NZ501781A - A method of generating transgenic plants encoding fructose 1,6 bisphosphate aldolase (fda) for increasing carbon assimilation for increasing solids uniformity for french fry and chipping industries - Google Patents

Abstract

Fructose-1,6-bisphosphate aldolase (FDA) is an enzyme reversibly catalysing the reaction converting triosephosphate into fructose-1,6-bisphosphate. Transgenic plants were generated that express the E. coli fda gene in the chloroplast to improve plant yield by increasing leaf starch biosynthetic ability in particular and sucrose production in general. Leaves from plants expressing the fda transgene showed a significantly higher starch accumulation, as compared to control plants expressing the null vector, particularly early in the photoperiod, but had lower leaf sucrose. Transgenic plants also had a significantly higher root mass. Furthermore, transgenic potatoes expressing fda exhibited higher uniformity of solids.

Description

WO 98/58069 PCT/US98/12447 EXPRESSION OF FRUCTOSE 1.6 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, 5 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 10 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 15 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 20 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 25 organs" (e.g., 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 30 leading to carbon assimilation) are glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), also known as triose phosphates (triose-P). The 1 Printed from Mimosa 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 cytosohc. Both isoenzymes appear 5 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 10 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 20 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, dned, and par-fried 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 25 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 2 Printed from Mimosa 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 5 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- (a) Inserting into the genome of a plant a recombinant, double-stranded DNA molecule comprising (l) 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, 15 (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, (b) obtaining transformed plant cells; and (c) regenerating from transformed plant cells genetically transformed plants that 20 have elevated FDA activity.

In another aspect of the present invention there is provided a recombinant, double-stranded DNA molecule comprising in sequence (i) 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.

In a further aspect of the present invention, the structural DNA sequence that 30 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 3 Printed from Mimosa OFFICE OF N.Z. 2 3 MAY 2001 h tCEIVED 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).

In particular, in one aspect, the present invention provides a recombinant, double-stranded DNA molecule containing [a] a promoter functional in plant cells, and [b] 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.

In another aspect, the present invention provides a transgenic plant cell containing in its genome the recombinant DNA molecule of the present invention and a transgenic plant containing such plant cells In further aspects, the present invention provides processes for increasing the yield in plants, for increasing the photosynthesis rate in plants, for increasing the growth rate in plants and for improving the solids uniformity in plants, which processes comprise transforming plant cells with the DNA molecule of the present invention and regenerating the transformed cells to produce a transgenic plant 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 4 (followed by page 4a) 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 5 are not overcooked. The results of such adjustments, however, are highly variable and may lead to poor quality product. Transgenic potatoes expressing 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 stops from higher solids uniformity tubers will require less time to 10 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 15 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 20 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).

Figure 2 shows a plasmid map for plant transformation vector pMON 17524 Figure 3 shows a plasmid map for plant transformation vector pMON 17542 Printed from Mimosa Figure 4 shows the change in diurnal fluctuations of sucrose, glucose, and starch levels in tobacco leaves expressing the fda transgene (pMON17524) and control (pMON17227) The light period is from 7:00 to 19:00 hours. Only fully expanded and non-senescmg leaves were sampled.

Figure S shows a plasmid map for plant transformation vector pMON13925.

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

Figure 7 shows a plasmid map for plant transformation vector pMON13936.

Figure 8 shows a plasmid map for plant transformation vector pMON 17581.

