the polypeptide either wholly or partially (for in ⁇ tance a fragment of the polypeptide may be sequenced) .
  • Amino acid sequence information may be used in obtaining nucleic acid encoding the polypeptide, for instance by designing one or more oligonucleotides (e.g. a degenerate pool of oligonucleotides) for use as probes or primers in hybridization to candidate nucleic acid.
  • the invention further provides use of the materials described herein for altering the quality and/or quantity of CWSP in a host cell. Particularly for altering the mannose : galactose ratio in a mannose/galactose containing compound in that host cell.
  • a method of influencing or affecting the CWSP content of a host cell comprising the step of causing or allowing expression of a heterologous nucleic acid ⁇ equence encoding a bio ⁇ ynthetic enzyme as discussed above within the cell.
  • the invention further provides a method of influencing or affecting the glycosyltransferase activity in a plant, the method comprising the step of causing or allowing expression of a heterologous nucleic acid sequence a ⁇ discussed above (e.g. encoding the fenugreek or guar galactosyltransferase or a variant thereof) within the cells of the plant.
  • a heterologous nucleic acid sequence a ⁇ discussed above e.g. encoding the fenugreek or guar galactosyltransferase or a variant thereof
  • the step may be preceded by the earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.
  • the foregoing di ⁇ cu ⁇ sion has been generally concerned with uses of the nucleic acids of the present invention for production of functional polypeptides, for instance for the purpose of increasing the galactosyltransferase activity in the cell.
  • a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • Anti ⁇ ense technology is also reviewed in Bourque, (1995) , Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
  • An alternative to anti-sense is to use a copy of all or part of the gene (galactosyltransferase or variant) inserted in sense, that i ⁇ the same, orientation a ⁇ the natural gene, to achieve reduction in expre ⁇ ion of the target gene by co- ⁇ uppre ⁇ sion.
  • a copy of all or part of the gene galactosyltransferase or variant
  • i ⁇ the same, orientation a ⁇ the natural gene to achieve reduction in expre ⁇ ion of the target gene by co- ⁇ uppre ⁇ sion.
  • ribozymes e.g. hammerhead ribozymes, which can catalyse the site- ⁇ pecific cleavage of RNA, ⁇ uch a ⁇ mRNA ( ⁇ ee e.g. Jaeger (1997) "The new world of ribozyme ⁇ ” Curr Opin Struct Biol 7:324-335, or Gibson &
  • the complete ⁇ equence corresponding to the coding sequence need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding ⁇ equence to optimise the level of anti- sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence.
  • the ⁇ equence employed may be about 500 nucleotide ⁇ or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be pos ⁇ ible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where pos ⁇ ible, ⁇ uch a ⁇ longer than about 600 nucleotide ⁇ , than about 700 nucleotide ⁇ , than about 800 nucleotide ⁇ , than about 1000 nucleotide ⁇ or more .
  • sequence employed in a down-regulation of gene expres ⁇ ion in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti- ⁇ en ⁇ e and sense RNA molecules to hybridise. There may be down regulation of gene expres ⁇ ion even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expres ⁇ ion to take place.
  • the pre ⁇ ent invention further provide ⁇ the use of Seq ID No 1 or 3, or the complement thereof, or a variant of any of these, for down- regulation of gene expres ⁇ ion, particularly down-regulation of expre ⁇ sion of a galacto ⁇ yltran ⁇ ferase gene or variant thereof, preferably in order to influence the galacto ⁇ yltransfera ⁇ e activity in a host cell, more preferably a plant cell, most preferably a plant.
  • the invention further provides use of an antibody to achieve the same.
  • Anti -sense or sense regulation may itself be regulated by employing an inducible promoter in an appropriate construct.
  • a yet further method of manipulating galactosyltransferase activity is to express an antibody to the enzyme in the plant. It has been demonstrated that functional antibodies and antibody fragments can be expressed intracellularly and can be targeted to sub-cellular compartments . Alteration of phenotype by this method has been demon ⁇ trated, for in ⁇ tance by Artsaenko et al (1995) Plant J 8: 745-750 and Owen et al (1992) Bio/Technology 10: 790-794.
  • a plant product derived from any of the transformed plants or plant cells, or produced by any of the methods, discussed above in relation to other aspects of the invention e.g. in which galactosyltransferase activity has been altered.
  • the plant product comprises an altered galactomannan, which is to say that the galactomannan contains an altered (preferably reduced) ratio of galactose to mannose and/or an altered backbone galactose distribution.
  • a commodity comprising the plant product described above (e.g. up to 5%, preferably 0.1 - 3%), particularly a human or animal foodstuff, or a cosmetic .
  • a frozen food product for instance an ice cream or water ice.
  • salad dres ⁇ ings sauces, gelled desserts and "reduced-fat” products.
  • Animal foodstuffs may include gel-based petfoods.
  • the food composition comprising altered galactomannan plus one other polysaccharide ⁇ elected from: xanthan; carrageenan; agarose .
  • Galactomannan ⁇ having altered hydrophilic and cryogelation properties may have particular application to industry a ⁇ additive ⁇ e.g. as stabiliser ⁇ , emul ⁇ ifiers, and in combination with other polysaccharides, to impart more complex rheologies .
  • a ⁇ additive ⁇ e.g. as stabiliser ⁇ , emul ⁇ ifiers, and in combination with other polysaccharides, to impart more complex rheologies .
  • Figure 1 shows an alignment of the fenugreek galactosyltransferase with the putative guar galactosyltransferase sequence.
  • Galtran2.pro fenugreek galactosyltransferase;
  • Guargalt.PRO guar sequence. Residues matching the fenugreek sequence exactly are boxed. Numbering corre ⁇ ponds to the guar sequence. Predicted membrane-spanning a-helix is underlined.
  • FIG. 2 typical data-sets correlating galactosyltransferase activity and a 51K protein. Triton X-100 solubilised extracts were separated on IEF gels containing the same detergent. Strips from each gel were treated to localise galactosyl-transfera ⁇ e activity and separated protein, and to plot the pH gradient.
  • a and B Alignment of galactosyltransfera ⁇ e profile activity with ⁇ econd-dimen ⁇ ion SDS-PAGE. Activity correlate ⁇ closely with a 51K protein [50K position arrowed] .
  • Figure 3 cDNA and deduced protein sequence of c.500 bp clone obtained by 3 'RACE.
  • the sequences of the degenerate gene-specific primer and an antisense primer [GTPA3] are double underlined.
  • Known sequences from the 51 K protein are underlined and italicised.
  • Figure 4 cDNA and deduced protein sequence from c.1000 bp clone.
  • the sequences of the 5' and 3' degenerate primers used to amplify the cDNA are double underlined.
  • the known protein sequence from the 51 K protein i ⁇ underlined and italici ⁇ ed.
  • Figure 5 cDNA and deduced protein ⁇ equence from c.1500 bp clone.
  • the primer ⁇ used to amplify the cDNA are double underlined.
  • the known protein sequence from the 51 K protein is underlined.
  • the orf beginning at bpl and ending at bp 1314 encodes a 438 aa protein.
  • Figure 6 Secondary structure prediction using neural network based program.
  • 6A show ⁇ predicted helix (H) ; extended sheet (E) ; other loop (blank) .
  • 6B show ⁇ predicted transmembrane helix (T) .
  • Figure 7 Digital autoradiogram of endo- ⁇ -D-mannanase digests of labelled polymeric products formed during galactosyltransferase assays of lOx concentrated supernatant ⁇ from three different Pichia colonies carrying truncated constructs .
  • a trace amount of the galactomannan active ⁇ -galactosidase from guar seeds was added to the digest in the centre lane.
  • Gal Galactose.
  • M2G galacto ⁇ ylmannobiose
  • M3G galactosylmannotriose
  • M5G2 digalacto ⁇ ylmannopentaose
  • 0 galactomanno-octasaccharides
  • N galactomanno-nona ⁇ accharides .
  • Annex lb tran ⁇ lation of the fenugreek cDNA sequence - Seq ID No 2
  • Annex 2b translation of the guar cDNA sequence - Seq ID No 4
  • Fenugreek plants were grown to flowering and fruiting under conditions which have been described elsewhere (Edwards et al . 1992) .
  • Membranes were prepared using a method similar to that described previously (Edwards et al . 1989). Endosperms were hand-isolated at a stage of seed development during which intensive galactomannan biosynthesi ⁇ wa ⁇ taking place [35-40 days after anthesis under our growth conditions] , and homogenised in a gla ⁇ Potter homogeni ⁇ er with 50 mM Tri ⁇ HCl buffer pH 7.5 containing 1 mM EDTA and 5 mM dithiothreitol [DTT] .
  • the ratio of buffer to plant ti ⁇ ue was 0.5 ml buffer per endosperm. Larger particles were removed by an initial centrifugation at 13000 g [10 min] , and the supernatant was spun at 100000 g for 1 hour. The pelleted membranes were resuspended in the same buffer [usually 100 ⁇ l per endosperm] .
  • the incubation mixture [total volume 100 ⁇ l; incubated at 30°C, usually for 1 h] comprised membranes [usually equivalent to 0.1 - 1 endosperm] , DTT [2.5 mM] , EDTA [0.5 mM] , MgCl2 [2.5 mM] , MnC12 [5 mM] , UDP-Gal [800 ⁇ M] and/or GDP-Man [80 ⁇ M] in 25 mM Tris HCl buffer, pH 7.5.
  • the GDP-Man and/or the UDP-Gal substrate was labelled with the appropriate nucleoside diphospho- [U- 14 C] -sugar ⁇ .
  • Specific radioactivitie ⁇ were adjusted to 25-250 Bq.nmol "1 and were checked by scintillation counting in each experiment.
  • glacial acetic acid [50 ⁇ l] was added and the mixture heated at 100°C for 2 min.
  • Carrier galactomannan [100 ⁇ l of a 0.2 % w/v solution of locust bean galactomannan] was then added, followed by methanol to a final concentration of 70 % v/v.
  • the mixture was heated [70°C for 10 min] and centrifuged [13000 g, 10 min] . the supernatant was discarded, and the pellet washed twice with hot 70% methanol as has been described (Edwards et al . 1989).
  • Mannan synthase could be assayed as above using labelled GDP-Man and unlabelled UDP-Gal. Under these conditions the product was a galactomannan, labelled in the mannosyl residues. It could be assayed also in the absence of unlabelled UDP-Gal, when the product was labelled (1-4) - ⁇ -mannan.
  • Membranes were i ⁇ olated a ⁇ above, and re ⁇ uspended [homogeniser] in 100 mM Tris HCl buffer pH 7.5 [12.5 ( ⁇ l per endosperm] containing EDTA [2 M] and DTT [10 mM] .
  • Samples of the resuspended membranes were mixed with an equal volume of 2% [w/v] detergent, placed on ice and homogenised briefly every 10 min for 30 min. Suspensions were then centrifuged at 100000 g for 1 hour. Supematants were retained, and pellets were resuspended in a 1:1 mixture of resuspension buffer and 2% detergent, with a volume equal to that of the supernatant. Standard assays for mannan synthase and galactosyltran ⁇ ferase were carried out on supernatant ⁇ and resuspended pellets.
  • a pure sample of the labelled compound was obtained by carrying out a larger scale incubation and column purification as above, and strip-loading TLC plates with the column eluate. After developing the plates, the labelled product was located by digital autoradiography and then purified by removing the appropriate area of silica gel from the plates and eluting the silica gel with water.
  • a pure ⁇ -galactosida ⁇ e from guar seeds catalysed the complete conversion of the purified labelled compound to labelled galactose, and when the reaction was carried out in a graded fashion there were no labelled intermediate ⁇ produced. Thu ⁇ the labelled product carried a single ⁇ -linked galactose residue.
  • Results were con ⁇ istent with a model for acceptor substrate binding, according to which the ⁇ -galacto ⁇ yltran ⁇ ferase has an acceptor ⁇ ub ⁇ trate binding requirement comprising six principal binding sites for mannosyl residues of the acceptor substrate. For transfer to occur, at least five of the sites must be occupied, and transfer occurs to the mannose residue at the third binding site [measured from the non-reducing end] .
  • manno-oligosaccharide acceptors allowed the assay of the galactomannan galactosyltransferase after digitonin solubilisation.
  • Standard procedure was to incubate the detergent extract with mannohexao ⁇ e [1 mM] , MnC12 [usually 10 mM] and labelled UDP-Gal [800 ⁇ M] , dilute, spin through DEAE cellulose columns, freeze-dry the eluent and dissolve in water [50 ⁇ l] as above. Scintillation counting of an aliquot of the resulting solution gave a measure of the total radioactivity eluted from the column.
  • the proportion of thi ⁇ activity pre ⁇ ent in the galactosylmanno-hexaose product of the galactosyltran ⁇ ferase reaction was estimated by TLC and quantitative digital autoradiography of a further aliquot. Development of a new method to associate enzyme activi ty wi th particular proteins in the digi tonin extract .
  • isoelectric focussing [IEF] of enzymatically active detergent extracts on vertical agarose minigels prepared in the presence of the solubilismg detergent. It was found that detergent- ⁇ olubili ⁇ ed protein ⁇ , pre ⁇ ent pre ⁇ umably in micelle ⁇ , moved into the gel and were focu ⁇ ed according to their apparent pi values. Moreover, mannan synthase and galactosyltran ⁇ ferase activities in digitonin extracts were retained after focussing. After focussing, gels were cut into 1cm wide strips parallel to the direction of current flow.
  • one ⁇ uch ⁇ trip could be cut into slices perpendicular to the direction of current flow, each slice eluted with IM KCl and the pH values of the resulting solutions measured.
  • the pH gradient [establishment, shape, stability] was monitored also during focu ⁇ ing by loading the IEF gels with ⁇ mall ⁇ amples of coloured "marker" proteins flanking the sample of detergent-solubilised enzyme.
  • the second approach was to place an IEF strip adjacent to the one sliced for enzyme activity determination along a long ⁇ ample well of an SDS gel and ⁇ ubjecting it to SDS-PAGE in a direction perpendicular to that of IEF.
  • Thi ⁇ 2 -dimensional IEF/SDS-PAGE approach gave an excellent visual correlation between protein ⁇ on the stained SDS-PAGE gel and enzyme activity.
  • isoelectric focus ⁇ ing gel ⁇ [8 x 10 cm] were prepared by a ⁇ embling a " ⁇ andwich" of a glass plate to which pre-marked GelBond agarose gel support medium 0.1 mm thick [FMC BioProducts] had been attached [Hoefer Technical bulletin No 134] , 1 mm spacers, and a notched alumina plate [Hoefer] in a Hoefer Gel Caster SE 245. GelBond was used to ensure that gel dimension ⁇ did not change during any manipulations and staining procedures.
  • the agarose separation gel was prepared by mixing I ⁇ oGel agaro ⁇ e [120 mg] , ⁇ orbitol [2.4 g] and water [10.36 ml] and heating on a boiling water bath for 10 min with frequent mixing to di ⁇ olve the agarose. After cooling to 65°C, the volume was made up to the original value.
  • the sample and reference wells were cleaned and dried using strips of filter paper, and the sample, overlay and IEF standards applied.
  • the sample consi ⁇ ted of 750 ⁇ l of detergent extract [detergent concentration 1%] prepared as de ⁇ cribed above, mixed with 45 ⁇ l of the ⁇ ame ampholyte mixture a ⁇ was used to prepare the separating gel, 65 ⁇ l glycerol and 5 ⁇ l bromophenol blue [0.05 % w/v in water] . It wa ⁇ pipetted into the ⁇ ample well of the gel.
  • An overlay was prepared from 40 ⁇ l of the ampholyte mixture, 40 ⁇ l of 2% detergent, 40 ⁇ l glycerol 5 ⁇ l of the bromophenol blue solution and 680 ⁇ l water. A portion of this was pipetted into the reference well, and the remainder was layered carefully over the ⁇ ample. The overlay i ⁇ le ⁇ den ⁇ e than the sample, but more dense than the cathode buffer, thus forming a barrier to direct mixing of the sample and the strongly alkaline cathode buffer. Coloured IEF standards [Bio-Rad] (2.5 ⁇ l) were pipetted directly into the reference well.
  • cathode buffer (20 mM NaOH) was carefully poured into the back (upper) chamber of the apparatus so that it did not mix with the overlay and sample, and anode buffer (6 mM phosphoric acid) poured into the lower chamber.
  • the IEF was run at 200 V for 30 minute ⁇ during which mo ⁇ t of the sample could be seen to enter the gel, and then at 600 V for 60 minutes. During this time the coloured IEF standard ⁇ could be seen to migrate, focus and stabilise in position, and the current taken fell from about 12 mA to a stable final value of around 2 mA.
  • the gel sandwich was removed from the apparatus and the gel, attached to GelBond, separated from the plates and spacers.
  • the two strips were then dehydrated in methanol for 15 min, dried between sheet ⁇ of filter paper and ⁇ tained with Coomassie Blue.
  • the ⁇ tained ⁇ trip ⁇ showed the complete range of IEF standards. They also revealed the positions of stained band ⁇ in the ⁇ ample, and ⁇ howed whether or not the ⁇ ample had focussed in bands running perfectly horizontally across the gel. Further strips were proce ⁇ sed to obtain enzyme activity, protein distribution and pH gradient as indicated above.
  • mannan synthase and galactosyltransferase activity peaks overlapped.
  • the mannan synthase gave a broad peak at about pi 6.0, tailing towards the origin of the gel where a large proportion of the activity remained, apparently unable to enter the gel.
  • all of the galactosyltransferase activity entered the gel, and gave a more symmetrical peak [pi 6.0] overlapping with that of the mannan synthase .
  • the peptides were electroblotted and ⁇ ubjected to
  • N-terminal sequencing to give internal sequence data from the 50K protein.
  • sequence information obtained was compared with international database information, there was extremely high homology between the obtained sequences and those of membrane bound provicilin storage protein precursors. This indicated either that the about 50K band identified on 2-D gels was not the galactosyltran ⁇ fera ⁇ e, or that the corresponding band excised from the one-dimensional SDS gel ⁇ contained more than one protein, the vicilin-related protein predominating .
