AU702520B2 - Genetic manipulation of plants to increase stored carbohydrates - Google Patents

Description

WO 96/06173 PCT/AU95/00527 "Genetic Manipulation of Plants to Increase Stored Carbohydrates" TECHNICAL FIELD The present invention relates to plants genetically modified to increase the level of stored carbohydrates in the plant, particularly during periods of high sink activity and low source activity. The invention also relates to the genetic constructs used to produce the engineered plants and the method of producing the engineered plants.

BACKGROUND ART The soluble storage carbohydrate found in plants, including sucrose, glucans, starch and fructans, are an important source of feed for animals, particularly grazing ruminants. These carbohydrates are stored nonstructurally which makes them readily available for digestion by animals and therefore an important source of digestible energy.

During periods of high sink activity and low source activity, such as during a drought, the level of stored carbohydrates falls as the non-structural storage carbohydrates are mobilised for use in seed filling.

The result of this mobilisation, particularly in relation to pasture grasses, is a significant loss of feed value to grazing ruminants due to the reduction in the levels of the stored carbohydrates. This reduction is caused by the enzymatic degradation of the stored carbohydrates.

This enzymatic degradation is assisted by the fact that the stored carbohydrates generally have a low degree of polymerization. For example, as noted by Radojevic et al 1994, during the period from late spring to early autumn in southern Australia, the declining feed quality of the grasses causes a corresponding reduction in the lactation by dairy herds and necessitates the use of supplementary feeds. This decline in digestibility is associated with a decline in the level of soluble carbohydrates.

Perennial rye grass lines which accumulate high concentrations of soluble carbohydrates from late spring SUBSTIUTE SHEET (Rule 26) WO 96/06173 PCT/AU95/00527 2 to early autumn do not suffer as large a decline in digestibility (Radojevic et al 1994). The result of this increased digestibility is a corresponding increase in milk production by dairy herds.

In addition to this, there are many pasture plants, such as white clover which do not possess any significant levels of stored carbohydrate.

There has, therefore, been a desire to develop methods for preventing the degradation of the stored carbohydrates during plant senescence and to increase the level of stored carbohydrates in pasture plants with low levels.

Glucosvltransferases of Streptococcus salivarius It is known that many strains of Streptococcus salivarius and Streptococcus mutans, produce extracellular a-D-glucosyltransferase (Gtfs), an enzyme which catalyses the formation of glucan from sucrose.

These Gtfs are also found in many other species of oral streptococci.

The Gtfs utilise the high free energy of the glycosidic bond of sucrose to synthesise glucans (Jacques NA, Giffard PM, 1991). Gtfs produce either soluble or insoluble products by transferring a glucose residue from sucrose to a growing glucan chain.

Gtfs which produce an insoluble product are generally considered to be primer-dependent (Walker GJ, Jacques NA, 1987). These primer-dependent Gtfs require a dextran (a-(1-6)-linked glucan) as a receptor for polymerisation to proceed at an appreciable rate. In contrast, Gtfs that produce soluble products may be either primer-dependent or primer-independent.

The genetic sequences for 10 qtf genes from a number of Streptococcus species have been ascertained (Gilmore KS, Russell RRB, Ferretti JJ). All the Gtfs coded by these genes possess highly conserved putative signal sequences that lead to the secretion of these enzymes.

PCTIAU95/00527 WO 96/06173 3 The remainder of each protein is arbitrarily divided into two domains the N-terminal two-thirds "catalytic domain" and the C-terminal one-third "glucan-binding domain".

S. salivarius ATCC 25975 has been shown to possess at least four different gtf genes (Giffard et al (1991); Giffard et al (1993)). Each of these genes codes for a highly hydrophilic monomeric glucosyltransferase that possesses unique enzymic properties. These Gtfs synthesize structurally different glucans from sucrose.

For example, the genes coding for GtfJ and GtfL produce enzymes which synthesize insoluble glucans. GtfJ is a primer-dependent enzyme producing essentially a linear u(1-3)-glucan while GtfL is a primer-independent enzyme that synthesizes a glucan containing 50% and a-(1i6)-linked glucosyl residues. In contrast, the qtfK and gtfM genes code for enzymes which produce a soluble glucan which possess (16)-linked glucosyl residues.

GtfK is primer stimulated while GtfM is primer independent.

DESCRIPTION OF THE INVENTION Up until now, a qtf gene in S. salivarius or any other Streptococcus species which produces a glucosyltransfererase that synthesises a glucan which is both soluble and primer independent has not been described.

The significance of a glucosyltransferase produced by S. salivarius, or any other streptococci, which is both primer independent and which synthesises a soluble glucan product is twofold. First, the primer independence of the Gtf means that the enzyme should be functional when expressed in plants while the glucan that is formed from sucrose in the plant should be readily stored without detriment to the plant, due to its solubility.

WO 96/06173 PCT/AU95/00527 4 An important characteristic of soluble glucans produced by Gtf synthesis is that they are poorly degraded by plant enzymes and are readily digested by the diverse microflora present in the rumen of grazing livestock.

The inventors of the present invention have isolated and characterised a novel gtf (GtfM) gene in S.

salivarius which codes for a primer independent Gtf which produces a glucan which is soluble, resistant to degradation by plant enzymes and readily digested by microflora present in the rumen of grazing livestock.

According to a first aspect of the present invention there is provided a plant containing bacterial DNA which codes for a glucosyltransferase which catalyses the formation of glucans from sucrose.

Preferably, the plant contains bacterial DNA which codes for a glucosyltransferase which is primer independent.

More preferably, the plant contains DNA which codes for a glucosyltransferase which catalyses the formation of soluble glucans.

More preferably, the bacterial DNA is obtained from Streptococcus salivarius.

According to a second aspect of the present invention there is provided a DNA comprising a sequence according to SEQ ID NO: 1.

According to a third aspect of the present invention there is provided a DNA sequence which is a variant of a DNA having a sequence according to SEQ ID NO: 1. In this respect a "variant" is a polynucleotide which corresponds to or comprises a portion of the DNA of the invention, or is "homologous" to the DNA of the invention. For the purposes of this description, "homology" between two polynucleotide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second. In particular, a polynucleotide is "homologous" to the DNA of the r WO 96/06173 PCT/AU95/00527 5 invention if there is greater than 70% identity in the DNA sequence.