Figure 9 shows potato tuber cross-sections of improved solids uniformity Segal Russet 15 Burbank lines (top row) versus unimproved nontransgemc 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 20 encoding an fda (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 25 and an increase m 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 30 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 enzypie. Thus, increased size resulting from overexpression of FDA may be seen in the seed, fruit, stem, 6 Printed from Mimosa Second, increasing FDA expression in the cytoso! 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 INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 3 MAY 2001 RECEIVED 4a 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 octopme synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the cauhmovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S and the figwort mosaic virus (FMV) 35S-promoters, the light-mducible promoter from the 7 Printed from Mimosa small subumt of ribulose-l,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, e.g., 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 CaMV35S 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 10 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 15 expression of the fda 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 20 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 25 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 tissue-specific) 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 30 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, 8 Printed from Mimosa 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 the fda 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 5 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 leaf-specific 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 10 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 ribulose-l,5-bisphosphate carboxylase (RUBISCO), isolated from eastern larch (Larix laricma) (Campbell et al., 1994), the cab gene, encoding the chlorophyll a/b-binding 15 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 (Truernit et al., 1995), and the thylacoid membrane proteins, isolated from 20 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 the fda 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 30 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 (Salanpubat and Belliard, 1987, 1989); the major tuber proteins including the 22 kDa protein complexes 9 Printed from Mimosa 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 6-5 conglycinin (Tierney, 1987) or other seed-specific promoters, such as the napin and phaseolin promoters, can be used to over-express an fda 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 10 used to express an fda 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 an fda gene is the promoter 15 for a glutelin gene from nee. more particularly the Osgt-1 promoter (Zheng et al., 1993), the maize granule-bound starch synthase (waxy) gene (zmGBS), the rice small subumt 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 an fda gene in wheat include those for the genes for the 20 ADPglucose pyrophosphorylase (ADPGPP) subunits, for the granule bound and other starch synthases, for the branching and debranching enzymes, for the embryogenesis-abundant 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 25 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 an fda 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 Printed from Mimosa WO 98/58069 PCT/US98/12447 accomplished by utilizing the root-specific subdomams 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 5 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 10 sequence. Rather, the non-translated leader sequence can be denved 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 HSP70 intron and the rice actin intron, both of which are known in the art. Another 15 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 poiyadenylation 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 (1) the 3' transcribed, non-20 translated regions containing the poiyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopahne synthase (NOS) gene, and (2) plant genes like the soybean storage protein genes and the small subunit of the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene.

Plastid-directed expression of fructose-1.6-bisphosphate aldolase activity 25 In one embodiment of the invention, the fda 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 30 removed during the import steps Examples of such chloroplast proteins include the small subunit of ribulose-l,5-biphosphate carboxylase (ssRUBISCO, SSU), 5-enolpyruvateshikimate-3-phosphate synthase (EPSPS), ferredoxin, ferredoxin 11 Printed from Mimosa PCT/U S98/12447 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 5 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. The fda 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 10 As used herein, the term "fructose 1, 6-bisphophate aldolase" means an enzyme (E.C 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). Various fda genes encoding the enzyme have been sequenced, as have numerous 15 proteins, such as the cytosolic enzyme from maize (GenBank Accession S07789;S 10638), cytosolic enzyme from rice (GenBank Accession JQ0543), cytosolic enzyme from spinach (GenBank Accession S31091 ,S22093), from Arabidopsis thaliana (GenBank Accession SI 1958), from spinach chloroplast (GenBank Accession S31090,A21815;S22092), from yeast (S cerevisiae) (GenBank Accession S07855, S37882, S12945; S39178; 20 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 reinhardtu (GenBank Accession S48639, S58485, S58486; S34367), 25 from Corynebacterium glutamicum (GenBank Accession S09283; X17313), from Campylobacter jejuni (GenBank Accession S52413), from Haemophilus influenzae (strain Rd KW20) (GenBank Accession C64074), from Streptococcus pneumonia (GenBank Accession AJ005697), from rice (GenBank Accession X53130), and from the maize anaerobically regulated gene (GenBank Accession XI2872).