  • the properties, including transfer-specificity of the Triton-solubili ⁇ ed enzyme were compared with and found identical with those of the digitonin-solubilised enzyme. This allowed the IEF / SDS-PAGE separation described above to be carried out using Triton X-100 in place of digitonin. Thi ⁇ gave greatly improved activity resolution and protein separation. Also following our observation that manno-oligosaccharides would serve a ⁇ acceptors for detergent-solubili ⁇ ed galactosyltransferase, polymeric galactomannan ⁇ with low, medium and high galactose-substitution were tested as acceptors [locust bean, guar and fenugreek galactomannans respectively] .
  • Locust bean galactomannan was an efficient acceptor, guar galactomannan was less efficient and fenugreek galactomannan was not an acceptor.
  • the IEF agarose gel offered the possibility of its use as an m si tu acceptor for gel-separated galactosyltransferase, and the design of a new rapid, sensitive, highly resolving procedure for localising the enzyme activity.
  • the entire strip could be incubated in the presence of labelled UDP-Gal, whereby galactosyltransferase focussed withm the strip would catalyse the transfer of labelled galactose residues to the galactomannan component of the separating gel. After thorough washing of the gel, any radioactivity remaining withm it was a measure of and a localisation of galactosyltransfera ⁇ e activity.
  • the material focussing at pi 6.2 to 6.8 was excised from an entire Triton X-100 IEF gel and the gel sections were applied as the sample to an SDS-PAGE gel. After running, the gel was blotted onto Problott membrane, and the blot wa ⁇ ⁇ tained lightly with Coomassie blue. The 51K and 49K bands were adequately separated, and the 51K band was exci ⁇ ed carefully from the blot and ⁇ ubjected to N-terminal sequencing. Sequence was obtained. To obtain internal ⁇ equence information from the 51K protein IEF and SDS-PAGE wa ⁇ carried out as above.
  • Example 2 Acquisition of a cDNA sequence encoding the 51K probable galactosyltransferase protein.
  • RNA was prepared from endosperm ⁇ hand-i ⁇ olated from developing fenugreek ⁇ eed ⁇ during the early ⁇ tages of galactomannan deposition [32-35 days after anthesis (Edwards et al . 1992)].
  • 3'RACE PCR [Frohman M A, Martin G R (1989) Rapid amplification of cDNA ends using nested primers .
  • Techniques 1: 165-170] was carried out using a degenerate primer [GT3S4] designed to an internal protein sequence, a c500 bp cDNA wa ⁇ amplified.
  • the partial terminal sequences of the clOOO bp clone are shown in Fig. 4.
  • perfect primer ⁇ were designed to the extreme 5' end of the clOOO bp clone and to the 3' untranslated region of the c500 bp clone.
  • RT-PCR carried out using a proof-reading thermostable DNA polymerase [Pfu - Stratagene] , resulted in the amplification of a cl500 bp cDNA which was cloned and fully sequenced.
  • the complete sequence, shown in Fig. 5, had an orf encoding a 438 amino acid polypeptide.
  • the deduced molecular weight was 51281 Daltons, and the deduced pi was 6.646, in close agreement with the values observed for the Triton X-100 ⁇ olubilised 51K protein.
  • RNA from developing fenugreek endosperms Seeds from pods harvested 32-35 days after anthesis were hand-dissected under aseptic conditions, and the endosperm tissue was dropped directly into liquid nitrogen. Endosperms from 100 ⁇ eed ⁇ [weight approx 1 g] were then ground in a mortar and pe ⁇ tle with liquid nitrogen, and RNA wa ⁇ prepared e ⁇ sentially according to the procedure of Lopez -Gomez R and Gomez-Lim M A (1992) A method for extracting intact RNA from frui ts rich in polysaccharides using ripe mango . HortScience 27: 440-442. This method, which involves an extraction buffer containing 20% ethanol, circumvented problems associated with the dissolution of galactomannan in extraction buffers. RNA yields were typically about 50 ⁇ g.
  • degenerate primers to amino acid sequence from the 51K protein .
  • a degenerate primer was de ⁇ igned to the extreme N-terminal part of the 'N-terminal [Table 1.1]' amino acid sequence and designated NTP2S.
  • a further degenerate primer pair [sense and antisense, designated GT3S4 and GT3A4 was designed to part of the 'internal 1 [Table 1.1]' sequence (see above).
  • PCR reaction mixture comprised 5 ⁇ l first strand cDNA, 25 pmol T7 primer, 25 pmol GT3S4 primer, 0 01 ⁇ mol of each dNTP, 2 5 U Taq polymerase [Pharmacia] and lOx Taq polymerase buffer [5 ⁇ l, Pharmacia] in a total volume of 50 ⁇ l
  • m 30 ⁇ l was heated to 95°C for 7 mm, and then held at 75°C whilst the remaining components were added
  • the complete mixture was heated at 50°C for 2 mm, followed by 72°C for 5 mm, ⁇ ubjected to 30 cycles of 94°C [1 mm]-
  • the PCR protocol [with "hot start” as above] comprised 30 cycles of 94°C [1 min] - 37°C [1 min] 72°C [2 min] , with a final period at 72°C [15 min] . This resulted in the amplification of a clOOO bp cDNA which was exci ⁇ ed from gel ⁇ , purified [Hybaid Recovery] and cloned, ⁇ ubcloned and sequenced from both ends.
  • PCR mixture ["hot-started" a ⁇ above] comprised first strand cDNA template, GTP5S and GTP6A primer ⁇ [25 pmol each], dNTP's [0.01 ⁇ mol each], Pfu DNA polymerase [2.5 U; Stratagene] and lOx Pfu buffer [5 ⁇ l; Stratagene] in a total volume of 50 ⁇ l .
  • the mixture was held at 50°C for 2 min, then at 72°C for 10 min before being subjected to 30 cycles of 94°C [1 min] - 50°C [1 min] - 72°C [4 min] and held at 72°C for 15 min. This resulted in the amplification of a cl500 bp fragment which wa ⁇ excised from the gel and purified [Hybaid Recovery] .
  • the 3' A overhangs necessary for ligation into the pCR2.1 vector were added sub ⁇ equently in a reaction containing purified DNA, dATP [0.01 ⁇ mole ⁇ ] Taq polymera ⁇ e [2.5U] and Taq buffer [Pharmacia] in a volume of 50 ⁇ l, heated to 72°C for 10 min.
  • the cDNA was then purified [Hybaid Recovery], sub-cloned and sequenced.
  • the ⁇ equence which contained an orf of 1314 bp, encompa ⁇ ed all known ⁇ equence from the c500bp and clOOO bp clones. It encoded a 438 amino acid protein, deduced molecular weight 51282 and deduced pi 6.646.
  • the deduced protein sequence included all amino acid sequence data obtained from the 51K protein, and was clearly the cDNA sequence which encoded it.
  • Such sequence ⁇ serve to anchor proteins to membranes, and are typical of many Golgi membrane bound proteins, including several glycosyltransferases [Paulson J C and Colley K J (1989) Glycosyl transf erases . Structure, localization and control of cell type specific glycosylation J Biol Chem 264: 17615- 17618] .
  • Example 3 Evidence that the 51K protein is the fenugreek galactomannan galactosyltransferase
  • Any expres ⁇ ed fenugreek galacto ⁇ yltran ⁇ fera ⁇ e activity would be ea ⁇ ily identified. It was recognised that expre ⁇ sion of the full-length DNA including the transmembrane helix "anchor" sequence might lead to the attachment of any expressed protein to cellular membranes of the host microorganism.
  • our strategy included the expression not only of the full length 51K protein but also of a truncated protein lacking the sequence from the N-terminus to just beyond the transmembrane helix.
  • the truncated protein if expressed, might be expected to be enzymatically active but not membrane-bound.
  • primers GTEXP1S and GTEXP3A amplified a cl400 bp band which wa ⁇ purified from gel ⁇ , dige ⁇ ted with Not 1 and Xho 1, re-purified and cloned into pPIC9 which had been previou ⁇ ly digested with the same re ⁇ triction enzymes.
  • Ampicillin-resistant clones were screened for the presence of insert ⁇ by PCR u ⁇ ing gene-specific primers and a primer designed to part of the ⁇ -factor sequence on the vector . This primer confirmed not only that apparently correct inserts were present but also confirmed their orientations.
  • Plasmid DNA prepared from positive clones was further checked for the presence of the correct inserts by digestion with Xhol and Not 1.
  • Primers GTEXP2S and GTEXP3A amplified a c 1300 bp fragment which was similarly treated.
  • Competent cells of Pichia pastoris GS115 were prepared and transformed using the EasyComp [Invitrogen] kit. Separate transformation ⁇ were carried out u ⁇ ing pPIC9FStul, pPIC9TStul and Stu 1 lineari ⁇ ed pPIC9 a ⁇ control.
  • putative positive transformant ⁇ were ⁇ elected on the basis of their ability to grow on histidme-free medium a ⁇ de ⁇ cribed in the Invitrogen Pichia expression kit manual. Putative positives were further screened by direct PCR amplification of colonies. Yea ⁇ t cell ⁇ were boiled for 10 mm prior to the addition of the PCR ingredients .
  • the assays [100 ⁇ l] contained 50 ⁇ l concentrated supernatant, 25 mM Tris-HCl buffer pH 7.5, 2 mM MnCl2, 0.2% [w/v] locu ⁇ t bean galactomannan and 800 ⁇ M labelled UDP-Gal, and were incubated at 30°C for 2 hours.
  • Example 4 identification of a homologous ⁇ equence from developing guar ( Cyamopsis tetragonoloba [L] Taub.) endosperms, and demonstration that it encode ⁇ a galactomannan galacto ⁇ yltran ⁇ fera ⁇ e
  • MN10_YEAST SWI ⁇ S-PROT: P50108
  • GM12_SCHPO SWISSPROT: Q09174
  • Degenerate sense and antisen ⁇ e primer ⁇ GT5S1 and GT5A1 - Table 3 were de ⁇ igned, following the fenugreek galacto ⁇ yltran ⁇ fera ⁇ e ⁇ equence, to a short region of very high homology between all three sequences. This covered amino acids 190 - 210 of the fenugreek galactosyltransferase sequence .
  • RNA wa ⁇ prepared, a ⁇ for fenugreek, from the developing endo ⁇ perms of guar seeds during the early ⁇ tage ⁇ of galactomannan deposition (30 to 35 days after anthesis, Edwards et al . 1992) .
  • First strand cDNA was synthesi ⁇ ed, as before, using the (dT) 17 -R 1 R 0 primer (Frohman and Martin, 1989) .
  • primer GT5S1 primer
  • T7 RNA polymerase promoter an 800 - 900 bp cDNA was amplified.
  • PCR amplification was carried out using sense and antisen ⁇ e primer ⁇ de ⁇ igned to the fenugreek galacto ⁇ yltransfera ⁇ e sequence between amino acid 210 and the C- terminus of the protein, paired with GT5S1 and T7 , using the purified 800- 900 bp cDNA as template.
  • One pair (GT5S4 and GT5A4 , Table 3) gave efficient amplification of cDNA bands.
  • GT5S4 in combination with T7 and GT5S1 in combination with GT5A4 resulted in the amplification of bands of the sizes expected if the 800 - 900 bp band amplified by 3 ' RACE wa ⁇ a sequence homologous to the fenugreek galactosyltransferase.
  • Amplified cDNA was recovered (Hybaid Recovery) and used as template for a second round amplification, using the degenerate primer (NTP2S, as used in Example 2) designed to the N-terminal protein sequence of the fenugreek galacto ⁇ yltran ⁇ era ⁇ e along with GT5A1. This resulted in the amplification of a 570 bp cDNA.
  • the sequence showed a continuous open reading frame from the start to base 1326 (Annex 2b) . Near the 5' end (base 24 onwards), the encoded protein sequence wa ⁇ closely similar to that at the the N-terminus of the fenugreek galactosyltransfera ⁇ e (ATKFGS in fenugreek, and AKFGS in guar) . In guar, thi ⁇ sequence was immediately preceded by a methionine residue, which may represent the start of tran ⁇ lation. On thi ⁇ assumption, the cDNA encoding the putative guar galactosyltransferase comprise ⁇ 1305 bp and encode ⁇ a 435 amino acid protein. The fenugreek galacto ⁇ yltran ⁇ fera ⁇ e and the putative guar galactosyltransferase are aligned in Fig 1.
  • Pichia constuct ⁇ were obtained for both the full-length and the truncated sequence, and culture filtrates were a ⁇ sayed for galactomannan galacto ⁇ yltran ⁇ ferase activity exactly a ⁇ de ⁇ cribed in
  • Example 1 Culture supe atants from control transformants (no insert) and from transformant ⁇ with full-length inserts did not contain measurable amounts of galactomannan galactosyltran ⁇ fera ⁇ e activity, whereas supematants from transformants with truncated insert ⁇ contained significant levels of activity (7.99 + 1.90 ⁇ mol . I "1 .h "1 ; 6 independent clones; supematants not concentrated).
  • Transgenic plants containing modified levels of the fenugreek or guar galactosyltransferase genes, or derivatives thereof, may be produced using methods known to those ⁇ killed in the art. Gene constructs will be expres ⁇ ed con ⁇ titutively or in a ti ⁇ ue-specific manner in the seed or endosperm, potentially at a specific developmental stage. Constructs may include antisense versions of e.g. guar galactosyltransferase. Transgenic Guar plants may then be produced, for instance using methods analogous to those discus ⁇ ed in WO 97/20937. Thi ⁇ will result in guar galactomannan with a higher man/gal ratio.
  • Example 5 Foodstuff ⁇ compri ⁇ ing modified galactomannan
  • Modified galactomannans may be extracted from transgenic plant ⁇ by method ⁇ analogous to those used in the art .
  • An ice cream based on the modified galactomannan may be provided as follows :
  • a water ice may be provided a ⁇ follows :
  • Annex lb tran ⁇ lation of the fenugreek cDNA sequence - Seq ID No 2 ATKFGSKNKSSPWLSNGCIFLLGAMSALLMI GLNSFIAPIPNSNPKFNSFTTKLKSLNFTTNTNFAGPDL LHDPSDKTFYDDPETCYTMMDKPMKNWDEKRKEWLFHHPSFAAGATEKILVITGSQPTKCDNPIGDHLLLR FYKNKVDYCRIHNHDIIYNNALLHPKMDSYWAKYPMVRAAMLAHPEVEWIWWVDSDAIFTDMEFKLPLWRY KDHNLVIHGWEELVKTEHSWTGLNAGVFLMRNCQWSLDFMDVWASMGPNSPEYEKWGERLRETFKTKWRD SDDQTALAYLIAMGEDKWTKKIYMENEYYFEGYWLEISKMYDKMGERYDEIEKRVEGLRRRHAEKVSERYG EMREEYVKNLGDMRRPFITHFTGCQPCNGHH
  • Annex 2b translation of the guar cDNA sequence - Seq ID No 4

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Abstract

Provided are genes encoding enzymes (e.g. galactosyltransferases from fenugreek and guar) which have a role in the biosynthesis of complex non-cellulosic cell wall polysaccharides such as galactomannan. Variants and other products based on the genes are also provided, as are antibodies to the enzymes, plus also methods of isolating or preparing any of these. Also disclosed are vectors and other methods and materials which may be used for cloning the genes (or related nucleic acids e.g. anti-sense versions) into hosts such as transgenic plants having modified polysaccharides. Further disclosed are products, compositions and methods employing these plants and polysaccharides.

Description

GENES & ENZYMES TECHNICAL FIELD
The present invention relates to nucleic acids and other materials having a role in the biosynthesis of complex non-cellulosic plant cell wall polysaccharides. It further relates to various applications for such materials.
RELEVANT PRIOR ART
Plant cell walls contain a number of non-cellulosic polysaccharides which play important roles in vivo both structurally and as energy stores. Some of these are discussed in Brett & aldron (1996) "Physiology and Biochemistry of Plant Cell Walls - 2nd Edition" Pub. Chapman & Hall, London, especially pages 4-43. Generally these occur in the cell wall matrix phase as pectins and hemicelluloses .
Two principal cell wall storage polysaccharides (CWSPs) are the hemicelluloses galactomannan (e.g. guar gum, locust bean gum) and xyloglucan (e.g. tamarind seed polysaccharide) . The various known characteristics of these CWSPs, including their structure, application to industry, and metabolism is summarised in Reid & Edwards (1995)
"Galactomannans and other cell wall storage polysaccharides in seeds" in "Food polysaccharides and their applications" Ed: Stephen, pp 155- 186; Pub. Marcel Dekker.
.Role of galactomannans in vivo
Galactomannans are found in the endosperm cells of leguminous seeds, and in the endosperms of the seeds of a small number of non-leguminous species. In general they act as storage reserves, being broken down following germination to monosaccharides which are used by the developing seedling. Their overall biological functions are more complex. The galactomannan of fenugreek has been shown to be multifunctional, imbibing large amounts of water during seed hydration, deploying it as a buffer to protect the germinating embryo from post- imbibition drought, and serving as a substrate reserve following successful germination.
Structure of galactomannans
Structurally galactomannans comprise a (1-4) -β-linked D-mannan backbone which carries single-unit α-D-galactosyl substituents attached (1-6) -α to backbone mannose. Mannose/Galactose [Man/Gal] ratios in galactomannans range from about 3.5 [low-galactose] to about 1.1 [high galactose] . In the legumes, Man/Gal ratio is constant and fixed for the galactomannan of a given species [genetic control] , and Man/Gal ratios are similar within taxonomic sub-groupings of the Leguminosae . Species with similar Man/Gal ratios may differ in the statistical distribution of galactose residues along the mannan backbone. Galactomannans are closely related structurally to other cell wall storage polysaccharides of seeds [mannans, glucomannans , galactoglucomannans] and to non- cellulosic polysaccharides of non-seed plant cell walls [glucomannans, galactoglucomannans] .
