GENETIC MANIPULATION OF PLANTS TO INCREASE STORED CARBOHYDRATES

Abstract: 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 through production of a glycosyl-transferase which catalyses the formation of soluble glucans. The invention also relates to the genetic constructs used to produce the engineered plants and the method of producing the engineered plants.

Description: "GeneticManipulationofPlants 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 non- structurally 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 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. Glucosvltransfβrases of Streptococcus aalivarius It is known that many strains of Streptococcus sal±varius and Streptococcus produce extracellular α-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 (α- ( 1→ ~ ) -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 gtf 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. 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". £ . 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 Gtf produce enzymes which synthesize insoluble glucans. GtfJ is a primer-dependent enzyme producing essentially a linear o.(1→3)-glucan while GtfL is a primer-independent enzyme that synthesizes a glucan containing 50% Q;-(1→3)- and 50% α- (1→6) -linked glucosyl residues. In contrast, the αtfK and α fM genes code for enzymes which produce a soluble glucan which possess α- (1→6) -linked glucosyl residues. GtfK is primer stimulated while GtfM is primer independent. DESCRIPTION OF THE IMVEMTION Up until now, a σtf gene in £ι. 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. 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 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 70% 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. 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 (e.g. so called 'health bars' ) ; 2) in pharmaceutical preparations again as a binder; and 3) in medical preparations to increase antigenic activity. 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. FTT 1 ? 1 The general strategy adopted to isolate a gene from ϋ_ salivarius encoding a Gtf which produces a primer independent and soluble glucan is as follows: A λ gene bank containing ^ salivarius DNA was prepared. Positive clones were detected by using an E. coli strain grown on agar containing sucrose. £j. coli which contained σtf DNA from _. salivarius could convert the sucrose in the medium into a polymer which resulted in opaque colonies. These opaque colonies were then picked and the _. salivarius DNA excised and subjected to restriction mapping to ascertain whether the DNA was from a previously described £«_ 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-λC-13 was found to contain a novel crtf 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°C, supplemented with ampicillin (100 μg ml "1 ) , isopropylthiogalactoside (IPTG) (ImM) , or 5-bromo-4-chloro-3-indolyl- / 3-D-galactoside (X-gal) (100 μg ml "1 ) as appropriate. Cultures of £___. salivarius were grown at 37°C in semi-defined medium (SDM) containing 25 mM glucose supplemented with 0.005μl Tween 80 ml "1 where appropriate. Bactβriophagβ and p age ids. All genetic constructs, excluding sequencing subclones, are listed in Table l. Bacteriophage-λ derivatives were grown either as 20 ml or 1 L-liquid lysates using ϋ . 