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 coll (Stribhng and 12 Printed from Mimosa 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 5 (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 10 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 15 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 dimenc with molecular mass of approximately 80 kDa, and their activity depends on divalent metal ions The class II 20 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) 25 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 {Plasmodium falciparium, Plasmodium berghei)-, Trypanosoma (Trypanosoma brucet); Chlamydomonas (Chlamydomas reinhardtii), 30 Candida {Candida albicans); Corynebacterium (Corynebacterium glutamicum); Campylobacter {Campylobacter jejuni)-, and Haemophilus {Haemophilus, influenza). 13 Printed from Mimosa 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 known fda sequences. Low 5 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°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 10 sodium chloride, about 0.5% to about 5% casein, about 0 02% SDS or about 0.1% N-laurylsarcosine, about 0 001 M to about 0 03 M sodium citrate, at hybridization temperatures between about 50°C and about 70°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°C The skilled individual will 15 recognize that numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate fda 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 isolate fda sequences having greater than about 60% identity with the disclosed E coh fda sequence, more preferably greater than about 20 70% identity, most preferably greater than about 80% identity Depending on growth conditions Euglena gracilis, Chlamydomonas mundarta, and Chlamydomomas rheinhardi produce either a class I or a class II aldolase (Cremona, 1968; Russell and Gibbs, 1967; Guemni et al., 1971).

The isolation of a class II fda gene from E coh is described in the following 25 examples Its DNA sequence is given as SEQ ID NO.l and shown in Figure 1 The amino acid sequence 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; 30 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. 14 Printed from Mimosa PCT/U S98/12447 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 5 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 10 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 15 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 20 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 25 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 expressed 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 of the fda gene can be changed without changing the 30 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 Printed from Mimosa 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 transaenes 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 glgC 16 PCT Publication WO 10 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 with fda 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 15 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, e g., a plasmid that is capable of replication in a bacterial host, e.g., E coh 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 20 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 non-transformed 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 nptll gene can be selected using an appropriate antibiotic 30 such as kanamycin or G418. Other commonly used selectable markers include the bar gene, which confers bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., 1988), which confers glyphosate resistance; a nitrilase gene, which confers resistance to 16 Printed from Mimosa bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS), which confers imidazoiinone 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 5 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, 10 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, 15 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 an fda 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 20 tumefaciens, as well as those disclosed, e g., 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, 25 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 an fda gene in monocots using microprojectile bombardment is composed of the following: a promoter 30 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 17 Printed from Mimosa expression of the gene, such as the Hsp70 intron (PCT Publication W093/19189); and a 3' poiyadenylation sequence such as the nopaline synthase 3' sequence (NOS 3'; 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-bascd plant transformation vector for use in transformation of dicotyledonous plants is plasmid vector pMC)N530 (Rogers et al., 1987). Plasmid pMC)N530 is a derivative of pMC)N505 prepared by transferring the 2.3 kb Stul-Hindlll fragment of pMON316 (Rogers et al., 1987) into pMON526 Plasmid pMON526 is a simple derivative of pMC)N505 in which the Smal site is removed by digestion with 10 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 poiyadenylation 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 Hindlll to 15 Smal 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 tn-parental mating procedure (Horsch and Klee, 1986) Plasmid pMON505 retains all the important features of pMC)N200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric 20 NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the spectinomycin/streptomycin resistance determinant for selection in E coh 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 coh. Plasmid pMON505 contains a single T-DNA border 25 derived from the right end of the pTiT37 nopaline-type T-DNA Southern blot analyses have shown that plasmid pMC)N505 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 30 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 18 Printed from Mimosa confemng 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 the fda 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 10 (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, e.g , Ammirato et al (1984); Shimamoto et al. (1989), Fromm (1990), Vasil et al. (1990); Vasil et al. (1992); Hayashimoto (1990); andDattaetal (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 (5') to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the 20 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 25 responsible for dnving 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 (e.g., plant protoplast or explant).

"Transformation" refers to a process of introducing an exogenous nucleic acid sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in 19 Printed from Mimosa 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 vl .6 (Thompson et al., 1994) The number of matching bases or amino acids is divided by the total number of bases or ammo 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 ammo 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 synthetic DNA that encodes any of the enzymes discussed herein.