The polynucleotides of the present invention exclude those polynucleotides in the environment in which they occur in nature. They include the polynucleotides in a form in which they are substantially free of other Streptococcus salivarius polynucleotide sequences, such as sequences in isolated form, including those in substantially purified form.

According to a fourth aspect of the present invention there is provided a protein comprising the amino acid sequence according to SEQ ID NO: 2.

According to a fifth aspect of the invention there is provided a polypeptide comprising an amino-acid sequence which is a variant of SEQ ID NO:2. A variant is a polypeptide which corresponds to or comprises a portion of the polypeptide of the invention, or is "homologous" to the peptide of the invention. For the purposes of this description, "homology" between two peptide sequences connotes a likeness short of identity, indicative of a derivation of the first sequence from the second. In particular, a polypeptide is "homologous" to the peptide of the invention if there is greater than identity in the amino acid sequence.

These homologous polypeptides can be produced by conventional site-directed mutagenesis of the corresponding DNA or by chemical synthesis, and fall within the scope of the invention, particularly where they retain the biological activity of a glucosyltransferase.

The proteins and polypeptides of the invention exclude those proteins and polypeptides in the environment in which they occur in nature. They include the proteins and polypeptides in a form in which they are substantially free of other Streptococcus salivarius polypeptide sequences, such as sequences in isolated form, including those in substantially purified form.

WO 96(06173 PCT/AU95/00527 6 According to a sixth aspect of the present invention there is provided the microorganism E. coli containing plasmid pGSG501.

According to a seventh aspect of the present invention there is provided the microorganism E. coli containing plasmid pGSG502.

According to a eighth aspect of the present invention there is provided a plant containing

DNA

comprising a sequence according to SEQ ID NO: 1.

According to an ninth aspect of the present invention there is provided a plant containing DNA which is a variant of DNA having a sequence according to SEQ ID NO: 1.

According to a tenth aspect of the present invention there is provided a plant expressing a protein comprising an amino acid sequence according to SEQ ID NO: 2 or a variant thereof.

DNA and variants thereof of the invention can be incorporated into a variety of plant types. These include plants, such as grasses, used as fodder for livestock. They also include cereal crops or other starchy food product types, (to provide grain or other food with increased fibre); and horticultural crops, such as tomatoes and fruits, to provide fruits with increased solids.

In addition plants expressing the DNA and variants thereof, of the invention may also produce dextran which can in turn be used: 1) as a binder for use in processed foods so called 'health bars'); 2) in pharmaceutical preparations again as a binder; and 3) in medical preparations to increase antigenic activity.

WO 96/06173 PCT/AU95/00527 7 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a restriction map of the inserts from pGSG501 and pGSG502.

BEST METHOD OF PERFORMING THE INVENTION The invention is further described with reference to the accompanying Example which is no way limiting on the scope of the present invention.

Example 1 The general strategy adopted to isolate a gene from S. salivarius encoding a Gtf which produces a primer independent and soluble glucan is as follows: A X gene bank containing S. salivarius DNA was prepared. Positive clones were detected by using an E. coli strain grown on agar containing sucrose.

E. coli which contained gt DNA from S. salivarius could convert the sucrose in the medium into a polymer which resulted in opaque colonies. These opaque colonies were then picked and the S. salivarius DNA excised and subjected to restriction mapping to ascertain whether the DNA was from a previously described S. salivarius gtf gene, or whether the DNA was novel. Three clones containing novel DNA were located. These were subjected to a radioactive assay to determine whether the DNA encoded for a primer independent or primer dependent Gtf.

One clone-XC-13 was found to contain a novel qtf gene which coded for a primer independent Gtf. The DNA from this clone was then isolated and sequenced.

The particular details of this methodology are now described below.

Bacterial strains and growth conditions.

Escherichia coli LE392 and NM522 and S. salivarius ATCC 25975 were used. E. coli strains were grown in Luria- Bertani (LB) medium at 37 0 C, supplemented with ampicillin (100 pg ml" 1 isopropylthiogalactoside (IPTG) (ImM), or 5-bromo-4-chloro-3-indolyl-3-D-galactoside (X-gal) (100 WO 96/06173 PCT/AU95/00527 8 Ig ml 1 as appropriate. Cultures of S. salivarius were grown at 370C in semi-defined medium (SDM) containing mM glucose supplemented with 0.

005 g 1 Tween 80 ml where appropriate.

Bacteriophage and phagemids. All genetic constructs, excluding sequencing subclones, are listed in Table 1. Bacteriophage-X derivatives were grown either as 20 ml or 1 L-liquid lysates using E. coli strain LE392 as the host and DNA purified according to the method of Silhavy et al (1984). Plasmids were propagated in E.

coli strains as described previously (Giffard et al, 1991).

Screening of Gene Bank. A bacteriophage-X gene bank of S. salivarius ATCC 25975 (Pitty et al, 1989) was screened by detecting plaques on a lawn of E. coli LE392 grown at 37 0 C on minimal agar medium containing 0.2% glucose and 50 Ag ml' 1 methionine as well as 1% (wt/vol) sucrose with or without 0.02% (wt/vol) dextran Potential Gtf clones were detected by their opacity including X C-13 containing the qtf M gene.

Twenty recombinant plaques were picked from minimal media plates containing sucrose and the EcoRl restriction patterns of these recombinants were analysed. Of these recombinants, only XC-13 exhibited a unique EcoRl restriction pattern and Gtf activity. A restriction map of XC-13 was constructed using double restriction digests. The Gtf gene encoded by XC-13 (GtfM) was located on an 8.3 kbp insert (see figure The 5.3 kbp XbaI fragment from XC-13 was subcloned into pIBI31 (pGSG501; see Table 1) and was positive for Gtf activity as was the 4.8 kbp XbaI/EcoR1 from XC-13 subcloned into pIBI31 (pGSG502; see Table 1).