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-λ gene bank of S___ salivarius ATCC 25975 (Pitty et al, 1989) was screened by detecting plaques on a lawn of J _ coli LE392 grown at 37°C on minimal agar medium containing 0.2% glucose and 50 μg ml "1 methionine as well as 1% (wt/vol) sucrose with or without 0.02% (wt/vol) dextran T-10. Potential Gtf clones were detected by their opacity including λ C-13 containing the gtf 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 λC-13 exhibited a unique EcoRl restriction pattern and Gtf activity. A restriction map of λC-13 was constructed using double restriction digests. The Gtf gene encoded by λC-13 (GtfM) was located on an 8.3 kbp insert (see figure 1) . The 5.3 kbp Xbal fragment from λC-13 was subcloned into pIBI31 (pGSG501; see Table 1) and was positive for Gtf activity as was the 4.8 kbp Xbal/EcoRl from λC-13 subcloned into pIBI31 (pGSG502; see Table 1). Table 1. Vncterial Strains, Phnges and P ngciiiids Bacteria, Pliage or Pliagemid Description Source or reference Bacterium: Streptococcus salivarius ATCC 25975 ATCC (Hamilton, 1967). Esclierichia coli LE392 F- el4 ( crΛ) sdR514 (r κ m κ ) supE44 supF58 Murray et al., 1977 lacYI or Δ(laclZY)6 galK2 galT22 etDl tφR55 Esclieric ia coli NM522 F7.ιc/*Δ(/.ιcZ) M15 roΛ *B*lsupE t i Δ(lac-proAB) Gough and Murray, 1983 {lιsdMS-mcrB)5 (r κ m κ McrBC ) Bacteriop age: λL47.1 Loenen and Bram ar, 1980 λA-8 XL47.1 with GlfJ encoding 8.5khp £<ιι.3A partial fragment Pitty et al., 1989 of S. salivarius ATCC 25975 λA-33 XL47.1 with GtfK encoding 9.6kbp 5«ι.3A partial fragment Pitty et al., 1989 of S. salivarius ATCC 25975 λC-13 λl_47.1 with 8.3kbp GtlTvI encoding 5πκ3A partial fragment This study I of S. salivarius ATCC 25975 λD-10 XL47.1 with l lkbp GtfL encoding _SYπ.3Λ partial fragment This study I of S. salivarius ATCC 25975 λD-40 XL47.1 with partial fragment of S. salivarius ATCC 25975 This study isolated from sucrose-containing medium Pliagemid: pIB130 Ap r , fl origin replication, (--galactυsidase, IBI Corporation T3 and T7 polymerase promoters plB131 Ap r , fl origin replication, P-galactυsidase, IBI Corporation T3 and Υl polymerase promoters pGSGlϋl (pGSlϋl) plDI3U with GlfJ encoding 6.8khp Sacl/Daml ll fragment of λA-8 Giffard et al., 1991 pGSG201 (pGS201) plBI30 with GtfK encoding 7.3kbp BgllUBaml ll fragment of λA-33 Giffard et al., 1991 pGSG401 plBI30 with GtfL encoding 6.2 bp flri..iI II/M.-il fragment of λD-10 This study pGSG402 pIBI31 with r..2kbp -9.ι..ιl II/Λto.-l fragment of λD-10 This study pGSG403 plBI30 with 4.8kbp Ecol fragment of λD-10 This study pGSG404 plBI30 with 4.1kbp EcolU fragment or λD-10 This study pGSG501 plBI31 with GlfM encoding 5.3kbp _V_.