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  • 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)
  • C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
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  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

• This invention relates to the field of modification of cell wall polysaccharide content and composition in plants. More particularly, this invention relates to enzymes involved in the synthesis of plant cell wall polysaccharides and nucleotide sequences encoding such enzymes.
• Plant cells are characterised by having a rigid cell wall. These cell walls are comprised primarily of polymers of simple sugar monomers linked in a variety of linear or branched polymers known as polysaccharides. The most abundant simple sugar monomer is glucose, and the most abundant polymer is cellulose. Cellulose is a linear, unbranched polymer, comprised of ⁇ - 1,4 linked glucose monomers. Other polysaccharides found in plant cell walls include hemicellulose, which is a group of polysaccharides comprised of ⁇ -1,4 linked glucose monomers having side chains which may include sugars other than glucose. These side chains frequently include xylose, fucose, arabinose, and galactose.
• Pectins are another type of polysaccharide found in plant cell walls. Pectins are acidic polysaccharides, which are generally comprised primarily of galacturonic acid and rhamnose sugar monomers. Amylose is an additional common plant polysaccharide which is not usually found as a major component of cell walls. It acts primarily as a storage material for glucose, rather than as a structural polymer. However, because amylose is comprised primarily of ⁇ -l,4-linked glucose monomers, it is considered to be a related polymer from a biochemical and physiological perspective.
• Plant polysaccharides have many uses. Certain plastics, such as cellulose acetate, and synthetic textiles, such as rayon, are made from cellulose. In addition, some biodegradable plastics and digestible medicine capsules, as well as medical fillers and fiber additives for food, can be made from plant polysaccharides. In foodstuffs, polysaccharides have a profound impact on food quality. Cell walls contribute to crispness in carrots, while degradation of cell walls is required for softening of fruits, such as peaches and tomatoes. In maize, increased amylose is desirable for cattle feed, but not for human consumption, and increased cell wall strength reduces digestibility. In fiber crops, such as timber, cellulose is the primary polymer of interest. Wood density, a fundamental measure of structural timber quality, is essentially a measure of cellulose content. In the paper pulping industry, efficiency is measured in terms of yield of cellulose. Clearly, the ability to increase cellulose content in timber is an important economic goal.
• the sugars which make up plant cell wall polysaccharides are produced in the photo synthetic organs of plants.
• the sugars so produced are commonly converted into sucrose, a disaccharide consisting of glucose and fructose. Sucrose is transported throughout the plant, to wherever sugar monomers are called for.
• sucrose a disaccharide consisting of glucose and fructose.
• Sucrose is transported throughout the plant, to wherever sugar monomers are called for.
• the photosynthetic organs are often referred to as a source, while tissues requiring large amounts of sugar monomers are referred to as a sink.
• Actively growing regions of the plant are generally sink tissues, as new cell wall synthesis requires large amounts of sugar monomers.
• sugar monomers which make up plant cell walls are primarily 5- or 6-carbon sugars. Different sugars are generally distinguished by stereospecific orientation of hydroxyl groups. Plants contain a variety of enzymes, such as isomerases or epimerases, which can rapidly change the orientation of these hydroxyls. In addition, there are a number of enzymes which can add or remove a single carbon from a sugar monomer. The result is a single pool of sugar monomers which the plant can freely inter-convert into whichever kind is needed for cell wall synthesis.
• Plant polysaccharides are thus biochemically and physiologically inter-related. All polymers compete for the same pool of sugar monomers, and all sugar monomers can be freely interconverted to other types. Degradation of any one polymer will provide building material for any other. Attempts to engineer changes in one polymer may therefore have pleiotropic effects on other polymers.
• the rate of cell wall synthesis is dependent on both the availability of sugar monomers to serve as building blocks for the polymers of the wall, and the enzymes which polymerise those building blocks into polymers.
• Enzymes which are directly responsible for the synthesis of the major cell wall polymers, such as cellulose, hemicellulose and pectin, may have a profound impact on the rate of cell wall synthesis.
• Source-sink relations may play an important role in limiting cell wall synthesis, if the availability of substrates becomes limiting.
• Polymer degrading enzymes may liberate sugar monomers from unnecessary polymers for use in building new, desired polymers.
• Enzymes which can isomerise sugars from one form into another can convert the sugars into whichever kind is needed.
• Each of the different types of cell wall polysaccharides effectively competes for the same pool of sugar monomers, and each represents a potential source of monomers for any ofthe other polymers.
• CEL Cellulose synthase
• SUS sucrose synthase
• ANX annexin
• UDP-glucose pyrophosphorylase also known as UTP- glucose- 1 -phosphate uridylyltransferase.
• Glucose is primarily stored in most plants as amylose. Plants routinely store amylose and degrade it to free up the glucose monomers, as needed. By inhibiting the efficiency of glucose storage, or by increasing the liberation of glucose from amylose, the availability of glucose monomers for cellulose biosynthesis can be increased.