11 Table 1. Bacterial Strainas, Pliages and Phagcniids Description Source or reference Bacteriaam: Streptococcus salivarius ATCC 25975 AC 1lmlo,16) Escherichitl coli LE392 F e14-(nicrA-j hrd(R5l4 (rKn)K-) supE44 supFS58 Murray et al., 1977 Esclzericliia coli NM522 F'lacP~A~kicZ) AIlS proA +B +sup)E hIN 4(lac-proA) Li~rsdMfS-lcrB)S (rKI1KMcrBC-) Bacteriophiage: 11-47.1 XA-8 XA-33 XC-13 Phiagemid: plB133 plB133 XL-47.1 withi Otfi encoding 8.5kbj') Sau3A pairtial fragment of S. salivarus A'rcc 25975 11-47.1 with GtfK encoding 9.6khbi Sau3A partial fragment of S. salivaritts ATCC 25975 11-47.1 with 8.3kbp OtjfMv encoding Sau3A partial fragment of S. salivatius ATrCC 25975 11-47.1 with llkbp GIfL encoding Sau3A partial fragment of S. salivatiiis ATCC 25975 11,47.1 with Sau3A partial fragment of S. salivatius ATCC 25975 isolated fromi sucrose-contaillilg miediumn Loenen and Brammar, 1980 Pitty el al., 1989 Pitty et al., 1989 This study This study This study pGSG 101 pGSG2O1 pGSG4O1 pGSG4O2 pGSG403 pGSG404 pGSG501 pGSG5O2 pGSG5O3 (p)GS1() (pGS201) Apr, ft origin replication, P-galactosidase, T3 and VI' polymnerase p~romoters Apr, fI origin repl ication, P-galactosidase, T13 and T7 polymnerase promoters 1p113 130 with Gtfl encoding 6.8kbp SacflamnIll fragment of IA-8 p113O3 with GtfK encoding 7.30bp flglIl/Barnil 11 fragment of 'AA-33 p1133 with Gtfl- encoding 6.2kb~p Bandll/XbtiI fragment of ID-10 plB1 3 1 with 6.2kbp Band l/N~ba I fragment of ID-10 p113O3 with 4.8kbp EcolkI fragment of ID-10) pIBl30 with 4.lkbp EcoRi fragment of XLD-I() pl113l with GINM encoding 5.3kbp Xbal fragment of IC-13 pIB133 with GtfM encoding 4.8klbp EcoRlIXbaI fragment of IC-13 1I1133 with 3.7kbp KnlXbl fragmient of IC-13 1131 Corporation 1131 Corporation Giffard et al., 1991 Giffard et al., 1991 This study This study This study This study This study This study This study WO 96/06173 PCT/AU95/00527 10 Detection of Gtf activity. Gtf activity was routinely detected using a qualitative microtitre reducing sugar test for liberated fructose, outlined in Jacques N.A. (1983). Gtf activity encoded by phagemids was released from E. coli cells by permeabalizing 1 ml of a stationary phase culture. This was achieved by vortexing the cells in the presence of 50 pl 0.1% (wt/vol) SDS and 100 pl chloroform for 20 seconds.

Quantification of Gtf activity utilized [U-glucosyl-1 4

C]-

labelled sucrose. One unit of enzyme activity was defined as the amount of Gtf that catalyzed the incorporation of 1pmol of the glucose moiety of sucrose in 75% (vol/vol) ethanol-insoluble polysaccharide per min.

The assay mix used for the quantification of Gtf activity was scaled up to 8 ml and incubated with 3.2ml of bacteriophage X lysates at 370C for 2h. After the 2h incubation, the assay mix was boiled for a further lh to inactivate the enzyme and the amount of glucan formed (cpm) determined by assaying duplicate 500l aliquots.

After cooling to 37°C, C. gracile endo-(1-6)-a-Dglucanase was added to a final concentration of 500mU/ml and the solution incubated at 370C. Duplicate aliquots (500xl) were removed and assayed for total remaining glucan at varying time intervals over a 5h period. Any reduction in glucan (cpm) during this period was attributed to hydrolysis by the endo-(1-6)-a-D-glucanase.

DNA sequence analysis. DNA sequence determination was carried out on CsC1 purified double-stranded DNA using the Pharmacia T7 sequencing kit according to the manufacturer's instructions. Custom-made oligonucleotide primers (17mers) were used and all sequencing was confirmed in both directions. DNA sequences were assembled and open reading frames (orfs) detected using the IBI-Pustell sequence analysis software version 2.03.

Southern Hybridizations. Chromosomal DNA from S. salivarius ATCC 25975 was extracted and purified as WO 96/06173 PCT/AU95/00527 11 previously described (Giffard et al, 1991). Southern hybridizations were done essentially as outlined by Silhavy et al (1984) and in accordance with standard techniques such as those described in Maniatis et al (1989).

Incorporation into plants. Incorporation of qtfM gene into plants is obtained by standard transgenic techniques. The gtfM gene is obtained from XC-13 or pGSGS01 by PCR. Various constructs are made using PCR primers that either do or do not contain a coding region that adds a vacuolar targeting sequence to the N- or Cterminus of the GtfM protein. These PCR constructs are cloned into a pUC18 based vector containing a Cauliflower Mosaic Virus (CaMV) 35S promoter. By this means the streptococcal promoter is replaced by a plant promoter.

Other methods of incorporating foreign DNA into plants are taught in Australian Patent Application No.

46881/89 by Ciba Geigy Ag. They include the use of Agrobacterium tumefaciens and the leaf disc transformation method and the use of Tobacco Mosaic Virus

(TMV).

WO 96/06173 PCTAU95/00527 12 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Simpson, Christine Lynn Giffard, Philip Morrison Jacques, Nicholas Anthony (ii) TITLE OF INVENTION: Genetic Manipulation of Plants to Increase Stored Carbohydrates (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Griffith Hack Co STREET: Level 8, 168 Walker Street CITY: North Sydney STATE: New South Wales COUNTRY: Australia ZIP: 2060 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: AU PM7643 FILING DATE: 24-AUG-1994 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 61 2 9957 5944 TELEFAX: 61 2 957 6288 TELEX: 26547 WO 96/06173 WO 96/6 173PCT/AU95/00527 13 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 4853 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGAsNISM: Streptococcus salivarius (xi) SEQUENCE DESCRIPTION: SEQ ID 110:1:

CAGAGATTTA.

GAGTAGAGAT

AAACTACACA

GCCITGGCAG

CCTAACGGTG

ACAACTGTTA

GTAAGTCACG

CAAGCACAAA

AGTGGCCAAG

GCTGGGCAAA

TI'GACCAATG

GCCAACCGCA

ATCACAGGGC

CCAAATGTGA

CCGTCTCAAC

GCCTCTAACT

AAGAACGCAG

GTGGATCAAA

GTTTATAACC

ACGGCTGATA

TCAACTGAGA

GTCAACTACC

ATGATGAGCT

TGAAAAGAAG

GACAACAGAA

AGGTTAAGAA

GTGGAAGCCT

ACGGCTTGCA.