Galactomannan biosynthesis
Galactomannan biosynthesis has been studied using three model leguminous species representative of those forming high-galactose, medium-galactose and low-galactose galactomannans. These are fenugreek [ Trigonella foenum-graecum, Man/Gal = 1.1], guar [ Cyamopsis tetragonoloba , Man/Gal = 1.6] and senna [Senna occiden tali s, Man/Gal = 3.3] (see Edwards et al (1992) Planta 187:67-74; also (1995) Planta 195: 489-495; also (1989) Planta 178: 41-51).
These workers used mixed membrane preparations prepared from endosperms, hand-dissected from developing seeds. The preparations were enzymatically active, catalysing the formation of labelled polysaccharide from GDP-14C-mannose, from GDP-14C-mannose plus unlabelled UDP-galactose [UDP-Gal] and from unlabelled GDP-mannose [GDP-Man] and UDP-14C-galactose . By acid hydrolysis, and in particular the use of pure structure-sensitive galactomannan-hydrolysing enzymes, the polysaccharide products formed from combinations of UDP-Gal and GDP-Man were shown unequivocally to be galactomannans, and the product formed from GDP-Man alone to be (1-4) -β-linked mannan. Thus galactomannan biosynthesis appears to be catalysed by the interaction of two membrane-bound enzymes - a GDP-Man dependent (1-4) -β-D- mannosyltransferase and a specific, UDP-Gal dependent α-D- galactosyltransferase .
The nature of the interaction between the mannan synthase and the galactosyltransferase was also investigated using the membrane preparations . This demonstrated that the mannan synthase can operate independently of the galactosyltransferase, that the galactosyltransferase cannot operate in the absence of simultaneous mannan synthase action and that (1-4) -β-D-mannan preformed at the site of synthesis using the mannan synthase is not accessible as a substrate for the galactosyltransferase. Thus an experimental model for galactomannan biosynthesis involves stepwise chain-elongation of the mannan chain towards the non-reducing end catalysed by the mannan synthase and transfer of galactose, catalysed by the galactosyltransferase, to a hypothetical galactosyl acceptor mannose residue at or close to the [elongating] non-reducing chain-end.
The regulation of Man/Gal ratio in galactomannan biosynthesis .
In fenugreek, guar and senna the activities of the mannan synthase and the galactosyltransferase in developing endosperms vary pari passu with galactomannan deposition, and the relative amounts of the two activities vary little during the period of deposition. In fenugreek and guar [high- and medium-galactose galactomannans] the Man/Gal ratios of the galactomannan present in the endosperm cell walls during galactomannan deposition remain constant at 1.1 and 1.6 respectively. In senna the Man/Gal ratio increases during late seed development from about 2 to 3.3, and this change is accompanied by the appearance and increase of the activity of a galactomannan-active -galactosidase . Thus in the high and medium-galactose species Man/Gal ratio is determined only by the pathway of biosynthesis. In the low-galactose species the Man/Gal ratio of the primary biosynthetic product is controlled by the biosynthetic process, and the primary biosynthetic product undergoes a post-depositional modification catalysed by a galactomannan-active α-galactosidase .
In vi tro galactomannan biosynthesis .
Labelled galactomannans with a range of Man/Gal ratios can be formed in vitro from UDP-Gal and GDP-Man and the membrane-preparations from fenugreek. This is because the rate of mannan-chain elongation in vitro is independent of the rate of galactosyl transfer. Published work suggests that galactosyl transfer depends on the availability of nascent mannan chain as acceptor substrate, and the enzyme system in vitro forms low-galactose galactomannans when saturating GDP-Man and UDP-Gal concentrations are supplied. By retaining UDP-Gal concentrations at saturating and progressively decreasing the rate of mannan chain extension by lowering the GDP-Man concentration, a range of labelled galactomannan products can be obtained with galactose- contents approaching, but not exceeding, those of the primary products of biosynthesis in vivo. The labelled galactomannans can be fragmented, using a pure structure-sensitive endo- (1-4) -β-D-mannanase, to give a series of diagnostic manno-and galactomanno-oligosaccharides, the relative amounts of which can be determined accurately using quantitative digital autoradiography after separation on thin layer chromatography [TLC] plates The results of digital autoradiography comprise a structural "fingerprint" of each in vitro galactomannan
Computer modelling galactomannan biosynthesis . The experimental model for the interaction of the mannan synthase and galactosyltransferase in galactomannan biosynthesis has been computer modelled with an inbuilt [second-order Markov chain] assumption that the probability of obtaining galactose-substitution at the galactosylacceptor mannose residue is influenced by the existing states of substitution at the nearest and second-nearest neighbour mannose residues only Also computer modelled is the substrate specificity of the structure-sensitive endo- (1-4) -β-D-mannanase Thus a computer algorithm is available which when supplied with a set of four numerical probabilities [P00, P10, P01, Pu, corresponding to the possible states of substitution at the nearest and second-nearest neighbour mannose residues] will simulate the synthesis of a galactomannan molecule according to the experimental model, and its hydrolysis by the structure-sensitive endo-mannanase, outputting the relative proportions of the diagnostic manno- and galactomanno-oligosacchaπdes released This algorithm has been used to process the quantitative endo-mannanase fragmentation data from the labelled m vitro galactomannans from fenugreek, guar and senna, with input of the experimental data and output, for each galactomannan of a set of four probabilities The results generate the following three statistical statements
1. The second-order Markov chain assumption built into the computer simulation of the biosynthetic process is adequate
2 The specificities of the biosynthetic enzyme systems from fenugreek, guar and senna are different, giving different statistical patterns of galactose-substitution along the mannan backbone
3. For each species the deduced statistical substitution rules define maximum permitted degrees of galactose-substitution which are approached by the degrees of galactose substitution exhibited by the primary products of galactomannan biosynthesis m vivo.
In biochemical terms
• The (galacto) mannan substrate subsite recognition of the galactosyltransferaseε from fenugreek, guar and senna must encompass at least three backbone mannosyl residues the one which is the site of reaction, and the two preceding ones, towards the reducing end of the chain Other backbone mannosyl residues may be recognised by the galactosyltransferase, but their states of substitution do not influence greatly the probability of obtaining galactosyl- substitution at the reacting mannosyl residue
• Galactosyltransferase specificity regulates the distribution of galactose residues along the galactomannan backbone and also sets a maximum limit the degree of galactosyl substitution attainable for the primary product of biosynthesis m each species
• This limit is achieved in vivo
Applications for CWSPs to industry The complex hydrophilic properties of galactomannans underlie also the industrial applications of galactomannans For example, m the food industry they are used as stabilisers, emulsifiers and m combination with other polysaccharides and proteins to impart more complex rheologies
The commercial functionality of galactomannans is dependent upon the Man/Gal ratio and, to a lesser extent, the galactose distribution along the mannan backbone Generally higher Man/Gal ratios are desirable Of the two principal commercial galactomannans locust bean gum [Man/Gal = 3 5, galactomannan of Ceratoma siliqua] is superior to guar gum
[Man/Gal = 1 6, galactomannan of Cyamopsi s tetragonoloba] , particularly m mixed polysaccharide interactions
EP 0 255 153 (Unilever NV/Unilever pic) discusses the use of recombmantly produced guar alpha-galactosidase for providing galactomannans having improved properties
WO 97/20937 (Damsco) discusses methods of m vivo modification of mannose/galactose ratios in galactomannans The Examples apparently disclose the cloning of a phosphomannose isomeraεe gene (involved m mannose-6 -phosphate generation) from guar, and also the use of senna alpha-galactosidase
However it is clear from the discussion above that galactosyltransferases are key enzymes m the regulation of galactose distribution along the backbone and m controlling the Man/Gal ratio Indeed the importance of glycosyltrans erases is acknowledged in WO 97/20937 at page 26 However, notwithstanding this, and the extensive research done on their mechanism using impure membrane preparations, no membrane bound transferases involved in the biosynthesis of non- cellulosic plant cell wall polysaccharides have been purified and no cDNA or genomic DNA sequences encoding such transferases have been identified
The difficulty m isolating such enzymes is discussed briefly m Reid & Edwards (1995) supra at page 164 and Brett & Waldron (1996) at page 79 In particular the plant cell wall is an extremely complex structure making it difficult to purify polysacchaπde-acting enzymes, or to associate them with the metabolism of any given wall component The isolation of enzymes which catalyse the biosynthesis of CCWPs is particularly difficult because they are tightly membrane -bound, to Golgi membranes
An assay for galactosyltransferase activity, m the form of membrane preparations , is disclosed m Edwards et al (1989) Planta 178 41-51 As described above, m this assay a radiolabelled sugar nucleotide [glycosyldonor] substrate is supplied, the acceptor [nascent mannan] substrate is believed to be formed by the simultaneous operation of an associated mannan synthase The labelled polysaccharide product is then isolated Strict controls are necessary to ensure that the "correct" polysaccharide (galactomannan) is assayed
However this assay is unsuitable for assaying the enzyme in solubilised form This m turn means it can not readily be used for the identification and therefore purification of the solubilised enzyme (for instance, to a level sufficient to provide sequence data which could be used to isolate corresponding nucleic acids)
Thus it will be seen from the foregoing that the provision of novel nucleic acids and other materials having a role m the biosynthesis of complex non-cellulosic plant cell wall polysaccharides and/or uses thereof would provide a contribution to the art
DISCLOSURE OF THE INVENTION
The present inventors have used novel techniques to identify and isolate a membrane-bound glycosyltransferase, and encoding nucleic acid, which catalyses the biosynthesis of a complex non-cellulosic plant cell wall polysaccharide The glycosyltransferase has demonstrated activity as a galactosyltransferase, involved in the biosynthesis of galactomannan. The polypeptide has a single membrane-spanning α-helix near the N- terminus which appears to serve to anchor the whole polypeptide to a biological membrane. This is the first time that a plant enzyme with activity appropriate for hemicellulose or pectin synthesis has been isolated, and that a nucleic acid sequence has been positively identified as encoding the same.
Briefly, the inventors showed that using the assay described above with detergent "solubilised" fenugreek material, mannan synthase activity was apparently retained at a low level, whilst galactosyltransferase activity was lost completely.
However they established that soluble acceptor molecules, (manno- oligosaccharides) could be used to mimic the nascent mannan chain. Labelled galacto-manno-oligosaccharide products were then carefully purified from other labelled substances and the galactosyl link in these oligosaccharides was shown to be the correct one for a galactosyltransferase involved in galactomannan synthesis.
Owing to the limited amounts of material available (endosperms were hand-dissected from fenugreek seeds about 5mm in diameter at the correct stage of development) only very small-scale purifications of the detergent-solubilised extract could be contemplated. It was found that if isoelectric focussing [IEF] agarose gels were prepared with the solubilising detergent incorporated and detergent-solubilised extract applied as sample, galactosyltransferase activity survived the IEF procedure and was focuεsed within the gel. Strips from the gel were analysed in parallel for activity and protein content.
Initially galactosyltransferase activity in the narrow strips cut from the IEF gels was assayed by incubating them in the presence of UDP-14C-Gal and a manno-oligosaccharide (usually mannohexaose) and carrying out a quantitative analysis of 14C present in galactosylmannohexaose after the enzyme reaction. Obtaining accurate analysis data required a multi-step procedure involving ion-exchange chromatography, TLC, digital autoradiography and scintillation counting.
Subsequently the inventors determined that low-galactose (and to a lesser extent medium-galactose) galactomannans would also serve as acceptors for the detergent-solubilised galactosyltransferase This effect was quite unexpected as earlier studies carried out using the membrane preparations of the prior art suggested that the limited number of acceptor groups available in these substrates would restrict their usefulness The inventors further established that activity could be assessed m si tu m the IEF gels This could be done using commercial agarose IEF gels which contained a blend of agarose and a galactomannan (apparently locust bean gum) This meant that gel strips incubated m the presence of UDP-Gal could be subjected to a simple washing procedure, after which the radioactivity remaining m the gel strips provided a measure of, and a localisation of, galactosyltransferase activity
Protein distribution within IEF gel strips was determined using two procedures In the first, the strips were cut into narrow slices, which were soaked m SDS-PAGE sample buffer and placed within individual sample wells of SDS-PAGE gels In the second, entire strips were soaked in SDS-PAGE sample buffer turned at right angles and applied as sample to SDS-PAGE gels, giving effectively a 2 -dimensional gel, the first dimension being the IEF separation carried out in the presence of the solubilismg detergent
By correlating enzyme activity and protein distribution after IEF m this way the inventors were able to identify a small number of potential "candidate" proteins Further analysis including Western blotting and the use of different solubilismg detergents identified a particular protein with molecular weight about 50K All protein sequence data required for cloning the corresponding cDNA was obtained from the about 50K protein recovered from SDS gels
Further analysis demonstrated that the fenugreek sequence encoded a 51K protein, with a single hydrophobic membrane-spanning helix near the N- terminal end This is typical of golgi -bound enzymes
The sequence apparently shares limited but significant homology with yeast galactosyltransferases, plus also low homologies with yeast mannan synthases and a plant β-mannanase
Identity was confirmed by cloning the cDNA in-frame into the genome of Pichia pastoris methylotrophic yeast, under the control of an alcohol oxidase promoter, and with the yeast α-secretion factor Two constructs were made, one with the full cDNA sequence, and the other with the sequence minus the N-terminal membrane-spanning domain, to avoid expressed protein becoming membrane-bound in the yeast. Culture filtrates were assayed for galactosyltransferase activity. Controls contained none, constructs with the full sequence had moderate activity, and constructs with the curtailed sequence gave extremely high activity.
Each nucleic acid encoding a glycosyltransferase provided by the present inventors may be used to manipulate (e.g. galactomannan) synthesis both in vi tro and in vivo thereby allowing galactomannans to be tailored for particular applications. Additionally it can be used, for instance, to alter the cell wall rheology, and hence mechanical properties (e.g. texture) of plant tissues, thereby permitting the production of improved plants and plant products for consumption or industrial use (e.g. fruits, vegetables, timber, paper etc.) .
The galactosyltransferase nucleic acid can also be used to prepare novel genes (variants) having altered properties with respect to the wild-type, or alternatively to facilitate the isolation of homologous genes from natural sources.
In the Examples below, the information provided by the novel fenugreek sequence has been used to assist in the isolation of a guar homolog, the activity of which was confirmed using the same assays as those discussed above.
These and other aspects of the present invention will now be discussed in more detail .
According to a first aspect of the present invention there is provided a nucleic acid molecule encoding a polypeptide which is capable of catalysing the biosynthesis of a complex non-cellulosic plant cell wall polysaccharide .
The polysaccharide may be a pectin or a hemicellulose, preferably the latter. Examples of hemicelluloses include xylan, glucomannan, mannan, galactomannan, glucuronoxylan, xyloglucan, callose or arabinogalactan.
The polypeptide is preferably a glycosyltransferase, which is to say that it catalyses, inter alia , the addition of monosaccharides (optionally from an activated precursor or donor e.g. a sugar nucleotide, such as a diphosphate precursor e.g. ADP- CDP- GDP- TDP- or UDP-sugar) to a polysaccharide chain (the 'acceptor') generally, but not exclusively, at the non-reducing end. Such enzymes are occasionally also termed polysaccharide synthases or synthetases by those skilled in the art.
Preferably the hemicellulose is one which contains galactose, and the glycosyltransferase is membrane-bound in vivo .
The activity of the encoded polypeptide may be tested, for instance, by observing the addition of radiolabelled sugar residues from exogenously supplied radioactive sugar nucleotides to saccharides, for instance oligosaccharides , or more preferably polysaccharides. Such methods are described in more detail below.
Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid) . Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term "isolated" encompasses all of these possibilities.
The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.
Most preferably the nucleic acid encodes a galactosyltransferase, which is capable of catalysing the biosynthesis of galactomannan.
Thus in one embodiment of this aspect of the invention, there is disclosed a nucleic acid comprising the nucleotide sequence shown in Seq ID No 1 (Annex la) . This sequence represents that of a cDNA molecule encoding a galactosyl transf rase gene from fenugreek. The encoded polypeptide (Seq ID No 2) is also shown in Annex lb.
In a further embodiment of the invention, there is disclosed a nucleic acid comprising the nucleotide sequence shown in Seq ID No 3 (Annex 2a) . This sequence represents that of a cDNA molecule encoding a galactosyl transferase gene from guar. The encoded polypeptide (Seq ID No 4) is also shown in Annex 2b. These sequences are compared in Fig
1.
Also disclosed are nucleic acids which are variants of the sequences provided. A variant nucleic acid molecule shares homology with, or is identical to, all or part of the coding sequence discussed above.
Generally, variants may encode, or be used to isolate or amplify nucleic acids which encode, polypeptides which are capable of catalysing the biosynthesis of a complex non-cellulosic plant cell wall polysaccharide by binding nucleotide sugar precursors and transfer sugar residues to polysaccharides in the golgi compartment (s) .
Such polypeptides may include not only galactosyltransferases, but also other (golgi located) glycosyltransferases e.g. those involved in galacto (gluco) mannan biosynthesis such as mannosyl and glucosyl transferases. Also included may be galactosyltransferases which act on pectin or xyloglucan.
Other polypeptides having the requisite characteristics may include arabinosyltrans erase, glucosyltransferase, xylosyltranεferase, mannosyltransferase, fucosyltransferase, rhamnosyltransferase, galacturonyltransferase and glucuronyltransferase .
Activities may conveniently be assessed using in si tu analysis in chromatographic gels (e.g. agarose gels) containing a suitable substrate (e.g. galactomannan for galactosyltransferase activity). Such methods of assessment form one part of the present invention.