«l fragment of λC-13 This study pGSG502 plBI31 with GtfM encoding 4.8kbp EcoR\/Xbal fragment of λC-13 This study pGSG503 plBI31 with 3.7kbp Kpn\IXba\ fragment of λC-13 This study 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 μl 0.1% (wt/vol) SDS and 100 μl chloroform for 20 seconds. Quantification of Gtf activity utilized [U-glucosyl- 14 C] - labelled sucrose. One unit of enzyme activity was defined as the amount of Gtf that catalyzed the incorporation of lμmol 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 λ lysates at 37°C 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 500μl aliquots. After cooling to 37°C, C. gracile endo- (l-»6) -α-D- glucanase was added to a final concentration of 500mU/ml and the solution incubated at 37°C. Duplicate aliquots (500μl) 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- (l-*6) -α-D-glucanase. DNA sequence analysis. DNA sequence determination was carried out on CsCl 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 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 gtfM gene into plants is obtained by standard transgenic techniques. The αtfM gene is obtained from λC-13 or pGSG501 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 C- terminus 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 Aqrobacterium tumefaciens and the leaf disc transformation method and the use of Tobacco Mosaic Virus (TMV) . SEQUENCE LISTING (1) GENERAL INFORMATION: (i) 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: (A) ADDRESSEE: Griffith Hack & Co (B) STREET: Level 8, 168 Walker Street (C) CITY: North Sydney (D) STATE: New South Wales (E) COUNTRY: Australia (F) ZIP: 2060 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: WO (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: AU PM7643 (B) FILING DATE: 24-AUG-1994 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 61 2 9957 5944 (B) TELEFAX: 61 2 957 6288 (C) TELEX: 26547 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4853 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Streptococcus salivarius (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CAGAGATTTA TGAAAAGAAG ATGATTTTTT CCTATTTGTA ATTTGTCTGA ATATATCATA 60 GAGTAGAGAT GACAACAGAA AAAAGGATGA TTGATATAGA TGGAAAATAA GGTACGTTTT 120 AAACTACACA AGGTTAAGAA AAACTGGGTA ACTATTGGGG TGACCACTCT CTCAATGGTT 180 GCCTTGGCAG GTGGAAGCCT CCTAGCTCAA GGAAAAGTAG AGGCCGATGA GACGAGCGCA 240 CCTAACGGTG ACGGCTTGCA GCAACTGAGT GAGGATGGGA CTGCCAGTCT AGTGACGACA 300 ACAACTGTTA CTGAGCAAGC TAGTGCTCAA GCAAGTGTGT CAGCAGTAGC AACAGCCAGC 360 GTAAGTCACG AAACAAGCTT CCAGGCGGCG ACAAGTGCAG TCAGCCAGGA GGCAACTGCT 420 CAAGCACAAA CTAGTCCAGT TGCCAGTCAA GAAGTGGCAG TATCTTCGCA AACTCAATCC 480 AGTGGCCAAG AGACACAGAC TACTGAACAG GTGTCACAAG GTCAGACATC AACTCAAGTA 540 GCTGGGCAAA CAAGTGCTCA GTCTACTCCA AGTGTGACAG AACAAGCAAG ACCTAGAGTC 600 TTGACCAATG CAGCGCCAGC AATTGCCACA CGCGCTGCTG ATAGCACTAT TCGTATCAAT 660 GCCAACCGCA ATACTAACAT CACGATTACG GCCAGCGGTA CGACACCAAA TGTAACCATT 720 ATCACAGGGC CAAACACGCC TAAACCAAAC GTGACGGTGA CAAGTCCAAA TGGCACAAGA 780 CCAAATGTGA CCATTGTAAC GCAGCCAAAT CAACCCAACA AACCTGTTCA ACCAAGTCAA 840 CCGTCTCAAC CTAACAAGCC GGTCCAACCA AATCAGCCAA