CTGAGCAAGC

AAACAAGCTT

CTAGTCCAGT

AGACACAGAC

CAAGTGCTCA

CAGCGCCAGC

ATACTAACAT

CAAACACGCC

CCATTGTAAC

CTAACAAGCC

TGAAGACTAT

CCA'ITGAGCT

GTAAACCTT

AGGCCTATGA

GCTGGTACCG

AAGA'ITATCG

TCAACTATAT

ATGACTTGGC

ATGATTT

AAAAGGATGA

AAACTGGGTA

CCTAGCTCAA

GCAACTGAGT

TAGTGCTCA.A

CCAGGCGGCG

TGCCAGTCAA

TACTGAACAG

CCTATTTGTA

TTGATATAGA,

ACTATTGGGG

GGAAAAGTAG

GAGGATGGGA

GCAAGTGTGT

ACAAGTGCAG

GAAGTGGCAG

GTGTCACAAG

GTCTACTCCA AGTGTGACAG AATrGCCACA CGCGCTGCTG CACGATrACG GCCAGCGGTA TAAACCAAAC GTGACGGTGA GCAGCCAAAT CAACCCAACA GGTCCAACCA AATCAGCCAA CGATGGCAAG CAGTACTATG TGATGGGCGT C TITATTATT GTATCGTGCC GATGCCATTC

ATTTGTCTGA

TGGAAAATAA

TGACCACTCT

AGGCCGATGA

CTGCCAGTCT

CAGCAGTAGC

TCAGCCAGGA

TATCTTCGCA.

GTCAGACATC

AACAAGCAAG

ATAGCACTAT

CGACACCAAA

CAAGTCCAAA

AACCTGTTCA

GTCTTGACTA

TTGAAAATGG

TCGATGAGAC

CAAATAACTC

AACACTTGGA

ATGGGAAGAA

GGCCAGACAA

ATAAGACCTA

AACGAGGCAT

ATATATCATA

GGTACGTT

CTCAATGGTI'

GACGAGCGCA

AGTGACGACA

AACAGCCAGC

GGCAACTGCT

AACTCAATCC

AACTCAAGTA

ACCTAGAGTC

TCGTATCAAT

TGTAACCATr

TGGCACAAGA

ACCAAGTCAA

TAAACCAGTA

CGTCGTGAAA

TGGAGCTATG

TATCTATGCG

TAATTTCTTG

TGGACAGCT

GGTGACACAG

TACGACAGAT

CGAGGAACGT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380

TACGTCAAGT

TCCAAAACAG

TCCACTTG

GAGCCAACAA

AGCTGCTGCC

AAGAGTTTTG

AT1ITTGAAGG

ATGACTIGGT

GGGTTTGGTA

GAAACGGTC

WO 96/06173 WO 96(6 173PCT/AU95/00527 14

ATTGGTCGTG

CAGCCTGGCT

GGAGGCGCTC

CTCATGAACC

AATGGTGGT'

GCAGAGCAAC

CCAAGTGCCA

TTGCAGATTG

GCCATTGCTC

GATACCAAGG

TTCTTGCGTA

AATCGTT=A

AGGGCAATAC

GGAACTCTGA

TGACCT1'CCT

GCACACCGAC

ACGAGTTGCT

TAAACTGGCT

ACTTTGACGG

CTTCTGATTA

ATITGTCAAT

GTGCTCAG7T

AGAGCAATTA

GTGAGCAGAA

CATGATAGTG AAGTGCAAGC ACAGATGGTr TCACC7TCAC GACATCGCGA AGGCTGATAA ATGTI'GACCA ACAAGGATAG GGCCAATACA TGGCTGAAAA CGCATCAAAT ATGTAGCAGG ATCATGTCAT CTGTGCGTTA GAAACACGCA ACCAAGGAAT GCTAATGATC GTCTGGTAGT TTGCTTCTCA GCAAATCGAC GGATTGGTI'C GCTATACGGA GGTCATTCAA CCGTIGAAGT GAAAACCAAG ATGCCCGAAC TCATCAGCAG CTCTTGATTC Grr.AAGACAC CAAGTCAGTA GAGTGGGGAA TCACTTCCTT CACTTGGCTr