A typical method will comprise the steps of :
(i) applying a sample comprising a mixture of proteins to a detector gel, said detector gel comprising in admixture (a) a chromatographic gel suitable for chromatographic εeparation of a mixture of proteins;
(b) an acceptor substrate for a glycosyltransferase, wherein the acceptor substrate is compatible with the chromatographic gel in that it does not impair the chromatographic properties of the gel, but is accesεible as a substrate for the proteins of the mixture,
(ii) chromatographically separating said mixture on the basis of size and/or charge (111) locating the glycosyltransferase, if present, within the gel, on the basis of glycosyltransfer to the εubstrate
Suitable 'compatible' substrates may include xyloglucan, xylan, glucomannan and pectin
Variants of the present invention can include not only novel, naturally occurring, nucleic acids, isolatable using the sequences of the present invention, but also artificial nucleic acids having novel sequences, which can be prepared by the skilled person m the light of the present disclosure
Thus a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided The fragments may encode particular functional parts of the polypeptide, e g portions lacking the transmembrane α-helix near the N-termmus (e g between residues 15 to 41 of the fenugreek sequence, or as underlined in Fig 1) which may have improved properties such as solubility or activity
Equally the fragments may have utility m probing for, or amplifying, the sequence provided or closely related ones Suitable lengths of fragment, and conditions, for such processes are discussed m more detail below
Also included are nucleic acids which have been extended at the 3 ' or 5' terminus Also included are sequences e g genomic sequenceε, having additional, non-expressed, portions ('introns')
Sequence variants which occur naturally may include homologous galactosyltransferases from other species, alleles (which will include polymorphisms or mutations at one or more bases) or pseudoalleles (which may occur at cloεely linked loci to the galactosyl transferase gene from fenugreek) Also included within the εcope of the preεent invention would be isogenes, or other homologouε geneε which may belong to the same family as the galactosyltranεferaεe gene (e.g galactoglucomannan synthases) Although these may occur at different genomic loci to the gene, they are likely to share conserved regions with it
Artificial variants (derivatives) may be prepared by those skilled m the art, for instance by site directed or random mutagenesiε, or by direct syntheεiε Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence shown in Seq ID No 1 or 3. Preferably it encodes a polypeptide which is capable of catalysing the biosyntheεiε of a complex non-celluloεic plant cell wall polyεaccharide .
The term 'variant' nucleic acid aε uεed herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expresεion product of the variant nucleic acid.
Some of the aεpects of the present invention relating to variants will now be discussed in more detail.
Homology and activi ty
Similarity or homology may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J". Mol . Biol . 215: 403-10, which is in standard use in the art, or, and this may be preferred, the standard program BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman.
Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shareε at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
Homology may be over the full-length of the relevant sequence shown herein, or may be over a part of it, preferably over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, 400 or more amino acids or codons, compared with Seq ID Nos 1 to 4 as appropriate.
Thus a variant polypeptide in accordance with the present invention may include within the sequence shown in Seq ID No 2 or 4 , a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes. In addition to one or more changeε within the amino acid sequence εhown, a variant polypeptide may include additional amino acids at the C- terminus and/or N-termmuε Naturally, changeε to the nucleic acid which make no difference to the encoded polypeptide (1 e 'degeneratively equivalent') are included
The activity of a variant polypeptide may be assessed by transformation into a host cell capable of expressing the nucleic acid of the invention Methodology for such transformation is described in more detail below
Production of derivatives
Thus a further aεpect of the invention there is disclosed a method of producing a derivative nucleic acid comprising the step of modifying the coding sequence of Seq ID No 1 or 3
Changes to a sequence, to produce a derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more ammo acids the encoded polypeptide
Changes may be desirable for a number of reasons , including introducing or removing the following featureε restriction endonuclease sequences, codon usage, other siteε which are required for poεt tranεlation modification, cleavage sites m the encoded polypeptide, motifs in the encoded polypeptide for glycosylation, lipoylation etc Leader or other targeting sequences (e g membrane or golgi locating sequences) may be added to the expressed protein to determine its location following expresεion All of these may asεist efficiently cloning and expressing an active polypeptide recombinant form (aε described below)
Other desirable mutations may be random or site directed mutagenesis m order to alter the activity (e g specificity) or stability of the encoded polypeptide
Changes may be by way of conservative variation, I e subεtitution of one hydrophobic reεidue εuch aε lεoleucme, valme, leucme or methionine for another, or the εubεtitution of one polar reεidue for another, such as argmine for lysme, glutamic for aspartic acid, or glutamine for asparagine Aε lε well known to thoεe skilled in the art, altering the primary structure of a polypeptide by a conservative subεtitution may not εignificantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which iε critical in determining the peptide' ε conformation. Figures 6A and 6B show the predicted secondary structures of the fenugreek polypeptide.
Also included are variants having non-conservative substitutions. As is well known to thoεe skilled in the art, subεtitutionε to regionε of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide' s three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
Identification of variants
In a further aεpect of the preεent invention there iε provided a method of identifying and/or cloning a nucleic acid variant from a plant which method employs Seq ID No 1 or 3 or a derivative thereof .
In each case, if need be, clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequenceε, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.
In one embodiment, nucleotide sequence information provided herein may be used in a data-baεe (e.g. of expreεεed εequence tags, or sequence tagged sites) search to find homologous sequences, such as those which may become available in due course, and expresεion products of which can be tested for activity as deεcribed below.
In a further embodiment, a variant in accordance with the preεent invention iε also obtainable by means of a method which includes: (a) providing a preparation of nucleic acid, e.g. from plant cellε, (b) providing a nucleic acid molecule having a nucleotide sequence εhown in or complementary to Seq ID No 1 or 3 or a derivative thereof, (c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with εaid nucleic acid molecule.
Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes . Restriction fragments may then be separated by electrophoresis on an agaroεe gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
Test nucleic acid may be provided from a cell aε genomic DNA, cDNA or RNA, or a mixture of any of theεe, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as is deεcribed hereinafter. Probing may optionally be done by meanε of εo-called 'nucleic acid chipε' (see Marεhall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review) .
When using genomic DNA, this method may be uεed to isolate promoters or other regulatory elements, the activity of which may be confirmed by analogy with the methodε below e.g. using promoterless conεtructε in which isolated fragments are operably linked to detectable geneε.
Preliminary experimentε may be performed by hybridiεing under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisationε identified as positive which can be investigated further.
For instance, screening may initially be carried out under conditions, which compriεe a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (e.g. Standard Saline Citrate ('SSC') = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration. Alternatively, a temperature of about 50°C or lesε and a high salt (e.g. 'SSPE'= 0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4) . Preferably the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE These conditions will allow the identification of sequences which have a substantial degree of homology (similarity) with the probe sequence, without requiring the perfect homology for the identification of a stable hybrid
Preferably, hybridisation conditions will be selected (e g using higher temperatures) which allow the identification of sequences having 70% or more (e g 80%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the probe, while discriminating against sequences which have a lower level of sequence identity with respect to the probe After low stringency hybridisation has been used to identify several nucleic acids having a substantial degree of similarity with the probe sequence, this subset is then subjected to high stringency hybridisation, so as to identify thoεe cloneε having a particularly high level of homology with respect to the probe sequences High stringency conditions compπεe a temperature of about 42 °C or leεε, a formamide concentration of leεs than about 20%, and a low salt (SSC) concentration Alternatively they may comprise a temperature of about 65°C or less, and a low salt (SSPE) concentration Preferred conditions for εuch screening comprise a temperature of about 42°C, a formamide concentration of about 20%, and a salt concentration of about 2 X SSC, or a temperature of about 65°C, and a salt concentration of about 0 2 SSPE
It is well known m the art to increase stringency of hybridisation gradually until only a few positive clones remain Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low Using these conditions nucleic acid libraries, e.g cDNA libraries representative of expreεεed sequences, may be searched Those skilled m the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on
Binding of a probe to target nucleic acid (e g DNA) may be measured using any of a variety of techniques at the dispoεal of thoεe skilled m the art For instance, probes may be radioactively, fluorescently or enzymatically labelled Other methods not employing labelling of probe include amplification using PCR (see below), RN'ase cleavage and allele specific oligonucleotide probing The identification of succesεful hybridiεation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host
Amplification of variants In a further embodiment, hybridisation of nucleic acid molecule to a variant may be determined or identified indirectly, e g using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of glycosyltranεferaεes are employed
Using RACE PCR, only one such primer may be needed (see "PCR protocolε, A Guide to Methods and Applications", Eds Innis et al, Academic Press, New York, (1990) )
Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may include
(a) providing a preparation of plant nucleic acid, e g from a εeed or other appropriate tissue or organ,
(b) providing a pair of nucleic acid molecule primers useful m (l e suitable for) PCR, at leaεt one εaid primer having a sequence shown in or complementary to a sequence shown Seq ID No 1 or 3 or a derivative thereof,
(c) contacting nucleic acid m said preparation with said pπmerε under conditions for performance of PCR, (d) performing PCR and determining the presence or absence of an amplified PCR product The presence of an amplified PCR product may indicate identification of a variant
Nucleic acids for probing or amplification An oligonucleotide for use m probing or PCR may be about 30 or fewer nucleotideε m length (e g 18, 21 or 24) Generally specific primers are upwards of 14 nucleotides length For optimum specificity and coεt effectiveneεs, primers of 16-24 nucleotides m length may be preferred Those skilled in the art are well versed m the design of primers for use in processes such as PCR If required, probing can be done with entire restriction fragments of the gene discloεed herein which may be 100' s or even 1000' s of nucleotides m length
It may be desirable to select primers or probes which are distinctive for particular parts of the sequence which are likely to be associated with particular activities e g it may be desirable to avoid using sequence from the helix region as these are more likely to cross react with sequences not forming part of the present invention.
As used hereinafter, unless the context demands otherwise, where "galactosyltransferase" is specified, the invention also covers corresponding applications employing the variants discussed above.
In one aspect of the present invention, the nucleic acid encoding the galactosyltransferase described above is in the form of a recombinant and preferably replicable vector.
"Vector" is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacteri um binary vector in double or single stranded linear or circular form which may or may not be self transmiεεible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organiεms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast or fungal) cells.
A vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell. The vector may be a bi-functional expresεion vector which functions in multiple hosts. In the case of genomic DNA, thiε may contain itε own promoter or other regulatory elementε and in the case of cDNA thiε may be under the control of an appropriate promoter or other regulatory elementε for expression in the host cell
By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3 ' direction on the sense strand of double-stranded DNA) . "Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter iε "under transcriptional initiation regulation" of the promoter.
Thus this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the fenugreek galactosyltransferase gene or a variant thereof.
Generally speaking, thoεe εkilled in the art are well able to conεtruct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Preεε.
Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis (see above discussion in respect of variants) , sequencing, introduction of DNA into cellε and gene expression, and analysis of proteins, are deεcribed in detail in Current Protocols in Molecular Biology, Second Edition, Auεubel et al . eds., John Wiley & Sonε , 1992. The diεcloεureε of Sambrook et al . and Auεubel et al . are incorporated herein by reference .
One embodiment of thiε aεpect of the present invention provides a gene conεtruct, preferably a replicable vector, compriεing an inducible promoter operatively linked to a nucleotide sequence provided by the present invention, such as Seq ID No 1 or 3.
The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expresεion under the control of an inducible promoter iε "εwitched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expresεion) in the abεence of the appropriate stimulus. Other inducible promoters cause detectable conεtitutive expreεsion in the abεence of the stimulus . Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus .
Particularly of interest in the present context are nucleic acid constructs which operate as plant vectors .
Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectorε . In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publiεherε, pp 121-148) .
Suitable promoters which operate in plants include the Cauliflower Mo'saic Virus 35S (CaMV 35S) ; the cauliflower meri 5 and the Arabidopsi s thaliana LEAFY promoter that is expressed very early in flower development. Other promoters include the rice actin promoter. Inducible promoters may include the GST-II-27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledons and dicotyledons. Other examples are diεcloεed at pg 120 of Lindεey & Joneε (1989) "Plant Biotechnology in Agriculture" Pub. OU Press, Milton Keynes, UK. The promoter may be selected to include one or more sequence motifε or elements conferring developmental and/or tissue - specific regulatory control of expresεion.
If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes εuch as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinoneε and glyphoεate) .
The present invention also provides methodε compriεing introduction of such a construct into a cell and/or induction of expression of a conεtruct within a cell, by application of a εuitable stimulus e.g. an effective exogenous inducer .
In a further aspect of the invention, there is disclosed a host cell containing a heterologous construct according to the present invention, especially a plant or a microbial cell (e.g. yeast cell) .
The term "heterologous" is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (e.g. encoding galactosyltransferase) have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention. A heterologous gene may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inεerted sequence may be additional to the endogenous gene or other sequence. Nucleic acid heterologous to a plant cell may be non- naturally occurring in cells of that type, variety or species. Thus the heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or specieε or variety of plant. A further poεεibility is for a nucleic acid sequence to be placed within a cell in which it or a homolog is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
The host cell (e.g. plant cell) is preferably transformed by the conεtruct, which iε to say that the construct becomes established within the cell, altering one or more of the cell's characteristics and hence phenotype e.g. with respect to CCWP production.
Nucleic acid can be transformed into plant cells using any εuitable technology, εuch as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Cul ture, Academic Presε) , electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Physiol . 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U. S . A . 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9: 1-11.
Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous specieε. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (see e.g. Hiei et al . (1994) The Plant Journal 6, 271-282)). Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone iε inefficient or ineffective. Alternatively, a combination of different techniqueε may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co- cultivation with Agrobacterium (EP-A-486233 ) .
The particular choice of a transformation technology will be determined by its efficiency to transform certain plant specieε as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
Thus a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a construct as described above into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid according to the present invention into the genome.
The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g comprising the galactosyltransferase sequence) especially a plant or a microbial cell. In the transgenic plant cell (i.e. transgenic for the nucleic acid in question) the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome.
Generally speaking, following transformation, a plant may be regenerated, e.g. from single cells, calluε tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vaεil et al . , Cell Cul ture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weisεbach and Weissbach, Methods for Plant Molecular Biology, Academic Preεε, 1989.
The generation of fertile tranεgenic plantε haε been achieved in the cerealε rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al . (1992) Bio /Technology 10, 667-674, Vam et al , 1995, Biotechnology Advances 13 (4) 653-671, Vasil, 1996, Nature Biotechnology 14 page 702)
Plants which include a plant cell according to the invention are also provided
A plant according to the present invention may be one which does not breed true one or more properties Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights It is noted that a plant need not be considered a "plant variety" simply because it contains stably with its genome a transgene, introduced into a cell of the plant or an anceεtor thereof
In addition to the regenerated plant, the present invention embraces all of the following a clone of such a plant, εeed, selfed or hybrid progeny and descendants (e g Fl and F2 descendants) and any part of any of these The invention alεo provides a plant propagule from εuch a plant, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on
Preferably the plant is an endoεpermic legume which contains galactomannan as a CWSP One example is the guar plant Some methods for transforming and regenerating such plants are discuεεed m WO97/20937 (Danisco)
The present invention also encompasεes the expression product of any of the galactosyltransferase or variant nucleic acid sequences disclosed above, and alεo methods of making the expresεion product by expreεεion from encoding nucleic acid therefore under εuitable conditions, which may be εuitable hoεt cells
Particularly included is a truncated polypeptide, lacking the transmembrane helix, which is soluble and not membrane-associated and which also has galactoεyltranεferase activity
Following expreεεion, the product may be lεolated from the expreεεion εyεtem (e g microbial) and may be uεed as desired, for instance formulation of a composition including at least one additional component
Alternatively the product may be used to perform its function m vivo and in particular in planta as diεcussed above.
Purified galactosyltranεferase protein, or a variant thereof, may be used to raise antibodies employing techniques which are standard in the art. Antibodies and polypeptides comprising antigen-binding fragments of antibodies may be used in identifying variants from other species as discussed further below.
Methods of producing antibodieε include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof . Antibodies may be obtained from immunised animals using any of a variety of techniques known in the a t, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al , 1992, Nature 357: 80-82) . Antibodies may be polyclonal or monoclonal. Single chain antibodies e.g. from Camelidae may be preferred (εee WO 94/25591 of Unilever) .
Antibodieε may be modified in a number of ways. Indeed the term "antibody" should be construed as covering any specific binding substance having a binding domain with the required specificity. Thus, thiε term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or εynthetic. Chimaeric moleculeε compriεing an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expresεion of Chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023. It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragmentε are (I) the Fab fragment conεiεting of VL, VH, CL and CHI domainε; (ii) the Fd fragment consisting of the VH and CHI domainε ; (iii) the Fv fragment consisting of the Vl and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al . , Nature 341, 544-546 (1989) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragmentε, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allowε the two domainε to aεsociate to form an antigen binding site (Bird et al , Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988) ; (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies" , multivalent or multispecific fragments constructed by gene fusion (WO94/13804 ; P Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) .
Diabodies are multimerε of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the asεociation of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804) .
As an alternative or supplement to immunising a mammal, antibodieε with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
Antibodieε raiεed to a polypeptide or peptide can be used in the identification and/or isolation of variant polypeptides, and then their encoding genes. Thus, the present invention provides a method of identifying or isolating a galactosyltransferase or variant thereof (as diεcuεsed above) , comprising screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind said galactosyltransferase polypeptide or variant thereof, or preferably has binding specificity for such a polypeptide. Specific binding members such as antibodieε and polypeptides comprising antigen binding domains of antibodies that bind and are preferably specific for a galactosyltransferase polypeptide or mutant or derivative thereof represent further aspects of the present invention, as do their use and methods which employ them.
Candidate polypeptideε for εcreening may for instance be the products of an expresεion library created uεing nucleic acid derived from an plant of interest, or may be the product of a purification proceεε from a natural εource . A polypeptide found to bind the antibody may be iεolated and then may be subject to amino acid sequencing. Any suitable technique may be used to sequence the polypeptide either wholly or partially (for inεtance a fragment of the polypeptide may be sequenced) . Amino acid sequence information may be used in obtaining nucleic acid encoding the polypeptide, for instance by designing one or more oligonucleotides (e.g. a degenerate pool of oligonucleotides) for use as probes or primers in hybridization to candidate nucleic acid.
In addition to the aspects above, the invention further provides use of the materials described herein for altering the quality and/or quantity of CWSP in a host cell. Particularly for altering the mannose : galactose ratio in a mannose/galactose containing compound in that host cell.
For inεtance it provideε a method of influencing or affecting the CWSP content of a host cell (preferably a plant cell) , comprising the step of causing or allowing expression of a heterologous nucleic acid εequence encoding a bioεynthetic enzyme as discussed above within the cell.