• the rate-limiting enzyme in the storage of glucose as amylose is ADP-glucose pyrophosphorylase (AGP), also known as ATP-glucose-1 -phosphate adenylyltransferase (Iglesias et al., J. Biol Chem. 268:1081-1086, 1993). Conversely, the enzyme most responsible for liberating glucose from amylose is amylase (AMA: Kawagoe and Delmer, Genetic Engineering 19:63-87, 1997).
• enzymes clearly will be important in the engineering of economically useful changes in cellulose biosynthesis.
• enzymes which may be useful in influencing plant cell wall polysaccharide biosynthesis.
• Other enzymes likely to be involved in cellulose biosynthesis include 1,4- ⁇ -cellobiohydrolase, ⁇ -glucosidase, calnexin, cellobiose epimerase, cellobiose phosphorylase, cellulase A, dextransucrase, invertase, phosphodiesterase, phosphoglucomutase, sucrose phosphate synthase, sucrose phosphorylase, UDP-glucose 4-epimerase and UDP-glucose dehydrogenase.
• Enzymes believed to be involved in hemicellulose biosynthesis include ⁇ -glucanase, arabinan synthase, GDP-fucose pyrophosphorylase, GDP-mannose pyrophosphorylase, 1,3 and 1 ,4- ⁇ -glucanases, 1,3 and 1,4- ⁇ -glucosidases, mannose-6-phosphate isomerase, wDP-hexose pyrophosphorylase, xyloglucan endotransglycosylase and xyloglucan synthase.
• Enzymes likely to be involved in pectin biosynthesis include ⁇ -galactosidase, ⁇ -glucuronidase, exopolygalacturonase, glucuronosyl- transferase, pectin methyl-esterase, polygalacturonase and UDP-hexose-1 -phosphate uridylyltransferase.
• Enzymes believed to be involved in amylose biosynthesis include ⁇ -glucosidase, amylopectin 6-glucanohydrolase, amylopectin-branching glycosyltransferase, ⁇ -amylase, branching enzyme, inulosucrase, isoamylase, isomaltase, levansucrase, starch phosphorylase and starch synthase.
• Enzymes likely to be involved in the interconversion of 5-carbon sugars include 2-dehydro-3-deoxy-gluconokinase, aldehyde reductase, arabinose isomerase, D-arabinitol dehydrogenase, D-xylulose reductase, endo-l,4- ⁇ -xylanase, exo-1,4- ⁇ -xylanase, L-arabinose isomerase, L-ribulokinase, L-xylulokinase, phospho-ribulokinase, ribose 5-phosphate isomerase, ribulose-phosphate-3 -epimerase, ribulose-phosphate-4- epimerase, transaldolase, transketolase, xylose isomerase and xylulokinase.
• Enzymes likely to be involved in interconversion of 6-carbon sugars include 6-phospho-fructo-l -kinase, 6-phospho-fructo-2-kinase, trehalose phosphate synthase, aldolase, aldose 1 -epimerase, D-fructokinase, D-galactokinase, fructose 1 ,6-diphosphatase, gluconolactonase, glucose 1-phosphatase, glucose 6-phosphatase, glucose 6-phosphate dehydrogenase, glucose- phosphate isomerase, hexokinase, phosphoglucomutase, trehalase, trehalose phosphatase and UDP-galactose dehydrogenase.
• the present invention provides polynucleotides isolated from eucalyptus and pine which encode enzymes involved in the synthesis of cell wall polysaccharides. Genetic constructs including such sequences and methods for the use of such constructs are also provided, together with transgenic plants having altered cell wall polysaccharide content and composition.
• the isolated polynucleotides comprise a nucleotide sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908; (b) complements of the sequences recited in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-1 13, 119-129, 139-143 and 149-908; (c) reverse complements of the sequences recited in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908; (d) reverse sequences of the sequences recited in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908; and (e) sequences having either 40%, 60%, 75% or 90%
• isolated polypeptides encoded by a polynucleotide of the present invention are provided.
• such polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 30-56, 81-104, 106, 108, 114- 118, 129-138 and 144-148, and variants thereof.
• the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more of the inventive polynucleotide sequences, or in combination with one or more known polynucleotides, together with transgenic cells comprising such constructs.
• the present invention provides genetic constructs comprising, in the 5 '-3' direction, a gene promoter sequence; an open reading frame coding for at least a functional portion of an enzyme encoded by a polynucleotide of the present invention or a variant thereof; and a gene termination sequence.
• the open reading frame may be orientated in either a sense or antisense direction.
• Genetic constructs comprising a non-coding region of a gene coding for an enzyme encoded by the above polynucleotides or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence and a gene termination sequence, are also provided.
• the gene promoter and termination sequences are functional in a host plant.
• the gene promoter and termination sequences are those of the original enzyme genes but others generally used in the art, such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers such as the Kozak sequence or Omega enhancer, and Agrobacterium twnefaciens nopalin synthase terminator may be usefully employed in the present invention.
