AU735383B2 - Expression of fructose 1,6 bisphosphate aldolase in transgenic plants - Google Patents

Description

WO 98/58069 PCT/US98/12447 EXPRESSION OF FRUCTOSE 16 BISPHOSPHATE ALDOLASE IN TRANSGENIC PLANTS This invention relates to the expression of fructose 1,6 bisphosphate aldolase (FDA) in transgenic plants to increase or improve plant growth and development, yield, vigor, stress tolerance, carbon allocation and storage into various storage pools, and distribution of starch. Transgenic plants expressing FDA have increased carbon assimilation, export and storage in plant source and sink organs, which results in growth, yield and quality improvements in crop plants.

Recent advances in genetic engineering have provided the prerequisite tools to transform plants to contain alien (often referred to as "heterologous") or improved endogenous genes. These genes can lead either to an improvement of an already existing pathway in plant tissues or to an introduction of a novel pathway to modify product levels, increase metabolic efficiency, and or save on energy cost to the cell. It is presently possible to produce plants with unique physiological and biochemical traits and characteristics of high agronomic and crop processing importance. Traits that play an essential role in plant growth and development, crop yield potential and stability, and crop quality and composition include enhanced carbon assimilation, efficient carbon storage, and increased carbon export and partitioning.

Atmospheric carbon fixation (photosynthesis) by plants represents the major source of energy to support processes in all living organisms. The primary sites of photosynthetic activity, generally referred to as "source organs", are mature leaves and, to a lesser extent, green stems. The major carbon products of source leaves are starch, which represents the transitory storage form of carbohydrate in the chloroplast, and sucrose, which represents the predominant form of carbon transport in higher plants. Other plant parts named "sink organs" roots, fruit, flowers, seeds, tubers, and bulbs) are generally not autotrophic and depend on import of sucrose or other major translocatable carbohydrates for their growth and development. The storage sinks deposit the imported metabolites as sucrose and other oligosaccharides, starch and other polysaccharides, proteins, and triglycerides.

In leaves, the primary products of the Calvin Cycle (the biochemical pathway leading to carbon assimilation) are glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), also known as triose phosphates (triose-P). The WO 98/58069 PCT/US98/12447 condensation of G3P and DHAP into fructose 1,6 bisphosphate (FBP) is catalyzed reversibly by the enzyme fructose 1,6 bisphosphate aldolase (FDA), and various isozymes are known. The acidic isoenzyme appears to be chloroplastic and comprises about 85% of the total leaf aldolase activity. The basic isoenzyme is cytosolic. Both isoenzymes appear to be encoded by the nuclear genome and are encoded by different genes (Lebherz et al., 1984).

In the leaf, the chloroplast FDA is an essential enzyme in the Calvin Cycle, where its activity generates metabolites for starch biosynthesis. Removal of more than 40% of the plastidic aldolase enzymatic activity by antisense technology reduced leaf starch accumulation as well as soluble proteins and chlorophyll levels but also reduced plant growth and root formation (Sonnewald et al., 1994). In contrast, the cytosolic FDA is part of the sucrose biosynthetic pathway where it catalyzes the reaction of FBP production.

Moreover, cytosolic FDA is also a key enzyme in the glycolytic and gluconeogenesis pathways in both source and sink plant tissues.

In the potato industry, production of higher starch and uniform solids tubers is highly desirable and valuable. The current potato varieties that are used for french fry production, such as Russet Burbank and Shepody, suffer from a non-uniform deposition of solids between the tuber pith (inner core) and the cortex (outer core). French fry strips that are taken from pith tissue are higher in water content when compared to outer cortex french fry strips; cortex tissue typically displays a solids level of twenty-four percent whereas pith tissue typically displays a solids level of seventeen percent. Consequently, in the french fry production process, the pith strips need to be blanched, dried, and parfried for longer times to eliminate the excess water. Adequate processing of the pith fries results in the over-cooking of fries from the high solids cortex. The blanching, drying, and par frying times of the french fry processor need to be adjusted accordingly to accommodate the low solids pith strips and the high solids cortex strips. A higher solids .potato with a more uniform distribution of starch from pith to cortex would allow for a more uniform finished fry product, with higher plant throughput and cost savings due to reduced blanch, dry and par-fry times.

Although various fructose 1,6 bisphosphate aldolases have been previously characterized, it has been discovered that overexpression of the enzyme in. a transgenic plant provides advantageous results in the plant such as increasing the assimilation, export WO 98/58069 PCT/US98/12447 and storage of carbon, increasing the production of oils and/or proteins in the plant and improving tuber solids uniformity.

The present invention provides structural DNA constructs that encode a fructose 1,6 bisphosphate aldolase (FDA) enzyme and that are useful in increasing carbon assimilation, export, and storage in plants.

In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method of producing genetically transformed plants that have elevated carbon assimilation, storage, export, and improved solids uniformity comprising the steps of: Inserting into the genome of a plant a recombinant, double-stranded DNA molecule comprising a promoter that functions in the cells of a target plant tissue, (ii) a structural DNA sequence that causes the production of an RNA sequence that encodes a fructose 1,6 bisphosphate aldolase enzyme, (iii) a 3' non-translated DNA sequence that functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3' end of the RNA sequence; obtaining transformed plant cells; and regenerating from transformed plant cells genetically transformed plants that have elevated FDA activity.

In another aspect of the present invention there is provided a recombinant, doublestranded DNA molecule comprising in sequence a promoter that functions in the cells of a target plant tissue, (ii) a structural DNA sequence that causes the production of an RNA sequence that encodes a fructose 1,6 bisphosphate aldolase enzyme, (iii) a 3' pon-translated DNA sequence that functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3' end of the RNA sequence.

In a further aspect of the present invention, the structural DNA sequence that causes the production of an RNA sequence that encodes a fructose 1,6 bisphosphate aldolase enzyme is coupled with a chloroplast transit peptide to facilitate transport of the enzyme to the plastid.

WO 98/58069 PCT/US98/12447 In accordance with the present invention, an improved means for increasing carbon assimilation, storage and export in the source tissues of various plants is provided. Further means of improved carbon accumulation in sinks (such as roots, tubers, seeds, stems, and bulbs) are provided, thus increasing the size of various sinks (larger roots, tubers, etc.) and subsequently increasing yield and crop productivity. The increased carbon availability to these sinks would also improve composition and use efficiency in the sink (oil, protein, starch and/or sucrose production, and/or solids uniformity).

Various advantages may be achieved by the aims of the present invention, including: First, increasing the expression of the FDA enzyme in the chloroplast would increase the flow of carbon through the Calvin Cycle and increase atmospheric carbon assimilation during early photoperiod. This would result in an increase in photosynthetic efficiency and an increase in chloroplast starch production (a leaf carbon storage form degraded during periods when photosynthesis is low or absent). Both of these responses would lead to an increase in sucrose production by the leaf and a net increase in carbon export during a given photoperiod. This increase in source capacity is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield, vigor, and stress tolerance.

Second, increasing FDA expression in the cytosol of photosynthetic cells would lead to an increase in sucrose production and export out of source leaves. This increase in source capacity is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield, vigor, and stress tolerance.

Third, expression of FDA in sink tissues can show several desirable traits, such as increased amino acid and/or fatty acid pools via increases in carbon flux through glycolysis (and thus pyruvate levels) in seeds or other sinks and increased starch levels as result of increased production of glucose 6-phosphate in seeds, roots, stems, and tubers where starch is a major storage nonstructural carbohydrate (reverse glycolysis). This increase in sink strength is a desirable trait in crop plants and would lead to increased plant growth, storage ability, yield, vigor, and stress tolerance.

Fourth, the invention is particularly desirable for use in the commercial production of foods derived from potatoes. Potatoes used for the production of french fries and other products suffer from a non-uniform distribution of solids between the tuber pith (inner WO 98/58069 PCT/US98/12447 core) and the cortex (outer core). Thus, french fry strips from the pith regions of such tubers have a low solids content and a high water content in comparison to cortex strips from the same tubers. Therefore, the french fry processor attempts to adjust the processing parameters so that the final inner strips are sufficiently cooked while the outer cortex strips are not overcooked. The results of such adjustments, however, are highly variable and may lead to poor quality product. Transgenic potatoes expressingfda will provide to the french-fry and potato chip processor a raw product that consistently displays a higher tuber solids uniformity with acceptable agronomic traits. In the french fry plant production process, inner pith fry strips from higher solids uniformity tubers will require less time to blanch, less time to dry to a specific solids content, and less time to par-fry before freezing and shipping to retail and institutional end-users.

Therefore, with respect to potatoes, the present invention provides 1) a higher quality, more uniform finish fry product in which french fries from all tuber regions, when processed, are nearly the same, 2) a higher through-put in the french fry processing plant due to lower processing times, and 3) processor cost savings due to lower energy input required for lower blanch, dry, and par-fry times. A raw tuber product that displays a higher solids uniformity will also produce a potato chip that has a reduced saddle curl, and a reduced tendency for center bubble, which are undesirable qualities in the potato chip industry. Reduced fat content would also result; this would contribute to improved consumer appeal and lower oil use (and costs) for the processor. The increase in solids uniformity will also translate to an increase in overall tuber solids. For both the french fry and chipping industries, this overall tuber solids increase will also result in higher throughput in the processing plant due to lower processing times, and cost savings due to lower energy input for blanching, drying, par-frying, and finish frying.

Figure 1 shows the nucleotide sequence and deduced amino acid sequence of a fructose 1,6 bisphosphate aldolase gene from E. coli (SEQ ID No:l 1).

Figure 2 shows a plasmid map for plant transformation vector pMON 17524.

Figure 3 shows a plasmid map for plant transformation vector pMON 17542.

WO 98/58069 PCT/US98/12447 Figure 4 shows the change in diurnal fluctuations of sucrose, glucose, and starch levels in tobacco leaves expressing thefda transgene (pMON 17524) and control (pMON17227).

The light period is from 7:00 to 19:00 hours. Only fully expanded and non-senescing leaves were sampled.

Figure 5 shows a plasmid map for plant transformation vector pMON 13925.

Figure 6 shows a plasmid map for plant transformation vector pMON 17590.

Figure 7 shows a plasmid map for plant transformation vector pMON 13936.

Figure 8 shows a plasmid map for plant transformation vector pMON17581.

Figure 9 shows potato tuber cross-sections of improved solids uniformity Segal Russet Burbank lines (top row) versus unimproved nontransgenic Russet Burbank (bottom row).

This invention is directed to a method for producing plant cells and plants demonstrating an increased or improved growth and development, yield, quality, starch storage uniformity, vigor, and/or stress tolerance. The method utilizes a DNA sequence encoding anfda (fructose 1.6 bisphosphate aldolase) gene integrated in the cellular genome of a plant as the result of genetic engineering and causes expression of the FDA enzyme in the transgenic plant so produced. Plants that overexpress the FDA enzyme exhibit increased carbon flow through the Calvin Cycle and increased atmospheric carbon assimilation during early photoperiod resulting in an increase in photosynthetic efficiency and an increase in starch production. Thus, such plants exhibit higher levels of sucrose production by the leaf and the ability to achieve a net increase in carbon export during a given photoperiod. This increase in source capacity leads to increased plant growth that in turn generates greater biomass and/or increases the size of the sink and ultimately providing greater yields of the transgenic plant. This greater biomass or increased sink size may be evidenced in different ways or plant parts depending on the particular plant species or growing conditions of the plant overexpressing the FDA enzyme. Thus, increased size resulting from overexpression of FDA may be seen in the seed, fruit, stem, WO 98/58069 PCT/US98/12447 leaf, tuber, bulb or other plant part depending upon the plant species and its dominant sink during a particular growth phase and upon the environmental effects caused by certain growing conditions, e.g. drought, temperature or other stresses. Transgenic plants overexpressing FDA may therefore have increased carbon assimilation, export and storage in plant source and sink organs, which results in growth, yield, and uniformity and quality improvements.

Plants overexpressing FDA may also exhibit desirable quality traits such as increased production of starch, oils and/or proteins depending upon the plant species overexpressing the FDA. Thus, overexpression of FDA in a particular plant species may affect or alter the direction of the carbon flux thereby directing metabolite utilization and storage either to starch production, protein production or oil production via the role of FDA in the glycolysis and gluconeogenesis metabolic pathways.

The mechanism whereby the expression of exogenous FDA modifies carbon relationships is believed to derive from source-sink relationships. The leaf tissue is a sucrose source, and if more sucrose resulting from the activity of increased FDA expression is transported to a sink, it results in increased storage carbon (sugars. starch, oil, protein, etc.) or nitrogen (protein, etc.) per given weight of the sink tissue.

The expression in a plant of a gene that exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region, which adds polyadenylate nucleotides to the 3' end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of RNA. This RNA is then used as a template for the production of the protein encoded therein by the cells protein biosynthetic machinery.

A number of promoters that are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S and the figwort mosaic virus (FMV) 3 5 S-promoters, the light-inducible promoter from the WO 98/58069 PCT/US98/12447 small subunit of ribulose-1,5-bisphosphate carboxylase (ssRUBISCO), a very abundant plant polypeptide, and the chlorophyll a/b binding protein gene promoters, etc. All of these promoters have been used to create various types of DNA constructs that have been expressed in plants; see, PCT publication WO 84/02913.

Promoters that are known to or are found to cause transcription of DNA in plant cells can be used in the present invention. Such promoters may be obtained from a variety of sources such as plants and plant viruses and include, but are not limited to, the enhanced promoter and promoters isolated from plant genes such as ssRUBISCO genes.

As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of fructose 1,6 bisphosphate aldolase enzyme to cause the desired increase in carbon assimilation, export or storage. Expression of the double-stranded DNA molecules of the present invention can be driven by a constitutive promoter, expressing the DNA molecule in all or most of the tissues of the plant. Alternatively, it may be preferred to cause expression of thefda gene in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc. The promoter chosen will have the desired tissue and developmental specificity. Those skilled in the art will recognize that the amount of fructose 1,6 bisphosphate aldolase needed to induce the desired increase in carbon assimilation, export, or storage may vary with the type of plant. Therefore, promoter function should be optimized by selecting a promoter with the desired tissue expression capabilities and approximate promoter strength and selecting a transformant that produces the desired fructose 1,6 bisphosphate aldolase activity or the desired change in metabolism of carbohydrates in the target tissues. This selection approach from the pool of transformants is routinely employed in expression of heterologous structural genes in plants because there is variation between transformants containing the same heterologous gene due to the site of gene insertion within the plant genome (commonly referred to as "position effect").

In addition to promoters that are known to cause transcription (constitutively or tissuespecific) of DNA in plant cells, other promoters may be identified for use in the current invention by screening a plant cDNA library for genes that are selectively or preferably expressed in the target tissues of interest and then isolating the promoter regions by methods known in the art. In particular, it may be desirable to use a bundle sheath cell specific (or cell enhanced expression) promoter for use with C4 plants such as corn, WO 98/58069 PCT/US98/12447 sorghum, and sugarcane to obtain the yield benefits of overexpression of FDA and not use a constitutive promoter or a promoter with mesophyll cell enhanced expression properties.

For the purpose of expressing thefda gene in source tissues of the plant, such as the leaf or stem, it is preferred that the promoters utilized in the double-stranded DNA molecules of the present invention have relatively high expression in these specific tissues.

For this purpose, one may also choose from a number of promoters for genes with leafspecific or leaf-enhanced expression. Examples of such genes known from the literature are the chloroplast glutamine synthetase GS2 from pea (Edwards et al., 1990), the chloroplast fructose-1,6-bisphosphatase (FBPase) from wheat (Lloyd et al., 1991), the nuclear photosynthetic ST-LS1 from potato (Stockhaus et al., 1989), and the phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes from Arabidopsis thaliana (Leyva et al., 1995). Also shown to be active in photosynthetically active tissues are the carboxylase (RUBISCO), isolated from eastern larch (Larix laricina) (Campbell et al., 1994); the cab gene, encoding the chlorophyll a/b-binding protein of PSII, isolated from pine (cab6; Yamamoto et al., 1994), wheat (Cab-1; Fejes et al., 1990), spinach (CAB-1; Luebberstedt et al., 1994), and rice (cablR: Luan et al., 1992); the pyruvate orthophosphate dikinase (PPDK) from maize (Matsuoka et al., 1993); the tobacco Lhcbl*2 gene (Cerdan et al., 1997); the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene (Truemit et al., 1995); and the thylacoid membrane proteins, isolated from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS; Oelmueller et al., 1992).

Other chlorophyll a/b-binding proteins have been studied and described in the literature, such as LhcB and PsbP from white mustard (Sinapis alba; Kretsch et al., 1995).

Homologous promoters to those described here may also be isolated from and tested in the target or related crop plant by standard molecular biology procedures.