GTCTTGACTA TAAACCAGTA 900 GCCTCTAACT TGAAGACTAT CGATGGCAAG CAGTACTATG TTGAAAATGG CGTCGTGAAA 960 AAGAACGCAG CCATTGAGCT TGATGGGCGT CTTTATTATT TCGATGAGAC TGGAGCTATG 1020 GTGGATCAAA GTAAACCTTT GTATCGTGCC GATGCCATTC CAAATAACTC TATCTATGCG 1080 GTTTATAACC AGGCCTATGA TACGTCAAGT AAGAGTTTTG AACACTTGGA TAATTTCTTG 1140 ACGGCTGATA GCTGGTACCG TCCAAAACAG ATTTTGAAGG ATGGGAAGAA TTGGACAGCT 1200 TCAACTGAGA AAGATTATCG TCCACTTTTG ATGACTTGGT GGCCAGACAA GGTGACACAG 1260 GTCAACTACC TCAACTATAT GAGCCAACAA GGGTTTGGTA ATAAGACCTA TACGACAGAT 1320 ATGATGAGCT ATGACTTGGC AGCTGCTGCC GAAACGGTTC AACGAGGCAT CGAGGAACGT 1380 ATTGGTCGTG AGGGCAATAC CACTTGGCTT CGCCAGTTGA TGTCAGACTT CATCAAAACA 1440 CAGCCTGGCT GGAACTCTGA GAGTGAGGAC AATCTCTTAG TTGGTAAAGA CCACTTGCAA 1500 GGAGGCGCTC TGACCTTCCT AAATAATAGT GCGACAAGTC ATGCCAATTC AGATTTCCGC 1560 CTCATGAACC GCACACCGAC TAACCAAACG GGGACACGCA AATATCATAT TGACCGTTCA 1620 AATGGTGGTT ACGAGTTGCT CTTGGCTAAC GATATCGACA ACTCTAACCC AGCTGTTCAG 1680 GCAGAGCAAC TAAACTGGCT CCACTACATC ATGAACATTG GTTCTATCCT TGGCAATGAT 1740 CCAAGTGCCA ACTTTGACGG TGTTCGTATC GATGCGGTGG ACAATGTGGA TGCGGATCTC 1800 TTGCAGATTG CTTCTGATTA CTTCAAGGAA AAATACCGTG TCGCAGATAA TGAAGCAAAT 1860 GCCATTGCTC ATTTGTCAAT CCTTGAAGCT TGGTCATACA ACGACCACCA ATACAACAAG 1920 GATACCAAGG GTGCTCAGTT GTCTATCGAC AATCCACTAC GTGAAACGCT TTTGACGACT 1980 TTCTTGCGTA AGAGCAATTA CCGTGGCAGC TTGGAGCGCG TGATTACTAA CTCTCTTAAC 2040 AATCGTTCAA GTGAGCAGAA ACACACGCCA CGTGATGCCA ATTATATTTT CGTGCGAGCC 2100 CATGATAGTG AAGTGCAAGC TGTTTTGGCT AATATCATCA GCAAGCAGAT TAATCCAAAA 2160 ACAGATGGTT TCACCTTCAC TATGGATGAG CTCAAACAGG CCTTCGAAAT CTACAATGCG 2220 GACATCGCGA AGGCTGATAA AAAGTACACC CAGTACAATA TCCCAGCTGC CTATGCCACA 2280 ATGTTGACCA ACAAGGATAG TATCACTCGT GTTTACTACG GGGACCTCTT TACCGACGAT 2340 GGCCAATACA TGGCTGAAAA ATCACCGTAC TATAATGCCA TCGATGCCCT GCTCCGTGCT 2400 CGCATCAAAT ATGTAGCAGG TGGTCAAGAC ATGAAGGTTA CTAAGCTTAA TGGCTATGAA 2460 ATCATGTCAT CTGTGCGTTA TGGTAAGGGG GCAGAAGAAG CCAACCAGCT TGGTACTGCT 2520 GAAACACGCA ACCAAGGAAT GCTGGTCCTT ACAGCCAACC GTCCAGATAT GAAGTTGGGA 2580 GCTAATGATC GTCTGGTAGT CAATATGGGA GCTGCCCACA AAAATCAGGC TTACCGTCCA 2640 TTGCTTCTCA GCAAATCGAC AGGTCTTGCG ACCTACCTCA AGGATTCTGA TGTACCAGCT 2700 GGATTGGTTC GCTATACGGA CAATCAAGGG AACTTGACCT TCACGGCAGA TGATATTGCT 2760 GGTCATTCAA CCGTTGAAGT TTCAGGTTAT TTGGCAGTTT GGGTGCCAGT GGGTGCCTCA 2820 GAAAACCAAG ATGCCCGAAC CAAGGCTTCG AGCACCAAGA AGGGTGAGCA GGTCTTTGAA 2880 TCATCAGCAG CTCTTGATTC ACAAGTCATT TACGAAGGCT TCTCAAACTT CCAAGATTTC 2940 GTTAAGACAC CAAGTCAGTA CACCAACCGT GTTATTGCTC AAAATGCCAA ACTCTTCAAA 3000 GAGTGGGGAA TCACTTCCTT TGAATTTGCG CCACAGTATG TGTCTAGCCA AGACGGCACT 3060 TTCTTGGATT CTATCATTGA AAATGGCTAC GCCTTCGAGG ACCGCTACGA TATTGCCATG 3120 AGCAAGAACA ACAAATACGG TTCACTCAAA GACCTCATGG ATGCCCTTCG TGCCCTTCAC 3180 GCAGAAGGTA TCTCAGCCAT TGCTGACTGG GTTCCAGATC AAATTTACAA TCTCCCTGGA 3240 AAAGAAGTGG TAACAGCTTC TCGTACCAAT AGCTACGGTA CACCACGTCC AAATGCTGAA 3300 ATCTACAATA GCCTCTACGC AGCTAAAACA CGTACCTTTG GAAATGACTT CCAAGGCAAG 3360 TACGGTGGTG CCTTCCTTGA TGAATTGAAG GCAAAATACC CAGCAATCTT TGAGCGCGTG 3420 CAGATTTCAA ACGGCCGTAA ATTGACTACC AATGAGAAAA TCACGCAATG GTCAGCCAAG 