• CMV Cauliflower Mosaic Virus
• enhancers such as the Kozak sequence or Omega enhancer
• Agrobacterium twnefaciens nopalin synthase terminator may be usefully employed in the present invention.
• Tissue-specific promoters may be employed in order to target expression to one or more desired tissues.
• the gene promoter sequence provides for transcription in xylem.
• the genetic construct may further include a marker for the identification of transformed cells.
• transgenic plant cells comprising the genetic constructs of the present invention are provided, together with plants comprising such transgenic cells, and fruits, seeds and other products, derivatives, or progeny of such forestry plants.
• Propagules of the transgenic plants transformed with the inventive polynucleotides are also mcluded in the present invention.
• the word "propagule” means any part of a plant that may be used in reproduction or propagation, sexual or asexual, including cuttings. Plant varieties, particularly registrable plant varieties according to Plant Breeders'
• a plant need not be considered a "plant variety" simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
• the target plant is a woody plant, preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species, more preferably from the group consisting of pine and eucalyptus species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
• a method for producing a plant having modified cellulose content comprising transforming a plant cell with a genetic construct of the present invention to provide a transgenic cell and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
• the present invention provides methods for modifying the activity of a polypeptide in a plant, comprising stably incorporating into the genome of the plant a genetic construct of the present invention.
• the target plant is a woody plant, preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species, more preferably from the group consisting of pine and eucalyptus species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
• Fig. 1 illustrates the level of native CEL enzyme activity in positive control mung bean (V. radiata) plants.
• Fig. 2 illustrates the level of CEL enzyme activity in mammalian 293 T cells transfected with E. grandis CEL as compared to that in non-transfected 293T cells.
• cellulose is formed by polymerization of glucose into a linear, unbranched, polymer comprised of ⁇ - 1,4 linked glucose monomers (Kawagoe and Delmer, Genetic Engineering, 19:63-87, 1997).
• Cellulose is the most important plant cell wall polysaccharide from both a structural, as well as industrial, perspective. Other polysaccharides are essential for healthy cell walls, as well as for many alternative industrial uses.
• Glucose monomers are most commonly stored in the plant in the form of amylose by the action of several enzymes, with the rate limiting step for storage being catalysed by AGP (Iglesias et al., J. Biol. Chem. 268:1081-1086).
• Glucose monomers are freed from this storage polymer by the action of the enzyme AMA.
• the free monomers are activated by the action of the enzyme UGP, and polymerised into cellulose macro-crystalline structures by the action of the cellulose synthase enzyme complex.
• Pure CEL enzyme has been shown to form ⁇ -1 ,4 glucose linkages in vitro, but has not been shown to be sufficient for polymerization of the large polymers which are fundamental to the structure of plant cell walls.
• the holoenzyme complex appears to be necessary for this latter function.
• the holoenzyme is believed to be comprised of the CEL enzyme in combination with the SUS enzyme and ANX, the whole complex being integrated into the plasma membrane and forming a "rosette" structure as seen in electron micrographs of plant cell membranes (Arioli et al., Science 279:717-720, 1998).
• the polysaccharide content of a plant may be increased or reduced, by incorporating additional copies of genes encoding enzymes involved in the synthesis of cell wall polysaccharides into the genome of the target plant.
• an increase or decrease in polysaccharide content may be obtained by transforming the target plant with antisense copies of such genes.
• the number of copies of genes encoding for different enzymes in the biosynthetic pathway of cell wall polysaccharides can be manipulated to modify the relative amount of each monosaccharide synthesized, thereby leading to the formation of cell walls having altered composition.
• the alteration of polysaccharide composition would be advantageous, for example, in tree processing for paper.
• isolated polynucleotides of the present invention were isolated from forestry plant sources, namely from Eucalyptus grandis and Pinus radiata, but they may alternatively be synthesized using conventional synthesis techniques.
• isolated polynucleotides of the present invention include polynucleotides comprising a sequence selected from the group consisting of sequences identified as SEQ ID NOS: 1 -29, 57-80, 105, 107, 109-1 13, 119-129, 139-143 and 149-908; complements of the sequences identified as SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908; reverse complements of the sequences identified as SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908; at least a specified number of contiguous residues ( ⁇ '-mers) of any of the above-menti
• the present invention also encompasses polynucleotides that differ from the disclosed sequences but which, due to the degeneracy of the genetic code, encode a polypeptide which is the same as that encoded by a polypeptide of the present mvention. Such polynucleotides are said to be "degeneratively equivalent" to a polynucleotide sequence disclosed herein.
• polynucleotides and polypeptides of the present invention were putatively identified by DNA and polypeptide similarity searches.
• SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908 are polynucleotide sequences
• SEQ ID NOS: 30-56, 81-104, 106, 108, 114-118, 129-138 and 144-148 are polypeptide sequences.