For the purpose of expressing thefda in sink tissues of the plant, for example the tuber of the potato plant; the fruit of tomato; or seed of maize, wheat, rice, or barley, it is preferred that the promoters utilized in the double-stranded DNA molecules of the present invention have relatively high expression in these specific tissues. A number of genes with tuber-specific or tuber-enhanced expression are known, including the class I patatin promoter (Bevan et al., 1986; Jefferson et al., 1990); the potato tuber ADPGPP genes, both the large and small subunits (Muller et al., 1990); sucrose synthase (Salanoubat and Belliard, 1987, 1989); the major tuber proteins including the 22 kDa protein complexes WO 98/58069 PCT/US98/12447 and proteinase inhibitors (Hannapel, 1990); the granule bound starch synthase gene (GBSS) (Rohde et al., 1990); and the other class I and II patatins (Rocha-Sosa et al., 1989; Mignery et al., 1988). Other promoters can also be used to express a fructose 1,6 bisphosphate aldolase gene in specific tissues, such as seeds or fruits. The promoter for Bconglycinin (Tierney, 1987) or other seed-specific promoters, such as the napin and phaseolin promoters, can be used to over-express anfda gene specifically in seeds. The zeins are a group of storage proteins found in maize endosperm. Genomic clones for zein genes have been isolated (Pedersen et al., 1982), and the promoters from these clones, including the 15 kDa, 16 kDa, 19 kDa, 22 kDa, 27 kDa, and gamma genes, could also be used to express anfda gene in the seeds of maize and-other plants. Other promoters known to function in maize, wheat, or rice include the promoters for the following genes: waxy, Brittle, Shrunken 2, branching enzymes I and II, starch synthases, debranching enzymes, oleosins, glutelins. and sucrose synthases. Particularly preferred promoters for maize endosperm expression, as well as in wheat and rice, of anfda gene is the promoter for a glutelin gene from rice. more particularly the Osgt-1 promoter (Zheng et al., 1993); the maize granule-bound starch synthase (waxy) gene (zmGBS); the rice small subunit ADPGPP promoter (osAGP) ;and the zein promoters, particularly the maize 27 kDa zein gene promoter (zm27) (see, generally, Russell et al., 1997). Examples of promoters suitable for expression of anfda gene in wheat include those for the genes for the ADPglucose pyrophosphorylase (ADPGPP) subunits, for the granule bound and other starch synthases, for the branching and debranching enzymes, for the embryogenesisabundant proteins, for the gliadins, and for the glutenins. Examples of such promoters in rice include those for the genes for the ADPGPP subunits, for the granule bound and other starch synthases, for the branching enzymes, for the debranching enzymes, for sucrose synthases, and for the glutelins. A particularly preferred promoter is the promoter for rice glutelin, Osgt-I. Examples of such promoters for barley include those for the genes for the ADPGPP subunits, for the granule bound and other starch synthases, for the branching enzymes, for the debranching enzymes, for sucrose synthases, for the hordeins, for the embryo globulins, and for the aleurone-specific proteins.

The solids content of root tissue may be increased by expressing anfda gene behind a root-specific promoter. An example of such a promoter is the promoter from the acid chitinase gene (Samac et al., 1990). Expression in root tissue could also be WO 98/58069 PCT/US98/12447 accomplished by utilizing the root-specific subdomains of the CaMV35S promoter that have been identified (Benfey et al., 1989).

The RNA produced by a DNA construct of the present invention may also contain a 5' non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs, as presented in the following examples, wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequence can be derived from an unrelated promoter or coding sequence.

In monocots, an intron is preferably included in the gene construct to facilitate or enhance expression of the coding sequence. Examples of suitable introns include the intron and the rice actin intron, both of which are known in the art. Another suitable intron is the castor bean catalase intron (Suzuki et al., 1994) Polvadenvlation signal The 3' non-translated region of the chimeric plant gene contains a polyadenylation signal that functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the RNA. Examples of suitable 3' regions are the 3' transcribed, nontranslated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and plant genes like the soybean storage protein genes and the small subunit of the carboxylase (ssRUBISCO) gene.

Plastid-directed expression of fructose- 1,6-bisphosphate aldolase activity In one embodiment of the invention, thefda gene may be fused to a chloroplast transit peptide, in order to target the FDA protein to the plastid. As used hereinafter, chloroplast and plastid are intended to include the various forms of plastids including amyloplasts. Many plastid-localized proteins are expressed from nuclear genes as precursors and are targeted to the plastid by a chloroplast transit peptide (CTP), which is removed during the import steps. Examples of such chloroplast proteins include the small subunit of ribulose-1,5-biphosphate carboxylase (ssRUBISCO, SSU), enolpyruvateshikimate-3-phosphate synthase (EPSPS), ferredoxin, ferredoxin WO 98/58069 PCT/US98/12447 oxidoreductase, the light-harvesting-complex protein I and protein II, and thioredoxin F. It has been demonstrated that non-plastid proteins may be targeted to the chloroplast by use of protein fusions with a CTP and that a CTP sequence is sufficient to target a protein to the plastid. Those skilled in the art will also recognize that various other chimeric constructs can be made that utilize the functionality of a particular plastid transit peptide to import the fructose-1,6-diphosphate aldolase enzyme into the plant cell plastid. Thefda gene could also be targeted to the plastid by transformation of the gene into the chloroplast genome (Daniell et al., 1998).

Fructose 1,6 bisphosphate aldolases As used herein, the term "fructose 1, 6-bisphophate aldolase" means an enzyme 4.1.2.13) that catalyzes the reversible cleavage of fructose 1,6-bisphosphate to form glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). Aldolase enzymes are divided into two classes, designated class I and class II (Witke and Gotz, 1993). Variousfda genes encoding the enzyme have been sequenced, as have numerous proteins, such as the cytosolic enzyme from maize (GenBank Accession S07789;S10638), cytosolic enzyme from rice (GenBank Accession JQ0543), cytosolic enzyme from spinach (GenBank Accession S31091;S22093), from Arabidopsis thaliana (GenBank Accession Sl 1958), from spinach chloroplast (GenBank Accession S31090;A21815;S22092), from yeast cerevisiae) (GenBank Accession S07855; S37882; S12945; S39178; S44523;X75781), from Rhodobacter sphaeroides (GenBank Accession B40767;D41080), from B. subtilis (GenBank Accession S55426; D32354; E32354: D41835), from garden pea (GenBank Accession S29048; S34411), from garden pea chloroplast (GenBank Accession S29047; S34410), from maize (GenBank Accession S05019), from Chlamydomonas reinhardtii (GenBank Accession S48639; S58485; S58486; S34367), from Corynebacterium glutamicum (GenBank Accession S09283; X17313), from Campylobacter'jejuni (GenBank Accession S52413), from Haemophilus influenzae (strain SRd KW20) (GenBank Accession C64074), from Streptococcus pneumonia (GenBank Accession AJ005697), from rice (GenBank Accession X53130), and from the maize anaerobically regulated gene (GenBank Accession X12872).

The class I enzymes may be isolated from higher eukaryotes, such as animals and plants, and in some prokaryotes, including Peptococcus aerogens, (Lebherz and Rutter, 1973), Lactobacillus casei (London and Kline, 1973), Escherichia coli (Stribling and WO 98/58069 PCT/US98/12447 Perham, 1973), Mycobacterium smegmatis (Bai et al., 1975), and most staphylococcal species (Gotz et al., 1979). The gene for the FDA enzyme may be obtained by known methods and has already been done so for several organisms, such as rabbit (Lai et al., 1974), human (Besmond et al., 1983), rat (Tsutsumi et al., 1984), Trypanosoma brucei (Clayton, 1985), and Arabidopsis thaliana (Chopra et al., 1990). These class I enzymes are invariably tetrameric proteins with a total molecular weight of about 160 kDa and function by imine formation between the substrate and a lysine residue in the active site (Alfounder et al., 1989).

In animal, three class I isozymes, classified as A, B, and C, are expressed in the cytosol of muscle, liver, and brain tissue respectively, and they differ from plant aldolases in their expression and compartmentation patterns (Joh et al., 1986). In the leaves of higher plants, FDA is a class I enzyme, and two different isoenzymes within the class have been documented. One is contained in the chloroplast and the other in the cytosol (Lebherz et al., 1984). The acidic plant isozyme appear to be chloroplastic and comprises about 85% of the total leaf aldolase activity. The basic plant isozyme is cytosolic, and both isozymes appear to be encoded by the nuclear genome and are encoded by different genes (Lebherz et al., 1984).

The class II type aldolases are normally dimeric with molecular mass of approximately 80 kDa, and their activity depends on divalent metal ions. The class II enzymes may be isolated from prokaryotes, such as blue-green algae and bacteria, and eukaryotic green algae and fungi (Baldwin et al., 1978). The gene for the FDA class II enzyme may be obtained by known methods and has already been done so from several organisms including Saccharomyces cerevisiae (Jack and Harris, 1971), Bacillus stearothermophilus (Jack, 1973), and Escherichia coli (Baldwin et al., 1978).

It is believed that highly homologous class II fructose 1, 6-bisphophate aldolases with similar catalyzing activity will also be found in other species of microorganism, such as Saccharomyces (Saccharomyces cerevisiae); Bacillus (Bacillus subtilis); Rhodobacter (Rhodobacter sphaeroides); Plasmodium (Plasmodiumfalciparium, Plasmodium berghei); Trypanosoma (Trypanosoma brucei); Chlamydomonas (Chlamydomas reinhardtii); Candida (Candida albicans); Corynebacterium (Corynebacterium glutamicum); Campylobacter (Campylobacterjejuni); and Haemophilus (Haemophilus..influenza).

WO 98/58069 PCT/US98/12447 Such sequences can be readily isolated by methods well known in the art, for example by nucleic acid hybridization. The hybridization properties of a given pair of nucleic acids are an indication of their similarity or identity. Nucleic acid sequences can be selected on the basis of their ability to hybridize with knownfda sequences. Low stringency conditions may be used to select sequences with less homology or identity.

One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium chloride, at temperatures ranging from about 20°C to about 55 0 C. High stringency conditions may be used to select for nucleic acid sequences with higher degrees of identity to the disclosed sequences. Conditions typically employed may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% Nlaurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50 0 C and about 70 0 C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50 0 C. The skilled individual will recognize that numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolatefda sequences having similarity to fda sequences known in the art and are not limited to those explicitly disclosed.herein.

Preferably, such an approach is used to isolatefda sequences having greater than about identity with the disclosed E.coli fda sequence, more preferably greater than about 70% identity, most preferably greater than about 80% identity.

Depending on growth conditions Euglena gracilis, Chlamydomonas mundana, and Chlamydomomas rheinhardi produce either a class I or a class II aldolase (Cremona, 1968; Russell and Gibbs, 1967; Guerrini et al., 1971).

The isolation of a class IIfda gene from E. coli is described in the following examples. Its DNA sequence is given as SEQ ID NO:1 and shown in Figure 1. The amino acid seqtence is shown in SEQ ID NO:2 and shown in Figure 1. This gene can be .used as isolated by inserting it into plant expression vectors suitable for the transformation method of choice as described. The E. coli FDA enzyme has an apparent pH optimum range near pH 7-9 and retains activity in the lower pH range of 5-7 (Baldwin et al., 1978; Alfounder et al., 1989).

Thus, many different genes that encode a fructose 1,6 bisphosphate aldolase activity may be isolated and used in the present invention.

WO 98/58069 PCT/US98/1 2447 Synthetic gene construction A carbohydrate metabolizing enzyme considered in this invention includes any sequence of amino acids, such as protein, polypeptide, or peptide fragment, that demonstrates the ability to catalyze a reaction involved in the synthesis or degradation of starch or sucrose. These can be sequences obtained from a heterologous source, such as algae, bacteria, fungi, and protozoa, or endogenous plant sequences, by which is meant any sequence that can be naturally found in a plant cell, including native (indigenous) plant sequences as well as sequences from plant viruses or plant pathogenic bacteria.

It will be recognized by one of ordinary skill in the art that carbohydrate metabolizing enzyme gene sequences may also be modified using standard techniques such as site-specific mutation or PCR, or modification of the sequence may be accomplished by producing a synthetic nucleic acid sequence and will still be considered a carbohydrate biosynthesis enzyme nucleic acid sequence of this invention. For example, "wobble" positions in codons may be changed such that the nucleic acid sequence encodes the same amino acid sequence, or alternatively, codons can be altered such that conservative amino acid substitutions result. In either case, the peptide or protein maintains the desired enzymatic activity and is thus considered part of this invention.

A nucleic acid sequence to a carbohydrate metabolizing enzyme may be a DNA or RNA sequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized in whole or in part. The structural gene sequences may be cloned, for example, by isolating genomic DNA from an appropriate source and amplifying and cloning the sequence of interest using a polymerase chain reaction (PCR). Alternatively, the gene sequences may be synthesized, either completely or in part, especially where it is desirable to provide plant-preferred sequences. Thus, all or a portion of the desired structural gene may be synthesized using codons preferred by a selected plant host. Plant-preferred codons may be determined, for example, from the codons used most frequently in the proteins Sexpressed in a particular plant host species. Other modifications of the gene sequences may result in mutants having slightly altered activity.

If desired, the gene sequence ofthefda gene can be changed without changing the protein sequence in such a manner as may increase expression and thus even more positively affect carbohydrate content in transformed plants. A preferred.manner for making the changes in the gene sequence is set out in PCT Publication WO 90/10076. A WO 98/58069 PCT/US98/12447 gene synthesized by following the methodology set out therein may be introduced into plants as described below and result in higher levels of expression of the FDA enzyme.

This may be particularly useful in monocots such as maize, rice, wheat, sugarcane, and barley.

Combinations with other transgenes The effect offda in transgenic plants may be enhanced by combining it with other genes that positively affect carbohydrate assimilation or content, such as a gene encoding for a sucrose phosphorylase as described in PCT Publication WO 96/24679, or ADPGPP genes such as the E. coli glgC gene and its mutant glgCl6. PCT Publication WO 91/19806 discloses how to incorporate the latter gene into many plant species in order to increase starch or solids. Another gene that can be combined withfda to increase carbon assimilation, export or storage is a gene encoding for sucrose phosphate synthase (SPS).

PCT Publication WO 92/16631 discloses one such gene and its use in transgenic plants.

Plant transformation/regeneration In developing the nucleic acid constructs of this invention, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector, a plasmid that is capable of replication in a bacterial host, E. coli.

Numerous vectors exist that have been described in the literature, many of which are commercially available. After each cloning, the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components of the desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell.

A recombinant DNA molecule of the invention typically includes a selectable marker so that transformed cells can be easily identified and selected from nontransformed cells. Examples of such include, but are not limited to, a neomycin phosphotransferase (nptll) gene (Potrykus et al., 1985), which confers kanamycin resistance. Cells expressing the nptII gene can be selected using an appropriate antibiotic such as kanamycin or G418. Other commonly used selectable markers include the bar gene, which confers bialaphos resistance; a mutant EPSP synthase gene (Uinchee et al., 1988), which confers glyphosate resistance; a nitrilase gene, which confers resistance to WO 98/58069 PCT/US98/12447 bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulphonylurea resistance (European Patent Application 154,204, 1985); and a methotrexate resistant DHFR gene (Thillet et al., 1988).

Plants that can be made to have enhanced carbon assimilation, increased carbon export and partitioning by practice of the present invention include, but are not limited to, Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nut, oat, oil seed rape, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, a vine, watermelon, wheat, yams, and zucchini.

A double-stranded DNA molecule of the present invention containing anfda gene can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, by Herrera-Estrella et al. (1983), Bevan (1984), Klee et al. (1985) and EPO publication 120,516. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen. DNA may also be inserted into the chloroplast genome (Daniell et al., 1998).

A plasmid expression vector suitable for the introduction of anfda gene in monocots using microprojectile bombardment is composed of the following: a promoter that is specific or enhanced for expression in the starch storage tissues in monocots, generally the endosperm, such as promoters for the zein genes found in the maize endosperm (Pedersen et al., 1982); an intron that provides a splice site to facilitate WO 98/58069 PCT/US98/12447 expression of the gene, such as the Hsp70 intron (PCT Publication W093/19189); and a 3' polyadenylation sequence such as the nopaline synthase 3' sequence (NOS Fraley et al., 1983). This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA.

A particularly useful Agrobacterium-based plant transformation vector for use in transformation of dicotyledonous plants is plasmid vector pMON530 (Rogers et al., 1987).

Plasmid pMON530 is a derivative of pMON505 prepared by transferring the 2.3 kb Stul- HindIII fragment of pMON316 (Rogers et al., 1987) into pMON526. Plasmid pMON526 is a simple derivative of pMON505 in which the Smal site is removed by digestion with Xmal, treatment with Klenow polymerase and ligation. Plasmid pMON530 retains all the properties of pMON505 and the CaMV35S-NOS expression cassette and now contains a unique cleavage site for Smal between the promoter and polyadenylation signal.

Binary vector pMON505 is a derivative of pMON200 (Rogers et al., 1987) in which the Ti plasmid homology region, LIH, has been replaced with a 3.8 kb HindIII to SmaI segment of the mini RK2 plasmid, pTJS75 (Schmidhauser and Helinski, 1985). This segment contains the RK2 origin of replication, oriV, and the origin of transfer, oriT, for conjugation into Agrobacterium using the tri-parental mating procedure (Horsch and Klee, 1986). Plasmid pMON505 retains all the important features of pMON200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the spectinomycin/streptomycin resistance determinant for selection in E. coli and A. tumefaciens, an intact nopaline synthase gene for facile scoring of transformants and inheritance in progeny, and a pBR322 origin of replication for ease in making large amounts of the vector in E. coli. Plasmid pMON505 contains a single T-DNA border derived from the right end of the pTiT37 nopaline-type T-DNA. Southern blot analyses have shown that plasmid pMON505 and any DNA that it carries are integrated into the plant genome, that is, the entire plasmid is the T-DNA that is inserted into the plant genome. One end of the integrated DNA is located between the right border sequence and the nopaline synthase gene and the other end is between the border sequence and the pBR322 sequences.

Another particularly useful Ti plasmid cassette vector is pMON 17227. This vector is described in PCT Publication WO 92/04449 and contains a gene encoding an enzyme WO 98/58069 PCT/US98/12447 conferring glyphosate resistance (denominated CP4), which is an excellent selection marker gene for many plants, including potato and tomato. The gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed from the FMV promoter as described therein.

When adequate numbers of cells (or protoplasts) containing thefda gene or cDNA are obtained, the cells (or protoplasts) are regenerated into whole plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice, maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), various floral crops, such as sunflower, and nut-bearing trees, such as almonds, cashews, walnuts, and pecans. See, Ammirato et al. (1984); Shimamoto et al. (1989); Fromm (1990); Vasil et al. (1990); Vasil et al. (1992); Hayashimoto (1990); and Datta et al. (1990).