3480 TATTTCAATG GAAGCAATAT CCAAGGTACT GGAGCTCGCT ATGTCCTACA AGATAACGCT 3540 ACCAACCAAT ACTTCAGCGT CAAAGCAGGT CAAACCTTCC TTCCTAAACA AATGACTGAA 3600 ATTACTGGAA GTGGTTTCCG TAGGGTTGGA GATGATGTCC AATACCTCTC AATTGGTGGC 3660 TACCTTGCTA AGAATACCTT TATTCAAGTC GGTGCCAACC AGTGGTATTA CTTTGATAAG 3720 AATGGCAACA TGGTCACAGG TGAGCAGGTC ATTGATGGCA AGAAATACTT CTTCCTAGAC 3780 AATGGTCTCC AGCTACGTCA TGTCCTTCGC CAAGGTAGTG ATGGTCATGT GTATTATTAC 3840 GATCCTAAAG GGGTTCAGGC CTTTAACGGA TTTTATGATT TTGCGGGTCC TCGCCAAGAC 3900 GTTCGCTACT TTGATGGCAA CGGTCAAATG TATCGTGGCC TCCACGACAT GTATGGCACA 3960 ACCTTCTATT TTGATGAAAA GACTGGTATT CAAGCCAAAG ACAAGTTCAT CCGTTTTGCG 4020 GACGGACGCA CGCGTTACTT CATCCCAGAT ACAGGAAATC TCGCAGTCAA CCGATTTGCG 4080 CAAAATCCTG AGAACAAGGC TTGGTATTAC CTCGATAGCA ACGGTTATGC CGTGACAGGA 4140 CTACAAACCA TTAACGGTAA GCAGTATTAC TTTGACAATG AAGGACGTCA GGTTAAGGGA 4200 CACTTTGTCA CTATCAATAA CCAACGTTAC TTCCTTGATG GTGATAGTGG TGAAATTGCT 4260 CCGTCACGCT TTGTGACGGA AAACAACAAG TGGTACTATG TCGATGGCAA TGGTAAACTG 4320 GTTAAAGGTG CTCAGGTCAT CAATGGTAAT CACTACTATT TCAACAATGA TTATAGCCAA 4380 GTCAAGGGTG CCTGGGCCAA CGGCCGTTAC TATGATGGTG ACTCAGGTCA GGCCGTAAGC 4440 AACCAATTCA TTCAAATTGC GGCTAACCAA TGGGCTTACC TTAACCAAGA TGGTCACAAA 4500 GTAACAGGTC TTCAAAATAT TAACAATAAA GTTTACTATT TTGGTAGCAA TGGTGCTCAA 4560 GTCAAAGGTA AATTGCTCAC TGTCCAAGGT AAGAAATGTT ACTTTGATGC CCACACAGGT 4620 GAGCAAGTGG TAAACCGCTT TGTCGAAGCT GCACGTGGCT GCTGGTATTA CTTTAACTCA 4680 GCTGGCCAAG CAGTGACTGG ACAACAGGTC ATCAATGGTA AACAACTTTA CTTCGACGGT 4740 TCAGGTCGTC AAGTTAAAGG ACGTTATGTT TATGTTGGTG GTAAACGACT CTTCTGCGAT 4800 GCCAAAACTG GTGAATTGAG ACAGCGTCGC TAATTAATAT GTACTTTAAA AAT 4853 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1577 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) 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 Trp 1 5 10 15 Val Thr He Gly Val Thr Thr Leu Ser Met Val Ala Leu Ala Gly Gly 20 25 30 Ser Leu Leu Ala Gin Gly Lys Val Glu Ala Asp Glu Thr Ser Ala Pro 35 40 45 Aβn Gly Asp Gly Leu Gin Gin Leu Ser Glu Asp Gly Thr Ala Ser Leu 50 55 60 Val Thr Thr Thr Thr Val Thr Glu Gin Ala Ser Ala Gin Ala Ser Val 65 70 75 80 Ser Ala Val Ala Thr Ala Ser Val Ser His Glu Thr Ser Phe Gin Ala 85 90 95 Ala Thr Ser Ala Val Ser Gin Glu Ala Thr Ala Gin Ala Gin Thr Ser 100 105 110 Pro Val Ala Ser Gin Glu Val Ala Val Ser Ser Gin Thr Gin Ser Ser 115 120 125 Gly Gin Glu Thr Gin Thr Thr Glu Gin Val Ser Gin Gly Gin Thr Ser 130 135 140 Thr Gin Val Ala Gly Gin Thr Ser Ala Gin Ser Thr Pro Ser Val Thr 145 150 155 160 Glu Gin Ala Arg Pro Arg Val Leu Thr Asn Ala Ala Pro Ala He Ala 165 170 175 Thr Arg Ala Ala Asp Ser Thr He Arg He Asn Ala Asn Arg Asn Thr 180 185 190 Asn He Thr He Thr Ala Ser Gly Thr Thr Pro Asn Val Thr He He 195 200 205 Thr Gly Pro Asn Thr Pro Lys Pro Asn Val Thr Val Thr Ser Pro Asn 210 215 220 Gly Thr Arg Pro Asn Val Thr He Val Thr Gin Pro Asn Gin Pro Asn 225 230 235 240 Lys Pro Val Gin Pro Ser Gin Pro Ser Gin Pro Asn Lys Pro Val Gin 245 250 255 Pro Asn Gin Pro Ser Leu Asp Tyr Lys Pro Val Ala Ser Asn Leu Lys 260 265 270 Thr He Asp Gly Lys Gin Tyr Tyr Val Glu Asn Gly