• the polynucleotides and polypeptides of the present invention have demonstrated similarity to enzymes that are known to be involved in the synthesis of cell wall polysaccharides. The putative identity of each of the inventive polynucleotides is shown below in Table 1.
• polynucleotide(s), means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides.
• An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner.
• An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised.
• a polynucleotide may consist of an entire gene, or any portion thereof.
• Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide” therefore includes all such operable anti-sense fragments.
• polypeptide encompasses amino acid chains of any length including full length proteins, wherein amino acid residues are linked by covalent peptide bonds.
• Polypeptides of the present invention may be naturally purified products, or may be produced partially or wholly using recombinant techniques.
• complement For the sequence 5' AGGACC 3', the complement, reverse complement and reverse sequence are as follows: complement 3' TCCTGG 5' reverse complement 3' GGTCCT 5' reverse sequence 5' CCAGGA 3'.
• the term "variant" covers any sequence which has at least about 40%), more preferably at least about 60%), more preferably yet at least about 75% and most preferably at least about 90% identical residues (either nucleotides or amino acids) to a sequence of the present invention.
• the percentage of identical residues is determined by aligning the two sequences to be compared, determining the number of identical residues in the aligned portion, dividing that number by the total length of the inventive, or queried, sequence and multiplying the result by 100.
• Polynucleotide or polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determmed against another polynucleotide, using computer algorithms that are publicly available.
• Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms.
• the similarity of polypeptide sequences may be examined using the BLASTP algorithm. Both the BLASTN and BLASTP software are available on the NCBI anonymous FTP server (ftp://ncbi.nlm.nih.gov) under /blast/executables/.
• the computer algorithm FASTA is available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/fasta/. Version 2.04, [February 1996], set to the default parameters described in the documentation and distributed with the algorithm, is prefened for use in the determination of variants according to the present invention.
• the use of the FASTA algorithm is described in Pearson WR and Lipman DJ, "Improved Tools for Biological Sequence Analysis,” Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Pearson WR, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymol. 183:63-98, 1990.
• the following running parameters are prefened for determination of alignments and identities using BLASTN that contribute to the E values and percentage identity of polynucleotides of the present invention: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -r 1 -v 30-b 30 -i queryseq -o results; and the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [F
• the following running parameters are prefened for determination of alignments and identities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences:
• BLASTP the following running parameters are prefened: blastall -p blastp -d swissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; and parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional
• the "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm align and identify similar portions of sequences.
• the hits are ananged in order of the degree of similarity and the length of sequence overlap.
• Hits to a database sequence generally represent an overlap over only a fraction ofthe sequence length ofthe queried sequence.
• the BLASTN and FASTA algorithms also produce "Expect" values for alignments.
• the Expect value (E) indicates the number of hits one can "expect” to see over a certain number of contiguous sequences by chance when searching a database of a certain size.
• the Expect value is used as a significance threshold for determining whether the hit to a database, such as the prefened EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
• the aligned and matched portions of the sequences then have a probability of 90% of being the same.
• the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.
• variant polynucleotides with reference to each of the polynucleotides of the present invention, preferably comprise sequences having the same number or fewer nucleic acids than each of the polynucleotides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide of the present invention. That is, a variant polynucleotide is any sequence that has at least a 99%> probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at the default parameters.
• a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide ofthe present invention that has at least a 99%o probability of being the same as the polynucleotide ofthe present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at the default parameters.
• variant polynucleotide hybridize to the polynucleotide of the present invention under stringent conditions.
• stringent conditions refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
• the present mvention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity as a polypeptide encoded by a polynucleotide of the present invention.
• polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are contemplated by and encompassed within the present invention.
• polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10% ofthe total sequence length are also contemplated by and encompassed within the present invention.
• polypeptides comprising sequences that differ from the polypeptide sequences recited in SEQ ID NOS: 30-56, 81-104, 106, 108, 114-118, 129-138 and 144-148 as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by an encompassed within the present invention, provided the variant polypeptide has activity in a cell wall polysaccharide synthesis pathway.
• variants of the inventive sucrose synthase (SUS) polypeptides are provided wherein the N-terminal serine phosphorylation site has been replaced by an acidic amino acid (such as Asp or Glu) by, for example, site directed mutagenesis. Nakai et al.
• SUS polypeptides mutated in this manner possess increased activity compared to wild-type SUS (Nakai et al., Plant Cell PhysioL 59:1337-1341, 1998).
• Polynucleotides encoding such variants of the inventive SUS polypeptides may therefore be employed in transgenic plants to increase cellulose production.
• the polynucleotides ofthe present invention may be isolated from various libraries, or may be synthesized using techniques that are well known in the art.
• the polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids.
• a plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology.
• One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5 nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5 nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.
• polynucleotides identified as SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908 are refened to as "partial" sequences, in that they do not represent the full coding portion of a gene encoding a naturally occurring polypeptide.