The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.

The term "promoter" or "promoter region" refers to a nucleic acid sequence, usually found upstream to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase or other factors necessary for start of transcription at the correct site. As contemplated herein, a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences.

The promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its .ability to produce mRNA.

"Regeneration" refers to the process of growing a plant from a plant cell plant protoplast or explant).

"Transformation" refers to a process of introducing an exogenous nucleic acid sequence a vector, recombinant nucleic acid molecule) into a cell or protoplast in WO 98/58069 PCT/US98/12447 which that exogenous nucleic acid is incorporated into a chromosome or is capable of autonomous replication.

A "transformed cell" is a cell whose DNA has been altered by the introduction of an exogenous nucleic acid molecule into that cell.

The term "gene" refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.

"Identity" refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson et al., 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 50 bases or amino acids in length, the number of matches are divided by 50 and multiplied by 100 to obtain a percent identity.

"C-terminal region" refers to the region of a peptide, polypeptide, or protein chain from the middle thereof to the end that carries the amino acid having a free carboxyl group.

The phrase "DNA segment heterologous to the promoter region" means that the coding DNA segment does not exist in nature in the same gene with the promoter to which it is now attached.

The term "encoding DNA" refers to chromosomal DNA, plasmid DNA, cDNA, or Ssynthetic DNA that encodes any of the enzymes discussed herein.

SThe term "genome" as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs of the present invention introduced into bacterial host cells can therefore be either chromosomally integrated or plasmidlocalized. The term "genome" as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular WO 98/58069 PCT/US98/12447 components of the cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized.

The terms "microbe" or "microorganism" refer to algae, bacteria, fungi, and protozoa.

The term "mutein" refers to a mutant form of a peptide, polypeptide, or protein.

"N-terminal region" refers to the region of a peptide, polypeptide, or protein chain from the amino acid having a free amino group to the middle of the chain.

"Overexpression" refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein.

The term "plastid" refers to the class of plant cell organelles that includes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids. These organelles are self-replicating and contain what is commonly referred to as the "chloroplast genome," a circular DNA molecule that ranges in size from about 120 kb to about 217 kb, depending upon the plant species, and which usually contains an inverted repeat region.

The phrase "simple carbohydrate substrate" means a monosaccharide or an oligosaccharide but not a polysaccharide; simple carbohydrate substrate includes glucose, fructose, sucrose, lactose. More complex carbohydrate substrates commonly used in media such as corn syrup, starch, and molasses can be broken down to simple carbohydrate substrates.

The term "solids" refers to the nonaqueous component of a tuber (such as in potato) or a fruit (such as in tomato) comprised mostly of starch and other polysaccharides, simple carbohydrates, nonstructural carbohydrated, amino acids, and .other organic molecules.

The following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications, truncations, etc., can be made to the methods and genes described herein while not departing from the spirit and scope of the present invention.

WO 98/58069 PCT/US98/12447

EXAMPLES

EXAMPLE 1 cDNA cloning and overexpression Unless otherwise stated, basic DNA manipulations and genetic techniques, such as PCR, agarose electrophoresis, restriction digests, ligations, E. coli transformations, colony screens, and Western blots were performed essentially by the protocols described in Sambrook et al. (1989) or Maniatis et al. (1982).

The E. colifda gene sequence (SEQ ID NO: 1) was obtained from Genbank (Accession Number X14682) and nucleotide primers with homology to the 5' and 3' end were designed for PCR amplification. E. coli chromosomal DNA was extracted and the E.

colifda gene was amplified by PCR using the 5' oligonucleotide 5'GGGGCCATGGCTAAGATTTTTGATTTCGTA3' (SEQ ID NO:3) and the 3' oligonucleotide 5'CCCCGAGCTCTTACAGAACGTCGATCGCGTTCAG3' (SEQ ID NO:4). The PCR cycling conditions were as follows: 94 C, 5 min (1 cycle); addition of polymerase; 94 C, 1 min.. 60 C, 1 min., 72 C, 2 min.30 sec. (35 cycles). The 1.08 kb PCR product was gel purified and ligated into an E.coli expression vector, pMON5723, to form a vector construct that was used for transformation of frozen competent E. coli JM 101 cells. The pMON5723 vector contains the E.coli recA promoter and the T7 gene 10 leader sequences, which enable high level expression in E.coli (Wong et al., 1988). After induction of the transformed cells, a distinct protein band of about 40 kDa was apparent on an SDS PAGE gel, which correlates with the size of the subunit polypeptide chain of the dimeric aldolase II. It was shown that most of the induced protein was present in the soluble phase. A gel slice containing the highly induced protein was isolated and antibodies were produced in a goat, which was injected with the homogenized gel slice (emulsified in Freund's complete adjuvant).

Thefda gene sequence was subsequently cloned into another E.coli expression vector, under the control of the taq promoter. Induction with IPTG of JM101 cells transformed with this vector showed the same 40 kDa overexpressed protein band. This new clone was used in an enzyme assay for FDA activity. Cells transformed with this vector construct were grown in a liquid culture, induced with IPTG, and grown for another 3 hours. Subsequently, a 3 mL cell culture was spun down, dissolved in 100mM Tris and sonicated. The cell pellet was spun down, and the crude cell extract supematant was WO 98/58069 PCT/US98/12447 assayed for FDA activity, using a coupled enzymatic assay as described by Baldwin et al.

(1978). This assay was routinely performed at 30 0

C.

The reaction was performed in a 1 mL final volume in excess presence of the enzymes triosephosphate isomerase (TIM) and alpha-glycerophosphate dehydrogenase (GDH) in a reaction mixture containing final concentrations of 100mM Tris pH 8.0, 4.75 mM fructose 1,6 bisphosphate, 0.15 mM NADH, 500 U/mL TIM, and 30 U/mL GDH.

The decrease in absorbance at 340nm, after addition of the cell extract supernatant, was recorded using an HP diode array spectrophotometer. One international unit of aldolase activity is that causing the oxidation of 2 itmol of NADH/min in this assay system.

Cell extracts containing the vector with thefda sequence showed a substantial increase in aldolase activity (13.1 I.U./mg protein) as compared to cells transformed with the control vector (0.15 I.U./mg protein). The activity was shown to be inhibited by EDTA, known to specifically inhibit class II aldolases.

EXAMPLE 2 Plant transformation and fda expression in tobacco Targeting of FDA protein E.coli fructose 1,6 bisphosphate aldolase was targeted to the plastid in plants in order to assess its influence on carbohydrate metabolism and starch biosynthesis in these plant organelles. To accomplish the import of the E.coli aldolase into the plastids, a vector was constructed in which the aldolase was fused to the Arabidopsis small subunit transit peptide (CTP1) (Stark et al., 1992) or the maize small subunit CTP (Russell et al., 1993), creating constructs in which the CTP-fda fusion gene was located between the promoter from the figwort mosaic virus (P-FMV35S; Gowda et al., 1989) and the 3'nontranslated region of the nopaline synthase gene (NOS Fraley et al., 1983) sequences. The vector construct containing the expression cassette [P- FMV/CTPl/fda/NOS3'] was subsequently used for tobacco protoplast transformation, which was performed as described in Fromm et al. (1987), with the following modifications. Tobacco cultivar Xanthi line D (Txd) cell suspensions were grown in 250mL flasks, at 25 0 C and 138 rpm in the dark. For maintenance, a sub-culture volume of 9 mL was removed and added to 40 mL of fresh Txd media containing MS salts, 3% sucrose, 0.2 g/L inositol, 0.13 g/L asparagine, 80 iL of a 50 mg/mL stock of PCPA, 5 ltL WO 98/58069 PCT/US98/12447 of a 1 mg/mL stock of kinetin, and 1 mL of 1000x vitamins (1.3 g/L nicotinic acid, 0.25 g/L thiamine, 0.25 g/L pyridoxine HCL, and 0.25 g/L calcium pantothenate) every 3 to 4 days. Protoplasts were isolated from 1-day-old suspension cells that came from a 2-dayold culture. Sixteen milliliters of cells were added to 40 mL of fresh Txd media and allowed to grow 24 hours prior to digestion and isolation of the protoplasts. The centrifugation stage for the enzyme mix has been eliminated. The electroporation buffer and protoplast isolation media were filter sterilized rather than autoclaved. The electroporation buffer did not have 4 mM CaCl 2 added. The suspension cells were digested in enzyme for 1 hour. Protoplasts were counted on a hemacytometer, counting only the protoplasts that look intact and circular. Bio-rad Gene Pulser cuvettes (catalog 165-2088) with a 0.4-cm gap and a maximum volume of 0.8 mL were used for the electroporations. Fifty to 100 .tg of DNA containing the gene of interest along with 5 tig of internal control DNA containing the luciferase gene were added per cuvette. The final protoplast density at electroporation was 2x10 6 /mL and electroporater settings were a 500 pFarad capacitance and 140 volts on the Bio-rad Gene Pulser. Protoplasts were put on ice after resuspension in electroporation buffer and remained on ice in cuvettes until minutes after electroporation. Protoplasts were added to 7 mL of Txd media 0.4 M mannitol and conditioning media after electroporation. At this stage coconut water was no longer used. The protoplasts were grown in 1- hour day/night photoperiod regime at 26°C and were spun down and assayed or frozen 20-24 hours after electroporation.

Western blot analysis performed on the protoplast extracts, obtained after transformation, showed processing into the mature FDA in the tobacco protoplasts.

Expression was detected of a protein migrating at approximately 40 kDa, which is the molecular weight of the aldolase subunit and the size of the protein also observed after overexpression of the aldolase in E. coli.

The expression cassette [P-FMV/CTP1/fda/NOS3'] was subsequently cloned into the NotI site of pMON 17227 (described in PCT Publication WO 92/04449), in the same orientation as the selectable marker expression cassette, to form the plant transformation vector pMON17524, as shown in Figure 2 (SEQ ID NO: An additional construct was made and used for tobacco protoplast transformation, fusing thefda gene to the Arabidopsis EPSPS transit peptide (CTP2), which is described in US patent 5,463,175. The transit peptide was cloned (through the SphI site) into the 24 WO 98/58069 PCT/US98/12447 SphI site located immediately upstream from the N-terminus of thefda gene sequence in the CTP -fda fusion (described above). This new CTP2-fda fusion gene was then cloned into a vector between the FMV promoter and the NOS 3' sequences. When this construct containing the CTP2/fda gene sequences was used for tobacco protoplast transformation, expression was detected of a protein migrating at approximately 40 kDa, which is the molecular weight of the aldolase subunit and the size of the protein also observed after overexpression of the aldolase in E. coli.

The NotI cassette [P-FMV/CTP2/fda/NOS3'] from this construct was then cloned into the NotI site of pMON 17227, in the same orientation as the selectable marker expression cassette, to form the plant transformation vector pMON 17542, which is shown in Figure 3 (SEQ ID NO:6).

For cytoplasmic expression of the FDA in tobacco protoplasts, a construct was made in which thefda gene sequence (without being coupled to a transit peptide) was cloned into a vector backbone, between the FMV promoter and the NOS 3' sequences.

Using this construct for tobacco protoplast transformation also showed expression of a protein of the same size, migrating at approximately 40 kDa.

fda expression in tobacco plants Two constructs, containing thefda gene, fused to the Arabidopsis small subunit CTP1 (pMON17524) (SEQ ID NO:5, Figure 2) and the Arabidopsis EPSPS (CTP2) transit peptide (pMON17542) (SEQ ID NO:6, Figure were used for tobacco plant transformation, as described in US patent 5,463,175. A vector without the CTP-fda sequences, pMON 17227 (described in PCT Publication WO 92/04449), was used as a negative control. The plant transformation vectors were mobilized into the ABI Agrobacterium strain. Mating of the plant vector into the ABI strain was done by the triparental conjugation system using the helper plasmid pRK2013 (Ditta et al., 1980).

Growth chamber-grown tobacco transformant lines were generated and first screened by Western blot analysis to identify expressors using goat antibody raised against E.coli-expressedfda. Subsequently, for pMON 17524-expressing tobacco lines, leaf nonstructural carbohydrates were analyzed (sucrose, glucose, and hydrolyzed starch into glucose) by means of a YSI Instrument, Model 2700 Select Biochemistry Analyzer.

Starting at flowering stage, leaf samples were also taken from these plants and analyzed for diurnal changes in leaf nonstructural carbohydrates.

WO 98/58069 PCT/US98/12447 Five hundred milligrams to 1 g fresh tobacco leaf tissue samples were harvested and extracted in 5 mL of hot Na-phosphate buffer (40 g/L NaH 2

PO

4 and 10 g/L Na 2

H

2

PO

4 in double de-ionized water) by homogenization with a Polytron. Test tubes were then placed in an 85 0 C water bath for 15 minutes. Tubes were centrifuged for 12 minutes at 3000 rpm and the supernatants saved for soluble sugar analysis. The pellet was resuspended in 5 mL of hot Na-phosphate buffer mixed with a Vortex and centrifuged as described above. The supernatant was carefully removed and added to the previous supernatant fraction for soluble sugar (sucrose and glucose) analysis by YSI using appropriate membranes.

The starch was extracted from the pellet using the Megazyme Kit (Megazyme, Australia). To the pellet, 200 pL of 50% ethanol and 3 mL of thermostable alpha-amylase (300U) were added and the mixture vortexed. Samples were then incubated in boiling water for 6 minutes and stirred after 2 and 4 minutes. Tubes were placed in 50 0 C water bath and 4 mL of 200 mM acetate buffer (pH 4.5) were added followed by 0.1 mL amyloglucosidase (20 Incubation occurred for 1 hour. Test tubes were then centrifuged for 15 minutes at 3000 rpm. Aliquots were taken from the supernatant and analyzed for glucose by YSI. The free glucose was adjusted to anhydrous glucose (as it occurs in starch by multiplying by the ratio 162/182). The total volume per tube was 7.1 mL.

As seen in Tablel, expression of thefda gene in tobacco correlated with a significant increase in leaf starch levels. However, referring to Figure 4, when a diurnal profile of starch levels was established in thefda-expressing leaves, this increase was apparent mainly early in the photoperiod, which is a phase when leaves are known to have peak photosynthetic activity. This increase in starch has no apparent negative effect on the plant because the increased starch is turned over during the dark period. There was no apparent increase in steady state levels of sucrose or glucose in tobacco leaves expressing E.colifda as compared to the control.

WO 98/58069 PCT/US98/12447 Table 1 Leaf Carbohydrate Levels of Plants Expressing thefda Transgene' (pMON17524) High Expressors Low Expressors Negative total protein) 0.01%) Control (mg/g fresh weight) STARCH 35.08 2.84 23.25 3.20 16.69 2.92 SUCROSE 0.97 +0.17 0.86 0.25 0.66 0.19 GLUCOSE 1.88 +0.17 1.58+0.20 1.68+0.26 1Leaf samples were harvested at midday.

A second set of transgenic tobacco plants transformed with the construct pMON17542 were grown in the greenhouse. Tobacco plants containing a vector without the CTP-fda sequences, pMON 17227, were used as negative control. Of all the pMON 17542-lines screened for expression by Western blot analysis, 18 were high expressors of the total cellular protein) and 15 lines were low expressors Fifteen plants containing the null vector, pMON17227, were used as control.

Fully expanded leaves from plants expressing thefda transgene and negative controls were tested for sucrose export by collecting phloem exudate from excised leaf systems. The phloem exudation technique is described in Groussol et al. (1986). Leaves were harvested at 11:30 AM and placed in an exudation medium, containing 5 mM EDTA at pH 6.0, and allowed to exude for a period of 4 hours under full light and high humidity. The exudation solution was immediately analyzed for sucrose level, as described above in the carbohydrate analysis method. As seen in Table 2, a significant increase in sucrose export out of source leaves was observed in plants expressing thefda transgene.

This increase in sucrose export byfda-expressing leaves is an illustration of an increase in source capacity, very likely due to an increased carbon flow through the Calvin Cycle (in response to increased triose-P utilization) and thus an increase in net carbon utilization by the leaf. As seen in Table 2, the increase in sucrose loading in the phloem correlates with the level of fda expression.

WO 98/58069 PCT/US98/12447 Table 2 Levels of Sucrose in Phloem Exudate from Excised Leaves offda Transgenic Tobacco Plants (pMON 17542) Water uptake sucrose in phloem exudate (pl/g F.Wt./h) (ng/leaf) (ng/g F.Wt.) fda high expressors 320 20 330 60 108 22 fda low expressors 340 10 210 10 77 3 Control 390 30 160+ 10 56 3 Referring to Table 3, preliminary analysis of plant growth and development revealed no significant differences in number of leaves or pods per plant, plant height, stem diameter, or apparent seed weight per plant, between plants expressing thefda gene and the vector control under the specific growing and analysis conditions. However, as seen in Table 4, thefda-transgenic plants had a significantly higher root mass. This may be an indication that, under these conditions, roots represented a more dominant sink that attracted excess carbohydrate produced by the source leaves. Furthermore, the present illustration shows that the increase in root mass in the presence of the E.colifda gene was accomplished with no apparent negative effect on shoot growth, inflorescence, or seed set.

Therefore, this increase in root growth and final root dry weight is a desirable plant trait because it would lead to a rapid seedling establishment following germination and greater plant ability to tolerate drought, cold stress, other environmental challenges, and transplanting. In different plants and under different growing conditions, other plant parts (such as seed, fruit, stem, leaf, tuber, bulb, etc.) are expected to show the weight increase observed in tobacco roots overexpressing thefda transgene.