Val Val Lys Lys 275 280 285 Asn Ala Ala He Glu Leu Asp Gly Arg Leu Tyr Tyr Phe Asp Glu Thr 290 295 300 Gly Ala Met Val Asp Gin Ser Lys Pro Leu Tyr Arg Ala Asp Ala He 305 310 315 320 Pro Asn Asn Ser He Tyr Ala Val Tyr Asn Gin Ala Tyr Asp Thr Ser 325 330 335 Ser Lys Ser Phe Glu His Leu Asp Asn Phe Leu Thr Ala Asp Ser Trp 340 345 350 Tyr Arg Pro Lys Gin He Leu Lys Asp Gly Lys Asn Trp Thr Ala Ser 355 360 365 Thr Glu Lys Asp Tyr Arg Pro Leu Leu Met Thr Trp Trp Pro Asp Lys 370 375 380 Val Thr Gin Val Asn Tyr Leu Asn Tyr Met Ser Gin Gin Gly Phe Gly 385 390 395 400 Asn Lys Thr Tyr Thr Thr Asp Met Met Ser Tyr Asp Leu Ala Ala Ala 405 410 415 Ala Glu Thr Val Gin Arg Gly He Glu Glu Arg He Gly Arg Glu Gly 420 425 430 Asn Thr Thr Trp Leu Arg Gin Leu Met Ser Asp Phe He Lys Thr Gin 435 440 445 Pro Gly Trp Asn Ser Glu Ser Glu Asp Asn Leu Leu Val Gly Lys Asp 450 455 460 His Leu Gin Gly Gly Ala Leu Thr Phe Leu Asn Asn Ser Ala Thr Ser 465 470 475 480 His Ala Asn Ser Asp Phe Arg Leu Met Asn Arg Thr Pro Thr Aβn Gin 485 490 495 Thr Gly Thr Arg Lys Tyr His He Asp Arg Ser Asn Gly Gly Tyr Glu 500 505 510 Leu Leu Leu Ala Asn Asp He Asp Asn Ser Asn Pro Ala Val G n Ala 515 520 525 Glu Gin Leu Asn Trp Leu His Tyr He Met Asn He Gly Ser He Leu 530 535 540 Gly Asn Asp Pro Ser Ala Asn Phe Asp Gly Val Arg He Asp Ala Val 545 550 555 560 Asp Asn Val Asp Ala Asp Leu Leu Gin He Ala Ser Asp Tyr Phe Lys 565 570 575 Glu Lys Tyr Arg Val Ala Asp Asn Glu Ala Asn Ala He Ala His Leu 580 585 590 Ser He Leu Glu Ala Trp Ser Tyr Asn Asp His Gin Tyr Asn Lys Asp 595 600 605 Thr Lys Gly Ala Gin Leu Ser He Asp Asn Pro Leu Arg Glu Thr Leu 610 615 620 Leu Thr Thr Phe Leu Arg Lys Ser Asn Tyr Arg Gly Ser Leu Glu Arg 625 630 635 640 Val He Thr Asn Ser Leu Asn Asn Arg Ser Ser Glu Gin Lys His Thr 645 650 655 Pro Arg Asp Ala Asn Tyr He Phe Val Arg Ala Hie Asp Ser Glu Val 660 665 670 Gin Ala Val Leu Ala Asn He He Ser Lys Gin He Asn Pro Lys Thr 675 680 685 Asp Gly Phe Thr Phe Thr Met Asp Glu Leu Lys Gin Ala Phe Glu He 690 695 700 Tyr Asn Ala Asp He Ala Lys Ala Asp Lys Lys Tyr Thr Gin Tyr Asn 705 710 715 720 He Pro Ala Ala Tyr Ala Thr Met Leu Thr Asn Lys Asp Ser He Thr 725 730 735 Arg Val Tyr Tyr Gly Asp Leu Phe Thr Asp Asp Gly Gin Tyr Met Ala 740 745 750 Glu Lys Ser Pro Tyr Tyr Asn Ala He Asp Ala Leu Leu Arg Ala Arg 755 760 765 He Lys Tyr Val Ala Gly Gly Gin Asp Met Lys Val Thr Lye Leu Asn 770 775 780 Gly Tyr Glu He Met Ser Ser Val Arg Tyr Gly Lys Gly Ala Glu Glu 785 790 795 800 Ala Asn Gin Leu Gly Thr Ala Glu Thr Arg Asn Gin Gly Met Leu Val 805 810 815 Leu Thr Ala Aβn Arg Pro Asp Met Lys Leu Gly Ala Asn Asp Arg Leu 820 825 830 Val Val Asn Met Gly Ala Ala His Lys Asn Gin Ala Tyr Arg Pro Leu 835 840 845 Leu Leu Ser Lys Ser Thr Gly Leu Ala Thr Tyr Leu Lys Asp Ser Asp 850 855 860 Val Pro Ala Gly Leu Val Arg Tyr Thr Asp Asn Gin Gly Asn Leu Thr 865 870 875 880 Phe Thr Ala Asp Asp He Ala Gly His Ser Thr Val Glu Val Ser Gly 885 890 895 Tyr Leu Ala Val Trp Val Pro Val Gly Ala Ser Glu Asn Gin Asp Ala 900 905 910 Arg Thr Lys Ala Ser Ser Thr Lys Lys Gly Glu Gin Val Phe Glu Ser 915 920 925 