• the partial polynucleotide sequences disclosed herein may be employed to obtain the conesponding full length genes for various species and organisms by, for example, screening DNA expression libraries using hybridization probes based on the polynucleotides of the present invention, or using PCR amplification with primers based upon the polynucleotides of the present invention.
• polynucleotide of the present invention upstream and downstream of the conesponding mRNA, as well as identify the conesponding genomic DNA, including the promoter and enhancer regions, of the complete gene.
• the present invention thus comprehends isolated polynucleotides comprising a sequence identified in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, or a variant of one of the specified sequences, that encode a functional polypeptide, including full length genes.
• Such extended polynucleotides may have a length of from about 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than about 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, more preferably yet a length of less than about 2,000 nucleic acids or base pairs.
• extended polynucleotides ofthe present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.
• Polynucleotides of the present invention also comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, complements, reverse sequences, and reverse complements of such sequences, and their variants.
• polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NOS: 30-56, 81-104, 106, 108, 114-118, 129-138 and 144-148, and their variants.
• x-mer refers to a sequence comprising at least a specified number ("x") of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, or the polypeptides identified as SEQ ID NOS: 30-56, 81-104, 106, 108, 114-118, 129-138 and 144-148.
• the value of x is preferably at least 20; more preferably, at least 40; more preferably yet, at least 60; and most preferably, at least 80.
• polynucleotides and polypeptides of the present invention comprise a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide or polypeptide identified as SEQ ID NOS: 1-908, and variants thereof.
• oligonucleotide probes and primers complementary to and or conesponding to SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, and variants of those sequences are also comprehended by the present invention.
• Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest.
• the term "oligonucleotide” refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction.
• An oligonucleotide probe or primer is described as "conesponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908, or a variant of one ofthe specified sequences.
• Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more preferably at least 98%> to 100%, of the nucleotides of the other strand.
• substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions.
• Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mM.
• Hybridization temperatures can be as low as 5°C, but are generally greater than about 22°C, more preferably greater than about 30°C, and most preferably greater than about 37°C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
• the DNA from plants or samples or products containing plant material can be either genomic DNA or DNA derived by preparing cDNA from the RNA present in the sample.
• DNA-RNA or RNA-RNA hybridization assays are also possible.
• the mRNA from expressed genes would then be detected instead of genomic DNA or cDNA derived from mRNA ofthe sample.
• RNA probes could be used.
• artificial analogs of DNA hybridizing specifically to target sequences could also be used.
• the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention.
• Probes and primers of the present mvention may be from about 8 to 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more preferably from about 15 to 40 base pairs in length.
• the probes can be easily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, and potential for formation of loops and other factors, which are well known in the art.
• kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence.
• Kits of the present invention may comprise one or more probes or primers conesponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NOS: 1-29, 57-80, 105, 107, 109-113, 119-129, 139-143 and 149-908.
• the present invention provides genetic that include an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention or a variant thereof.
• the "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the metabolic step, i.e., the portion of the molecule that is capable of binding one or more reactants or is capable of improving or regulating the rate of reaction.
• the functional portion can be determined by targeted mutagenesis and screening of modified protein products with protocols well known in the art. Normally, the functional portion is 10-20 amino acids, but can be shorter or longer.
• the active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high substrate specificity.
• polypeptide encoded by a polynucleotide includes polypeptides encoded by a nucleotide sequence which includes the partial isolated DNA sequences of the present invention.
• the open reading frame may be inserted in the genetic construct in a sense or antisense orientation, such that transformation of a target plant with the genetic construct will produce a change in the amount or structure of the polypeptide compared to the wild-type plant. Transformation with a genetic construct comprising an open reading frame in a sense orientation will generally result in modified expression of the selected gene, while transformation with a genetic construct comprising an open reading frame in an antisense orientation also generally results in modified expression ofthe selected gene.
• a population of plants transformed with a genetic construct comprising an open reading frame of the present invention in either a sense or antisense orientation may be screened for increased or reduced expression ofthe gene in question using techniques well known to those of skill in the art, and plants having the desired phenotypes may thus be isolated.
• expression of a gene involved in the biosynthesis of polysaccharides may be inhibited by inserting a portion of an open reading frame of the present invention, in either sense or antisense orientation, in the genetic construct.
• Such portions need not be full- length but preferably comprise at least 25 and more preferably at least 50 residues of a polynucleotide of the present invention.
• a much longer portion or even the full length polynucleotide conesponding to the complete open reading frame may be employed.
• the portion of the open reading frame does not need to be precisely the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene.
• a sequence derived from one species may be used to inhibit expression of a gene in a different species.
• the inventive genetic constructs comprise a polynucleotide including a non-coding region of a gene coding for a polypeptide encoded by a polynucleotide of the present invention, or a polynucleotide sequence complementary to such a non-coding region.
• non-coding regions which may be usefully employed in such constructs include introns and 5 '-non-coding leader sequences. Transformation of a target plant with such a genetic construct may lead to a reduction in the amount of polysaccharide synthesized by the plant by the process of co-suppression, in a manner similar to that discussed, for example, by Napoli et al. (Plant Cell 2:279-290, 1990) and de Carvalho Niebel et al. (Plant Cell 7:347-358, 1995).