WO 98/58069 PCT/US98/12447 Table 3 Assessment of Certain Plant Growth and Development Parameters in Tobacco Expressing thefda Transgene' (pMON17542) #pods/plant #leaves/plant Plant height Seed weight (cm) (g/plant) high expressors 162 40 25.4 0.8 65.3 3.1 18.8 2.4 Control 156 28 24.4 0.5 65.8 5.1 17.3 2.6

I

To achieve this analysis, 14 high-expressor lines were compared to 15 control plants.

Measurements were made prior to seed harvest (most pods have reached maturity). The number of leaves was confirmed by counting the number of nodes to account for leaf drop.

Table 4 Tobacco Root Dry Weight of Plants Expressing the E.colifda Transgene I (pMON17542) Root Dry Weight (g/plant) fda high expressors 64.0 3.9 fda low expressors 62.7 5.4 Control 31.7+ 1.6 1 Roots from 5 high and 7 low expressing lines and 6 control plants were excised and washed carefully then placed in a 65 0 C drying oven for at least 48 hours. Roots were removed from the oven and allowed to equilibrate in the laboratory for 2 hours before dry weight determination.

EXAMPLE 3 Plant transformation and fda expression in corn plants Targeting ofFDA protein Vectors containing thefda gene with and without the plastid targeting peptide were made for transformation in corn and are also suitable for other monocots, including rice, wheat, barley, sugarcane, triticale, etc.

WO 98/58069 PCT/US98/12447 For the cytosolic expression ofthefda gene in corn plants, a construct was made in which thefda gene sequence was fused to the backbone of a vector containing the enhanced CaMV 35S promoter (e35S; Kay et al., 1987), the HSP70 intron (US patent 5,593,874), and the NOS3' polyadenylation sequence (Fraley et al., 1983). This created a NotI cassette [P-e35S/HSP70 intron/fda/NOS3'] that was cloned into the NotI site of pMON30460, a monocot transformation vector, to form the plant transformation vector pMON13925, as shown in Figure 5. pMON30460 contains an expression cassette for the selectable marker neomycin phosphotransferase typell gene (nptll) [P-35S/NPTII /NOS3'] and a unique NotI site for cloning the gene of interest. The final vector (pMON13925) was constructed so that the gene of interest and the selectable marker gene were cloned in the same orientation. A vector fragment containing the expression cassettes for these gene sequences could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation.

For the chloroplast-targeted expression of thefda gene in corn plants, a construct was made in which thefda gene sequence, coupled to the maize RUBISCO small subunit CTP (Russell et al., 1993), was fused to the backbone of a vector containing the enhanced (CaMV) 35S promoter, the HSP70 intron, and the NOS3' polyadenylation sequences. This created a NotI cassette [P-e35S/HSP70 intron/mzSSuCTP/fda/NOS3'] that was cloned into the NotI site (in the same orientation as the selectable marker cassette /NOS3']) of the monocot transformation vector pMON30460, to form the vector pMON17590, as shown in Figure 6. From this vector a fragment containing thefda gene expression cassette and the selectable marker cassette could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation.

For the cytosolic endosperm-specific expression of the aldolase gene in corn, the fda gene sequence was cloned into a vector (in the same orientation as the selectable marker cassette[P-35S/NPTII /NOS3']) containing the glutelin gene promoter P-osgtl S(Zheng et al., 1993), the HSP70 intron, and the NOS3' polyadenylation sequences to form the vector pMON13936, as shown in Figure 7. From this vector a fragment containing the fda gene expression cassette [P-osgtl/HSP70intron/fda/NOS3'] and the selectable marker cassette could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation.

WO 98/58069 PCT/US98/12447 Maize plant transformation Transgenic maize plants transformed with the vectors pMON13925 (described above) or pMON 17590 (described above) were produced using microprojectile bombardment, a procedure well-known to the art (Fromm, 1990; Gordon-Kamm et al., 1990; Walters et al., 1992). Embryogenic callus initiated from immature maize embryos was used as a target tissue. Plasmid DNA at Img/mL in TE buffer was precipitated onto tungsten particles using a calcium chloride spermidine procedure, essentially as described by Klein et al. (1988). In addition to the gene of interest, the plasmids also contained the neomycin phosphotransferase II gene (nptll) driven by the 35S promoter from Cauliflower Mosaic Virus. The embryogenic callus target tissue was pretreated on culture medium osmotically buffered with 0.2M mannitol plus 0.2M sorbitol for approximately four hours prior to bombardment (Vain et al., 1993). Tissue was bombarded two times with the DNA-coated tungsten particles using the gunpowder version of the BioRad Particle Delivery System (PDS) 1000 device. Approximately 16 hours following bombardment, the tissue was subcultured onto a medium of the same composition except that it contained no mannitol or sorbitol, and it contained an appropriate aminoglycoside antibiotic, such as G418", to select for those cells that contained and expressed the 35S/nptlI gene. Actively growing tissue sectors were transferred to fresh selective medium approximately every 3 weeks. About 3 months after bombardment, plants were regenerated from surviving embryogenic callus essentially as described by Duncan and Widholm (1988).

Aldolase activity from transgenic maize In order to measure leaf aldolase activity, corn leaf samples were taken and immediately frozen on dry ice. Aldolase enzyme was extracted from the leaf tissue by grinding the leaf tissue at 4 0 C in 1.2 mL of the extraction buffer (100 mM Hepes, pH mM MgCl 2 5mM MnC12, 100 mM KC1, 10 mM DTT, 1% BSA, 1 mM PMSF, g/mL leupeptin, 10 ug/mL aprotinin). The extract was centrifuged at 15,000 x g, at 4°C for 3 minutes, and the non-desalted supernatant was assayed for enzyme activity. This extraction method gave about 60% recovery of E. coli FDA activity.

Total aldolase activity was determined in 0.98 mL of reaction mixture that consisted of 100 mM EPPS-NaOH, pH 8.5, 1 mM fructose-bisphosphate,.0.1 mM NADH, mM MgCl2, 4 units of alpha-glycerophosphate dehydrogenase, and 15 units of WO 98/58069 PCT/US98/12447 triosephosphate isomerase. The reaction was initiated by addition of 20 pL of leaf extract.

The resulting data, generated from a single experiment, are presented in Table Table Aldolase Activity from Transgenic Maize Leaves Lines A340/min/20pL Activity H99 (control) 0.113 100 pMON 17590 0.233 206 pMON13925 0.251 222 A phenotype was visible in the primary transformants (RO plants) expressing the E. coli FDA when the protein was targeted to the chloroplast. The leaves were chlorotic but seed set was normal. RI plants were grown in both field and in greenhouse experiments. Starch was not detectable in the leaves using an iodine staining and pollination was delayed. It is believed that the phenotype in these corn plants may be the result of the promoter (e35S) used in both the pMON17590 and pMON13925 vectors not being preferred for causing FDA expression in corn. Because e35S is believed to cause mesophyll enhanced expression and the Calvin Cycle in a C4 plant such as corn occurs predominantly in the bundle sheath cells, the use of a promoter directing enhanced expression in the bundle sheath cells (such as the ssRUBISCO promoter) may be preferred. Vectors containing such a promoter and driving expression of FDA have been prepared and are being tested in maize.

In particular, the maize RuBISCO small subunit (PmzSSU, a bundle sheath cellspecific promoter) has been used to construct vectors for cell-specificfda expression in maize. A class I aldolase (fdal), anfda without an iron sulfur cluster and with different properties fromfdall, was utilized to improve carbon metabolism in C4 crops maize) The gene for the class I aldolase was amplified from the genome of Staphylococcus aureus and activity was comfirmed. Transformation vectors were then constructed to express both classes of aldolase (fdal andfdall) in a cell-specific manner in maize. The following cassettes have been made: pMON13899: PmzSSU/hsp70/mzSSU CTP/fdal pMON13 9 90PmzSSU/hsp70/mzSSU CTP/fdall WO 98/58069 PCT/US98/12447 pMONI 3988:P35S/hsp70/fdal.

These vectors were used for corn transformation as described generally above. The biochemical and physiological analysis of the primary transformants should allow for the identification ofaldolase gene overexpression that will lead to increase growth and development and yield in maize.

Also, two vectors were used for transformation of corn which would target the expression of the E. colifda II gene in the maize endosperm. The vector pMON 13936 uses the rice gtl promoter to drive expression of aldolase in the cytoplasm of the endosperm cells. Another vector (pMON 36416) uses the same promoter with the maize RuBISCO small subunit transit peptide to localize the protein in the amyloplasts.

Homozygous lines of the cytosolic aldolase transformants have been identified (Homozygosity of 37 plants was confirmed using western blot analysis) and seed from these plants were collected for grain composition analysis (moisture, protein, starch, and oil). Of the 53 pMON 36416 primary transformants screened for amylopast-targeted aldolase expression, 11 were positive. These plants will be tested for homozygosity selection/propagation and kernels from the homozygotes will be used for composition analysis.

EXAMPLE 4 Plant transformation and fda expression in potato plants Targeting offda expression The plant expression vector, pMON 17542 (described earlier), in which thefda gene is expressed behind the FMV promoter and the aldolase enzyme is fused to the chloroplast transit peptide CTP2, was used for Agrobacterium-mediated potato transformation.

A second potato transformation vector was constructed by cloning the NotI cassette [P-FMV/CTP2/fda/NOS3'] (described earlier) into the unique NotI site of pMON23616. pMON23616 is a potato transformation vector containing the nopaline-type T-DNA right border region (Fraley et al., 1985), an expression cassette for the neomycin phosphotransferase typell gene [P-35S/NPTII /NOS3'] (selectable marker), a unique NotI site for cloning the gene expression cassette of interest, and the T-DNA left border region (Barker et al., 1983). Cloning of the NotI cassette [P-FMV/CTP2/fda/NOS3'] (described earlier) into the NotI site of pMON23616 results in the potato transformation vector WO 98/58069 PCT/US98/12447 pMON17581, as shown in Figure 8. The vector pMON17581 was constructed such that the gene of interest and the selectable marker gene were transcribed in the same direction.

Potato plant transformation The plant transformation vectors were mobilized into the ABI Agrobacterium strain. Mating of the plant vector into the ABI strain was done by the triparental conjugation system using the helper plasmid pRK2013 (Ditta et al., 1980). The vector pMON 17542 was used for potato transformation via Agrobacterium transformation of Russet Burbank potato callus, following the method described in PCT Publication WO 96/03513 for glyphosate selection of transformed lines.

After transformation with the vector pMON 17542, transgenic potato plantlets that came through selection on glyphosate were screened for expression of E. coli aldolase by leaf Western blot analysis. Out of 112 independent lines assayed. 50fda-expressing lines were identified, withfda expression levels ranging between 0.12% and 1.2 of total extractable protein.

The plant transformation vector PMON 17581 was used for Agrobacteriummediated transformation of HS3 1-638 potato callus. HS3 1-638 is a Russet Burbank potato line previously transformed with the mutant ADPglucose pyrophosphorylase (glgC16) gene from E.coli Patent 5,498,830). The potato callus was transformed following the method described in PCT Publication WO 96/03513, substituting kanamycin (administered at a concentration of 150-200 mg/L) for glyphosate as a selective agent.

The transgenic potato plants were screened for expression of thefda gene by assaying leaf punches from tissue culture plantlets. Western blot analysis (using antibodies raised against the E. coli aldolase) of leaf tissue from the pMON17581-transformed lines identified 12 expressing lines out of 56 lines screened. Expression was detected of a protein migrating at approximately 40 kDa, which is the molecular weight of the E. coli (classll) aldolase subunit and the size of the protein observed after overexpression of the Saldolase in E. coli.

Specific gravity measurements of transgenic potato plants From the 50fda-expressing potato lines obtained after transformation with pMON 17542, 7 of the highest expressing lines were micropropagated in tissue culture, and 8 copies of each line were planted in pots 14 inches in diameter and 12 inches deep, containing a mixture of: Metro 350 potting media, fine sand, /4 Ready Earth WO 98/58069 PCT/US98/12447 potting media. Wild-type Russet Burbank plantlets from tissue culture were planted as controls. All plants were cultivated for approximately 5 months in the greenhouse in which daytime temperature was approximately 21-23°C while nighttime temperature was approximately 13 C. Plants were watered every other day throughout their active growing period and fertilized with Peter's 20-20-20 commercial fertilizer once a week, at levels similar to commercial applications. Fertilization was carried out only for the first 2 /2 months, at which point fertilization was stopped completely. Plants were allowed to naturally senesce, and at approximately 50% senescence, tubers were harvested.

For each line at harvest, all tubers from all 8 pots were pooled and a total weight l0 was obtained. Then for each line, tubers 30 g or greater were pooled and specific gravity was determined on this group of tubers. Specific gravity is the weight of the tubers in air divided by the weight in air minus the weight in water. Results of these weight measurements are presented in Table 6.

Table 6 Specific gravity measurements from transgenic potato plants Line Total Overall Combined Increase in Combined Weight of Specific Weight Yield Weight Total Weight Tubers over 30g Gravity Increas of Tubers (Tubers over of Total Weight) e over 30g RB 6609 4477 67.70% 1.087 40652 5138 neg 1307 neg 25.40% 1.08 40611 7170 8.5% 4533 1.3% 63.20% 1.083 40608 7470 13.0% 1070 neg 14.30% 1.081 40632 7776 21.8% 5453 21.8% 70.10% 1.088 40614 8688 31.5% 5468 22.2% 62.90% 1.083 40631 8800 33.2% 6188 38.2% 70.30% 1.084 40610 9746 47.0% 7777 73.0% 80% 1.087 This table summarizes the tuber yield and specific gravity for all seven lines grown in the greenhouse. The results indicate that, in comparison to the control, all but one of thefda lines show an increase in overall tuber yield, and that in four lines, there is a corresponding increase in percentage of tubers that weigh more than 30 g. For combined tubers over g, the-percent of total weight is near that of the control, and for two lines is greater than the control. This indicates that five out of the six of the lines show higher overall yield and are not making smaller tubers. In other words, with the increase in overall yield, there is a corresponding increase in percentage of bigger tubers (over 30 However, there is no increase in specific gravity of the tubers.

WO 98/58069 PCT/US98/12447 In conclusion, it appears that expression offda in potato produces greater numbers of tubers per plant without a sacrifice in tuber size. This represents a yield benefit in that the farmer could potentially be able to produce the same amount of tubers using less acreage. Similar experiments will also be performed by co-expression offda with other carbohydrate metabolizing genes, such as glgC 16, in order to determine how such combinations will affect tuber yield, tuber solids deposition and overall tuber specific gravity.

Aldolase activity from transgenic potato After being cultivated for 3 months (post planting) in the greenhouse, leaf samples were taken from 6 of the highest fda-expressing potato lines, obtained after transformation with pMON17542, and assayed for aldolase activity.

In order to measure potato leaf aldolase activity, duplicate leaf samples from each line were taken and immediately frozen on dry ice. Aldolase was extracted from 0.2 g of leaf tissue by grinding at 4 0 C in 1.2 mL of the extraction buffer: 100 mM Hepes, pH 5 mM MgCI 2 5 mM MnCl 2 100 mM KC1, 10 mM DTT, 1% BSA, ImM PMSF, itg/mL leupeptin, 10 pg/mL aprotinin. The extract was assayed for aldolase activity as described earlier.

Six independent transgenic potato lines expressingfda were tested for aldolase activity. The expression offda in leaves is an indicator of the expression in the whole plant because the FMV promoter used to drive expression of the respective encoding DNAs directs gene expression constitutively in most, if not all, tissues of potato plants.

Table 7 summarizes the quantitative protein expression data for each of the lines, and the percent activity for each individual line.

WO 98/58069 PCT/US98/12447 Lines Control 40608 40610 40652 40632 40631 40611 Table 7 Aldolase Activity from Transgenic Russet Burbank Potato Leaves Exp.#1 Exp.#2 Act(U/gFW) %Act Act(U/gFW) %Act 4.461 100 4.732 100 6.969 156 8.055 170 8.489 190 7.326 155 5.812 130 6.367 135 5.257 118 4.244 90 5.764 129 4.968 105 5.715 128 5.836 123 Average Activity 100 163 173 132 104 117 126 Solids uniformity in transgenic potato Twenty-five Russet Burbank lines expressingfda (potato lines designated "Maestro"), obtained after transformation with pMON 17542, and fifteen Russet Burbank Simple Solid lines, also containing glgCl6 (PCT Publication WO 91/19806 and US Patent 5,498,830), expressingfda (potato lines designated "Segal"), obtained after transformation with pMON 17581, were field tested at two different sites. For each field site, 36 plants per line (three repetitions of 12 plants per line) were evaluated for tuber solids distribution.

At harvest, tubers were pre-sorted at each field site into a ten to twelve ounce category, and nine tubers from each replicated plot were analyzed in groups of three.

For a typical 10-12 ounce tuber having a diameter of 7-8 cm, starch distribution was evaluated by removing the center longitudinal slice (13 mm) from each tuber. Slices were then peeled and laid flat on a cutting board where the inner tuber region (pith region) was removed by a 14-mm cork punch. The tissue from pith to cortex (perimedullary region) was removed by an up-to-a 2-inch cork punch. The remaining cortex tissue was approximately an 8-mm wide ring from the outermost region of the slice.

Specific gravity was then determined by weighing both the pooled pith punches and pooled cortex punches in air and then in water: Specific gravity Air Wt./(Air Wt.-Water Wt.) WO 98/58069 PCT/US98/12447 After calculating specific gravity, solids levels were determined by the following equation: -214.9206 2 18 .1 8 52 *Sp. Gravity) The degree of solids uniformity (Solids Uniformity Index) is determined by calculating the pith to cortex solids ratio (pith solids divided by cortex solids). The three groups of three tubers per plot were averaged, at which point the average of three plot replications was calculated per field site.