In addition to the aspects above, the invention further provides a method of influencing or affecting the glycosyltransferase activity in a plant, the method comprising the step of causing or allowing expression of a heterologous nucleic acid sequence aε discussed above (e.g. encoding the fenugreek or guar galactosyltransferase or a variant thereof) within the cells of the plant.
In each case the step may be preceded by the earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.
The foregoing diεcuεsion has been generally concerned with uses of the nucleic acids of the present invention for production of functional polypeptides, for instance for the purpose of increasing the galactosyltransferase activity in the cell.
However the information discloεed herein may alεo be uεed to reduce the activity of galactoεyltransferases in cells in which it is desired to do so.
For inεtance down-regulation of expreεsion of a target gene may be achieved using anti-sense technology.
In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al , (1988) Nature 334, 724-726; Zhang et al , (1992) The Plant Cell 4, 1575-1588, Engliεh et al . , (1996) The Plant Cell 8, 179-188.
Antiεense technology is also reviewed in Bourque, (1995) , Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
Thuε a nucleotide εequence which iε complementary to any of the coding εequenceε diεcussed above (including variants) forms one part of the present invention.
"Complementary to" meanε capable of baεe pairing with, whereby A iε the complement of T (and U) ; G is the complement of C.
An alternative to anti-sense is to use a copy of all or part of the gene (galactosyltransferase or variant) inserted in sense, that iε the same, orientation aε the natural gene, to achieve reduction in expreεεion of the target gene by co-εuppreεsion. See, for example, van der Krol et al . , (1990) The Plant Cell 2, 291-299; Napoli et al . ,
(1990) The Plant Cell 2, 279-289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588, and US-A-5 , 231 , 020. Further refinements of the gene silencing or co-suppreεsion technology may be found in W095/34668 (Biosource) ; Angell & Baulcombe (1997) The EMBO Journal 16,12:3675- 3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553.
Further options for down regulation of gene expression include the use of ribozymes, e.g. hammerhead ribozymes, which can catalyse the site- εpecific cleavage of RNA, εuch aε mRNA (εee e.g. Jaeger (1997) "The new world of ribozymeε" Curr Opin Struct Biol 7:324-335, or Gibson &
Shillitoe (1997) "Ribozymes : their functions and strategies for their use" Mol Biotechnol 7: 242-251.)
The complete εequence corresponding to the coding sequence (in reverse orientation for anti-sense) need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding εequence to optimise the level of anti- sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence.
The εequence employed may be about 500 nucleotideε or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be posεible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where posεible, εuch aε longer than about 600 nucleotideε, than about 700 nucleotideε, than about 800 nucleotideε, than about 1000 nucleotideε or more .
It' may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, although total complementarity or similarity of sequence is not essential . One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expresεion in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.
The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-εenεe and sense RNA molecules to hybridise. There may be down regulation of gene expresεion even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expresεion to take place.
Thuε the preεent invention further provideε the use of Seq ID No 1 or 3, or the complement thereof, or a variant of any of these, for down- regulation of gene expresεion, particularly down-regulation of expreεsion of a galactoεyltranεferase gene or variant thereof, preferably in order to influence the galactoεyltransferaεe activity in a host cell, more preferably a plant cell, most preferably a plant.
The invention further provides use of an antibody to achieve the same.
Anti -sense or sense regulation may itself be regulated by employing an inducible promoter in an appropriate construct. A yet further method of manipulating galactosyltransferase activity is to express an antibody to the enzyme in the plant. It has been demonstrated that functional antibodies and antibody fragments can be expressed intracellularly and can be targeted to sub-cellular compartments . Alteration of phenotype by this method has been demonεtrated, for inεtance by Artsaenko et al (1995) Plant J 8: 745-750 and Owen et al (1992) Bio/Technology 10: 790-794.
In a further aspect of the present invention there is disclosed a plant product derived from any of the transformed plants or plant cells, or produced by any of the methods, discussed above in relation to other aspects of the invention (e.g. in which galactosyltransferase activity has been altered) .
Preferably the plant product comprises an altered galactomannan, which is to say that the galactomannan contains an altered (preferably reduced) ratio of galactose to mannose and/or an altered backbone galactose distribution.
In a further aspect of the present invention there is provided a commodity comprising the plant product described above (e.g. up to 5%, preferably 0.1 - 3%), particularly a human or animal foodstuff, or a cosmetic .
Particularly envisaged in terms of human foodstuffs is a frozen food product, for instance an ice cream or water ice. Also of interest are salad dresεings, sauces, gelled desserts and "reduced-fat" products.
Animal foodstuffs may include gel-based petfoods.
The food composition comprising altered galactomannan plus one other polysaccharide εelected from: xanthan; carrageenan; agarose .
Galactomannanε having altered hydrophilic and cryogelation properties may have particular application to industry aε additiveε e.g. as stabiliserε, emulεifiers, and in combination with other polysaccharides, to impart more complex rheologies .
The variouε aspects of the invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments falling within the scope of the present invention will occur to those skilled in the art in the light of these. FIGURES
Figure 1: shows an alignment of the fenugreek galactosyltransferase with the putative guar galactosyltransferase sequence. Galtran2.pro = fenugreek galactosyltransferase; Guargalt.PRO = guar sequence. Residues matching the fenugreek sequence exactly are boxed. Numbering correεponds to the guar sequence. Predicted membrane-spanning a-helix is underlined.
Figure 2: typical data-sets correlating galactosyltransferase activity and a 51K protein. Triton X-100 solubilised extracts were separated on IEF gels containing the same detergent. Strips from each gel were treated to localise galactosyl-transferaεe activity and separated protein, and to plot the pH gradient.
A and B. Alignment of galactosyltransferaεe profile activity with εecond-dimenεion SDS-PAGE. Activity correlateε closely with a 51K protein [50K position arrowed] .
C. SDS-PAGE separation [welled gel] of slices from a further strip of the same IEF gel [50K position arrowed] . The two peak activity slices [εee A], indicated with aεterisks, are enriched in a 51K protein.
D and E. Second dimension SDS-PAGE and Western blot of an identical gel challenged with an antiserum raised against pea vicilin. The position of the 51K protein iε arrowed in each.
Figure 3: cDNA and deduced protein sequence of c.500 bp clone obtained by 3 'RACE. The sequences of the degenerate gene-specific primer and an antisense primer [GTPA3] are double underlined. Known sequences from the 51 K protein are underlined and italicised.
Figure 4: cDNA and deduced protein sequence from c.1000 bp clone. The upper sequence iε from the 5' end of the clone, the lower from the 3' end. The sequences of the 5' and 3' degenerate primers used to amplify the cDNA are double underlined. The known protein sequence from the 51 K protein iε underlined and italiciεed.
Figure 5: cDNA and deduced protein εequence from c.1500 bp clone. The primerε used to amplify the cDNA are double underlined. The known protein sequence from the 51 K protein is underlined. The orf beginning at bpl and ending at bp 1314 encodes a 438 aa protein.
Figure 6: Secondary structure prediction using neural network based program. 6A showε predicted helix (H) ; extended sheet (E) ; other loop (blank) . 6B showε predicted transmembrane helix (T) .
Figure 7: Digital autoradiogram of endo- β-D-mannanase digests of labelled polymeric products formed during galactosyltransferase assays of lOx concentrated supernatantε from three different Pichia colonies carrying truncated constructs . A trace amount of the galactomannan active α-galactosidase from guar seeds was added to the digest in the centre lane. Gal = Galactose. Abbreviations for diagnostic galactomanno-oligosacc arides (Reid et al, 1995) : M2G = galactoεylmannobiose; M3G = galactosylmannotriose; M5G2 = digalactoεylmannopentaose; 0 = galactomanno-octasaccharides; N= galactomanno-nonaεaccharides .
SEQUENCE ANNEXES
Annexe la fenugreek cDNA εequence - Seq ID No 1
Annex lb: tranεlation of the fenugreek cDNA sequence - Seq ID No 2
Annexe 2a: guar cDNA sequence - Seq ID No 3
Annex 2b: translation of the guar cDNA sequence - Seq ID No 4
EXAMPLES
Example 1- Identification of a polypeptide and acquisition of amino acid sequence
Isolation of membranes capable of catalysing galactomannan biosynthesis in vi tro from developing fenugreek seeds .
Fenugreek plants were grown to flowering and fruiting under conditions which have been described elsewhere (Edwards et al . 1992) . Membranes were prepared using a method similar to that described previously (Edwards et al . 1989). Endosperms were hand-isolated at a stage of seed development during which intensive galactomannan biosynthesiε waε taking place [35-40 days after anthesis under our growth conditions] , and homogenised in a glaεε Potter homogeniεer with 50 mM Triε HCl buffer pH 7.5 containing 1 mM EDTA and 5 mM dithiothreitol [DTT] . Uεually the ratio of buffer to plant tiεεue was 0.5 ml buffer per endosperm. Larger particles were removed by an initial centrifugation at 13000 g [10 min] , and the supernatant was spun at 100000 g for 1 hour. The pelleted membranes were resuspended in the same buffer [usually 100 μl per endosperm] .
Standard assays for mannan synthase and galactosyl transferase in isolated membranes
These have been described (Edwards et al, 1989, 1992, 1995). Briefly, the incubation mixture [total volume 100 μl; incubated at 30°C, usually for 1 h] comprised membranes [usually equivalent to 0.1 - 1 endosperm] , DTT [2.5 mM] , EDTA [0.5 mM] , MgCl2 [2.5 mM] , MnC12 [5 mM] , UDP-Gal [800 μM] and/or GDP-Man [80 μM] in 25 mM Tris HCl buffer, pH 7.5. The GDP-Man and/or the UDP-Gal substrate was labelled with the appropriate nucleoside diphospho- [U-14C] -sugarε . Specific radioactivitieε were adjusted to 25-250 Bq.nmol"1 and were checked by scintillation counting in each experiment. At the end of the incubation time glacial acetic acid [50 μl] was added and the mixture heated at 100°C for 2 min. Carrier galactomannan [100 μl of a 0.2 % w/v solution of locust bean galactomannan] was then added, followed by methanol to a final concentration of 70 % v/v. The mixture was heated [70°C for 10 min] and centrifuged [13000 g, 10 min] . the supernatant was discarded, and the pellet washed twice with hot 70% methanol as has been described (Edwards et al . 1989).
Mannan synthase could be assayed as above using labelled GDP-Man and unlabelled UDP-Gal. Under these conditions the product was a galactomannan, labelled in the mannosyl residues. It could be assayed also in the absence of unlabelled UDP-Gal, when the product was labelled (1-4) -β-mannan.
Galactoεyltransferase waε aεεayed uεing labelled UDP-Gal and unlabelled GDP-Man. It could not be assayed in the absence of GDP-Man, since the galactosyl residues were tranεferred only to newly transferred mannose residues (Edwards et al . 1989, 1992, 1995)
Detergent treatment of the membranes
Membranes were iεolated aε above, and reεuspended [homogeniser] in 100 mM Tris HCl buffer pH 7.5 [12.5 (μl per endosperm] containing EDTA [2 M] and DTT [10 mM] . Samples of the resuspended membranes were mixed with an equal volume of 2% [w/v] detergent, placed on ice and homogenised briefly every 10 min for 30 min. Suspensions were then centrifuged at 100000 g for 1 hour. Supematants were retained, and pellets were resuspended in a 1:1 mixture of resuspension buffer and 2% detergent, with a volume equal to that of the supernatant. Standard assays for mannan synthase and galactosyltranεferase were carried out on supernatantε and resuspended pellets.
Of the detergents used [digitonin, CHAPS, octyl glucoside, Triton X-100, NP-40], only digitonin gave appreciable mannan synthaεe activity in the 100000 g supernatant.
In a typical digitonin experiment, the activity surviving digitonin treatment was 12.4%, of which 35% was in the supernatant and 65% in the pellet. None of the detergents gave any galactosyltransferase activity, either in pellets or supematants .
Demonstration of galactomannan galactosyl transferase in digi tonin extracts using manno-oligosaccharide acceptors .
Our observation that mannan εynthase activity was retained in digitonin extracts without associated galactosyltranεferaεe activity indicated either that the galactoεyltranεferaεe activity had been denatured preferentially or that the functional association between the mannan synthase and the galactosyltransferase had been disrupted by detergent treatment to the extent that the nascent mannan backbone was no longer available to the galactosyltransferase as acceptor substrate. If the latter were true it was possible that replacement acceptor substrates could be added to the extracts to mimic the mannan backbone. Initially digitonin extracts exhibiting soluble mannan synthase activity were incubated as for the standard assay, with added mannohexaose [1 mM] , no GDP-Man, and labelled UDP-Gal [800 μM] . At the end of the incubation period the mixture was diluted by the addition of water [100 μl] and then spun through εmall columnε (approximately 200 μl) of DEAE-celluloεe [Whatman DE52] anion-exchanger which had been equilibrated with buffer identical to that used in the incubation. Thiε procedure removed almoεt all of the unused labelled UDP-Gal substrate, which is negatively charged and binds to the cationic DEAE cellulose. After freeze-drying, the column eluate was dissolved in water (50 μl) and 20μl sampleε were εpotted onto silicagel TLC plates (Merck 5553) . The plates were developed three times in a solvent composed of n-propanol, nitromethane and water (5:2:3 by vol), dried and analysed by digital autoradiography . The appearance of a radioactive εpot running εlightly slower than mannohexaose indicated that labelled galactose had been transferred from UDP-Gal to the mannohexaose. A pure sample of the labelled compound was obtained by carrying out a larger scale incubation and column purification as above, and strip-loading TLC plates with the column eluate. After developing the plates, the labelled product was located by digital autoradiography and then purified by removing the appropriate area of silica gel from the plates and eluting the silica gel with water. A pure α-galactosidaεe from guar seeds catalysed the complete conversion of the purified labelled compound to labelled galactose, and when the reaction was carried out in a graded fashion there were no labelled intermediateε produced. Thuε the labelled product carried a single α-linked galactose residue. Further analysis of the labelled product with a pure structure-sensitive endo-mannanase and a commercially available exo-β-mannoεidaεe from snail [Sigma M9400] confirmed that the galactose reεidue had been tranεferred α- (1-6) to mannohexaoεe . The effectiveness of manno-oligoεaccharides of different chain-length as galactosyl acceptors waε compared [M5<<M6<M7<M8~M9, M10] and the nature of the productε formed in each case was investigated using the three enzymes mentioned above, TLC and digital autoradiography. Results were conεistent with a model for acceptor substrate binding, according to which the α-galactoεyltranεferase has an acceptor εubεtrate binding requirement comprising six principal binding sites for mannosyl residues of the acceptor substrate. For transfer to occur, at least five of the sites must be occupied, and transfer occurs to the mannose residue at the third binding site [measured from the non-reducing end] .
Thus manno-oligosaccharide acceptors allowed the assay of the galactomannan galactosyltransferase after digitonin solubilisation. Standard procedure was to incubate the detergent extract with mannohexaoεe [1 mM] , MnC12 [usually 10 mM] and labelled UDP-Gal [800 μM] , dilute, spin through DEAE cellulose columns, freeze-dry the eluent and dissolve in water [50 μl] as above. Scintillation counting of an aliquot of the resulting solution gave a measure of the total radioactivity eluted from the column. The proportion of thiε activity preεent in the galactosylmanno-hexaose product of the galactosyltranεferase reaction was estimated by TLC and quantitative digital autoradiography of a further aliquot. Development of a new method to associate enzyme activi ty wi th particular proteins in the digi tonin extract .
The small amounts of tisεue available from the hand-dissected endosperm tissues, and the presence of the detergent in the extracts, made conventional procedures for protein purification or enrichment impracticable. A method to asεociate mannan synthase and galactosyltransferaεe activitieε with diεcrete polypeptideε separated on SDS-PAGE gelε was therefore devised. The method, with its extenεion and refinements as described below, was used successfully to pinpoint the galactomannan galactosyltransferase protein. Its effectivenesε iε strongly dependent upon its ability to give an exact correlation between enzyme activity profiles from IEF carried out in a first dimenεion, and SDS-PAGE carried out in the εecond dimension.
Generally, the method involved isoelectric focussing [IEF] of enzymatically active detergent extracts on vertical agarose minigels prepared in the presence of the solubilismg detergent. It was found that detergent-εolubiliεed proteinε, preεent preεumably in micelleε, moved into the gel and were focuεεed according to their apparent pi values. Moreover, mannan synthase and galactosyltranεferase activities in digitonin extracts were retained after focussing. After focussing, gels were cut into 1cm wide strips parallel to the direction of current flow. To determine the shape of the pH gradient, one εuch εtrip could be cut into slices perpendicular to the direction of current flow, each slice eluted with IM KCl and the pH values of the resulting solutions measured. The pH gradient [establishment, shape, stability] was monitored also during focuεεing by loading the IEF gels with εmall εamples of coloured "marker" proteins flanking the sample of detergent-solubilised enzyme. To measure galactosyltransferase activity on the IEF gels, further strips were cut into sliceε [uεually 2mm] and each slice was aεsayed for activity.
In this way activity could be localised within the IEF gels. To determine which proteins were focussed at particular pointε within the gel, two related experimental approacheε were used. In the first an IEF gel strip adjacent to the one sliced for activity determination was sliced in exactly the εame way and each εlice waε treated with SDS-PAGE sample buffer and placed in an individual εample well of an SDS gel. Staining of the gel after SDS-PAGE then allowed a visual correlation of enzyme activity with polypeptide diεtribution. The second approach was to place an IEF strip adjacent to the one sliced for enzyme activity determination along a long εample well of an SDS gel and εubjecting it to SDS-PAGE in a direction perpendicular to that of IEF. Thiε 2 -dimensional IEF/SDS-PAGE approach gave an excellent visual correlation between proteinε on the stained SDS-PAGE gel and enzyme activity.