GAGTGAGGAC

AAATAATAGT

TAACCAAACG

CTTGGCTAAC

CCACTACATC

TGTTCGTATC

CTrCAAGGAA

CCITGAAGCT

GTCTATCGAC

CCGTGGCAGC

ACACACGCCA

TGTTTTGGCT

TATGGATGAG

AAAGTACACC

TATCACTCGT

ATCACCGTAC

TGGTCAAGAC

TGGTAAGGGG

GCTGGTCCTT

CAATATGGGA

AGGTCTTGCG

CAATCAAGGG

TTCAGG'rrAT

CAAGGCTTCG

ACAAGTCA'T

CACCAACCGT

TGAATTTGCG

AAATGGCTAC

TTCACTCAAA

TGCTGACTGG

TCGTACCAAT

CGCCAGTTGA

AATCTCTI'AG

GCGACAAGTC

GGGACACGCA

GATATCGACA

ATGAACATIG

GATGCGGTGG

AAATACCGTG

TGGTCATACA

AATCCACTAC

TTGGAGCGCG

CGTGATGCCA

AATATCATCA

CTCAAACAGG

CAGTACAATA

GITTTACTACG

TATAATGCCA

ATGAAGGTTA

GCAGAAGAAG

ACAGCCAACC

GCTGCCCALCA

ACCTACCTCA

AACTTGACCT

TTGGCAGTTT

AGCACCAAGA

TACGAAGGCT

GTTrATTGCTC

CCACAGTATG

GCCITCGAGG

GACCTCATGG

G'FrCCAGATC

AGCTACGGTA

TGTCAGACTT

'FrGGTAAAGA

ATGCCAATTC

AATATCATAT

ACTCTAACCC

GTTCTATCCT

ACAATGTGGA

TCGCAGATAA

ACGACCACCA

GTGAAACGCT

TGATTACTAA

ATI'ATATITT

GCAAGCAGAT

CCTI'CGAAAT

TCCCAGCTGC

GGGACCTCTT

TCGATGCCCT

CTAAGCTTAA

CCAACCAGCT

GTCCAGATAT

AAAATCAGGC

AGGATTCTGA

TCACGGCAGA

GGGTGCCAGT

AGGGTGAGCA

TCTCAAACTT

AAAATGCCAA

TGTCTAGCCA

ACCGCTACGA

ATGCCCTTCG

AAATTTACAA

CACCACGTCC

GAAATGACTT

CAGCAATCTT

CATCAAAACA

CCACTTGCAA

AGATCCGC

TGACCGTTCA

AGCTGTTCAG

TGGCAATGAT

TGCGGATCTC

TGAAGCAAAT

ATACAACAAG

TrGACGACT

CTCTCTTAAC

CGTGCGAGCC

TAATCCAAAA

CTACAATGCG

CTATGCCACA

TACCGACGAT

GCTCCGTGCT

TGGCTATGAA

TGGTACTGCT

GAAGTTGGGA

TI'ACCGTCCA

TGTACCAGCT

TGATATTGCT

GGGTGCCTCA

GGTCTI'GAA

CCAAGA=~C

ACTCTTCAAA

AGACGGCACT

TATTGCCATG

TGCCCTTCAC

TCTCCCTGGA

AAATGCTGAA

CCAAGGCAAG

TGAGCGCGTG

1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420

TTCTTGGATT

AGCAAGAACA

GCACLAAGGTA

AAAGAAGTGG

ATCTACAATA

TACGGTGGTG

CTATCATTGA

ACAAATACGG

TCTCAGCCAT

TAACAGCTTC

GCCTCTACGC

CCTI'CCTTGA

AGCTAAAACA CGTACCTTTG TGAATTGAAG GCAAAATACC WO 96/06173 WO 96/6 173PCT/AU95/00527 is CAGA=rCAA ACGGCCGTAA TA CAATG GAAGCAATAT ACCAACCAAT ACTTCAGCGT ATI'ACTGGAA GTGG CCG