Analyses of several previous solids uniformity field trials (data not shown) have demonstrated nontransgenic, wild-type Russet Burbank potato to have a typical pith to cortex tuber solids ratio within the range of 68% to 72%, depending on growing region and agricultural practices. Tables 8-1 1 provide the pith to cortex solids ratios by plant line number, with a higher pith to cortex solids ratio indicating a greater degree of solids uniformity.

Tables 8 and 9 represent the data from one field site (site 1) for Segal and Maestro, respectively, and illustrate that the majority of Segal and Maestro lines have higher pith to cortex solids ratios than that of 68.4% for the Russet Burbank control, with some lines approaching an 82% pith to cortex solids ratio.

Tables 10 and 11 represent the data from another field site (site 2) for Segal and Maestro, respectively, and also illustrate that the majority of Maestro and Segal lines have higher pith to cortex solids ratios than that of the Russet Burbank control, with some lines approaching an 88% pith to cortex solids ratio. In the site 2 field trial, the Russet Burbank control had an atypical, abnormally high pith-to-cortex solids uniformity ratio of 79.3%, which was most likely due to environmental growing conditions. The site 2 results demonstrate that expression in Russet Burbank potato of E. colifda, alone or with coexpression of glgC 16, increases tuber solids uniformity even in a growing season when tuber solids uniformity is already extremely high in nontransgenic Russet Burbank. That is, thefda gene continues to perform when agricultural conditions are already conducive to an abnormally high solids uniformity level.

WO 98/58069 WO 9858069PCTIUS 98/12447 Table 8. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio.

Segal Russet Burbank Lines. Site I Line Ratio S-29 79.1 S-9 75.8 71.3 71.3 S-21 70.5 70.2 S-18 70.0 RB control 68.4 S-32 68.3 S-16 65.6 Table 9. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio.

Maestro Russet Burbank Lines. Site 1 Line Ratio M-13 74.0 M-12 73.6 M-1 73.4 M-3 73.0 M-6 72.4 M-9 71.2 M-1 1 70.6 M-18 70.5 M-17 69.9 M-19 69.4 M-5 69.3 68.9 RB control 68.4 M-8 68.3 M-43 67.7 M-23 67.3 M-7 67.0 M-39 66.6 M-22 66.0 65.4 M-27 61.4 WO 98/58069 WO 9858069PCT/US98/1 2447 Table 10. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio Segal Russet Burbank Lines. Site 2 Line Ratio S-33 87.4 S-54 87.1 86.8 S-29 85.1 S-21 84.3 S-16 83.2 S-20 81.5 S-18 80.7 S-32 80.6 RB control 79.3 S-09 79.0 Table 11. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio Maestro Russet Burbank Lines. Site 2 Line Ratio M-04 87.7 M-18 83.9 M-17 83.8 M-03 83.7 M-09 83.4 83.2 M-29 82.9 M-44 82.3 M-08 82.2 M-43 81.6 M-22 81.1 80.8 M-0O1 80.5 80.2 M-45 79.6 M-39 79.5 M-27 79.5 RB control 79.3 M-13 78.9 M-22 78.8 M- 19 78.7 M-07 78.2 M-12 77.9 M-23 77.3 M-06 76.5 75.0 M-1 1 74.1 WO 98/58069 PCT/US98/12447 The effect of aldolase on pith to cortex solids ratios in the Segal lines is slightly more dramatic than in Maestro lines. We believe this phenotype is due to expression of fda in a background in which the Russet Burbank host expresses glgC 16 at a relatively low to moderate level, and that the combination offda plus glgC 16 provides improved benefits. Cross sectional tuber slices (Figure 9) of three Segal lines with improved solids uniformity illustrate a greater deposition of starch within the inner regions of the tuber.

Specifically, an increase in cortex volume accompanied by relocation of the xylem ring towards the center of the tuber, plus a more opaque pith tissue due to an increase in starch density, are evident in the transgenic lines. This physiological alteration may be due to an increase in sucrose translocation from source to sink, which may influence phloem element distribution during tuber development or sucrose availability for starch biosynthesis across the tuber.

Example Plant transformation and FDA expression in cotton plants The E. colifda vectors pMON17524 [FMV/CTPl/fda] (Figure 2) and pMON 17542 [FMV/CTP2/fda] (Figure 3) were transformed into cotton using Agrobacterium as described by Umbeck et al. (1987) and in US Patent 5004863. The protein was targeted to the chloroplast using either the Arabidopsis SSU CTP 1 (pMON 17524) or the Arabidopsis EPSPS (pMON 17542) chloroplast transit peptide.

Aldolase expression in cotton Five-week-old calli transformed with both vectors were analyzed by Western blot analyses and by aldolase assays. Western blot analysis indicated a large amount of protein at the position of the full-length FDA standard and a lesser amount at the same position in the control callus extracts. It appeared that the protein was fully processed. To verify that FDA was expressed in the tissue and for comparison of activity, calli transformed with the two vectors were extracted in a buffer that would prevent loss of activity of the transgene product. BSA was added to final concentration of 1 mg/mL, which limited the analysis of processing on import by Western blot. Aldolase assays were performed plus or minus mM EDTA, which inhibits the E. coli enzyme but not the plant enzyme. The results of the assays are shown in Table 12.

MOBT086P Table 12 Aldolase Activity in Cotton Calli and Cotton Leaf A A340 e-3mg protein/5 min Colony# -EDTA +EDTA Fold Increase Controls Cotton Leaf (Coker) 4.0 4.2 Uninoculated Calli 7.7 5.6 1.3X Inoculated Calli (E35S/GUS) #1 6.8 6.1 #2 3.5 FDA calli pMON 17542 #1 3.5 2.3 #3 5.5 2.6 2.1X #5 9.2 3.8 2.4X #4 19.8 3.6 pMON17524 #2 15.2 5.8 2.6X #3 12.5 4.0 3.1X 14.4 2.9 4.9X #6 4.1 1.2 20 The results indicate that there is good expression of thefda gene in cotton callus. Almost all calli had at least twofold higher aldolase activity, and the increase was sensitive to inhibition by EDTA. Processing appeared complete by Western blot analysis using these samples.

With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, we note that unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that we intend each of those words to be so interpreted in construing the foregoing description and/or the following claims.

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Thompson et al. (1994) Nucl. Acids Res. 22:4673-4680.

Tierney et al. (1987) Planta 172:356-363.

Truernit et al. (1995) Planta 196 (3):564-570.

Tsutsumi et al. (1984) J. Biol. Chem. 259, 14572-14575.

Umbeck et al. (1987) Biotechnology. 5, 263-266.

Vain et al. (1993) Plant Cell Reports 12: 84-88.

Vasil et al. (1990) Bio/Technology 8:429-434.

Vasil et al. (1992) Bio/Technology 10:667-674.

Walters et al. (1992) Plant Molecular Biology 18: 189-200.

Witke and Goetz (1993) Journal of Bacteriology 175(22): 7495-7499 Wong et al. (1988) Gene 68: 193-203.

WO 98/58069 PCTIUS98/1 2447 Yamamioto et al. (1994) Plant and Cell Physiology 35(5):773-778.

Zheng et al. (1993) Plant J. 4: 3357-3366.

WO 98/58069 PCT/US98/12447 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Gerard Barry NordineCheikh Ganesh Kishore (ii) TITLE OF INVENTION: Expression of Fructose 1,6 Bisphosphate Aldolase in Transgenic Plants (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Arnold, White Durkee STREET: P.O. Box 4433 CITY: Houston STATE: Texas COUNTRY: United States of America ZIP: 77210-4433 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US Unknown FILING DATE: Concurrently Herewith CLASSIFICATION: Unknown (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US Prov. App. Serial No. 60/049,995 FILING DATE: June 17, 1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: Patricia A. Kammerer REGISTRATION NUMBER: 29,775 REFERENCE/DOCKET NUMBER: MOBT086 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (713) 787-1400 TELEFAX: (713) 787-1440 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1080 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/58069 WO 9858069PCTJUS98/1 2447 TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGTCTAAGA TTTTTGATTT CGTAAAACCT GGCGTAATCA CTGGTGATGA CGTACAGAAA

GTTTTCCAGG

120

GACTCCATCA

180

TTCTCCAACG

240

GGTGCTGCTA

300

TATGGTGTTC

360

GACGGTCTGT

420

TCTCACATGA

480

TACCTGGAGC

540

GGTGAAGAAG

600

GAAGACGTTG

660

GCGTCCTTCG

720

ATCCTGCGTG

780

AACTTCGTAT

840

TACGGCGTAG

900

CTGAACTACT

960

GAAGATCAGC

1020

TAGCAAAAGA

ACGCCGTACT

GTGGTGCTTC

TCCTGGGCGC

CGGTTATCCT

TGGACGCGGG

TCGACCTGTC

GCATGTCCAA

ACGGCGTGGA

ATTACGCATA

GTAACGTACA

ATTCTCAGGA

TCCACGGTGG

TAAAAATGAA

ACAAAGCGAA

CGAACAAGAA

AAACAACTTC

GGAAACCGCT

CTTTATCGCT

GATCTCTGGT

GCACACTGAC

TGAAAAACAC

TGAAGAATCT

AATCGGCATG

CAACAGCCAC

CACCGAACTG

CGGTGTTTAC

ATATGTTTCC

TTCCGGTTCT

CATCGATACC

CGAAGCTTAT

ATACTACGAT

GCACTGCCAG CAGTAAACTG GCTAAAGTTA

AAGCGCCGGT

GGTAAAGGCG

TGAAATCTGA

GCGCATCACG TTCACCAGAT CACTGCGC!GA

AGAAACTGCT

TTCGCAGC!TA

CCGGTAAGCC

CTGCAAGAGA ACATCGAAAT ACTCTGGAAA TCGAACTGGG ATGGACGC!TT

CTGCACTGTA

AGCAAAATCA

GCCCGCGTTT

AAGCCGGGTA

ACGTGGTTCT

AAGAAACACA

ACCTGCCGCA

ACTGCTCAGG

AAATCAAAGA

GATACCCAAT

GGGCAACCTG

CTGCAGGGTC

AGCTGGGTAA

CCGCGCGTAT

GGCTGCGTGC

48

CGTCGGTACT

TATCGTTCAG

CGTTCCGCAG

GGCTGAACAT

GCCGTGGATC

GCTGTTCTCT

CTGCTCTAAA

TTGCACCGGT

CACCCAGCCG

CACCATCGCA

GACTCCGACC

CAACAGCCTG

CTCCGTAAGC

GGAAGGCGTT

CCCGAAAGGC

CGGTCAGACT

WO 98/58069 PCT/US98/12447 TCGATGATCG CTCGTCTGGA GAAAGCATTC CAGGAACTGA ACGCGATCGA CGTTCTGTAA 1080 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 359amino acids TYPE: amino

STRANDEDNESS:

TOPOLOGY: Linear (xi) SEQUENCE DESCR: Met Ser Lys Ile P Asp Asp Val Gin L 2( Ala Leu Pro Ala V, 3! Val Leu Glu Thr A: Phe Ser Asn Gly G 6! Ser Asp Val Pro G: 8 Ala His His Val H 9 Ile Leu His Thr A 1 Asp Gly Leu Leu A 1 Lys Pro Leu Phe S 1 Leu Gin Glu Asn I 1 Ser Lys Ile Gly M 1 Gly Glu Glu Asp G 1 Leu Tyr Thr Gin P 2 IPTION: SEQ ID NO:2 he Asp Phe Val Lys Pro Gly Val Ile Thr Gly ys al 5 la 0 ly 5 In 0 is 5 sp 10 sp 25 er 40 le 55 et 70 ly 85 ro 00 Val Asn Ala Ala Gly Gin His Ala Ser Glu Thr Val Glu Phe Cys Lys Ser Ala Met Cys Gly His Ile Leu Asp Asp Gin Val Val Phe Ala Ala Ala Glu Met Cys Glu Asn Val Val Gly Lys Ile Ile Glu Lys Lys lle Ser Ile Ser Asp 10 Ala 25 Thr 40 Ala 55 Ala 70 Leu 85 His 100 Lys 115 His 120 Asp 145 Lys 160 Glu 175 His 190 Tyr 205 Lys Asp Pro Gly Gly Tyr Leu Phe Leu Tyr Leu Met Ala Asn Ile Ile Gly Ile Val Pro Ala Glu Glu Cys Ala Thr Asn Asn Val Val Ser Pro Trp Thr Glu Arg Thr Ser Glu Phe Ala Gin Lys Gly Val 105 Ile 120 Gly 135 Ser 150 Met 165 Gly 180 Ala 195 Leu 210 WO 98/58069 PCT/US98/12447 Ser Val Ile Pro Thr Met Leu Gly Pro Leu Lys His Leu His Ala Asn Asn Asn Arg Glu Ile Gly Arg Asn Gin Ile Tyr Pro Val Lys Ser Val Asp Ser Glu Asp Tyr Lys Trp Ala Ile Asn Val Phe Val Trp Ala Pro Gin Asn Ala 220 Val 235 Ser 250 His 265 Ser 280 Ala 295 Tyr 310 Asn 325 Thr 340 Ala 355 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGGCCATGG CTAAGATTTT TGATTTCGTA INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCCCGAGCTC TTACAGAACG TCGATCGCGT TCAG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: WO 98/58069 PTU9/24 PCTIUS98/12447 LENGTH: 10847 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:S: 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501

CGATAAGCTT

TTCGATTGCT

GCAATGGTGT

CAACGCAAAT

AGCTTATCCG

TAATTGGCTC

GCGTGCATGC

CTCTGGTCTT

ACAGGTCCTT

GGTCTTTTGG

TATGGGTGCC

TTGGTAACGG

GCTGCAACTG

CGATAGCACT

GTGTGTTGAA

GGTGATCGTC

CACCTACAGG

TTGCTGGTCT

ACTCGTGACC

CGTTGAGACT

GTAAGCTCAC

GCTTTCCCAT

CCTTAACGTT

AGGAAATGGG

GAAGACGTGG

TGTTCCAGAA

CTGTTGCAGC

GAACTCCGTG

CAAGCTCAAC

GTGGTCGTCC

GCTACCCACC

CGTTTCTGAA

GCTTCCCAGA

CTCTCCGACA

CGGATCCAGC

CAATGCATCA

ATTATGGCAT

ATTTACTGTG

TGGAT -GAGA

TTAATATTAT

ATAAGAGATA

GCCTCTAATG

CATTATGCTT

TGCAAATGTT

GAACTTTCCT

GCTTTATAAT

TTTTTTAATG

CGACCTGCAG

AGCAGCATTC

TATTCAATT

TTTGTAAGGA

GATGTAATTG

TCAATTGAAG

GCAGAACCCA

CTCCCTTATC

ATTTCGTCGT

TGAGCTTCGT

TTCACGGTGC

TCTGGAACCG

CATGTTTGGA

AAGGTGAAGA

AGAATCCGTA

TGGACTCCTT

GTTGCCGTTT

TTCATTGGTG

CCCACTTCGC

TTCCAGTTAC

GTACCTATGG

CAACACCCCA

ACACTGAAAA

GATGCTGACG

CGGTCAAGTG

TGGTTGCTGC

TTGATGAACC

TGCCGACATC

CTGACTTGCG

GACCGTGCTC

TGCATTCGCT

TTAAGGAAAG

GGTGTTGATT

TGACGGTAAG

TCGATCACCG

AACCCTGTTA

GTTCATGGAT

CTAAGGCTGC

TTTCGTTCGT

GTTTCATTGC

TGGGAAAACT

TTTTTTATTC

AGAGTTAATG

TTGTTTTTTC

TGCAAALCATT

ACCGAAGTTA

ATTCACTAGG

ACTGAATACA

TTATGTAATT

TATAGTTATA

CATTTTATGA

CCACTCGAAG

CAGATTGGGT

GGTATCGCCA

AGAATTCTCA

GAGGAAGATC

TTTCTCCGAT

TCTCTTATCT

GGTTTCTCTG

CGTGGGGATT

CCTCTTAAGG

AAGCAGCCGT

TCCGTATTCC

GGTCTCGCTA

TGTTATCAAC

AGGAAGGTGA

GCTCCTGAGG

GACTATGGGT

ACGCTTCTCT

GAAATGGGTG

CTTGCGTGGA

CTTCCGCTCA

GGTATCACCA

GATGCTTCAA

GTGTGCGTAC

ATTGATGTTC

CTTGCTTGTT

CAACCCGTAC

GAAGTGATCA

TGTTCGTTCT

CTTCTATGAT

GAAGGTGCTA

CGACCGTCTT

GCGATGAAGG

GGTCTCGGTA

TATCGCTATG

CTGTTGATGA

TTGATGGCTG

TTGATGAGCT

ATCATCGGTT

GCACACACCA

GTTTTTCTTG

GGTTTTCGCT

AATGATATGG

TCTTATTTGT

TTGTTTTGAG

ATATGAGGAG

CAACAAATAT

AGTATGTCCT

TTCCAGAATC

CTCATGGATT

CTTGCCAATT

CGGCCGCGTT

TCAATCAACA

AAACCAAGAA

GTCCAAAGCC

AAAATTTTCA ATCCCCATTC GGCGCAAGTT AGCAGAATCT CCAATCTCTC GAAATCCAGT AAGACGCAGC AGCATCCACG GAAGAAGAGT GGGATGACGT TCATGTCTTC TGTTTCCACG CCAGCAACTG CTC!GTAAGTC AGGTGACAAG TCTATCTCCC GCGGTGAAAC TCGTATCACC ACTGGTAAGG CTATGCAAGC TACTTGGATC ATTGATGGTG CTCCTCTCGA TTTCGGTAAC C!TTGTTGGTG TTTACGATTT C!ACTAAGCGT CCAATGGGTC TGCAGGTGAA GTCTGAAGAC CCAAAGACTC CAACGCCAAT AGTGAAGTCC GCTGTTCTGC CTGTTATCGA GCCAATCATG GGTTTTGGTG CTAACCTTAC C!ATCCGTCTT GAAGGTCGTG CAGGTGATCC ATCCTCTACT CCAGGTTCCG ACGTCACCAT TGGTCTCATC TTGACTCTGC ACCCACGTCT TGCTGGTGGA TCTACTTTGA AGGGTGTTAC CGACGAGTAT CCAATTCTCG CCGTTATGAA CGGTTTGGAA TCTGCTGTCG CAAACGGTCT TGAGACTTCT CTCGTCGTGC ACGCTTCTGG AGCAGCTGTC AGCTTCCTCG TTATGGGTCT TGCTACTATG ATCGCTACTA GTCTTGGAGC TAAGATCGAA CAAGAATTCG AGCTCGGTAC TCGACAACGT TCGTCAAGTT GAATCCTACT GAGTTCGAGT TACCATTTGT TGTGCTTGTA ATCGAACTGT GAAATGGAAA TCCTTTTGTT CATTCTCAAA TGTGTGTTGA ATTTGAAATT TAAAATGTG TCAAATCGTG TAAAACACTT GTAGTTGTAC ATTTTCAGAC CTAGAAAAGC CTTGTGTTTT AGACATTTAT CTTGTCAGAT TCTAATCATT TGTAGTTGAG TATGAAAATA GATTGACAAC ATGCATCAAT CAAGCTTGAG CTCAGGATTT AGGTACGAGC CATATCACTT GGAACTCCCA TCCTCAAAGG TCAACAAGGT CAGGGTACAG WO 98/58069 WO 9858069PCTIUS98/12447 2551 2602.