In more detail; isoelectric focusεing gelε [8 x 10 cm] were prepared by aεεembling a "εandwich" of a glass plate to which pre-marked GelBond agarose gel support medium 0.1 mm thick [FMC BioProducts] had been attached [Hoefer Technical bulletin No 134] , 1 mm spacers, and a notched alumina plate [Hoefer] in a Hoefer Gel Caster SE 245. GelBond was used to ensure that gel dimensionε did not change during any manipulations and staining procedures. To aid subsequent division of gelε, the reverεe [hydrophobic, adjacent to the glaεε] εide of the Gelbond film waε pre-marked uεing a fine marker-pen with guide lines to facilitate accurate cutting into strips and slices. The agarose separation gel was prepared by mixing IεoGel agaroεe [120 mg] , εorbitol [2.4 g] and water [10.36 ml] and heating on a boiling water bath for 10 min with frequent mixing to diεεolve the agarose. After cooling to 65°C, the volume was made up to the original value. For digitonin gelε 600 μl of 2% [w/v] digitonin [Sigma D1407] waε added before boiling to give a final concentration of 0.1% in the gels. For Triton X-100 gels (see below) , 600 μl of a 2% [w/v] solution of the detergent [Boehringer 789 704] was added after cooling to 65°C, due to the low [65°C] cloud point of this detergent, again giving a final detergent concentration in the gel of 0.1%. The in-gel detergent concentrations were above the critical micelle concentrationε [CMC] of the detergentε [0.09% for digitonin and 0.02% for Triton X-100] and were used to maintain protein solubility during IEF. Finally [at 65°C] 600 μl of ampholytes [a 4:1 (vol:vol) mixture of pH 5.0 - 8.0 Ampholine, Sigma A5799 and pH 3.5 -
10.0 Ampholine, Sigma A5174] were added to the agarose mixture to give a concentration of 2% in the gel. The gel "εandwich" waε pre-warmed in an oven, the gel mixture [at 65°C] was added using a syringe, and a reference well comb [Hoefer] was inserted. This comb gives a 6.7 cm wide sample well, with a small 0.5 cm wide reference well alongside. The gel was left to set for l hour before it was assembled into a Hoefer SE 250 vertical gel apparatus which was cooled by water circulation to approximately 4°C. Cooling was also carried out during IEF to ensure adequate dissipation of heat generated and minimise loss of enzyme activity. The sample and reference wells were cleaned and dried using strips of filter paper, and the sample, overlay and IEF standards applied. The sample consiεted of 750 μl of detergent extract [detergent concentration 1%] prepared as deεcribed above, mixed with 45 μl of the εame ampholyte mixture aε was used to prepare the separating gel, 65 μl glycerol and 5 μl bromophenol blue [0.05 % w/v in water] . It waε pipetted into the εample well of the gel. An overlay was prepared from 40 μl of the ampholyte mixture, 40 μl of 2% detergent, 40 μl glycerol 5 μl of the bromophenol blue solution and 680 μl water. A portion of this was pipetted into the reference well, and the remainder was layered carefully over the εample. The overlay iε leεε denεe than the sample, but more dense than the cathode buffer, thus forming a barrier to direct mixing of the sample and the strongly alkaline cathode buffer. Coloured IEF standards [Bio-Rad] (2.5 μl) were pipetted directly into the reference well. Finally, cathode buffer (20 mM NaOH) was carefully poured into the back (upper) chamber of the apparatus so that it did not mix with the overlay and sample, and anode buffer (6 mM phosphoric acid) poured into the lower chamber. The IEF was run at 200 V for 30 minuteε during which moεt of the sample could be seen to enter the gel, and then at 600 V for 60 minutes. During this time the coloured IEF standardε could be seen to migrate, focus and stabilise in position, and the current taken fell from about 12 mA to a stable final value of around 2 mA. After running, the gel sandwich was removed from the apparatus and the gel, attached to GelBond, separated from the plates and spacers. It was then cut up into εtripε parallel to the direction of current flow. The two extreme end εtripε were cut to include side-strips from the sample area. Thus one of them also included the reference standards. These two sectionε were fixed in 10% trichloroacetic acid [TCA] / 40% methanol for 15 min. During thiε time, two non-coloured standards at pi 6.0 and pi 6.5 became visible aε opaque bandε . Thiε allowed them to be used in some experiments aε markerε for the peak of activity of galactosyltransferase [pi 6.0 in digitonin and pi 6.5 in Triton X-100] . The two strips were then dehydrated in methanol for 15 min, dried between sheetε of filter paper and εtained with Coomassie Blue. The εtained εtripε showed the complete range of IEF standards. They also revealed the positions of stained bandε in the εample, and εhowed whether or not the εample had focussed in bands running perfectly horizontally across the gel. Further strips were proceεsed to obtain enzyme activity, protein distribution and pH gradient as indicated above.
In the digitonin- solubilised enzyme preparations, mannan synthase and galactosyltransferase activity peaks overlapped. The mannan synthase gave a broad peak at about pi 6.0, tailing towards the origin of the gel where a large proportion of the activity remained, apparently unable to enter the gel. By contrast, all of the galactosyltransferase activity entered the gel, and gave a more symmetrical peak [pi 6.0] overlapping with that of the mannan synthase . There was a good correlation between the galactosyltransferase activity and a protein band with an apparent molecular weight [Mr] of about 5OK. The corresponding [about 50K] band was identifiable on one-dimensional SDS gels of digitonin-solubiliεed enzyme. Such gelε were therefore electroblotted blotted onto "Problott" [Applied Bioεystems] membrane, and the excised blotted band was subjected to N-terminal sequencing. Repeated attempts gave no sequence, indicating that the protein was blocked to sequencing at the N-terminus. To obtain internal εequence data, the band waε exciεed from one-dimensional SDS gels and subjected to' digestion with endo-proteinase GluC followed by separation of product peptides on SDS gels [Cleveland et al (1977) J Biol Chem 252: 1102-1106] . The peptides were electroblotted and εubjected to
N-terminal sequencing to give internal sequence data from the 50K protein. When the sequence information obtained was compared with international database information, there was extremely high homology between the obtained sequences and those of membrane bound provicilin storage protein precursors. This indicated either that the about 50K band identified on 2-D gels was not the galactosyltranεferaεe, or that the corresponding band excised from the one-dimensional SDS gelε contained more than one protein, the vicilin-related protein predominating .
Extension of the above method for use wi th other detergents , and refinements giving more rapid galactosyl transferase localisation in IEF gels wi th higher precision
Following our obεervation that galactosyltransferaεe activity was retained in digitonin extracts, and could be asεayed uεing mannohexaoεe as described above, other detergents which, unlike digitonin, had abolished mannan synthase activity almost entirely were investigated. All thoεe tested [Triton X-100, NP-40, CHAPS, octyl glucoside] gave some retention of activity, but Triton X-100 and NP-40 gave very high retentions, approximately double that observed for digitonin. The properties, including transfer-specificity of the Triton-solubiliεed enzyme were compared with and found identical with those of the digitonin-solubilised enzyme. This allowed the IEF / SDS-PAGE separation described above to be carried out using Triton X-100 in place of digitonin. Thiε gave greatly improved activity resolution and protein separation. Also following our observation that manno-oligosaccharides would serve aε acceptors for detergent-solubiliεed galactosyltransferase, polymeric galactomannanε with low, medium and high galactose-substitution were tested as acceptors [locust bean, guar and fenugreek galactomannans respectively] . Locust bean galactomannan was an efficient acceptor, guar galactomannan was less efficient and fenugreek galactomannan was not an acceptor. When the labelled products of transfer of galactose residues to guar and locust bean gums were subjected to hydrolysis using the structure-sensitive endo-β-mannanase, the distribution of label m the fragment oligosaccharideε waε conεistent with transfer to relatively unsubstituted regions of the mannan backbone.
It was found that the commercial agarose preparation ["IsoGel" - FMC Bio-products] sold for isoelectric focussing is an agarose-galactomannan blend On enzymatic digesting a sample of the blend with the structure-sensitive endo-mannanase the "fingerprint" of galactomannan-deπved oligosacchaπdes observed on TLC was consistent with a low-galactose galactomannan, probably locust bean gum. The presence of a low-galactose galactomannan the IEF agarose gel offered the possibility of its use as an m si tu acceptor for gel-separated galactosyltransferase, and the design of a new rapid, sensitive, highly resolving procedure for localising the enzyme activity. To localise galactosyltransferase activity in an IEF gel strip, the entire strip could be incubated in the presence of labelled UDP-Gal, whereby galactosyltransferase focussed withm the strip would catalyse the transfer of labelled galactose residues to the galactomannan component of the separating gel. After thorough washing of the gel, any radioactivity remaining withm it was a measure of and a localisation of galactosyltransferaεe activity.
In practice a complete gel strip [on GelBond] cut parallel to the direction of current flow was pre-mcubated m strong buffer [200 mM Tπs-HCl pH 7.5] for 10 mm in order to bring it to the correct pH for galactosyltransferase assay. The whole strip was then incubated in a mixture containing 50 mM Tris-HCl pH 7.5 , 10 mM MnC12, 0.2% [w/v] Triton X-100 and 800 μM 14C-labelled UDP-Gal for 3 hourε at 30°C. The strip was then fixed in 40% [v/v] methanol / 10% [v/v] acetic acid for 20 minutes and washed overnight in 40%methanol. This procedure removed virtually all unincorporated label, and retained the labelled galactomannan product with the gel. The following day the gel strip waε cut into 2 mm strips perpendicular to the direction of current flow. Each strip was removed from the GelBond, transferred to a microcentrifuge tube, washed once with hot [60°C, 10 min] and twice with cold [room temp, 20 min] 40% methanol, dissolved in 20 μl concentrated HCl, and subjected to liquid scintillation counting.
In the case of Triton X-100 gels analysed using the procedure described above for galactosyltransferase localisation, the galactosyltransferase focussed at about pi 6.5. Correlation of activity with protein bands on SDS gels as above gave excellent register of activity with protein at about Mr 50K. However in contrast to the digitonin gels, the protein at about 50K was resolved into two main components. The correlation between the galactosyltransferase activity and one of these two components [Mr 51K] was very close. Western immunoblotting showed that this protein did not cross react with an anti-vicilin poyclonal antiseru . The second major band at about 50K [Mr 49K] cross-reacted strongly with the antiserum, indicating that it was the provicilin storage protein precursor mentioned above [Fig. 2] .
To purify a small quantity of the 51K protein and obtain protein sequence, the material focussing at pi 6.2 to 6.8 was excised from an entire Triton X-100 IEF gel and the gel sections were applied as the sample to an SDS-PAGE gel. After running, the gel was blotted onto Problott membrane, and the blot waε εtained lightly with Coomassie blue. The 51K and 49K bands were adequately separated, and the 51K band was exciεed carefully from the blot and εubjected to N-terminal sequencing. Sequence was obtained. To obtain internal εequence information from the 51K protein IEF and SDS-PAGE waε carried out as above. The gels were stained lightly, and the 51K band was excised and subjected to digeεtion in gel (Cleveland et al . 1977) with endoproteinase GluC. The product polypeptides were separated by SDS-PAGE, blotted and subjected to N-terminal εequencing. Only one of the polypeptides waε present in sufficient quantity to give a sequence [Internal 1, Table 1] . Further sequence data waε obtained by in-gel digeεtion with endoproteinase LysC, separation of the resultant peptides by HPLC and direct εequencing [Internal 2 and 3, Table 1] . All protein sequence data were compared with international protein databases, and there were no significant sequence homologies .
Table 1. N-terminal and internal εequence information from 51K possible galactomannan galactosyltranεferase .
Identification Sequencing data [see text]
N-terminal ATKFGSKN-S-PWL Internal 1 GY- EISKMYDKMGE-YD Internal 2 FGFIHPNLLDK Internal 3 SVSPLPFGYPAASP
Example 2. Acquisition of a cDNA sequence encoding the 51K probable galactosyltransferase protein.
Degenerate primers were designed to the amino acid sequence information acquired from the 51K probable galactosyltransferase protein:
These were:
GY-LEISKMYDKMGE-YD
5' AAGATGTATGACAAGATGGG 3' (sense primer GT3S4) A C T A
5' CCCATCTTGTCATACATCTT 3' (antisense primer GT3A4) A G T
ATKFGSKN-S-PWL
5' GCIACIAAATTTGGIA 3' (sense primer NTP2S) G C T
RNA was prepared from endospermε hand-iεolated from developing fenugreek εeedε during the early εtages of galactomannan deposition [32-35 days after anthesis (Edwards et al . 1992)]. When 3'RACE PCR [Frohman M A, Martin G R (1989) Rapid amplification of cDNA ends using nested primers . Techniques 1: 165-170] was carried out using a degenerate primer [GT3S4] designed to an internal protein sequence, a c500 bp cDNA waε amplified. When cloned and sequenced [Fig. 3] it was found to encode further amino acid εequence from the 51K protein, adjoining that uεed to design the degenerate primer, and all the other internal sequence information shown in Table 1.1. In a procedure incorporating elements of 5'RACE [Frohman and Martin, 1989], and PCR amplification using degenerate primers, a clOOO bp cDNA was amplified. When cloned and partially sequenced from both ends it encoded at the 5' end all the N-terminal amino acid sequence in Table 1, it overlapped at the 3' end with the c500 bp clone to the extent of the primer, and it encoded further amino acid sequence from "internal 1" [Table 1] . The partial terminal sequences of the clOOO bp clone are shown in Fig. 4. To obtain a single cDNA encompasεing the whole εequence, perfect primerε were designed to the extreme 5' end of the clOOO bp clone and to the 3' untranslated region of the c500 bp clone. RT-PCR, carried out using a proof-reading thermostable DNA polymerase [Pfu - Stratagene] , resulted in the amplification of a cl500 bp cDNA which was cloned and fully sequenced. The complete sequence, shown in Fig. 5, had an orf encoding a 438 amino acid polypeptide. The deduced molecular weight was 51281 Daltons, and the deduced pi was 6.646, in close agreement with the values observed for the Triton X-100 εolubilised 51K protein.
In more detail :
Prepara tion of RNA from developing fenugreek endosperms . Seeds from pods harvested 32-35 days after anthesis were hand-dissected under aseptic conditions, and the endosperm tissue was dropped directly into liquid nitrogen. Endosperms from 100 εeedε [weight approx 1 g] were then ground in a mortar and peεtle with liquid nitrogen, and RNA waε prepared eεsentially according to the procedure of Lopez -Gomez R and Gomez-Lim M A (1992) A method for extracting intact RNA from frui ts rich in polysaccharides using ripe mango . HortScience 27: 440-442. This method, which involves an extraction buffer containing 20% ethanol, circumvented problems associated with the dissolution of galactomannan in extraction buffers. RNA yields were typically about 50 μg.
Design of degenerate primers to amino acid sequence from the 51K protein . A degenerate primer was deεigned to the extreme N-terminal part of the 'N-terminal [Table 1.1]' amino acid sequence and designated NTP2S. A further degenerate primer pair [sense and antisense, designated GT3S4 and GT3A4 was designed to part of the 'internal 1 [Table 1.1]' sequence (see above).
Use of 3 'RACE PCR to obtain a c500 bp clone . 3' Rapid amplification of cDNA ends [3' RACE] was carried out essentially according to Frohman and Martin (1989) . First strand cDNA synthesis from fenugreek endosperm RNA was primed using the (dTl^-R^R,. primer described by Frohman and Martin (1989) , and PCR was carried out using the degenerate primer GT3S4 and a T7 RNA polymerase promoter primer [5' TAATACGACTCACTATAGGG 3'] recognising part of the R.-R0 sequence The PCR reaction mixture comprised 5 μl first strand cDNA, 25 pmol T7 primer, 25 pmol GT3S4 primer, 0 01 μmol of each dNTP, 2 5 U Taq polymerase [Pharmacia] and lOx Taq polymerase buffer [5 μl, Pharmacia] in a total volume of 50 μl To obtain a "hot start" the template, m 30 μl, was heated to 95°C for 7 mm, and then held at 75°C whilst the remaining components were added The complete mixture was heated at 50°C for 2 mm, followed by 72°C for 5 mm, εubjected to 30 cycles of 94°C [1 mm]- 50°C [1 mm] - 72°C [1 5 mm] , and then held at 72°C for 15 mm Agarose gel electrophoresis of the PCR mixture gave a weak signal at c500bp Reamplification by PCR using the same primers and conditions gave a very εtrong εignal on gelε at c500 bp The remainder of the PCR reamplification mixture was purified [Hybaid Recovery DNA purification kit] and cloned into the commercial [Invitrogen] plasmid pCR 2 1 using the 3' A overhangs resulting from the action of Taq DNA polymerase The cDNA fragment was subcloned and sequenced The encoded ammo acid sequence mcluded further known ammo acid sequence from the "internal 1" peptide uεed to design the degenerate primer, and sequences corresponding to the "internal 2" and "internal 3" sequenceε obtained directly from the 51K protein [Fig 3]
Use of a modified 5 'RACE PCR protocol to obtain a cl OOObp clone Initially 5' RACE waε carried out essentially according to Frohman and Martin (1989) First strand cDNA synthesiε from fenugreek RNA waε primed using random hexamers , and polyA tailed at the 3' end using terminal transferase Second strand syntheεiε waε primed using the (dT) 17-R1-Rα primer described by Frohman and Martin (1989) and PCR amplification was carried out using the T7 promoter primer described above and a perfect primer [5' CATTTCACCATAACGTTCACTCAC 3 'designated GTPA3] designed to part of the sequence of the c500bp clone [Fig 3] The procedure of Frohman and Martin was modified by carrying out the second strand syntheεis and PCR amplification in separate stages In the first stage, "hot-started" as above, poly A tailed first strand cDNA [5 μl] , (dT) 17-R1-RD primer [2 5 pmol], dNTP's [0 01 μmol each], with Taq polymerase [2 5 U] and Taq polymerase buffer [Pharmacia] were heated at 45°C for 2 mm and then 72°C for 10 mm In the second stage, T7 primer and primer GTPA3 were added to the above mixture whilst it was held at 72°C The combined mixture was then subjected to 30 cycles of 94°C [1 mm] -50°C [1 mm] - 72°C [1.5 mm], and then held at 72°C for 15 mm This procedure resulted in the amplification of DNA covering a wide range of molecular sizeε, which waε purified free of primerε and low molecular weight productε [Hybaid Recovery] , and PCR amplified using the degenerate N-terminal primer NTP2S and the degenerate internal antisense primer GT3A4 [see above] . The PCR protocol [with "hot start" as above] comprised 30 cycles of 94°C [1 min] - 37°C [1 min] 72°C [2 min] , with a final period at 72°C [15 min] . This resulted in the amplification of a clOOO bp cDNA which was exciεed from gelε, purified [Hybaid Recovery] and cloned, εubcloned and sequenced from both ends. The sequence encoded at the 5' end the full "N-terminal" [Table 1.1] sequence from the 51K protein and, at the 3' end, the part of the "internal 1" [Table 1.1] sequence used to design the primer plus all the other amino acids towards the N-terminus of the "internal 1" peptide [Fig. 4] .