• Ribozymes are synthetic RNA molecules that comprise a hybridizing region complementary to two regions, each of which comprises at least 5 contiguous nucleotides in a mRNA molecule encoded by one of the inventive polynucleotides. Ribozymes possess highly specific endonuclease activity, which autocatalytically cleaves the mRNA.
• the genetic constructs of the present invention further comprise a gene promoter sequence and a gene termination sequence, operably linked to the DNA sequence to be transcribed, which control expression of the gene.
• the gene promoter sequence is generally positioned at the 5' end of the DNA sequence to be transcribed, and is employed to initiate transcription of the DNA sequence.
• Gene promoter sequences are generally found in the 5' non-coding region of a gene but they may exist downstream of the open reading frame, in introns (Luehrsen KR, Mol. Gen. Genet. 225:81-93, 1991) or in the coding region, as for example in a plant defence gene (Douglas et al., EMBO J. 70:1767-1775, 1991).
• the gene promoter sequence When the construct includes an open reading frame in a sense orientation, the gene promoter sequence also initiates translation of the open reading frame.
• the gene promoter sequence For DNA constructs comprising either an open reading frame in an antisense orientation or a non-coding region, the gene promoter sequence consists only of a transcription initiation site having a RNA polymerase binding site.
• gene promoter sequences which may be usefully employed in the DNA constructs of the present invention are well known in the art.
• the gene promoter sequence, and also the gene termination sequence may be endogenous to the target plant host or may be exogenous, provided the promoter is functional in the target host.
• the promoter and termination sequences may be from other plant species, plant viruses, bacterial plasmids, and the like.
• gene promoter and termination sequences are from the inventive sequences themselves.
• Factors influencing the choice of promoter include the desired tissue specificity of the construct, and the timing of transcription and translation.
• constitutive promoters such as the 35S Cauliflower Mosaic Virus (CaMV 35S) promoter
• CaMV 35S 35S Cauliflower Mosaic Virus
• Use of a tissue specific promoter will result in production of the desired sense or antisense RNA only in the tissue of interest.
• the rate of RNA polymerase binding and initiation can be modulated by external stimuli, such as light, heat, anaerobic stress, alteration in nutrient conditions and the like.
• Temporally regulated promoters can be employed to effect modulation of the rate of RNA polymerase binding and initiation at a specific time during development of a transformed cell.
• the original promoters from the enzyme gene in question, or promoters from a specific tissue-targeted gene in the organism to be transformed, such as eucalyptus or pine are used.
• Other examples of gene promoters which may be usefully employed in the present invention include mannopine synthase (mas), octopine synthase (ocs), and those reviewed by Chua et al. (Science 244:174-181, 1989).
• the gene termination sequence which is located 3' to the DNA sequence to be transcribed, may come from the same gene as the gene promoter sequence or may be from a different gene.
• Many gene termination sequences known in the art may be usefully employed in the present invention, such as the 3' end of the Agrobacterium tumefaciens nopaline synthase gene.
• prefened gene terminator sequences are those from the original enzyme gene or from the target species to be transformed.
• the genetic constructs of the present invention may also contain a selection marker that is effective in plant cells, to allow for the detection of transformed cells containing the inventive construct.
• markers which are well known in the art, typically confer resistance to one or more toxins.
• One example of such a marker is the NPTII gene whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al., in Weissbach, A and H, eds., Methods for Plant Molecular Biology, Academic Press Inc.: San Diego, CA, 1988).
• Transformed cells can thus be identified by their ability to grow in media containing the antibiotic in question.
• the presence ofthe desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots.
• a transcription initiation site is additionally included in the genetic construct when the sequence to be transcribed lacks such a site.
• the genetic construct of the present invention may be linked to a vector having at least one replication system, for example E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the conectness ofthe manipulation determmed.
• the genetic constructs of the present invention may be used to transform a variety of plants, both monocotyledonous (e.g., grasses, corn, grains, oat, wheat and barley), dicotyledonous (e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple), and Gymnosperms (e.g., Scots pine; see Aronen, Finnish Forest Res. Papers, Vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11 :84-89, 1993), and larch (Huang et al, In Vitro Cell 27:201-207, 1991).
• monocotyledonous e.g., grasses, corn, grains, oat, wheat and barley
• dicotyledonous e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple
• the inventive genetic constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter each year by the addition of woody tissue.
• the target plant is selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata.
• pines such as Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clausa, Pinus contorta, Pinus coulte ⁇ , Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus jeffreyi, Pinus lambertiana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, Pinus taeda, Pinus virginiana; other gymnosperms, such as Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, Abies procera, Chamaecyparis lawsonion
• Huniperus virginiana Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, Larix siberica, Libocedrus decurrens, Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea ponnes, Picea rubens, Picea sitchensis, Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, Tsuga canadensis, Tsuga heterophylla, Tsuga mertensiana, Thuja occidentalis, Thuja plicata; and Eucalypts, such as Eucalyptus alba, Eucalyptus bancroftii, Eucalyptus botyroides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucal
• transformation of a plant with a genetic construct of the present invention will result in a modification in polysaccharide synthesis in the plant.