2652.

27021 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 AGTCTCCAAA CCATTAGCCA AATCAAAGTA AACTACTGTT AGAAAAAGAC ATCCACCGAA AATCTTGTCA ACATCGAGCA ATGGTGCAGA ATTGTTAGGC GCAAAGATAA AGCAGATTCC

GAAAAGAGCT

TGACGACCAC

TGAAGGATCA

GCTTCCTCTA

CACTATGGTC

CCACCCGCAA

AGAGTTAACT

GACTCTCTCT

GCATGCAGGC

ACTGGTGATG

CGCACTGCCA

TGGAAACCGC

GGTGGTGCTT

GGGTGCTGCT

TGGCTGAACA

AAGAAACTGC

CTTCGCAGCT

CTGAAGAATC

CGCATGTCCA

TGGTGAAGAA

ACACCCAGCC

AGCCCGCGTT

CAAGCCGGGT

AATATGTTTC

TTCCACGGTG

CTACGGCGTA

GGGAAGGCGT

CAGCTGGGTA

TCCGCGCGTA

AGAAAGCATT

ACCGGATCCA

GATTGAATCC

AATTACGTTA

GAGATGGGTT

TAGAAAACAA

TCATCTATGT

GTGGTGGCCG

GTGAATGTAG

TATTGCTTTC

AATGtACTTT

GAAAAAAAAT

CTCATTGCTG

AATATATCCT

GGTTTCTACG

TGAGCCATGT

TGATCCACAT

CGTTCGCGCG

GGGTTCGAGA

GGGCGCAGCC

GGTTAAAAGA

AATGCTGGAT

CCCCTCATCT

GTCCTGACAG

AAAA~GAATTC

TCAGATACTG

TGCTCTCTTC

GCTCCTTTCA

GGCTAACAAC

GCATGCAGGT

TACCTTCCTG

CATGGCTAAG

ACGTACAGAA

GCAGTAAACT

TGCTAAAGTT

CCTTTATCGC

ATCCTGGGCG

TTATGGTGTT

TGCCGTGGAT

AC CGGTAAG C

TCTGCAAGAG

AAATCGGCAT

GACGGCGTGG

GGAAGACGTT

TCACCATCGC

AACGTGGTTC

CAAGAAACAC

GTTCCGGTTC

GTAAAAATGA

TCTGAACTAC

ACCCGAAAGG

TGGCTGCGTG

CCAGGAACTG

ATTCCCGATC

TGTTGCCGGT

AGCATGTAAT

TTTATGATTA

AATATAGCGC

TACTAGATCG

CATCGATCGT

ACACGTCGAA

GCCTATAAAT

CATTTTATAA

TGGTAATTAC

ATCCATGTAG

GCCGCCGCTG

CAGAACTGAG

GCACCTTCCC

GGGACTTTTc t'GGCGCCAGC

AGGGGGGGCA

CTGGTTAAAA

CAGGTTAGCG

TTTCTGCCTG

GTCATCACTC

AAAGCTACAG

CCAGCACATG

GACTTAAAGT

GCTGGCTTGT

GCACCTACCA

TCTAGTACAA

CCCACTCACT

CCTCTATATA

AACCAATCCT

CGCTACTATG

ACGGACTTAA

GACATTACTT

GTGGCCTCCG

ACCTTACCGA

ATTTTTGATT

AGTTTTCCAG

GCGTCGGTAC

AAAGCGCCGG

TGGTAAAGGC

CGATCTCTGG

CCGGTTATCC

CGACGGTCTG

CGCTGTTCTC

AACATCGAAA

GACTCTGGAA

ACAACAGCCA

GATTACGCAT

AGCGTCCTTC

TGACTCCGAC

AACCTGCCGC

TACTGCTCAG

ACATCGATAc

TACAAAGCGA

CGAAGATcAG

CCGGTCAGAC

AACGCGATCG

GTTCAAACAT

CTTGCGATGA

AATTAACATG

GAGTCCCGCA

GCAAACTAGG

GGGATCGATC

GAAGTTTCTC

ATAAAGATTT

ACGACGGATC

TAACGCTGCG

TCTTTCTTTT

ATTTCCCGGA

CCGCTTTGCA

CCGGTTAGGC

CCCAACACGG

CTAGCTTGGC

TGGGGGGATG

CCCCCCTTCG

ACAAGGTTTA

GTGGCCGAAA

TGGACAGCCC

TGCCCCTCAA

GAGATCAATG

CATCATGGTC

TAGTGGGCAT

GGGGACCAGA

AAAGCATCTT

GTGGGGAACA

AATGCGTATG

AGAAGGCATT

TCTAGAAGAT

GTTGCCTCTC

GTCCTCCGCT

CCATCACAAG

ATTGGAAAGA

TTCCGGTGGT

TCGTAAAACC

GTAGCAAAAG

TGACTCCATC

TTATCGTTCA

GTGAAATCTG

TGCGCATCAC

TGCACACTGA

TTGGACGCGG

TTCTCACATG

TCTGCTCTAA

ATCGAACTGG

CATGGACGCT

ACACCGAACT

GGTAACGTAC

CATCCTGCGT

ACAACAGCCT

GAAATCAAAG

CGATACCCAA

ACGAAGCTTA

CCGAACAAGA

TTCGATGATC

ACGTTCTGTA

TTGGCAATAA

TTATCATATA

TAATGCATGA

ATTATACATT

ATAAATTATC

CCCGGGCGGC

ATCTAAGCCC

CCGAATTAGA

GTAATTTGTC

GACATCTACA

TCTCCATATT

CATGAAGCCA

CCCGGTGGAG

AGATAATTTC

TGAGCGACGG

TGCCATTTTT

GGAGGCCCGC

GCGTGCGCGG

TAAATATTGG

AACGGGCGGA

CTCAAATGTC

GTGTCAAGGA

AAGAATCTTC

AGTAAGTTTC

CTTTGAAAGT

CAAAAAAGGA

TGCCTTTATT

AAATAACGTG

ACGAACGCAG

CATTCCCATT

CTCCACAATG

CGGCTCAGGC

GCCTTCCCAG

CAACGGCGGA

AGAAGTTTGA

CGCGTCAACT

TGGCGTAATC

AAAACAACTT

AACGCCGTAC

GTTCTCCAAC

ACGTTCCGCA

GTTCACCAGA

CCACTGCGCG

GTGAAAAACA

ATCGACCTGT

ATACCTGGAG

GTTGCACCGG

TCTGCACTGT

GAGCAAAATC

ACGGTGTTTA

GATTCTCAGG

GAACTTCGTA

ACTCCGTAAG

TGGGCAACCT

TCTGCAGGGT

AATACTACGA

GCTCGTCTGG

AGAGCTCGGT

AGTTTCTTAA

ATTTCTGTTG

CGTTATTTAT

TAATACGCGA

GCGCGCGGTG

CGCCACTCGA

CCATTTGGAC

ATAATTTGTT

GTTTTATCAA

TTTTTGAATT

GACCATCATA

TTTACAATTG

CTTGCATGTT

CATTGAGAAC

GGCAACGGAG

GGGGTGAGGC

GTTACCGGGA

TCACGCGCCA

TTTAAAAGCA

AACCCTTGCA

AATAGGTGCG

TCGCGCCCCT

WO 98/58069 WO 9858069PCTIUS98/12447 5451 5502.

5551 56021 5651 5701 5751 5801 5851 5902.

5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001 8051 8101 8151 8201 8251 8301

CATCTGTCAG

CCCCAGGCTT

TTTCGCCGAT

GCCTGCCCCT

GTCAACGTCC

ATCCACAACG

CGTTTCTGGC

GGGCAACCAG

GAGAGCCTTC

TCGTCGCCGC

GTGCCGGCAG

CGCGACGATG

TCGCTCAAGC

CAGGCCATTA

GGCGTTCGCG

CTTCCGGCGG

GTAGATGACG

CAGCCTAACT

CCTCGGCGAG

TACCTTGTCT

CTCGACCTGA

AAGAATTGGA

ACCCTTGGCA

CGGCGCATCT

GCTCCTGTCG

AGCAGAATGA

AAAACGTCTG

TTCGTAAAGT

GGATCTGCAT

GTATTAACGA

CCGCATCCAT

CATGTTCATC

GGTATCATTA

GTGACCAAAC

CCAGACATTA

AGGCAGACAT

AGCTGCCTCG

GCTCCCGGAG

AAGCCCGTCA

ATGACCCAGT

GCATCAGAGC

GCACAGATGC

CGCTCACTGA

GCTCACTCAA

AGGAAAGAAC

AGGCCdCGTT

CACAAAAATC

AAGATACCAG

CGACCCTGCC

GTGGCGCTTT

CGTTCGCTCC

GCTGCGCCTT

GACTTATCGC

GTATGTAGGC

ACACTAGAAG

TTCGGAAAIA

TAGCGGTGG7

GATCTCAAGA

TAGTCGCGCC

GTCCACATCA

TTGCGAGGCT

CATCTGTCAA

GCCCCTCATC

CCGGCGGCCG

GCGTTTGCAG

CCCGGTGAGC

AACCCAGTCA

ACTTATGACT

CGCTCTGGGT

ATCGGCCTGT

CTTCGTCACT

TCGCCGGCAT

ACGCGAGGCT

CATCGGGATG

ACCATCAGGG

TCGATCACTG

CACATGGAAC

GCCTCCCCGC

ATGGAAGCCG

GCCAATCAAT

GAACATATCC

CGGGCAGCGT

TTGAGGACCC

ATCACCGATA

CGACCTGAGC

CTGGAAACGC

CGCAGGATGC

AGCGCTGGCA

ACCGCCAGTT

ATCAGTAACC

CCCCCATGAA

AGGAAAAAAC

ACGCTTCTGG

CTGTGAATCG

CGCGTTTCGG

ACGGTCACAG

GGGCGCGTCA

CACGTAGCGA

AGATTGTACT

GTAAGGAGAA

CTCGCTGCGC

AGGCGGTAAT

ATGTGAGCAA

GCTGGCGTTT

GACGCTCAAG

GCGTTTCCCC

GCTTACCGGA

CTCATAGCTC

AAGCTGGGCT

ATCCGGTAAC

CACTGGCAGC

GGTGCTACAG

GACAGTATTT

GAGTTGGTAG

TTTTTTGTTT

AGATCCTTTG

CCTCAAGTGT CAATACCGCA GGGCACTTAT TCTGTGGGAA ACTCGCGTAA AAiTCAGGCGT GGCCAGCTCC ACGTCGCCGG CCGAAATCGA CGCCGCGCCG GGTGAGTCGG CCCCTCAAGT TGTCAGTGAG GGCCAAGTTT TCCGCGTGGT GCCGCGGTGT CTCGCACACG GCTTCGACGG GGCCATAGAC GGCCGCCAGC CCAGCGGCGA GTCGGAAAGG GTCGATCGAC CGATGCCCTT GCTCCTTCCG GTGGGCGCGG GGCATGACTA GTCTTCTTTA TCATGCAACT CGTAGGACAG CATTTTCGGC GAGGACCGCT TTCGCTGGAG CGCTTGCGGT ATTCGGAATC TTGCACGCCC GGTCCCGCCA CCAAACGTTT CGGCGAGAAG GGCGGCCGAC GCGCTGGGCT ACGTCTTGCT GGATGGCCTT CCCCATTATG ATTCTTCTCG CCCGCGTTGC AGGCCATGCT GTCCAGGCAG ACAGCTTCAA GGATCGCTCG CGGCTCTTAC GACCGCTGAT CGTCACGGCG ATTTATGCCG GGGTTGGCAT GGATTGTAGG CGCCGCCCTA GTTGCGTCGC GGTGCATGGA GCCGGGCCAC GCGGCACCTC GCTAACGGAT TCACCACTCC TCTTGCGGAG ALACTGTGAAT GCGCAAACCA ATCGCGTCCG CCATCTCCAG CAGCCGCACG TGGGTCCTGG CCACGGGTGC GCATGATCGT GGCTAGGCTG GCGGGGTTGC CTTACTGGTT CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC AACAACATGA ATGGTCTTCG GTTTCCGTGT GGAAGTCAGC GCCCTGCACC ATTATGTTCC TGCTGGCTAC CCTGTGGAAC ACCTACATCT TTGACCCTGA GTGATTTTTC TCTGGTCCCG GTTTACCCTC ACAACGTTCC AGTAACCGGG CGTATCGTGA GCATCCTCTC TCGTTTCATC CAGAAATTCC CCCTTACACG GAGGCATCAA CGCCCTTAAC ATGGCCCGCT TTATCAGAAG AGAAACTCAA CGAGCTGGAC GCGGATGAAC CTTCACGACC ACGCTGATGA GCTTTACCGC TGATGACGGT GAAAACCTCT GACACATGCA CTTGTCTGTA AGCGGATGCC GGGAGCAGAC GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC TAGCGGAGTG TATACTGGCT TAACTATGCG GAGAGTGCAC CATATGCGGT GTGAALATACC AATACCGCAT CAGGCGCTC'r TCCGCTTCCT TCGGTCGTTC GGCTGCGGCG AGCGGTATCA ACGGTTATCC ACAGAATCAG GGGATAACGC AA.GGCCAGCA AAAGGCCAGG AACCGTAAAA TTCCATAGGC TCCGCCCCCC TGACGAGCAT TCAGAGGTGG CGAAACCCGA CAGGACTATA CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC TACCTGTCCG CCTTTCTCCC TTCGGGAAGC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT GTGTGCACGA ACCCCCCGTT CAGCCCGACC TATCGTCTTG AGTCCAACCC GGTAAGACAC AGCCACTGGT AACAGGATTA GCAGAGCGAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT GGTATCTGCG CTCTGCTGAA GCCAGTTACC CTCTTGATCC GGCAAACAAA CCACCGCTGG GCAAGCAGCA GATTACGCGC AGAAAAAAAG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG WO 98/58069 WO 9858069PCTIUS98 1 2447 8351 8401 8451 8501 8551 8601 8651 8701 8751 8801 8851 8901 8951 9001 9051 9101 9151 9201 9251 9301 9351 9402.

9451 9501 9551 9601 9651 9701 9751 9801 9851 9901 9951 10001 10051 10101 10151 10201 10251 10301 10351 10401 10451 10501 10551 10601 10651 .10701 10751.