TACCTTGCTA

AATGGCAACA

AATGGTCTCC

GATCCTAAAG

GTTCGCTACT

ACCTTCTATT

GACGGACGCA

CAAAATCCTG

CTACAAACCA

CACTTGTCA

CCGTCACGCT

GTTAAAGGTG

AGAATACCTI

TGGTCACAGG

AGCTACGTCA

GGGTTCAGGC

TrGATGGCAA

TTGATGAAAA

CGCGTrACTT

AGAACAAGGC

TTAACGGTAA

CTATCAATAA

ATTGACTACC

CCAAGGTACT

CAAAGCAGGT

TAGGGTTGGA

TATTCAAGTC

TGAGCAGGTC

TGTCCTTCGC

C'I1AACGGA

CGGTCAAATG

GACTGGTATT

CATCCCAGAT

TTGGTATTAC

GCAGTATI'AC

CCAACGTTAC

AATGAGAAAA

GGAGCTCGCT

CAAACCTrCC

GATGATGTCC

GGTGCCAACC

ATI'GATGGCA

CAAGGTAGTG

TITTATGATT

TATCGTGGCC

CAAGCCAAAG

ACAGGAAATC

CTCGATAGCA

TTTGACAATG

TTCCTTGATG

TGGTACTATG

CACTACTATT

TATGATGGTG

TGGGCITACC

GTTTACTATT

AAGAAATGTr

GCACGTGGCT

ATCAATGGTA

TATGTTGGTG

TAATTAATAT

TCACGCAATG

ATGTCCTAC-A

ITCCTAAACA

AATACCTCTC

AGTGGTATTA

AGAAATACT

ATGGTCATGT

TTGCGGGTCC

TCCACGACAT

ACAAGTTCAT

TCGCAGTCAA

ACGGTTATGC

AAGGACGTCA

GTGATAGTGG

TCGATGGCAA

TCAACAATGA

ACTCAGGTCA

'rrAACCAAGA

TTGGTAGCAA

ACTTTGATGC

GCTGGTATTA

AACAACT=A

GTAAACGACT

GTACI=AAA

GTCAGCCAAG

AGATAACGCT

AATGACTGAA

AAITGGTGGC

CTTTGATAAG

CTTCCTAGAC

GTATTATTAC

TCGCCAAGAC

GTATGGCACA

CCGTrITTGCG

CCGATI'IGCG

CGTGACAGGA

GGTTAAGGGA

TGAAATI'GCT

TGGTAAACTG

TTATAGCCAA

GGCCGTAAGC

TGGTCACAAA

TGGTGCTCAA

CCACACAGGT

CTTAACTCA

CITCGACGGT

CTTCTGCGAT

AAT

3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4853 'rrGTGACGGA AAACAACAAG CTCAGGTCAT CAATGGTAAT GTCAAGGGTG CCTGGGCCAA CGGCCGTTAC AACCAATrCA TTCAAATTGC GGCTAACCAA GTAACAGGTC TTCAAAATAT TAACAATAAA GTCAAAGGTA AATTGCTCAC TGTCCAAGGT GAGCAAGTGG TAAACCGCTT TGTCGAAGCT GCTGGCCAAG CAGTGACTGG ACAACAGGTC TCAGGTCGTC AAGITAAAGG ACGTTATGTT GCCAAAACTG GTG-AATI'GAG ACAGCGTCGC WO 96/06173 WO 96/6 173PCT/AU95/00527 16 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1577 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Streptococcus salivarius (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Glu Asn Lys Val Arg Phe Lys Leu His Lys Val Lys Lys Asn Tip 1 5 10 Val Ser Asn Val Ser Ala Pro Gly Thr 145 Glu Thr Asn Thr Gly 225 Lys Thr Leu Gly Thr Ala Thr Val Gln 130 Gln Gln Arg Ile Gly 210 Thr Pro Ile Leu Asp Thr Val Ser Ala 115 Glu Val Ala Ala Thr 195 Pro Arg Val Gly Val Ala Gln Gly Leu Thr Thr Ala Thr Ala Val 100 Ser Gln Thr Gln Ala Gly Arg Pro 165 Ala Asp 180 Ile Thr Asn Thr Pro Asn Gln Pro 245 Thr Thr Gly Lys Gln Gln 55 Val Thr 70 Ala Ser Ser Gln Glu Val Thr Thr 135 Gln Thr 150 Arg Val Ser Thr Ala Ser Pro Lys 215 Val Thr 230 Ser Gln Leu Val 40 Leu Glu Val Glu Ala 120 Glu Ser Leu Ile Gly 200 Pro Ile Pro Ser 25 Glu Ser Gln Ser Ala 105 Val Gln Ala Thr Arg 185 Thr Asn Val Ser Lys 265 met Ala Glu Ala His 90 Thr Ser Val Gln Asn 170 Ile Thr Val Thr Gln 250 Val Asp Asp Ser 75 Glu Ala Ser Ser Ser Ala Asn Pro Thr Gln 235 Pro Ala Leu Glu Thr Gly Thr Ala Gln Thr Ser Gln Ala Gln Thr 125 Gln Gly 140 Thr Pro Ala Pro Ala Asn Asn Val 205 Val Thr 220 Pro Asn Asn Lys Ala Gly Ser Ala Ala Ser Ala Ser Phe Gln Gln Thr 110 Gln Ser Gln Thr Ser Val Ala Ile 175 Arg Asn 190 Thr Ile Ser Pro Gln Pro Pro Val 255 Gly Pro Leu Val Ala Ser Ser Ser Thr 160 Ala Thr Ile Asn Asn 240 Gln Pro Asn Gln Pro Ser Leu Asp Tyr 260 Pro Val Ala Ser Asn Leu Lys 270 WO 96/06173 PCT/AU95/00527 17 Thr Ile Asp Gly Lys 275 Asn Gly 305 Pro Ser Tyr Thr Val 385 Asn Ala Asn Pro His 465 His Thr Leu Glu Gly 545 Asp Glu Ser Thr Ala 290 Ala Asn Lys Arg Glu 370 Thr Lys Glu Thr Gly 450 Leu Ala Gly Leu Gin 530 Asn Asn Lys Ile Lys 610 Ala Met Asn Ser Pro 355 Lys Gin Thr Thr Thr 435 Trp Gin Asn Thr Leu 515 Leu Asp Val Tyr Leu 595 Gly lie Val Ser Phe 340 Lys Asp Val Tyr Val 420 Trp Asn Gly Ser Arg 500 Ala Asn Pro Asp Arg 580 Glu Ala Glu Asp Ile 325 Glu Gin Tyr Asn Thr 405 Gin Leu Ser Gly Asp 485 Lys Asn Trp Ser Ala 565 Val Ala Gin Gin Tyr Tyr 280 Leu Asp Gly 295 Gin Ser Lys 310 Tyr Ala Val His Leu Asp Ile Leu Lys 360 Arg Pro Leu 375 Tyr Leu Asn 390 Thr Asp Met Arg Gly Ile Arg Gin Leu 440 Glu Ser Glu 455 Ala Leu Thr 470 Phe Arg Leu Tyr His Ile Asp Ile Asp 520 Leu His Tyr 535 Ala Asn Phe 550 Asp Leu Leu Ala Asp Asn Trp Ser Tyr 600 Leu Ser Ile 615 Val Glu Asn Gly Arg Pro Tyr Asn 345 Asp Leu Tyr Met Glu 425 Met Asp Phe Met Asp 505 Asn Ile Asp Gin Glu 585 Asn Asp Leu Leu Asn 330 Phe Gly Met Met Ser 410 Glu Ser Asn Leu Asn 490 Arg Ser Met Gly Ile 570 Ala Asp Asn Tyr Tyr 315 Gin Leu Lys Thr Ser 395 Tyr Arg Asp Leu Asn 475 Arg Ser Asn Asn Val 555 Ala Asn His Pro Tyr 300 Arg Ala Thr Asn Trp 380 Gin Asp Ile Phe Leu 460 Asn Thr Asn Pro Ile 540 Arg Ser Ala Gin Leu 620 Val 285 Phe Ala Tyr Ala Trp 365 Trp Gin Leu Gly Ile 445 Val Ser Pro Gly Ala 525 Gly Ile Asp Ile Tyr 605 Arg Val Asp Asp Asp Asp 350 Thr Pro Gly Ala Arg 430 Lys Gly Ala Thr Gly 510 Val Ser Asp Tyr Ala 590 Asn Glu Lys Glu Ala Thr 335 Ser Ala Asp Phe Ala 415 Glu Thr Lys Thr Asn 495 Tyr Gin Ile Ala Phe 575 His Lys Thr Lys Thr Ile 320 Ser Trp Ser Lys Gly 400 Ala Gly Gin Asp Ser 480 Gin Glu Ala Leu Val 560 Lys Leu Asp Leu WO 96/06173 WO 