RT-PCR amplification of a single cDNA encoding the full protein sequence. Perfect primers were designed to the 5' terminus of the clOOO bp clone [5' GCGACGAAATTTGGTTCCAA 3', designated GTP5S] and to part of the 3' untranslated region of the c500 bp clone [5'
GCTAATATCATCACCACCTTC 3', designated GTP6A] , [Fig. 5] and RT-PCR was carried out on fenugreek endosperm RNA, using the proofreading Pfu [Stratagene] DNA polymerase. First strand synthesis was primed uεing the primer. The PCR mixture , ["hot-started" aε above] comprised first strand cDNA template, GTP5S and GTP6A primerε [25 pmol each], dNTP's [0.01 μmol each], Pfu DNA polymerase [2.5 U; Stratagene] and lOx Pfu buffer [5 μl; Stratagene] in a total volume of 50 μl . The mixture was held at 50°C for 2 min, then at 72°C for 10 min before being subjected to 30 cycles of 94°C [1 min] - 50°C [1 min] - 72°C [4 min] and held at 72°C for 15 min. This resulted in the amplification of a cl500 bp fragment which waε excised from the gel and purified [Hybaid Recovery] . The 3' A overhangs necessary for ligation into the pCR2.1 vector were added subεequently in a reaction containing purified DNA, dATP [0.01 μmoleε] Taq polymeraεe [2.5U] and Taq buffer [Pharmacia] in a volume of 50 μl, heated to 72°C for 10 min. The cDNA was then purified [Hybaid Recovery], sub-cloned and sequenced. The εequence, which contained an orf of 1314 bp, encompaεεed all known εequence from the c500bp and clOOO bp clones. It encoded a 438 amino acid protein, deduced molecular weight 51282 and deduced pi 6.646. The deduced protein sequence included all amino acid sequence data obtained from the 51K protein, and was clearly the cDNA sequence which encoded it.
Protein database searching gave no significant homology with the deduced sequence. Secondary structure predictions carried out using the neural network based algorithms of Rost B and Sander C (1993) J Mol Biol 232: 584-599; Proteins (1994) 19: 55-72; Proteinε (1994) 20: 216- 226; and Rost B et al (1995) Prot Sci 4: 521-533 [Fig. 6A and 6B] . Using a method designed specifically for prediction of transmembrane helices [Rost B, Casadio R, Fariεelli P, Sander C (1995) Prot Sci 4: 521-533] a εingle transmembrane helix near the N-terminus of the protein was predicted [Fig. 6B] . Such sequenceε serve to anchor proteins to membranes, and are typical of many Golgi membrane bound proteins, including several glycosyltransferases [Paulson J C and Colley K J (1989) Glycosyl transf erases . Structure, localization and control of cell type specific glycosylation J Biol Chem 264: 17615- 17618] .
Example 3. Evidence that the 51K protein is the fenugreek galactomannan galactosyltransferase
To establiεh with certainty a functional link between the 51K protein and the galactomannan galactosyltransferaεe, a εtrategy waε deviεed to insert the encoding DNA εequence into a micro-organism. Any expresεed fenugreek galactoεyltranεferaεe activity would be eaεily identified. It was recognised that expreεsion of the full-length DNA including the transmembrane helix "anchor" sequence might lead to the attachment of any expressed protein to cellular membranes of the host microorganism. Thus our strategy included the expression not only of the full length 51K protein but also of a truncated protein lacking the sequence from the N-terminus to just beyond the transmembrane helix. The truncated protein, if expressed, might be expected to be enzymatically active but not membrane-bound.
It was decided to attempt to insert the cDNA sequences in-frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase [AOX] promoter and the yeast α secretion factor. Pichia constructs were obtained for both the full-length and the truncated sequence, and culture filtrates were assayed for the activity of the fenugreek galactosyltransferaεe using locust bean galactomannan [low galactose] as acceptor substrate. Controls [no insert] gave no activity, full-length constructs gave moderate levels of activity, and truncated constructs gave very high levels of activity [Table 2] .
Table 2 Galactomannan galactosyltransferase activities in lOx concentrated 44 hour culture supematants from Pichia transformantε , in relation to the activity in a typical Triton X-100 extract of fenugreek membranes [not concentrated]
Figure imgf000049_0001
Fragmentation of the labelled galactomannan product of the reaction, separation of the labelled oligoεaccharideε by TLC and digital autoradiography gave a pattern of labelled galactomanno- oligosaccharides identical with those obtained using the detergent- solubilised galactomannan galactosyltranεferase [Fig 7] . The type of galactosyltransferase activity present in the culture supematants from the Pi chia transformantε waε identical with that of the εolubiliεed fenugreek galactoεyltranεferaεe, providing proof that the 51K protein encoded the fenugreek galactomannan galactoεyltranεferaεe . The levelε of εecreted activity were high. Full length constructs gave activities approaching those in typical detergent extracts [see above] , whilεt truncated constructs gave very much higher levelε of activity. This indicated either that the presence of the membrane -anchoring helical domain of the full-length protein hampered expression and/or secretion, or that the modified protein lacking the membrane anchor had a higher specific activity under our in vitro assay conditions .
PCR amplification of cDNA encoding the complete protein sequence and a trunca ted sequence lacking the transmembrane helix, wi th sequence extensions permi tting insertion of the sequences in- frame into the genome of Pichia pastoris under the control of an AOX promoter and the yeast α-secretion factor.
Primers , des ignated GTEXP1S , GTEXP2 S and GTEXP3A were des igned and synthesised to allow amplification of the entire εequence and of a truncated sequence with sequence extens ions allowing cloning in - f rame in the multiple cloning s ite of the Pi chia expression vector pPIC9 [ Invitrogen] , us ing the Xhol and-- Not l restriction s ites :
Sense primer GTEXP1S : 5 ' GTA TCT CTC GAG AAA AGA GCG ACG AAA TTT GGT TCC AAA 3 '
A T K F G S K - Senεe primer GTEXP2S :
5 ' GTA TCT CTC GAG AAA AGA AAC TCC AAC CCA AAA TTC AAC 3 '
N S N P K F N - (Xhol s i tes are underl ined)
- Y P A A S P 3 ' ATG GGG CGA CGT AGT GGT ATT TCC CGC CGG CGC TTA ATT 5 ■ (Not l s ite underl ined)
Using plasmid DNA with the full-length cl500 bp sequence [Fig. 5] as template, primers GTEXP1S and GTEXP3A amplified a cl400 bp band which waε purified from gelε, digeεted with Not 1 and Xho 1, re-purified and cloned into pPIC9 which had been previouεly digested with the same reεtriction enzymes. Ampicillin-resistant clones were screened for the presence of insertε by PCR uεing gene-specific primers and a primer designed to part of the α-factor sequence on the vector . This primer confirmed not only that apparently correct inserts were present but also confirmed their orientations. Plasmid DNA prepared from positive clones was further checked for the presence of the correct inserts by digestion with Xhol and Not 1. Primers GTEXP2S and GTEXP3A amplified a c 1300 bp fragment which was similarly treated.
Transformation of Pichia . The pPIC9 constructs with the full length and truncated sequences, pPIC9F and pPIC9T respectively, were each amplified, and samples of each plasmid DNA were linearised with Stu 1 [pPIC9FΞtul, pPIC9TStul] . Competent cells of Pichia pastoris GS115 were prepared and transformed using the EasyComp [Invitrogen] kit. Separate transformationε were carried out uεing pPIC9FStul, pPIC9TStul and Stu 1 lineariεed pPIC9 aε control. In each case, putative positive transformantε were εelected on the basis of their ability to grow on histidme-free medium aε deεcribed in the Invitrogen Pichia expression kit manual. Putative positives were further screened by direct PCR amplification of colonies. Yeaεt cellε were boiled for 10 mm prior to the addition of the PCR ingredients .
Assay for galactomannan galactosyltransferase activi ty associated wi th Pichia transformants . Putative positive transformants, asεumed to be Mut+ [fast growing] as would be expected from the restriction enzyme used to cleave the pPIC9 vectors before transformation [Invitrogen Pichia expression kit manual], were inoculated, using single colonies, into 10 ml of BMGY [no methanol] medium m 50 ml conical tubes and grown at 30°C with continuous rotatory shaking [200 rpm] for 24 hours [A600 about 2.7] . Cells were harvested by centrifugation. The supenatantε were decanted and the cellε resuspended m BMMY [containing methanol] medium to give an A600 value of 1.0 Samples [50 ml] were further cultured at 30°C for 70 hours, samples being withdrawn at 0, 20, 44 and 70hours Methanol waε added to 0.5% at every sampling.
All samples were centrifuged, and supernatantε were collected, concentrated [xlO] using Vivapore [Vivaεcience] membrane concentrators [7.5K cut-off], and aεsayed for galactosyltransferase activity using locust bean galactomannan as galactosylacceptor. The assays [100 μl] contained 50 μl concentrated supernatant, 25 mM Tris-HCl buffer pH 7.5, 2 mM MnCl2, 0.2% [w/v] locuεt bean galactomannan and 800 μM labelled UDP-Gal, and were incubated at 30°C for 2 hours. At the end of the incubation time glacial acetic acid [50 μl] was added and the mixture was heated at 100°C for 2 mm. The galactomannan acceptor waε precipitated by adding methanol to a final concentration of 70%, washed exhaustively with hot 70% methanol as described previously, and either subjected to liquid scintillation counting or fragmentation using the structure sensitive endo-mannanase from A niger. Concentrated supernatantε from pPIC9 controlε contained no activity, whilst those from full-length constructs contained low activity, and those from truncated constructs showed very high activity comparable with the activities present in detergent extracts from membrane preparations.
Typical activity data are shown m Table 2. When labelled galactomannan products were digested with the A niger endo-β-mannanase, the only labelled productε of the reaction were diagnostic galactomannan oligosaccharides [Fig. 7] .
Example 4 - identification of a homologous εequence from developing guar ( Cyamopsis tetragonoloba [L] Taub.) endosperms, and demonstration that it encodeε a galactomannan galactoεyltranεferaεe
On further databaεe searching, the fenugreek galactosyltransferaεe showed limited homology at the protein level with several yeast sequences known or believed to be galactosyltransferases , notably
MN10_YEAST (SWIΞS-PROT: P50108) and GM12_SCHPO (SWISSPROT: Q09174) . Degenerate sense and antisenεe primerε (GT5S1 and GT5A1 - Table 3) were deεigned, following the fenugreek galactoεyltranεferaεe εequence, to a short region of very high homology between all three sequences. This covered amino acids 190 - 210 of the fenugreek galactosyltransferase sequence .
Table 3. Primerε uεed to obtain the guar εequence
GT5S1 5' GAG TGG ATI TGG TGG GTI GAC 3'
A T
GT5A1 5' TCI ACC CAC CAI ATC CAT TC 3'
C
GT5S4 5' AGG CAT GCA GAG AAA GTG AGT 3'
GT5A4 5' ACT CAC TTT CTC TGC ATG CCT 3'
GT5A5 5' TTT TCG TCC CAG TTT TTC AT 3'
C A A C
GP1A 5' GGC GTT CGT TGG GAT CGT AT 3'
GP2S 5' GTA TCA CAT TCA CTC ACT CC 3'
RNA waε prepared, aε for fenugreek, from the developing endoεperms of guar seeds during the early εtageε of galactomannan deposition (30 to 35 days after anthesis, Edwards et al . 1992) . First strand cDNA was synthesiεed, as before, using the (dT) 17-R1R0 primer (Frohman and Martin, 1989) . When 3 ' RACE was carried out using this first strand cDNA, primer GT5S1, and the T7 RNA polymerase promoter, an 800 - 900 bp cDNA was amplified. To test whether the 800 - 900 bp band was likely to be a homologue of fenugreek galactosyltranεferaεe, PCR amplification was carried out using sense and antisenεe primerε deεigned to the fenugreek galactoεyltransferaεe sequence between amino acid 210 and the C- terminus of the protein, paired with GT5S1 and T7 , using the purified 800- 900 bp cDNA as template. One pair (GT5S4 and GT5A4 , Table 3) gave efficient amplification of cDNA bands. GT5S4 in combination with T7 and GT5S1 in combination with GT5A4 resulted in the amplification of bands of the sizes expected if the 800 - 900 bp band amplified by 3 ' RACE waε a sequence homologous to the fenugreek galactosyltransferase.
-5 ' RACE protocol (Frohman and Martin, 1989) was carried out, modified aε deεcribed in Example 2. First strand cDNA reverse-transcribed from guar RNA waε primed uεing random hexamers , and polyA tailed at the 3' end using terminal transferaεe. Second εtrand synthesis was primed using the (dT) 17-R1-R0 primer. A firεt round of PCR amplification was carried out using the R.. primer (Frohman and Martin 1989) and GT5A4 (Table 3) . Amplified cDNA was recovered (Hybaid Recovery) and used as template for a second round amplification, using the degenerate primer (NTP2S, as used in Example 2) designed to the N-terminal protein sequence of the fenugreek galactoεyltranε eraεe along with GT5A1. This resulted in the amplification of a 570 bp cDNA.
Alternative second round amplifications were attempted using primer Rz and antisenεe primers designed to the fenugreek galactosyltransferase εequence between amino acid 190 and the N-terminus. This was in order to amplify sequence extending 5' of the terminus of the fenugreek sequence. One of these primerε (GT5A5, deεigned to amino acidε 96 - 116, Table 3) resulted in the amplification of a 400 bp cDNA.
All of the above cDNA' ε were gel purified [Hybaid Recovery] , cloned, and εubcloned. Sequence data obtained from them waε aligned to give a composite clone. Perfect sense and antisense primers were designed to sequences near the 5' end and the 3' end respectively of the composite εequence, and uεed in RT-PCR reactionε using guar RNA as template and the Pfu proof-reading DNA polymerase (Stratagene) . The combination GP2S and GP1A (Table 3) resulted in the amplification of a single c 1400 bp cDNA. This was gel purified, and ligated into the commercial plasmid pCR 2.1 TOPO (Invitrogen), and E coli cells (TOP 10F' , Invitrogen) were transformed with the ligation mixture, and transformants were amplified. Single colonies were PCR checked and a single positive colony waε used to prepare plasmid DNA, which was uεed aε template for the full εequencing of the c 1400 bp inεert. A series of plasmid based and gene-specific sequencing primers waε used. The base sequence of the insert iε shown in Annex 2a.
The sequence showed a continuous open reading frame from the start to base 1326 (Annex 2b) . Near the 5' end (base 24 onwards), the encoded protein sequence waε closely similar to that at the the N-terminus of the fenugreek galactosyltransferaεe (ATKFGS in fenugreek, and AKFGS in guar) . In guar, thiε sequence was immediately preceded by a methionine residue, which may represent the start of tranεlation. On thiε assumption, the cDNA encoding the putative guar galactosyltransferase compriseε 1305 bp and encodeε a 435 amino acid protein. The fenugreek galactoεyltranεferaεe and the putative guar galactosyltransferase are aligned in Fig 1.
Clearly the two sequences are highly similar (77% similarity; differences highlighted in Fig. 1) . As for the fenugreek galactosyltranεferaεe (Example 2), secondary structure predictionε [Rost B and Sander C (1993) J Mol Biol 232: 584-599; (1994) Proteinε 19: 55-72; (1994) Proteins 20: 216-226; Rost B et al (1995) Prot Sci 4: 521-533] revealed a εingle, membrane-εpanning helix near the N-terminuε (underlined in Fig. 1) . To eεtablish whether or not the guar sequence waε functionally a galactomannan galactoεyltranεferaεe, the full-length protein (residues 1 - 435, Fig. 1) and a truncated protein lacking the membrane spanning helix (residues 43 - 435, Fig 1) were separately overexpresεed in Pichia pastoris aε described for the fenugreek sequence in Example 2. The experimental strategy employed was exactly as in Example 2.
Pichia constuctε were obtained for both the full-length and the truncated sequence, and culture filtrates were aεsayed for galactomannan galactoεyltranεferase activity exactly aε deεcribed in
Example 1. Culture supe atants from control transformants (no insert) and from transformantε with full-length inserts did not contain measurable amounts of galactomannan galactosyltranεferaεe activity, whereas supematants from transformants with truncated insertε contained significant levels of activity (7.99 + 1.90 μmol . I"1.h"1; 6 independent clones; supematants not concentrated). When labelled galactomannan products formed by the catalytic action of the expressed guar protein were digested with the structure-εensitive A niger endo-β- mannanase, the only digestion products were again oligosaccharides diagnoεtic of legume-seed galactomannan.
Example 5 - production of transgenic plants
Transgenic plants containing modified levels of the fenugreek or guar galactosyltransferase genes, or derivatives thereof, may be produced using methods known to those εkilled in the art. Gene constructs will be expresεed conεtitutively or in a tiεεue-specific manner in the seed or endosperm, potentially at a specific developmental stage. Constructs may include antisense versions of e.g. guar galactosyltransferase. Transgenic Guar plants may then be produced, for instance using methods analogous to those discusεed in WO 97/20937. Thiε will result in guar galactomannan with a higher man/gal ratio.
Example 5 - Foodstuffε compriεing modified galactomannan
Modified galactomannans may be extracted from transgenic plantε by methodε analogous to those used in the art .