10801

AACGAAAACT

CTTCACCTAG

GTATATATGA

GCACCTATCT

CCCCGTCGTG

GTGCTGCAAT

GCAATAAACC

TTTATCCGCC

GTAGTTCGCC

CGGGAGCACA

CGGCGCGGCT

CGAAGTATCG

TCGAACCGAC

GGCCTGAAGC

GCTTGATGAA

CGGCTTCCCC

GTTGTGCACG

GCAATTTGGA

CAGCCACGAT

CATAGCGTTG

TCCTGAACAG

ACTCGCCGCC

TCCCGCATTT

CGCTGCCGAC

TACTTGAAGC

CGCGCAGATC

CAAGGTAGTC

TCGCGGCGCG

ACGCTCGTCG

GGCGAGTTAC

GGTCCTCCGA

TGAGGGATCA

GCCAAGGGAT

TGGGTGGTTG

GGGGAACCCT

TTTTCACGCC

TAGGTTTACC

GGCGGGAAAC

AGCATTCCAG

TCAAATTGGT

GTAAGGAAGA

CTCCAAACCA

CAAAGTAAAC

AAAAGACATC

CTTGTCAACA

GTGCAGAATT

AAGATAAAGC

AAGAGCTGTC

CGACCACAAA

AGGATCATCA

CACGTTAAGG

ATCCTTTTAA

GTAAACTTGG

CAGCGATCTG

TAGATAACTA

GATACCGCGA

AGCCAGCCGG

TCCATCCAGT

AGTTAATAGT

GGATGACGCC

TAATTCAGGA

ACTCAACTAT

GTTGCTGGCC

CACACAGTGA

ACAACGCGGC

TGGAGAGAGC

ACGACATCAT

GAATGGCAGC

CGACATTGAT

CCTTGGTAGG

GATCTATTTG

CGACTGGGCT

GGTACAGCGC

TGGGCAATGG

TAGGCAGGCT

AGTTGGAAGA

GGCAAATAAT

GCTTAACTCA

TTTGGTATGG

ATGATCCCCC

TCGAGGATTT

AGCCACAGCA

CTTTTTGGAA

AACAGAAGTC

GTGGTTGGCA

CTTTTAAATA

CGCCAATATA

GACAATCTGA

ATTGGGTTCA

ATCGCCAAAA

ATTCTCAGTC

TTAGCCAAAA

TACTGTTCCA

CACCGAAGAC

TCGAGCAGCT

GTTAGGCGCA

AGATTCCTCT

CTGACAGCCC

AGAATTCCCT

GATACTGAAC

GATTTTGGTC

ATTAAAAATG

TCTGACAGTT

TCTATTTCGT

CGATACGGGA

GACCCACGCT

AAGGGCCGAG

CTATTAATTG

TTGCGCAACG

TAACAATTCA

GTTAAACATC

CAGAGGTAGT

GTACATTTGT

TATTGATTTG

GAGCTTTGAT

GAGATTCTCC

TCCGTGGCGT

GCAATGACAT

CTGdCTATCT

TCCAGCGGCG

AGGCGCTAAA

GGCGATGAGC

AGTAACCGGC

AGCGCCTGCC

TATCTTGGAC

ATTTGTTCAC

GTCTAACAAT

AGCGTTAGAT

CTTCATTCAG

ATGTTGTGCA

TTCGGCGCTG

GCCCACTCGA

TGCTGCTCCG

ATTATCGTAC

TGCACATACA

TCCGTTA.TTC

TCCTGTCAAA

TCCCCATCAA

ATCAACAAGG

CCAAGAAGGA

CAAAGCCTCA

GCTACAGGAG

GCACATGCAT

TTAAAGTTAG

GGCTTGTGGG

CCTACCAAAA

AGTACAAGTG

ACTCACTAAT

CTATATAAGA

CAATCCTrCT

ATGAGATTAT

AAGTTTT.AAA

ACCAATGCTT

TCATCCATAG

GGGCTTACCA

CACCGGCTCC

CGCAGAAGTG

TTGCCGGGAA

TTGTTGCCAT

TTCAAGCCGA

ATGAGGGAAG

TGGCGTCATC

ACGGCTCCGC

CTGGTTACGG

CAACGACCTT

GCGCTGTAGA

TATCCAGCTA

TCTTGCAGGT

TGCTGACAAA

GAGGAACTCT

TGAAACCTTA

GAAATGTAGT

AAAATCGCGC

GGCCCAGTAT

AAGAAGATCG

TACGTGAAAG

TCGTTCAAGC

GCTGCAGGCA

CTCCGGTTCC

AAAAAGCGGT

CGCTACGTCC

CCTCTAGCCG

TCGTCAGGCT

GGAATGCCAA

AATGGACGAA

TAATAAACGC

CACTGATAGT

GCTTGAGCTC

TACGAGCCAT

ACTCCCATCC

ACAAGGTCAG

ATCAATGAAG

CATGGTCAGT

TGGGCATCTT

GACCAGACAA

GCATCTTTGC

GGGAACAAAA

GCGTATGACG

AGGCATTCAT

AGAAGATCTA

CAAAAAGGAT

TCAATCTAAA

AATCAGTGAG

TTGCCTGACT

TCTGGCCCCA

AGATTTATCA

GTCCTGCAAC

GCTAGAGTAA

TGCTGCAGGT

CACCGCTTCG

CGGTGATCGC

GAGCGCCATC

AGTGGATGGC

TGACCGTAAG

TTGGAAACTT

AGTCACCATT

AGCGCGAACT

ATCTTCGAGC

AGCAAGAGAA

TTGATCCGGT

ACGCTATGGA

GCTTACGTTG

CGAAGGATGT

CAGCCCGTCA

CTTGGCCTCG

GCGAGATCAC

CGACGCCGCT

TCGTGGTGTC

CAACGATCAA

TAGCTCCTTC

GCKACCGCGT

ACCCAGACGA

TTCCGACGTT

GCACTCCCGA

CGGATAAACC

TCTTTTCTCT

TTAAACTGAA

AGGATTTAGC

ATCACTTTAT

TCAAAGGTTT

GGTACAGAGT

AATCTTCAAT

AAGTTTCAGA

TGAAAGTAAT

AAAAGGAATG

CTTTATTGCA

TAACGTGGAA

AACGCAGTGA

TCCCATTTGA

AGCTTAT

INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 10901 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: Linear WO 98/58069 WO 9858069PCTIUS98/12447 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701

CGATAAGCTT

TTCGATTGCT

GCAATGGTGT

CA.ACGCAAAT

AGCTTATCCG

TAATTGGCTC

GCGTGCATGC

CTCTGGTCTT

ACAGGTCCTT

GGTCTTTTGG

TATGGGTGCC

TTGGTAACGG

GCTGCAACTG

CGATAGCACT

GTGTGTTGAA

GGTGATCGTC

CACCTACAGG

TTGCTGGTCT

ACTCGTGACC

CGTTGAGACT

GTAAGCTCAC

GCTTTCCCAT

CCTTAACGTT

AGGAAATGGG

GAAGACGTGG

TGTTCCAGAA

CTGTTGCAGC

GAACTCCGTG

CAAGCTCAAC

GTGGTCGTCC

GCTACCCACC

CGTTTCTGA-A

GCTTCCCAGA

CTCTCCGACA

CGGATCCAGC

CAATGCATCA

ATTATGGCAT

ATTTACTGTG

TGGATGGAGA

TTAATATTAT

ATAAGAGATA

GCCTCTAATG

CATTA TGCTT

TGCAAATGTT

GAACTTTCCT

GCTTTATAAT

TTTTTTAATG

CGACCTGCAG

AGCAGCATTC

TATTCAAATT

TTTGTAAGGA

AGTCTCCAAA

AATCAAAGTA

AGAAAAAGAC

AATCTTGTCA

GATGTAATTG

TCAATTGAAG

GCAGAACCCA

CTCCCTTATC

ATTTCGTCGT

TGAGCTTCGT

TTCACGGTGC

TCTGGAACCG

CATGTTTGGA

AAGGTGAAGA

AGAATCCGTA

TGGACTCCTT

GTTGCCGTTT

TTCATTGGTG

CCCACTTCGC

TTCCAGTTAC

GTACCTATGG

CAACACCCCA

ACACTGAAAA.

GATGCTGACG

CGGTCAAGTG

TGGTTGCTGC

TTGATGAACC

TGCCGACATC

CTGACTTGCG

GACCGTGCTC

TGCATTCGCT

TTAAGGAAAG

GGTGTTGATT

TGACGGTAAG

TCGATCACCG

AACCCTGTTA

GTTCATGGAT

CTAAGGCTGC

TTTCGTTCGT

GTTTCATTGC

TGGGAAAACT

TTTTTTATTC

AGAGTTAATG

TTGTTTTTTC

TGCAAACATT

ACCGAAGTTA

ATTCACTAGG

ACTGAATACA

TTATGTAATT

TATAGTTATA

CATTTTATGA

CCACTCGAAG

CAGATTGGGT

GGTATCGCCA

AGAATTCTCA

CCATTAGCCA

AACTACTGTT

ATCCACCGAA

ACATCGAGCA

GAGGAAGATC

rTTCTCCGAT rCTCTTATCT

GGTTTCTCTG

CGTGGGGATT

CCTCTTAAGG

PAGCAGCCGT

rCCGTATTCC

GGTCTCGCTA

TGTTATCAAC

P.GGAAGGTGA

GCTCCTGAGG

GACTATGGGT

ACGCTTCTCT

GAAATGGGTG

CTTGCGTGGA

CTTCCGCTCA

GGTATCACCA

GATGCTTCAA

GTGTGCGTAC

ATTGATGTTC

CTTGCTTGTT

CAACCCGTAC

GAAGTGATCA

TGTTCGTTCT

CTTCTATGAT

GAAGGTGCTA

CGACCGTCTT

GCGATGAAGG

GGTCTCGGTA

TATCGCTATG

CTGTTGATGA

TTGATGGCTG

TTGATGAGCT

ATCATCGGTT

GCACACACCA

GTTTTTCTTG

GGTTTTCGCT

AATGATATGG

TCTTATTTGT

TTGTTTTGAG

ATATGAGGAG

CAACAAATAT

AGTATGTCCT

TTCCAGAATC

CTCATGGAT'r

CTTGCCAATT

CGGCCGCGTT

TCAATCAACA

AAACCAAGAA

GTCCAAAGCC

AAAGCTACAG

CCAGCACATG

GACTTAA1AGT

GCTGGCTTGT

AAAATTTTCA

GGCGCAAGTT

CCAATCTCTC

AAGACGCAGC

GAAGAAGAGT

TCATGTCTTC

CCAGCAACTG

AGGTGACAAG

GCGGTGAAAC

ACTGGTAAGG

TACTTGGATC

CTCCTCTCGA

CTTGTTGGTG

CACTAAGCGT

TGCAGGTGAA

CCAAAGACTC

AGTGAAGTCC

CTGTTATCGA

GGTTTTGGTG

CATCCGTCTT

CAGGTGATCC

CCAGGTTCCG

TGGTCTCATC

ACCCACGTCT

TCTACTTTGA

CGACGAGTAT

CCGTTATGAA

TCTGCTGTCG

TGAGACTTCT

ACGCTTCTGG

AGCTTCCTCG

TGCTACTATG

GTCTTGGAGC

CAAGAATTCG

TCGACAACGT

GAATCCTACT

TACCATTTGT

ATCGAACTGT

TCCTTTTGTT

TGTGTGTTGA

TAAAAATGTG

TAAAACACTT

ATTTTCAGAC

CTTGTGTTTT

CTTGTCAG.AT

TGTAGTTGAG

GATTGACAAC

CAAGCTTGAG

AGGTACGAGC

GGAACTCCCA

TCAACAAGGT

GAGATcAATG

CATCATGGTC

TAGTGGGCAT

GGGGACCAGA

ATCCCCATTC

AGCAGAATCT

GAAATCCAGT.

AGCATCCACG

GGGATGACGT

TGTTTCCACG

CTCGTAAGTC

TCTATCTCCC

TCGTATCACC

CTATGCAAGC

ATTGATGGTG

TTTCGGTAAC

TTTACGATTT

CCAATGGGTC

GTCTGAAGAC

CAACGCCAAT

GCTGTTCTGC

GCCAATCATG

CTAACCTTAC

GAAGGTCGTG

ATCCTCTACT

ACGTCACCAT

TTGACTCTGC

TGCTGGTGGA

AGGGTGTTAC

CCAATTCTCG

CGGTTTGGAA

CAAACGGTCT

CTCGTCGTGC

AGCAGCTGTC

TTATGGGTCT

ATCGCTACTA

TAAGATCGAA

AGCTCGGTAC

TCGTCAPLGTT

GAGTTCGAGT

TGTGCTTGTA

GAAATGGAAA

CATTCTCAAA

ATTTGAAATT

TCAAATCGTG

GTAGTTGTAC

CTAGAAAAGC

AGACATTTAT

TCTAATCATT

TATGAAAATA

ATGCATCAAT

CTCAGGATTT

CATATCACTT

TCCTCAAAGG

CAGGGTACAG

AAGAATCTTC

AGTAAGTTTC

CTTTGAAAGT

CAAAAAAGGA

WO 98/58069 WO 9858069PCT/US98/12447 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5152.

5201 5251 5301 5351 5401 5451 5501 5551 5601

ATGGTGCAGA

GCAAAGATAA

GAAAAGAGCT

TGACGACCAC

TGAAGGATCA

CGATAAGCTT

TTCGATTGCT

GCAATGGTGT

CAACGCAAAT

AGCTTATCCG

TAATTGGCTC

GCGTGCATGC

AATCACTGGT

ACTTCGCACT

GTACTGGAAA

CAACGGTGGT

CGCAGGGTGC

CAGATGGCTG

CGCGAAGAAA

AACACTTCGC

CTGTCTGAAG

GGAGCGCATG

CCGGTGGTGA

CTGTACACCC

AATCAGCCCG

TTTACAAGCC

CAGGAATATG

CGTATTCCAC

TAAGCTACGG

ACCTGGGAAG

GGGTCAGCTG

ACGATCCGCG

CTGGAGAAAG

CGGTACCGGA

TTAAGATTGA

GTTGAATTAC

TTATGAGATG

GCGATAGAAA

GGTGTCATCT

TCGAGTGGTG

GGACGTGAAT

TGTTTATTGC

TCAAAATGTA

AATTGAAAAA

CATACTCATT

ATTGALATATA

TGTTGGTTTC

GAACTGAGCC

GGAGTGATCC

AGGCCGTTCG

GGGAGGGTTC

GCCAGGGCGC

AGCAGGTTAA

TGCAAATGCT

TGCGCCCCTC

CCCTCATCTG

TTATCCCCAG

GCGTTTTCGC

ATTGTTAGGC

AGCAGATTCC

GTCCTGACAG

AAAAGAATTC

TCAGATACTG

GATGTAATTG

TCAATTGAAG

GCAGAACCCA

CTCCCTTATC

ATTTCGTCGT

TGAGCTTCGT

AGGCcatggC

GATGACGTAC

GCCAGCAGTA

CCGCTGCTAA

GCTTCCTTTA

TGCTATCCTG

AACATTATGG

CTGCTGCCGT

AGCTACCGGT

AATCTCTGCA

TCCAAAATCG

AGAAGACGGC

AGCCGGAAGA

CGTTTCACCA

GGGTAACGTG

TTTCCAAGAA

GGTGGTTCCG

CGTAGTAAAA

GCGTTCTGAA

GGTAACCCGA

CGTATGGCTG

CATTCCAGGA

TCCAATTccc

ATCCTGTTGC

GTTAAGCATG

GGTTTTTATG

ACAAAATATA

ATGTTACTAG

GCCGCATCGA

GTAGACACGT

TTTCGCCTAT

CTTTCATTTT

AAATTGGTAA

GCTGATCCAT

TCCTGCCGCC

TACGCAGAAC

ATGTGCACCT

ACATGGGACT

CGCGGGGCGC

GAGAAGGGGG

AGCCCTGGTT

AAGACAGGTT

GGATTTTCTG

ATCTGTCATC

TCAGTAGTCG

GCTTGTCCAC

CGATTTGCGA

GCACCTACCA

I'CTAGTACAA

CCCACTCACT

CCTCTATATA

A~ACCAATCCT

GAGGAAGATC

I'TTCTCCGAT

TCTCTTATCT

GGTTTCTCTG

CGTGGGGATT

CCTCTTAAGG

TAJGATTTTT

AGAAAGTTTT

A.ACTGCGTCG

AGTTAAAGCG

TCGCTGGTAA

GGCGCGATCT

TGTTCCGGTT

GGATCGACGG

AAGCCGCTGT

AGAGAACATC

GCATGACTCT

GTGGACAACA

CGTTGATTAC

TCGCAGCGTC

GTTCTGACTC

ACACAACCTG

GTTCTACTGC

ATGAACATCG

CTACTACAAA

AAGGCGAAGA

CGTGCCGGTC

ACTGAACGCG

GATCGTTCAA

CGGTCTTGCG

TAATAATTAA

ATTAGAGTCC

GCGCGCAAAC

ATCGGGGATC

TCGTGAAGTT

CGAAATAAAG

AAATACGACG

ATAATAACGC

TTACTCTTTC

GTAGATTTCC

GCTGCCGCTT

TGAGCCGGTT

TCCCCCCAAC

TTTCCTAGCT

CAGCTGGGGG

GGCACCCCCC

AAAAACAAGG

AGCGGTGGCC

CCTGTGGACA

ACTCTGCCCC

CGCCCCTCAA

ATCATCTGTG

GGCTGGCCAG

PAAG CATCTT TGCCTTTATT GTGGGGAACA AAATAACGTG AATGCGTATG ACGAACGCAG AGAAGGCATT CATTCCCATT TCTAGAAGAT CTAAGCTTAT AAAATTTTCA ATCCCCATTC GGCGCAAGTT AGCAGAATCT CCAATCTCTC GAAATCCAGT A.AGACGCAGC AGCATCCACG GAAGAAGAGT GGGATGACGT TCATGTCTTC TGTTTCCACG GATTTCGTAA AACCTGGCGT CCAGGTAGCA AAAGAAAACA GTACTGACTC CATCAACGCC CCGGTTATCG TTCAGTTCTC AGGCGTGAAA TCTGACGTTC CTGGTGCGCA TCACGTTCAC ATCCTGCACA CTGACCACTG TCTGTTGGAC GCGGGTGAAA TCTCTTCTCA CATGATCGAC GAAATCTGCT CTAAATACCT GGAAATCGA-A CTGGGTTGCA GCCACATGGA CGCTTCTGCA GCATACACCG AACTGAGCAA CTTCGGTAAC GTACACGGTG CGACCATCCT GCGTGATTCT CCGCACAACA GCCTGAACTT TCAGGAAATC AAAGACTCCG ATACCGATAC CCAATGGGCA GCGAACGAAG CTTATCTGCA TCAGCCGAAC AAGAAATACT AGACTTCGAT GATCGCTCGT ATCGACGTTC TGTAAGAGCT ACATTTGGCA ATAAAGTTTC ATGATTATCA TATAATTTCT CATGTAATGC ATGACGTTAT CGCAATTATA CATTTAATAC TAGGATAAAT TATCGCGCGC GATCCCCGGG CGGCCGCCAC TCTCATCTAA GCCCCCATTT ATTTCCGAAT TAGAATAATT GATCGTAATT TGTCGTTTTA TGCGGACATC TACATTTTTG TTTTTCTCCA TATTGACCAT CGGACATGAA GCCATTTACA TGCACCCGGT GGAGCTTGCA AGGCAGATAA TTTCCATTGA ACGGTGAGCG ACGGGGCAAC TGGCTGCCAT TTTTGGGGTG GATGGGAGGC CCGCGTTACC TTCGGCGTGC GCGGTCACGC TTTATAAATA TTGGTTTAAA GAAAAACGGG CGGAAACCCT GCCCCTCAAA TGTCAATAGG TCAALGTGTCA AGGATCG.CGC GTGTCAATAC CGCAGGGCAC GGAAACTCGC GTAAAATCAG CTCCACGTCG CCGGCCGAAA WO 98/58069 WO 9858069PCTIUS98/1 2447 5651 5701 5751 5801 5851 5901.