• an increase in the production of cellulose in a plant may be obtained by introducing a genetic construct comprising an open reading frame encoding the enzyme CEL in a sense orientation.
• transformation of a plant with a genetic construct comprising either an open reading frame encoding CEL in an antisense orientation or a non-coding (untranslated) region of a CEL gene will lead to a reduction in the cellulose content ofthe transformed plant.
• Techniques for stably incorporating genetic constructs into the genome of target plants are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like.
• the choice of technique will depend upon the target plant to be transformed. For example, dicotyledonous plants and certain monocots and gymnosperms may be transformed by Agrobacterium Ti plasmid technology, as described, for example by Bevan (Nucleic Acids Res. 12:8711-8721, 1984).
• Targets for the introduction of the genetic constructs of the present invention include tissues, such as leaf tissue, disseminated cells, protoplasts, seeds, embryos, meristematic regions; cotyledons, hypocotyls, and the like.
• the prefened method for transforming eucalyptus and pine is a biolistic method using pollen (see, for example, Aronen, Finnish Forest Res. Papers, 595:53, 1996) or easily regenerable embryonic tissues.
• cells having the inventive genetic construct incorporated in their genome may be selected by means of a marker, such as the kanamycin resistance marker discussed above.
• Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques well known in the art.
• the cell wall is allowed to reform under appropriate osmotic conditions.
• an appropriate germination or callus initiation medium is employed.
• an appropriate regeneration medium is used for explants. Regeneration of plants is well established for many species.
• RNA in target plant cells can be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the polynucleotides incorporated into the genome of the target plant host.
• a target plant may be transformed with more than one genetic construct ofthe present invention, thereby modulating the activity of more than one cell wall polysaccharide enzyme, affecting enzyme activity in more than one tissue, or affecting enzyme activity at more than one expression time.
• a genetic construct may be assembled containing more than one open reading frame coding for a polypeptide encoded by a polynucleotide of the present invention or more than one non-coding region of a gene coding for such a polypeptide.
• polynucleotides of the present inventive may also be employed in combination with other known sequences encoding polypeptides involved in the synthesis of cell wall polysaccharides. In this manner, it may be possible to modify a biosynthetic pathway of cell wall polysaccharides in a non-woody plant to produce a new type of woody plant.
• Example 1 Isolation and Characterization of cDNA Clones from Eucalyptus grandis Eucalyptus grandis cDNA expression libraries (from various tissues, including flowers, leaves, phloem, roots, seeds, shoot buds and xylem) were constructed and screened as follows. mRNA was extracted from the plant tissue using the protocol of Chang et al. (Plant
• a cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according to the manufacturer's protocol.
• the resulting cDNAs were packaged using a Gigapack II Packaging Extract (Stratagene) employing 1 ⁇ l of sample DNA from the 5 ⁇ l ligation mix.
• Mass excision of the library was done using XLl-Blue MRF' cells and XLOLR cells (Stratagene) with ExAssist helper phage
• the excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, MD) and plated out onto LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside (IPTG).
• cDNA clones were sequenced first from the 5' end and, in some cases, also from the 3' end. For some clones, internal sequence was obtained using subcloned fragments. Subcloning was performed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector.
• the determined cDNA sequences are provided in SEQ ID NO: 2, 3, 6, 7, 9, 12-15, 18,
• Pinus radiata cDNA expression libraries (from various tissues, including cell cultures, fascicle meristems, phloem, pollen sacs, roots, seedlings, shoot buds, strobilus and xylem) were constructed and screened as described above in Example 1. DNA sequence for positive clones was obtained using forward and reverse primers on a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer.
• the determined cDNA sequences are provided in SEQ ID NO: 1, 4, 5, 8, 10, 11, 16, 17, 20, 22, 24, 25, 27, 59, 67-70, 74-77, 80, 109-113, 140, 143, 162-185, 196, 199-204, 234-251, 257-263, 265-272, 294-318, 331-365, 367-372, 378- 381, 386-389, 394-398, 435-481, 504-506, 513-521, 529, 530, 548-551, 555, 574-586, 590, 591, 613-620 and 772-908.
• Example 3 Polynucleotide and Amino Acid Analysis
• the determined cDNA sequences described above were compared to and aligned with known sequences in the EMBL database (as updated to May 1999).
• the polynucleotides identified in SEQ ID NO: 1-29, 57-80, 105, 107, 109-111, 115-125, 135-139 and 145-904 were compared to polynucleotides in the EMBL database using the BLASTN algorithm Version 2.0.6 [Sep-16-1998] set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -GO -E0 -rl -v30 -b30 -i queryseq -o results.