Ser Ala Ala Leu Asp Ser Gin Val He Tyr Glu Gly Phe Ser Asn Phe 930 935 940 Gin Asp Phe Val Lys Thr Pro Ser Gin Tyr Thr Asn Arg Val He Ala 945 950 955 960 Gin Asn Ala Lys Leu Phe Lys Glu Trp Gly He Thr Ser Phe Glu Phe 965 970 975 Ala Pro Gin Tyr Val Ser Ser Gin Asp Gly Thr Phe Leu Asp Ser He 980 985 990 He Glu Asn Gly Tyr Ala Phe Glu Asp Arg Tyr Asp He 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 He Ser Ala He Ala Asp Trp Val Pro Asp 1025 1030 1035 1040 Gin He 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 He 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 He Phe 1090 1095 1100 Glu Arg Val Gin He Ser Asn Gly Arg Lys Leu Thr Thr Asn Glu Lys 1105 1110 1115 1120 He Thr Gin Trp Ser Ala Lys Tyr Phe Asn Gly Ser Asn He 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 Lye Ala Gly Gin Thr Phe Leu Pro Lys Gin Met Thr Glu He 1155 1160 1165 Thr Gly Ser Gly Phe Arg Arg Val Gly Asp Asp Val Gin Tyr Leu Ser 1170 1175 1180 He Gly Gly Tyr Leu Ala Lys Asn Thr Phe He Gin 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 He 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 He Gin Ala Lys Asp Lys Phe He Arg Phe Ala Asp Gly Arg Thr Arg 1300 1305 1310 Tyr Phe He Pro Asp Thr Gly Asn Leu Ala Val Asn Arg Phe Ala Gin 1315 1320 1325 Asn Pro Glu Asn Lys Ala Trp Tyr Tyr Leu Asp Ser Asn Gly Tyr Ala 1330 1335 1340 Val Thr Gly Leu Gin Thr He Aβn Gly Lys Gin Tyr Tyr Phe Asp Asn 1345 1350 1355 1360 Glu Gly Arg Gin Val Lys Gly His Phe Val Thr He Asn Asn Gin Arg 1365 1370 1375 Tyr Phe Leu Asp Gly Asp Ser Gly Glu He Ala Pro Ser Arg Phe Val 1380 1385 1390 Thr Glu Asn Asn Lys Trp Tyr Tyr Val Asp Gly Aβn Gly Lys Leu Val 1395 1400 1405 Lye Gly Ala Gin Val He Aβn Gly Aβn Hie Tyr Tyr Phe Aβn Asn Asp 1410 1415 1420 Tyr Ser Gin Val Lys Gly Ala Trp Ala Asn Gly Arg Tyr Tyr Asp Gly 1425 1430 1435 1440 Asp Ser Gly Gin Ala Val Ser Asn Gin Phe He Gin He Ala Ala Aβn 1445 1450 1455 Gin Trp Ala Tyr Leu Asn Gin Aβp Gly His Lys Val Thr Gly Leu Gin 1460 1465 1470 Asn He Asn Asn Lys Val Tyr Tyr Phe Gly Ser Asn Gly Ala Gin Val 1475 1480 1485 Lys Gly Lys Leu Leu Thr Val Gin Gly Lys Lys Cys Tyr Phe Aβp Ala 1490 1495 1500 Hie Thr Gly Glu Gin Val Val Aβn Arg Phe Val Glu Ala Ala Arg Gly 1505 1510 1515 1520 Cys Trp Tyr Tyr Phe Asn Ser Ala Gly Gin Ala Val Thr Gly Gin Gin 1525 1530 1535 Val He Asn Gly Lys Gin Leu Tyr Phe Aβp 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 Thr Gly Glu Leu Arg Gin Arg Arg 1570 1575 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 I mun 1990; ϋ£: 2452-8. 5. 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. 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: THE CLAIMS I. A plant containing bacterial DNA which codes for a glucosyltransferase which catalyses the formation of glucans from sucrose. 2. A plant according to claim 1 wherein the bacterial DNA is primer independent. 3. A plant according to claim 1 where the glucosyltransferase catalyses the formation of soluble glucans. 4. A DNA comprising a sequence according to SEQ ID NO: 1. 5. A DNA having a sequence which is a variant of SEQ ID. NO: 1. 