An ice cream based on the modified galactomannan may be provided as follows :
Ingredient Amount
Galactomannan 0.35 Liquid sugar 15
Skimmed Milk (30% solidε)15.9 Butter fat 9
Eεpiron 300 5
MGp 0.3 Flavour 0.01
Colour 0.004
Water to 100
A water ice may be provided aε follows :
Ingredient Amount Galactomannan 0.1
Liquid sugar 15.7
Liquid dextrose 4
Citric acid 0.2
Flavour 2.6
Colour 0.0075
Water to 100
Sequence Annexes
Annexe la: fenugreek cDNA εequence - Seq ID No 1
gcgacgaaatttggttccaaaaacaaatcctctccatggctctcaaatggttgcatcttcctcctaggtgc aatgtcagc tcttcttatgatttgggggctcaattccttcatcgctccaatcccaaactccaacccaaaattcaactcct tcaccacca aactcaaatccttaaacttcaccacaaacaccaactttgctggtcctgatttgttacatgacccttcagac aaaaccttc tatgatgatccagaaacatgttacaccatgatggacaaaccaatgaaaaattgggatgagaagcgtaaaga atggctatt tcatcatccctcattcgcggctggagcaaccgaaaagatacttgttataacgggttcacagccgacaaagt gtgacaacc ccatcggagaccaccttttactaaggttctataaaaacaaggttgattattgtcgtatacacaaccacgac ataatctac aacaatgcattgttgcacccaaaaatggactcttactgggccaagtatcctatggttcgggccgcaatgtt ggcccatcc ggaagtagaatggatatggtgggtcgactctgatgccatctttaccgatatggaattcaagttaccgttat ggcgttaca aggatcacaaccttgtgattcatggttgggaagagttggttaagacagagcatagttggaccgggcttaac gcgggtgtt ttcttgatgaggaattgtcaatggtcgttggattttatggatgtttgggccagtatgggcccaaacagccc ggaatacga gaaatggggggagagacttagagaaacttttaagacaaaagtggtacgtgattcagatgatcagacggcgc ttgcttact tgatcgcgatgggagaggacaagtggacaaagaagatctatatggagaatgagtattattttgaagggtat tggttagag atttcaaagatgtatgataaaatgggtgagagatatgatgagatagaaaaaagagtggaagggttaaggag gaggcatgc agagaaagtgagtgaacgttatggtgaaatgagagaggagtatgttaagaatttaggggatatgagaagac cttttatta cacattttacagggtgccaaccttgtaatggtcatcataatccaatatatgctgcagatgattgctggaat ggcatggag agagctctcaattttgctgataatcaggtgttgcgcaagtttggtttcattcatccaaatctattggataa gtctgtttc tccattaccatttggataccccgctgcatcaeca
Annex lb: tranεlation of the fenugreek cDNA sequence - Seq ID No 2 ATKFGSKNKSSPWLSNGCIFLLGAMSALLMI GLNSFIAPIPNSNPKFNSFTTKLKSLNFTTNTNFAGPDL LHDPSDKTFYDDPETCYTMMDKPMKNWDEKRKEWLFHHPSFAAGATEKILVITGSQPTKCDNPIGDHLLLR FYKNKVDYCRIHNHDIIYNNALLHPKMDSYWAKYPMVRAAMLAHPEVEWIWWVDSDAIFTDMEFKLPLWRY KDHNLVIHGWEELVKTEHSWTGLNAGVFLMRNCQWSLDFMDVWASMGPNSPEYEKWGERLRETFKTKWRD SDDQTALAYLIAMGEDKWTKKIYMENEYYFEGYWLEISKMYDKMGERYDEIEKRVEGLRRRHAEKVSERYG EMREEYVKNLGDMRRPFITHFTGCQPCNGHHNPIYAADDCWNGMERALNFADNQVLRKFGFIHPNLLDKSV SPLPFGYPAASP
Annexe 2a: guar cDNA sequence - Seq ID No 3
GTATCACATTCACTCACTCCCATGGCCAAATTTGGTTCCAGAAACAAATCCCCTAAATGGA TCTCCAACGGTTGCTGCTTCCTCCTAGGAGCATTCACTGCTCTTCTTCTGCTCTGGGGTTTA TGCTCCTTCATCATCCCCATCCCAAACACCGACCCCAAGCTCAACTCCGTCGCCACCAGTT TGAGATCCCTTAACTTTCCCAAAAACCCCGCTGCCACCTTGCCTCCCAACTTGCAGCACGA CCCTCCTGACACCACCTTCTACGACGACCCCGAAACCAGTTATACCATGGACAAACCAAT GAAAAACTGGGACGAGAAGCGTAAGGAGTGGTTGCTGCATCATCCTTCGTTTGGCGCCGC AGCACGCGATAAGATTCTCCTGGTGACAGGTTCTCAGCCGAAACGGTGCCATAACCCGAT CGGCGACCACCTCCTGTTGCGGTTTTTCAAGAACAAGGTGGATTACTGCCGGCTGCACAAC TACGACATAATTTACAACAACGCGCTTCTGCATCCTAAAATGAACTCTTATTGGGCCAAGT ATCCAGTGATTCGGGCGGCGATGATGGCCCATCCGGAAGTGGAGTGGGTGTGGTGGGTGG ACTCGGACGCGGTTTTCACGGACATGGAGTTCAAGCTTCCGTTAAAGCGTTATAAGAACC ACAATCTGGTGGTTCACGGTTGGGAAGGATTGGTACGGTTGAACCATAGCTGGACGGGTC TAAACGCGGGCGTATTCTTGATTCGGAATTGCCAGTGGTCGTTGGAGTTCATGGATGTG TGGGTGAGCATGGGGCCACAGACTCCGGAATACGAGAAATGGGGGGAGAGGTTGAGAGAGA CATTCAAGGACAAGGTGCTGCCTGATTCGGACGATCAGACGGCGCTGGCTTACCTGATCG
CGACGGATAATAAGGACACGTGGAGGGAGAAGATCTTCTTGGAGAGCGAGTACTACTTCG AAGGGTACTGGCTGGAGATCGTGAAGACGTACGAGAACATAAGCGAGAGGTATGATGAG GTGGAGAGGAAGGTGGAAGGGTTGAGGAGGAGGCATGCGGAAAAGGTGAGCGAGAAAT ACGGTGCGATGAGGGAGGAGTATCTGAAGGACAACAAGAGGAGGCCCTTTATCACGCAC TTTACTGGGTGTCAACCCTGTAATGGCCACCATAATCCTGCTTATAATGCTAATGATTGCT
GGAATGGCATGGAGAGGGCTCTTAATTTCGCTGATAATCAAATCTTGCGTACTTACGGTTA TCACCGTCAAAATTTACTCGACAAGTCTGTTTCACCCTTACCTTTTGGTTACCCTGCTGCAT AATAATGTACTACTACTGATAACGACAGTTATTTAAAATTTATTATACGATCCCAACGAAC GCC
Annex 2b: translation of the guar cDNA sequence - Seq ID No 4
VSHSLTPMAKFGSRNKSPKWISNGCCFLLGAFTALLLLWGLCSFIIPIPNTDPKLNSVATSLRSLNFPKNP AATLPPNLQHDPPDTTFYDDPETSYTMDKPMKNWDEKRKEWLLHHPSFGAAARDKILLVTGSQPKRCHNPI GDHLLLRFFKNKVDYCRLHNYDII NNALLHPKMNSYWAKYPVIRAAMMAHPEVEWVWWVDSDAVFTDMEF KLPLKRYKNHNLWHGWEGLVRLNHSWTGLNAGVFLIRNCQWSLEFMDVWVSMGPQTPEYEKWGERLRETF KDKVLPDSDDQTALAYLIATDNKDTWREKIFLESEYYFEGYWLEIVKTYENISERYDEVERKVEGLRRRHA EKVSEKYGAMREEYLKDNKRRPFITHFTGCQPCNGHHNPAYNANDCWNGMERALNFADNQILRTYGYHRQN LLDKSVSPLPFGYPAA.. CTTTDNDSYLKFIIRSQRT

Claims

Claims
1 An isolated nucleic acid encoding a polypeptide which is capable of catalysing the bioεynthesiε of a complex non-cellulosic plant cell wall polysaccharide.
2 A nucleic acid as claimed in claim 1 wherein the polysaccharide is a hemicellulose.
3 A nucleic acid as claimed in claim 1 or claim 2 wherein the polypeptide iε a glycosyltransferase .
4 A nucleic acid as claimed in claim 3 wherein the polypeptide is a galactosyltransferaεe .
5 A nucleic acid as claimed in any one of claims 2 to 4 wherein the polysaccharide iε galactomannan.
6 A nucleic acid as claimed in any one of the preceding claimε having a sequence comprising Seq ID No 1 or iε degeneratively equivalent thereto.
7 A nucleic acid as claimed in any one of the preceding claims having a sequence comprising Seq ID No 3 or is degeneratively equivalent thereto.
8 A nucleic acid as claimed in any one of claims 1 to 5 which is a homologous variant of Seq ID No 1.
9 A nucleic acid as claimed in any one of claims 1 to 5 which is a homologouε variant of Seq ID No 3.
10 A nucleic acid as claimed in claim 8 or claim 9 wherein the variant is an allelic or pseudoallelic variant of Seq ID No 1 or Seq ID No 3.
11 A nucleic acid as claimed in claim 8 having a sequence which is a derivative of Seq ID No 1 by way of addition, insertion, deletion or substitution of one or more nucleotides and which encodes a polypeptide having altered activity with respect to Seq ID No 2. 12 A nucleic acid as claimed in claim 8 wherein the derivative encodes a functional portion of Seq ID No 2
13 A nucleic acid comprising at least 15 nucleotides having a sequence comprising, or being degeneratively equivalent to, part of Seq ID No 1
14 A nucleic acid as claimed in claim 9 having a sequence which is a derivative of Seq ID No 3 by way of addition, insertion, deletion or substitution of one or more nucleotides and which encodes a polypeptide having altered activity with respect to Seq ID No 4
15 A nucleic acid as claimed in claim 14 wherein the derivative encodes a functional portion of Seq ID No 2
16 A nucleic acid comprising at least 15 nucleotides having a sequence comprising, or being degeneratively equivalent to, part of Seq ID No 3
17 A nucleic acid which is complementary to the nucleic acid of any one of claims 6 to 16
18 A method for identifying or cloning a glycosyltransferase from a plant which method employs a nucleic acid molecule having a nucleotide sequence comprising, or complementary to, all or part of Seq ID No 1 or Seq ID No 3 , or a derivative of either
19 A method as claimed in claim 18 comprising the step of searching a data-base to find εequenceε which are homologouε to Seq ID No 1 or Seq ID No 3
20 A method aε claimed in claim 18 comprising the steps of
(a) providing a preparation of nucleic acid,
(b) providing a nucleic acid molecule having a nucleotide εequence comprising, or complementary to, all or part of the nucleic acid of claim 6 or claim 7,
(c) contacting nucleic acid said preparation with εaid nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue said preparation, and (d) identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule 21 A method as claimed in claim 20 wherein the hybridisation conditions are selected to allow the identification of sequences having about 70% or more sequence identity with the nucleic acid molecule.
22 A method as claimed in claim 18 comprising use of two primers to amplify a nucleic acid encoding a glycosyltransferase, at least one of the primers having a εequence comprising, or complementary to part of Seq ID No 1 or Seq ID No 3 or a derivative of either.
23 A method as claimed in claim 22 comprising the steps of:
(a) providing a preparation of plant nucleic acid,
(b) providing a pair of nucleic acid molecule primers suitable for PCR, at leaεt one of the primers having a sequence comprising, or complementary to part of the nucleic acid of claim 6 or claim 7, (c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,
(d) performing PCR and determining the presence or abεence of an amplified PCR product.
24 A nucleic acid molecule for use as a probe or primer in the method of any one of claims 20 to 23, said molecule having a sequence compriεing, or being complementary to, part of the nucleic acid of claim 6 or claim 7.
25 A recombinant vector comprising either the nucleic acid of any one of claims 1 to 17.
26 A vector as claimed in claim 25 which is capable of replicating in a suitable host.
27 A vector as claimed in claim 25 or claim 26 wherein the nucleic acid is operably linked to a promoter or other regulatory element for tranεcription in a host cell
28 A vector as claimed in claim 27 further comprising any one or more of the following: a terminator sequence; a polyadenylation sequence; an enhancer sequence; a marker gene.
29 A vector as claimed in claim 27 or claim 28 wherein the promoter is an inducible promoter. 30 A vector as claimed in any one of claims 25 to 29 which is a plant vector.
31 A vector as claimed in claim 30 comprising a selectable genetic marker which conferε a εelectable phenotype εelected from: reεistance to antibiotics or herbicides .
32 A method comprising the step of introducing a vector as claimed in any one of claims 26 to 31 into a cell.
33 A method for transforming a plant cell, comprising a method as claimed in claim 32, and further comprising the step of causing or allowing recombination between the vector and the plant cell genome to introduce the nucleic acid into the genome.
34 A host cell compriεing a vector aε claimed in any one of claims 26 to 31.
35 A host cell transformed with a vector as claimed in any one of claims 26 to 31.
36 A host cell as claimed in claim 34 or claim 35 which is a plant cell.
37 A host cell as claimed in claim 36 which is in a plant.
38 A method for producing a transgenic plant comprising a method as claimed in claim 33 and further comprising the step of regenerating a plant from the transformed cell.
39 A plant comprising the cell of claim 36 or claim 37.
40 A plant as claimed in claim 39 produced by the method of claim 38.
41 A plant aε claimed in claim 40 which iε an endoεpermic legume.
42 A plant which iε the progeny of a plant as claimed in claim 40 or claim 41, and comprising the cell of claim 36 or claim 37.
43 A part or propagule of the plant of any one of claimε 39 to 42. 44 A polypeptide encoded by the nucleic acid of any one of claimε 1 to 16.
45 A method of producing a polypeptide comprising the step of causing or allowing the expresεion from a nucleic acid of any one of claimε 1 to 16 in a εuitable host cell.
46 A composition comprising the polypeptide of claim 44.
47 An antibody or fragment thereof, or a polypeptide comprising the antigen-binding domain of the antibody, capable of specifically binding the polypeptide of claim 44.
48 A method of producing the antibody or fragment as claimed in claim 47 comprising the step of immunising a mammal with a polypeptide according to claim 44.
49 A method of identifying and/or isolating a glycoεyltransferase compriεing the εtep of εcreening candidate polypeptides with a polypeptide comprising the antigen-binding domain of the antibody of claim 47.
50 A method for the in vi tro εynthesis of a polysaccharide comprising the use of the polypeptide of claim 44.
51 A method for altering the quality or quantity of a polysaccharide in a host cell by influencing the glycosyltransferaεe activity in that cell, the method compriεing uεe of any one or more of the following: all or part of the nucleic acid of any one of claims 1 to 16; the polypeptide of claim 44; the antibody or fragment or polypeptide comprising the antigen-binding site thereof of claim 47.
52 A method as claimed in claim 51 wherein the polysaccharide iε a complex non-celluloεic plant cell wall polyεaccharide .
53 A method as claimed in claim 51 or claim 52 wherein the quality altered is galactose compoεition of the polysaccharide.
54 A method as claimed in claim 53 wherein the quality altered iε the mannoεe:galactoεe ratio in a mannoεe/galactoεe containing polysaccharide in the cell. 55 A method as claimed in any one of claims 51 to 54 comprising the step of causing or allowing expresεion of a nucleic acid according to any one of claims 1 to 17 within the cell.
56 A method as claimed in any one of claimε 51 to 55 comprising reducing the glycosyltransferase activity in the cell.
57 A method as claimed in claim 56 compriεing the εtep of causing or allowing the transcription of part of the nucleic acid of any one of claims 1 to 16 in the cell such as to co-suppress the expression of an endogenous glycosyltranεferaεe .
58' A method aε claimed in claim 56 comprising the step of causing or allowing the transcription of nucleic acid of claim 17 in the cell.
59 A method as claimed in claim 56 comprising the step of causing or allowing the expression of a polypeptide comprising the antigen-binding domain of the antibody of claim 47.
60 A method as claimed in any one of claimε 51 to 59 wherein the cell iε a plant cell.
61 A method as claimed in claim 60 wherein the plant cell is part of a plant.
62 A method aε claimed in any one of claimε 51 to 61 wherein the glycosyltransferase is a galactosyltransferase.
63 A complex non-cellulosic plant cell wall polysaccharide the quality of which has been altered in accordance with the method of claim 61 or claim 62.
64 A plant product derived from any one of the plants of claims 39 to 42 or the plant cell of claims 36 or claim 37, said product comprising a complex non-celluloεic plant cell wall polyεaccharide of claim 63.
65 A commodity comprising the altered cell wall εtorage polyεaccharide of claim 63.
66 A commodity aε claimed in claim 65 which is selected from: a human or animal foodεtuff; a coεmetic. 67 A foodstuff as claimed in claim 66 which is a frozen food product .
68 A frozen food product as claimed in claim 67 which is an ice cream or water ice.
PCT/GB1999/001610 1998-05-21 1999-05-21 Galactosyltransferase from plants involved in galactomannan biosynthrsis Ceased WO1999060103A2 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004046197A3 (en) * 2002-11-14 2005-02-10 Pioneer Hi Bred Int Genes for galactomannan production in plants and methods of use
WO2007047675A3 (en) * 2005-10-14 2008-02-28 Cornell Res Foundation Inc Nucleic acids and proteins associated with galactomannan synthesis in coffee

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GB9524752D0 (en) * 1995-12-04 1996-02-07 Danisco Modification process
GB9713852D0 (en) * 1997-06-30 1997-09-03 Univ Cambridge Tech Plant genes and uses thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004046197A3 (en) * 2002-11-14 2005-02-10 Pioneer Hi Bred Int Genes for galactomannan production in plants and methods of use
US7265265B2 (en) 2002-11-14 2007-09-04 Pioneer Hi-Bred International, Inc. Genes for galactomannan production in plants and methods of use
WO2007047675A3 (en) * 2005-10-14 2008-02-28 Cornell Res Foundation Inc Nucleic acids and proteins associated with galactomannan synthesis in coffee
US8217226B2 (en) 2005-10-14 2012-07-10 Nestec, S. A. Nucleic acids and proteins associated with galactomannan synthesis in coffee

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