9606173PCT/AU9S/00527 is Leu Thr Thr Phe Leu 625 Val Pro Gin Asp Tyr 705 Ile Arg Giu Ile Gly 785 Ala Leu Val Leu Val 865 Phe Tyr Arg Ser Gin 945 Ile Arg Ala Giy 690 Asn Pro Vai Lys Lys 770 Asn Thr Val Leu 850 Pro Thr Leu Thr Aia 930 Asp Thr Asp Val 675 Phe Aia Ala Tyr Ser 755 Tyr Giu Gin Ala Asn 835 Ser Ala Aia Aia Lys 915 Al a Phe Asn Ala 660 Leu Thr Asp Ala Tyr 740 Pro Val Ile Leu Asn 820 Met Lys Gly Asp Val 900 Ala Leu Val Ser 645 Asn Ala Phe Ile Tyr 725 Gly Tyr Ala Met Gly 805 Arg Giy Ser Leu Asp 885 Trp Ser Asp Lys Arg 630 Leu Tyr Asn Thr Ala 710 Ala Asp Tyr Gly Ser 790 Thr Pro Ala Thr Val 870 Ile Val Ser Ser Thr 950 Lys Ser Aen Asn Ile Ile met 695 Lye Thr Leu Asn Gly 775 Ser Ala Asp Ala Gly 855 Arg Ala Pro Thr Gin 935 Pro Asn Phe Ile 680 Asp Ala Met Phe Ala 760 Gin Vai Glu Met His 840 Leu Tyr Giy Val Lye 920 Val Ser Arg Vai 665 Ser Giu Asp Leu Thr 745 Ile Asp Arg Thr Lye 825 Lys Ala Thr His Gly 905 Lye Ile Gin Tyr Arg 635 Ser Ser 650 Arg Aia Lye Gin Leu Lye Lys Lye 715 Thr Asn 730 Asp Asp Asp Ala Met Lys Tyr Gly 795 Arg Asn 810 Leu Gly Aen Gin Thr Tyr Asp Aen 875 Ser Thr 890 Ala Ser Giy Giu Tyr Giu Tyr Thr 955 Giu His Ile Gin 700 Tyr Lys Giy Leu Val 780 Lys Gin Ala Ala Leu 860 Gin Val Glu Gin Gly 940 Asn Gin Asp Asn 685 Ala Thr Asp Gin Leu 765 Thr Gly Gly Asn Tyr 845 Lye Giy Giu Asn Val 925 Phe Arg Lys Ser 670 Pro Phe Gin Ser Tyr 750 Arg Lye Ala Met Asp 830 Arg Asp Asn Val Gin 910 Phe Ser Val His 655 Giu Lys Glu Tyr Ile 735 Met Ala Leu Giu Leu 815 Arg Pro Ser Leu Ser 895 Asp Giu Asn Ile 640 Thr Val Thr Ile Asn 720 Thr Ala Arg Aen Giu 800 Val Leu Leu Asp Thr 880 Gly Ala Ser Phe Ala 960 Gly Ser Leu Giu Arg Gin Aen Ala Lye Leu Phe Lye Giu Trp Gly 970 Ile Thr Ser Phe Giu Phe 975 WO 96/06173 PCT/AU95/00527 19 Ala Pro Gin Tyr Val Ser Ser Gin Asp Gly Thr Phe Leu Asp Ser Ile 980 985 990 Ile Glu Asn Gly Tyr Ala Phe Glu Asp Arg Tyr Asp Ile Ala Met Ser 995 1000 1005 Lys Asn Asn Lys Tyr Gly Ser Leu Lys Asp Leu Met Asp Ala Leu Arg 1010 1015 1020 Ala Leu His Ala Glu Gly Ile Ser Ala Ile Ala Asp Trp Val Pro Asp 1025 1030 1035 1040 Gin Ile Tyr Asn Leu Pro Gly Lys Glu Val Val Thr Ala Ser Arg Thr 1045 1050 1055 Asn Ser Tyr Gly Thr Pro Arg Pro Asn Ala Glu Ile Tyr Asn Ser Leu 1060 1065 1070 Tyr Ala Ala Lys Thr Arg Thr Phe Gly Asn Asp Phe Gin Gly Lys Tyr 1075 1080 1085 Gly Gly Ala Phe Leu Asp Glu Leu Lys Ala Lys Tyr Pro Ala Ile Phe 1090 1095 1100 Glu Arg Val Gin Ile Ser Asn Gly Arg Lys Leu Thr Thr Asn Glu Lys 1105 1110 1115 1120 Ile Thr Gin Trp Ser Ala Lys Tyr Phe Asn Gly Ser Asn Ile Gin Gly 1125 1130 1135 Thr Gly Ala Arg Tyr Val Leu Gin Asp Asn Ala Thr Asn Gin Tyr Phe 1140 1145 1150 Ser Val Lys Ala Gly Gin Thr Phe Leu Pro Lys Gin Met Thr Glu Ile 1155 1160 1165 Thr Gly Ser Gly Phe Arg Arg Val Gly Asp Asp Val Gin Tyr Leu Ser 1170 1175 1180 Ile Gly Gly Tyr Leu Ala Lys Asn Thr Phe Ile Gln Val Gly Ala Asn 1185 1190 1195 1200 Gin Trp Tyr Tyr Phe Asp Lys Asn Gly Asn Met Val Thr Gly Glu Gin 1205 1210 1215 Val Ile Asp Gly Lys Lys Tyr Phe Phe Leu Asp Asn Gly Leu Gin Leu 1220 1225 1230 Arg His Val Leu Arg Gin Gly Ser Asp Gly His Val Tyr Tyr Tyr Asp 1235 1240 1245 Pro Lys Gly Val Gin Ala Phe Asn Gly Phe Tyr Asp Phe Ala Gly Pro 1250 1255 1260 Arg Gin Asp Val Arg Tyr Phe Asp Gly Asn Gly Gin Met Tyr Arg Gly 1265 1270 1275 1280 Leu His Asp Met Tyr Gly Thr Thr Phe Tyr Phe Asp Glu Lys Thr Gly 1285 1290 1295 Ile Gin Ala Lys Asp Lys Phe Ile Arg Phe Ala Asp Gly Arg Thr Arg 1300 1305 1310 Tyr Phe Ile Pro Asp Thr Gly Asn Leu Ala Val Asn Arg Phe Ala Gin 1315 1320 1325 &)fq'i A ITflCIflC-107 WO 96/06173 Asn Pro Giu Asn Lys Ala Ti-p Tyr Tyr LeU ASP Ser Asn Gly Tyr Ala 1330 1335 1340 Val Thr Gly Leu Gin Thr Ile Asn Gly Lys Gin Tyr Tyr Phe Asp Asn 1345 1350 1355 1360 Giu Gly Arg Gin Val Lys Gly His Phe Val Th- Ile Asn Asn Gin Arg 1365 1370 1375 Tyr Phe Leu Asp Giy Asp Ser Giy Giu Ile Ala Pro Ser Arg Phe Val 1380 1385 1390 Thr Giu Asn Asn Lys Ti-p Tyr Tyr Val Asp Gly Asn Gly Lys Leu Val 1395 1400 1405 Lys Gly Ala Gin Val Ile Asn Gly Asn His Tyr Tyr Phe Asn Asn Asp 1410 1415 1420 Tyr Ser Gin Vai Lys Gly Ala Ti-p Ala Asn Gly Arg Tyr Tyr Asp Gly 1425 1430 1435 1440 Asp Ser Giy Gin Ala Val Ser Asn Gin Phe Ile Gin Ile Ala Ala Asn 1445 1450 1455 Gin Ti-p Ala Tyr Leu Asn Gin Asp Gly His Lys Val Thr Gly Leu Gin 1460 1465 1470 Asn Ile Asn Asn Lys Val Tyr Tyr Phe Gly Ser Asn Gly Ala Gin Val 1475 1480 1485 Lys Giy Lys Leu Leu Thr Val Gin Gly Lys Lys Cys Tyr Phe Asp Ala 1490 1495 1500 His Thr Gly Giu Gin Val Val Asn .Arg Phe Val Giu Ala Ala Arg Gly 1505 1510 1515 1520 Cys Ti-p Tyr Tyr Phe Asn Ser Ala Gly Gin Ala Val Thi- Gly Gin Gin 1525 1530 1535 Val Ile Asn Gly Lys Gin Leu Tyr Phe Asp Gly Ser Gly Arg Gin Val 1540 1545 1550 Lys Gly Arg Tyr Val Tyr Val Gly Gly Lys Arg Leu Phe Cys Asp Ala 1555 1560 1565 Lys Thi- Gly Glu Leu Arg Gin Arg Arg 1570 1575 WO 96/06173 PCT/AU95/00527 21 LIST OF REFERENCES 1. Radojevic et al. 1994 Aust J Agric Res 45, 901-12.