5951 6001 6051 6101 6151 S 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001 8051.

8101 8151 8201 8251 8301 8351 8401 8451 8501

TCGAGCCTGC

AAGTGTCAAC

TGGTATCCAC

ACGGCGTTTC

GCGAGGGCAA

CCTTGAGAGC

ACTATCGTCG

ACAGGTGCCG

GGAGCGCGAC

GCCCTCGCTC

GAAGCAGGCC

TGCTGGCGTT

CTCGCTTCCG

GCAGGTAGAT

TTACCAGCCT

GCCGCCTCGG

CCTATACCTT

CCACCTCGAC

CTCCAAGAAT

ACCAACCCTT

CACGCGGCGC

TCGTGCTCCT

GGTTAGCAGA

CTGCAAAACG

GTGTTTCGTA

TTCCGGATCT

ATCTGTATTA

CCCGCCGCAT

CGGGCATGTT

CATCGGTATC

TCAAGTGACC

GAAGCCAGAC

GAACAGGCAG

CCGCAGCTGC

TGCAGCTCCC

AGACAAGCCC

AGCCATGACC

TGCGGCATCA

TACCGCACAG

TCCTCGCTCA

ATCAGCTCAC

ACGCAGGAAA

AAAAAGGCCG

GCATCACAAA

TATAAAGATA

GTTCC6ACCC

AAGCGTGGCG

AGGTCGTTCG

GACCGCTGCG

ACACGACTTA

CGAGGTATGT

GGCTACACTA

TACCTTCGGA

CTGGTAGCGG

AAAGGATCTC

GTGGAACGAA

GGATCTTCAC

TAAAGTATAT

CCCTCATCTG

GTCCGCCCCT

AACGCCGGCG

TGGCGCGTTT

CCAGCCCGGT

CTTCAACCCA

CCGCACTTAT

GCAGCGCTCT

GATGATCGGC

AAGCCTTCGT

ATTATCGCCG

CGCGACGCGA

GCGGCATCGG

GACGACCATC

AACTTCGATC

CGAGCACATG

GTCTGCCTCC

CTGAATGGAA

TGGAGCCAAT

GGCAGAACAT

ATCTCGGGCA

GTCGTTGAGG

ATGAATCACC

TCTGCGACCT

AAGTCTGGAA

GCATCGCAGG

ACGAAGCGCT

CCATACCGCC

CATCATCAGT

ATTACCCCCA

AAACAGGAAA

ATTAACGCTT

ACATCTGTGA

CTCGCGCGTT

GGAGACGGTC

GTCAGGGCGC

CAGTCACGTA

GAGCAGATTG

ATGCGTAAGG

CTGACTCGCT

TCAAAGGCGG

GAACATGTGA

CGTTGCTGGC

AATCGACGCT

CCAGGCGTTT

TGCCGCTTAC

CTTTCTCATA

CTCCAAGCTG

CCTTATCCGG

TCGCCACTGG

AGGCGGTGCT

GAAGGACAGT

AAAAGAGTTG

TGGTTTTTTT

AAGAAGATCC

AACTCACGTT

CTAGATCCTT

ATGAGTA7LAC

TCAACGCCGC

CATCTGTCAG

GCCGGCCGCG

GCAGGGCCAT

GAGCGTCGGA

GTCAGCTCCT

GACTGTCTTC

GGGTCATTTT

CTGTCGCTTG

CACTGGTCCC

GCATGGCGGC

GGCTGGATGG

GATGCCCGCG

AGGGACAGCT

ACTGGACCGC

GAACGGGTTG

CCGCGTTGCG

GCCGGCGGCA

CAATTCTTGC

ATCCATCGCG

GCGTTGGGTC

ACCCGGCTAG

GATACGCGAG

GAGCAACAAC

ACGCGGAAGT

ATGCTGCTGG

GGCATTGACC

AGTTGTTTAC

AACCCGTATC

TGAACAGAAA

AAACCGCCCT

CTGGAGAAAC

ATCGCTTCAC

TCGGTGATGA

ACAGCTTGTC

GTCAGCGGGT

GCGATAGCGG

TACTGAGAGT

AGAAAATACC

GCGCTCGGTC

TAATACGGTT

GCAAAAGGCC

GTTTTTCCAT

CAAGTCAGAG

CCCCCTGGAA

CGGATACCTG

GCTCACGCTG

GGCTGTGTGC

TAACTATCGT

CAGCAGCCAC

ACAGAGTTCT

ATTTGGTATC

GTAGCTCTTG

GTTTGCAAGC

TTTGATCTTT

AAGGGATTTT

TTAAATTAAA

TTGGTCTGAC

GCCGGGTGAG

TGAGGGCCAA

GTGTCTCGCA

AGACGGCCGC

AAGGGTCGAT

TCCGGTGGGC

TTTATCATGC

CGGCGAGGAC

CGGTATTCGG

GCCACCAAAC

CGACGCGCTG

CCTTCCCCAT

TTGCAGGCCA

TCAAGGATCG

TGATCGTCAC

GCATGGATTG

TCGCGGTGCA

CCTCGCTAAC

GGAGAACTGT

TCCGCCATCT

CTGGCCACGG

GCTGGCGGGG

CGAACGTGAAL

ATGAATGGTC

CAGCGCCCTG

CTACCCTGTG

CTGAGTGATT

CCTCACAACG

GTGAGCATCC

TTCCCCCTTA

TAACATGGCC

TCAACGAGCT

GACCACGCTG

CGGTGAAAAC

TGTAAGCGGA

GTTGGCGGGT

AGTGTATACT

GCACCATATG

GCATCAGGCG

GTTCGGCTGC

ATCCACAGAA

AGCAAAAGGC

AGGCTCCGCC

GTGGCGAAAC

GCTCCCTCGT

TCCGCCTTTC

TAGGTATCTC

ACGAACCCCC

CTTGAGTCCA

TGGTAACAGG

TGAAGTGGTG

TGCGCTCTGC

ATCCGGCAAAL

AGCAGATTAC

TCTACGGGGT

GGTCATGAGA

AATGAAGTTT

AGTTACCAAT

TCGGCCCCTC

GTTTTCCGCG

CACGGCTTCG,

CAGCCCAGCG

CGACCGATGC

GCGGGGCATG

AACTCGTAGG

CGCTTTCGCT

AATCTTGCAC

GTTTCGGCGA

GGCTACGTCT

TATGATTCTT

TGCTGTCCAG

CTCGCGGCTC

GGCGATTTAT

TAGGCGCCGC

TGGAGCCGGG

GGATTCACCA

GAATGCGCAA

CCAGCAGCCG

GTGCGCATGA

TTGCCTTACT

GCGACTGCTG

TTCGGTTTCC

CACCATTATG

GAACACCTAC

TTTCTCTGGT

TTCCAGTAAC

TCTCTCGTTT

CACGGAGGCA

CGCTTTATCA

GGACGCGGAT

ATGAGCTTTA

CTCTGACACA

TGCCGGGAGC

GTCGGGGCGC

GGCTTAACTA

CGGTGTGAAA

CTCTTCCGCT

GGCGAGCGGT

TCAGGGGATA

CAGGAACCGT

CCCCTGACGA

CCGACAGGAC

GCGCTCTCCT

TCCCTTCGGG

AGTTCGGTGT

CGTTCAGCCC

ACCCGGTAAG

ATTAGCAGAG

GCCTAACTAC

TGAAGCCAGT

CAAACCACCG

GCGCAGAAAA

CTGACGCTCA

TTATCAAAAA

TAAATCAATc

GCTTAATCAG

WO 98/58069 WO 9858069PCTIUS98/12447 8551 8601 8651 8701 8751 8801 8851 8901 8951 9001 9051 9101 9151 9201 9251 9301 9351 9401 9451 9501 9551 9601 9651 9701 9751 9801 9851 9901 9951 10001 10051 10101 10151 10201 10251 10301 10351 10401 10451 10501 10551 10601 10651 10701 10751 10801 10851 10901

TGAGGCACCT

GACTCCCCGT

CCCAGTGCTG

ATCAGCAATA

CAACTTTATC

GTAAGTAGTT

AGGTCGGGAG

TTCGCGGCGC

TCGCCGAAGT

CATCTCGAAC

TGGCGGCCTG

TAAGGCTTGA

ACTTCGGCTT

CATTGTTGTG

AACTGCAATT

GAGCCAGCCA

AGAACATAGC

CGGTTCCTGA

TGGAACTCGC

GTTGTCCCGC

ATGTCGCTGC

GTCATACTTG

CTCGCGCGCA

TCACCAAGGT

CGCTTCGCGG

TGTCACGCTC

TCAAGGCGAG

CTTCGGTCCT

GCGTTGAGGG

ACGAGCCAAG

CGTTTGGGTG

CCGAGGGGAA

AACCTTTTCA

CTCTTAGGTT

TGAAGGCGGG

TAGCAGCATT

TTATTCAAAT

GTTTGTAAGG

GAGTCTCCAA

CAATCAAAGT

CAGAAAAAGA

TAATCTTGTC

AATGGTGCAG

TGCA7LAGATA

GGAAAAGAGC

GTGACGACCA

TTGAAGGATC

ATCTCAGCGA

CGTGTAGATA

CAATGATACC

AACCAGCCAG

CGCCTCCATC

CGCCAGTTAA

CACAGGATGA

GGCTTAATTC

ATCGACTCAA

CGACGTTGCT

AAGCCACACA

TGAAACAACG

CCCCTGGAGA

CACGACGACA

TGGAGAATGG

CGATCGACAT

GTTGCCTTGG

ACAGGATCTA

CGCCCGACTG

ATTTGGTACA

CGACTGGGCA

AAGCTAGGCA

GATCAGTTGG

AGTCGGCAAA

CGCGGCTTAA

GTCGTTTGGT

TTACATGATC

CCGATCGAGG

ATCAAGCCAC

GGATCTTTTT

GTTGAACAGA

CCCTGTGGTT

CGCCCTTTTA

TACCCGCCAA

AAACGACAAT

CCAGATTGGG

TGGTATCGCC

AAGAATTCTC

ACCATTAGCC

AAACTACTGT

CATCCACCGA

AACATCGAGC

AATTGTTAGG

AAGCAGATTC

TGTCCTGACA

CAAAAGAATT

ATCAGATACT

TCTGTCTATT

ACTACGATAC

GCGAGACCCA

CCGGAAGGGC

CAGTCTATTA

TAGTTTGCGC

CGCCTAACAA

AGGAGTTAAA

CTATCAGAGG

GGCCGTACAT

GTGATATTGA

CGGCGAGCTT

GAGCGAGATT

TCATTCCGTG

CAGCGCAATG

TGATCTGGCT

TAGGTCCAGC

TTTGAGGCGC

GGCTGGCGAT

GCGCAGTAAC

ATGGAGCGCC

GGCTTATCTT

AAGAATTTGT

TAATGTCTAA

CTCAAGCGTT

ATGGCTTCAT

CCCCATGTTG

ATTTTTCGGC

AGCAGCCCAC

GGAATGCTGC

AGTCATTATC

GGCATGCACA

AATATCCGTT

TATATCCTGT

CTGATCCCCA

TTCAATCAAC

AAAACCAAGA

AGTCCAAAGC

AAAAGCTACA

TCCAGCACAT

AGACTTAAAG

AGCTGGCTTG

CGCACCTACC

CTCTAGTACA

GCCCACTCAC

CCCTCTATAT

GAACCAATCC

TCGTTCATCC ATAGTTGCCT GGGAGGGCTT ACCATCTGGC CGCTCACCGG CTCCAGATTT CGAGCGCAGA AGTGGTCCTG ATTGTTGCCG GGAAGCTAGA AACGTTGTTG CCATTGCTGC TTCATTCAAG CCGACACCGc CATCATGAGG GAAGCGGTGA TAGTTGGCGT CATCGAGCGC TTGTACGGCT CCGCAGTGGA TTTGCTGGTT ACGGTGACCG TGATCAACGA CCTTTTGGAA CTCCGCGCTG TAGAAGTCAC GCGTTATCCA GCTAAGCGCG ACATTCTTGC AGGTATCTTC ATCTTGCTGA CAAAAGCAAG GGCGGAGGAA CTCTTTGATC TAAATGAAAC CTTAACGCTA GAGCGAAATG TAGTGCTTAC CGGCAAAATC GCGCCGAAGG TGCCGGCCCA GTATCAGCCC GGACAAGAAG ATCGCTTGGC TCACTACGTG AAAGGCGAGA CAATTCGTTC AAGCCGACGC AGATGCTGCA GGCATCGTGG TCAGCTCCGG TTCCCAACGA TGCAAAAAAG CGGTTAGCTC GCTGCGCTAC GTCCGCKACC TCGACCTCTA GCCGACCCAG TCCGTCGTCA GGCTTTCCGA GTACGGAATG CCAAGCACTC TACAAATGGA CGAACGGATA ATTCTAATAA ACGCTCTTTT CAAACACTGA TAGTTTAAAC TCAAGCTTGA GCTCAGGATT AAGGTACGAG CCATATCACT AGGAACTCCC ATCCTCAAAG CTCALACAAGG TCAGGGTACA GGAGATCAAT GAAGAATCTT GCATCATGGT CAGTAAGTTT TTAGTGGGCA TCTTTGAAAG TGGGGACCAG ACAAAAAAGG AAAAGCATCT TTGCCTTTAT AGTGGGGAAC AAAATAACGT TAATGCGTAT GACGAACGCA AAGAAGGCAT TCATTCCCAT TTCTAGAAGA TCTAAGCTTA

Claims (19)

1. A recombinant, double-stranded DNA molecule containing a promoter functional in plant cells, and a DNA sequence coding for a polypeptide having the enzymatic activity of a fructose- 1.6-bisphosphate aldolase and operatively linked to the promoter in sense orientation.

2. The DNA molecule according to claim 1. wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase is derived from a prokaryotic organism.

3. The DNA molecule according to claim 2. wherein the prokaryotic organism is Escherichia coli.

4. The DNA molecule according to claim 1. wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase has at least about 60% identity with a prokaryotic DNA sequence coding for fructose-1,6- bisphosphate aldolase class II. The DNA molecule according to claim 1. wherein the DNA sequence coding for the polvpeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase is a sequence capable of hybridizing with the coding region depicted as SEQ ID NO. 1 under conditions in which sodium chloride concentrations are between 0.15M and 0.9M and temperatures are in the range 20 0 C to

6. The DNA molecule according to claim 1, wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase has at least about 60% identity with the coding region depicted as SEQ ID NO. 1.

7. The DNA molecule according to claim 1, wherein the DNA sequence coding for the polypeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase has VEET MOBT086P

8. The DNA molecule according to claim 1, wherein the DNA sequence coding for a polypeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase has at least about 80% identity with the coding region depicted as SEQ ID NO. 1.

9. The DNA molecule according to claim 1, wherein the DNA sequence coding for the polypeptide having the enzymatic activity of a fructose-1,6-bisphosphate aldolase has the coding region depicted as SEQ ID NO. 1, or encodes the same peptide as SEQ ID NO. 1 in accordance with the degeneracy of the genetic code. A transgenic plant cell containing in its genome a recombinant DNA molecule according to any one of claims 1-9. 10 11. A transgenic plant containing plant cells according to claim

12. The transgenic plant of claim 11. wherein the plant exhibits a property selected from the group consisting of increased photosynthesis rates, increased yields, increased growth rates and improved solids uniformity compared with plants that do not contain the recombinant DNA molecule. 15 13. The transgenic plant according to claim I or 12, which is a crop plant.

14. The transgenic plant according to any one of claims 11 to 13, selected from the group consisting of corn, wheat, rice, tomato, potato, carrots, sweet potato, yams, artichoke, alfalfa, peanut,. barley, cotton, soybean, canola, sunflower, sugarbeet, apple, pear, orange peach, sugarcane, strawberry, raspberry, banana, grape, plantain, tobacco, lettuce. cassava, cruciferous vegetables, forestry species and horticultural species. The transgenic plant of any one of claims 11 to 14, wherein the plant is a potato.

16. A food product derived from the potato of claim

17. The food product of claim 16. which is a french fry or a potato chip. .MOBT086P

18. Propagation material derived from the transgenic plant of any one of claims 11 to

19. A process for increasing the photosynthesis rate in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9. and regenerating the transformed cells to produce a transgenic plant.

20. A process for increasing the yield in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9, and regenerating the transformed cells to produce a transgenic plant.

21. A process for increasing the growth rate in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 9, and 10 regenerating the transformed cells to produce a transgenic plant.

22. A process for improving the solids uniformity in plants which comprises transforming plant cells with a DNA molecule according to any one of claims I to 9, and regenerating the transformed cells to produce a transgenic plant.

23. In a method for the processing of potatoes into fries or chips, the improvement 15 comprising, utilizing a potato that overexpresses the fda transgene providing a higher solids uniformity in such potato. DATED this 7 day of May

2001. MINSANIO OMPANY, By its Patent Attorneys, E. F. WE~LTTOi-&,QO., (Bruce Wellington)