• 105, 107, 119-121, 123-128 and 139-143 were determined to have less than 40% identity to sequences in the EMBL database using the computer algorithm BLASTN, as described above.
• the cDNA sequences of SEQ ID NO: 57, 59, 66, 79 and 122 were determined to have less than 60%) identity to sequences in the EMBL database using BLASTN, as described above.
• the cDNA sequences of SEQ ID NO: 61, 71, 73 and 76 were determined to have less than 75%o identity to sequences in the EMBL database using BLASTN, as described above.
• the cDNA sequence of SEQ ID NO: 63 was determined to have less than 90% identity to sequences in the EMBL database using BLASTN, as described above.
• Enzyme ' assays for SUS were performed using the methods described by Sebkova, V. et al. (Plant PhysioL, 108:75-83, 1995). The data shown in Table 3 demonstrates enzyme activity for the expressed proteins.
• the K Sucrose of E. grandis is compared with the data reported by Delmer DP (J. Biol. Chem. 247:3822-3828, 1972) and Nakai et &l(BioSci. Biotech. Biochem. 61 :1500-1503).
• a sense construct containing the sequence of the coding region for cellulose synthase (CEL; SEQ ID NO: 50) from Eucalyptus grandis was inserted into the protein expression vector pcDNA3 (Invitrogen, Carlsbad, CA).
• the resulting construct was transfected into mammalian 293T cells (DuBridge RB et al., Mol. Cell. Biol. 7[l]:379-387, 1987), and recombinant protein was induced by the addition of IPTG. Proteins were solubilised from membranes, and the level of CEL activity was determined as described by Kudlicka K and Brown RM Jr. (Plant Phys. 115:643-656, 1997).
• a positive control for activity native CEL enzyme was solubilised from mung bean (Vigna radiata) plants.
• the determined levels of CEL activity for V. radiata are shown in Fig. 1.
• the levels of CEL activity found in mammalian 293T cells transfected with the Eucalyptus CEL expression clone were found to be similar to those obtained from V. radiata (Fig. 2).
• CEL activity was absent in non- transfected control 293T cells.
• Transformation of tobacco plants with a Pinus radiata CEL gene is performed as follows. Genetic constructs comprising sense and anti-sense constructs containing a polynucleotide including the coding region of CEL (SEQ ID NO: 8) from Pinus radiata are constructed and inserted into Agrobacterium tumefaciens by direct transformation using published methods (See, An G, Ebert PR, Mitra A, Ha SB, "Binary Vectors," in Gelvin SB and Schilperoort RA, eds., Plant Molecular Biology Manual, Kluwer Academic Publishers: Dordrecht, 1988).
• the constructs of sense polynucleotides are made by cloning PBK-CMV plasmid cDNA inserts into pART7 plasmids, followed by cloning of the Notl-digested 35S- Insert-OCS 3 'UTR- fragments from the pART7 vectors into pART27 plant expression vectors (See Gleave A, "A versatile binary vector system with a T-D ⁇ A organizational structure conducive to efficient integration of cloned D ⁇ A into the plant genome," Plant Molecular Biology 20:1203-1207, 1992). The presence and integrity of the transgenic constructs are verified by restriction digestion and D ⁇ A sequencing.
• Tobacco (Nicotia a tabacum cv. Samsun) leaf sections are transformed with the sense and anti-sense CEL constructs using the method of Horsch et al. (Science 227:1229-1231, 1985). Transformed plants containing the appropriate CEL construct are verified using Southern blot experiments. Expression of Pinus CEL in transformed plants is confirmed by isolating total RNA from each independent transformed plant line created with the CEL sense and anti-sense constructs. The RNA samples are analysed in Northern blot experiments to determine the level of expression ofthe transgene in each transformed line.
• CEL enzyme encoded by the Pinus CEL gene and by the endogenous tobacco CEL gene, is analysed for each transformed plant line created with the CEL sense and anti-sense constructs. Crude protein extracts are prepared from each transformed plant and assayed using the methods of Robertson et al. (Biochem J. 306:745-750, 1995) and Pear et al. (Proc. Natl. Acad. Sci. USA 93:12637-12642, 1996).
• the concentration of cellulose in the transformed tobacco plants is determined using the method of Smith and Harris (Plant Phys. 107:1399-1409, 1995). Briefly, whole tobacco plants, of an average age of 38 days, are frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. The cellulose content of 100 mg of frozen powder from an empty vector-transformed control plant line, at least one independent transformed plant line containing the sense construct for CEL and at least one independent transformed plant lines containing the anti-sense construct for CEL are determined using a glucan estimation kit from Megazyme (Warriewood, New South Wales, Australia) using the protocols supplied by the manufacturer. SEQ ID NOS: 1-908 are set out in the attached Sequence Listing. The codes for nucleotide and amino acid sequences used in the attached Sequence Listing, including the symbols "n” and "Xaa”, conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1.

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