2. Jacques NA, Giffard PM, "The Glycosyltransferases of Oral Streptococci" Todays Life Science 1991; 3: 40-6.

3. Walker GJ, Jacques NA, "Polysaccharides of Oral Streptococci" In: Reizer J, Peterkofsky A, Eds.

"Sugar Transport and Metabolism in Gram-Positive Bacteria". Chichester: Ellis Horwood, 1987; 39-68.

4. Gilmore KS, Russell RRB, Ferretti JJ, "Anaylsis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesises soluble glucans". Infect Immun 1990; 58: 2452-8.

Giffard PM, Simpson CL, Milward CP, Jacques NA, "Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC25975". J. Gen. Microbiol. 1991; 137:2577-93.

6. Giffard PM, Allen DM, Milward CP, Simpson CL, Jacques NA, "Sequence of the GtfK of Streptococcus salivarius ATCC25975 and the evolution of the gtf genes of oral streptococci". J Gen Microbiol 1993; 139:1511-22.

7. Pitty LS, Giffard PM, Gilpin ML, Russell RRB and Jacques NA, 1989. "Cloning and expression of glycosyltransferase C gene (gtfC) from Streptococcus mutans LM7. Infect Immun 55: 2176-2182.

8. Silliary TS, Berman ML, and Enquist LW, 1984.

"Experiments with Gene fusions". Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

WO 96/06173 PCT/AU95/00527 22 9. Jacques N.A. 1983. Membrane perturbation by cerulenin modulates glucosyltransferase secretion and acetate uptake by Streptococcus salivarius. J.

Gen. Micriobial. 129 3293-3302.

10. Maniatis T, Fritsch EF, and Sambrook J. (1989).

"Molecular Cloning; a laboratory manual. Second edition. Cold Spring Harbor Laboratory Press, N.Y.

Claims (23)

1. An isolated DNA comprising a sequence shown in SEQ ID NO:1.

2. A DNA which consists of the sequence shown in SEQ ID NO: 1.

3. A DNA which comprises a portion of the sequence shown in SEQ ID NO:1, and which codes for a primer independent glucosyltransferase which catalyses the formation of soluble glucans from sucrose.

4. A DNA which consists of a portion of the sequence shown in SEQ ID NO:1, and which codes for a primer independent glucosyltransferase which catalyses the formation of soluble glucans from sucrose. A DNA which comprises a sequence which has greater than 70% identity with the sequence shown in SEQ ID NO:1, wherein the DNA encodes a glucosyltransferase which catalyses the formation of soluble glucans from sucrose.

6. A DNA which consists of a sequence which has greater 25 than 70% identity with the sequence shown in SEQ ID NO:1, wherein the DNA encodes a glucosyltransferase which catalyses the formation of soluble glucans from sucrose.

7. An isolated protein which consists of the sequence shown in SEQ ID NO:2.

8. A protein which comprises the sequence shown in SEQ ID NO:2. JASpeci\200 299\250 299\29722.doc I

9. A protein which comprises a portion of the sequence shown in SEQ ID NO:2, wherein the protein catalyses the formation of soluble glucans from sucrose.

10. An protein which consists of a portion of the sequence shown in SEQ ID NO:2, wherein the protein catalyses the formation of soluble glucans from sucrose.

11. A protein which comprises a sequence which has greater than 70% identity with the sequence shown in SEQ ID NO:2, wherein the protein catalyses the formation of soluble glucans from sucrose.

12. A protein which consists of a sequence which has greater than 70% identity with the sequence shown in SEQ ID NO:2, wherein the protein catalyses the formation of soluble glucans from sucrose.

13. A plant which contains bacterial DNA which codes for a primer independent glucosyltransferase which catalyzes the formation of soluble glucans from sucrose.

14. A plant which contains a DNA according to any one of claims 1 to 6. 25 A plant which contains a primer independent glucosyltransferase which catalyses the formation of S. soluble glucans from sucrose in the plant.

16. A plant which contains a protein according to any one of claims 7 to 12.

17. A plant according to any one of claims 13 to 16, wherein the plant is a pasture plant. JASpeci\200 299\250 299\29722.doc

18. A method of increasing the level of stored carbohydrate in a plant with low levels of carbohydrate, the method comprising inserting DNA comprising a sequence which encodes a protein according to any one of claims 7 to 10, into the plant so that the plant expresses the protein, wherein the protein increases the level of stored carbohydrate in the plant.

19. A method of increasing the level of stored carbohydrate in a plant with low levels of carbohydrate, the method comprising inserting DNA comprising a sequence which encodes a protein according to claim 11 or claim 12, into the plant so that the plant expresses the protein, wherein the protein increases the level of stored carbohydrate in the plant. A method of preventing degradation of stored carbohydrate during senescence in a plant, the method comprising inserting DNA comprising a sequence which encodes a protein according to any one of claims 7 to into the plant so that the plant expresses the protein, wherein the protein prevents degradation of stored carbohydrate in the plant during senescence. I ar *aaa

21. A method of preventing degradation of stored carbohydrate during senescence in a plant, the method comprising inserting DNA comprising a sequence which encodes a protein according to claim 11 or claim 12, into the plant so that the plant expresses the protein, wherein the protein prevents degradation of stored carbohydrate in the plant during senescence.

22. The method of any one of claims 18 to 21 wherein the plant is a pasture plant. J:\Speci\200 299\250 299\29722.doc

23. The plasmid pGSG501 containing XC-13 DNA.

24. The plasmid pGSG502 containing XC-13 DNA.

25. Dextran, when produced by a plant according to any one of claims 13 to 17.

26. A pasture plant according to any one of claims 13 to 17, the plant being substantially as herein defined with reference to the Examples. DATED this 5th day of January 1999 NICHOLAS ANTHONY JACQUES, CHRISTINE LYNN SIMPSON and PHILIP MORRISON GIFFARD by their Patent Attorneys GRIFFITH HACK C. C*e* e J:\Speci\200 299\250 299\29722.doc