6. A protein comprising an amino acid sequence according to SEQ ID NO: 2. 7. A protein comprising an amino-acid sequence which is a variant of SEQ ID NO:2. 8. The plasmid pGSG501 containing λC-13 DNA. 9. The plasmid pGSG502 containing λC-13 DNA. 10. A plant containing DNA comprising a sequence according to SEQ ID NO: 1. II. A plant containing DNA having a sequence which is a variant of DNA SEQ ID NO: 1. 12. A plant expressing a protein comprising an amino acid sequence according to SEQ ID NO: 2. 13. A plant expressing a protein comprising an amino acid sequence which is a variant of SEQ ID NO: 2. O 96 06173 AMENDED CLAIMS PCT/AU95/00527 [received by the International Bureau on 18 January 1996 (18.01.96); original claims 2,3 cancelled; original claims 1 ,5,7,11 ,13 amended and renumbered; new claims 12-14 added; claim 4 renumbered as claim 2 claim 6 renumbered as claim 4 claims 8-10 renumbered as claims 6-8 claim 12 renumbered as claim 10 (2 pages)] 1. A plant containing primer independent bacterial DNA which codes for a glucosyltransferase which catalyses the formation of soluble glucans from sucrose. 5 2. A DNA comprising a sequence according to SEQ ID NO: 1. 3. A DNA having a sequence which is a variant of SEQ ID. NO: 1, in which minor alterations have been made compared to SEQ ID. NO: 1 resulting in a sequence which 10 is not identical to SEQ ID. NO: 1. 4. A protein comprising an amino acid sequence according to SEQ ID NO: 2. 5. A protein comprising an amino-acid sequence which is a variant of SEQ ID NO: 2, in which minor 15 alterations have been made compared to SEQ ID NO: 2 resulting in a sequence which is not identical to SEQ ID NO. 2. 6. The plasmid pGSG50l containing λC-13 DNA. 7. The plasmid pGSG502 containing λC-13 DNA. 20 8. A plant containing DNA comprising a sequence according to SEQ ID NO: 1. 9. A plant containing DNA according to claim 3. 10. A plant expressing a protein comprising an amino acid sequence according to SEQ ID NO: 2. 25 11. A plant expressing a protein comprising an amino acid sequence according to claim 5. 12. A method of improving the level of stored carbohydrate in a pasture plant with low levels, comprising inserting a DNA according to claim 2 or 3 into the plant so that the plant expresses a protein according to claim 4 or 5 in active form. 13. A method of preventing degradation of stored carbohydrate during plant senescence comprising inserting a DNA according to claim 2 or 3 into the plant so that the plant expresses a protein according to claim 4 or 5 in active form. 14. Dextran, when produced from a plant according to claim 1, or any one of claims 8 to 11. AMH!..:* ^ r ;

Inventors: GIFFARD PHILIP MORRISON, JACQUES NICHOLAS ANTHONY, SIMPSON CHRISTINE LYNN, GIFFARD, PHILIP, MORRISON, JACQUES, NICHOLAS, ANTHONY, SIMPSON, CHRISTINE, LYNN
Applicants: GIFFARD PHILIP MORRISON, JACQUES NICHOLAS ANTHONY, SIMPSON CHRISTINE LYNN, GIFFARD, PHILIP, MORRISON, JACQUES, NICHOLAS, ANTHONY, SIMPSON, CHRISTINE, LYNN
Application year: 1995
Main classification: C12N15
IPC codes: C12N9, C12N15