The 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 plasmid-localized. The term "genome" as it applies to plant cells encompasses no.t only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular Printed from Mimosa 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 10 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 15 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 20 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 25 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 30 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. 21 Printed from Mimosa 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 coll 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 coli fda 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 coh chromosomal DNA was extracted and the E. coh fda 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 mm (1 cycle), addition of polymerase; 94 C, 1 min.. 60 C, 1 mm , 72 C, 2 min 30 sec (35 cycles). The 1 08 kb PCR product was gel purified and ligated into an E coh expression vector, pMON5723, to form a vector construct that was used for transformation of frozen competent E.coli JM101 cells. The pMON5723 vector contains the E coh recA promoter and the T7 genelO leader (G10L) sequences, which enable high level expression in E coh (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 dimenc 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).

The fda gene sequence was subsequently cloned into another E.coh 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 lOOmM Tris and sonicated The cell pellet was spun down, and the crude cell extract supernatant was 22 Printed from Mimosa PCT/U S98/12447 assayed for FDA activity, using a coupled enzymatic assay as described by Baldwin et al. (1978). This assay was routinely performed at 30°C.

The reaction was performed in a 1 mL final volume in excess presence of the enzymes triosephosphate isomerase (TIM) and alpha-glycerophosphate dehydrogenase 5 (GDH) in a reaction mixture containing final concentrations of lOOmM 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 (I.U.) of aldolase activity is that causing the oxidation of 2 (imol of NADH/min in this assay 10 system.

Cell extracts containing the vector with the fda sequence showed a substantial increase in aldolase activity (13.1 I U /mg protein) as compared to cells transformed with the control vector (0.151 U./mg protein). The activity was shown to be inhibited by EDTA, known to specifically inhibit class II aldolases 15 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 20 plant organelles. To accomplish the import of the E colt 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 35S promoter from the figwort mosaic virus (P-FMV35S; Gowda et al., 1989) and the 3'-25 nontranslated region of the nopaline synthase gene (NOS 3', Fraley et al., 1983) sequences. The vector construct containing the expression cassette [P-FMV/CTPl//t/a/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 250-30 mL flasks, at 25°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 fiL 23 Printed from Mimosa of a 1 mg/mL stock of kinetin, and 1 mL of lOOOx vitamins (1.3 g/L nicotinic acid, 0.25 g/L thiamine, 0.25 g/L pyndoxme 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-day-old culture. Sixteen milliliters of cells were added to 40 mL of fresh Txd media and 5 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 CaCl2 added The suspension cells were digested in enzyme for 1 hour. Protoplasts were counted on a hemacytometer, counting 10 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 ng of DNA containing the gene of interest along with 5 jag of internal control DNA containing the luciferase gene were added per cuvette. The final protoplast density at electroporation was 2xl0^/mL and electroporater settings were a 500 15 |aFarad 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 10 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 photopenod regime at 26°C 20 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 25 overexpression of the aldolase in E. coli.

The expression cassette [P-FMV/CTPl//cfo/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: 5).

An additional construct was made and used for tobacco protoplast transformation, fusing the fda 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 Printed from Mimosa SphI site located immediately upstream from the N-terminus of the fda gene sequence in the CTPl -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 CTP2Ifda gene sequences was used for tobacco protoplast transformation, 5 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 coll.

The NotI cassette [P-FMV/CTP2//cfa/NOS3'] from this construct was then cloned into the NotI site of pMON 17227, in the same orientation as the selectable marker 10 expression cassette, to form the plant transformation vector pMON17542, which is shown in Figure 3 (SEQ IDNO.6).

For cytoplasmic expression of the FDA in tobacco protoplasts, a construct was made in which the fda gene sequence (without being coupled to a transit peptide) was cloned into a vector backbone, between the FMV promoter and the NOS 3' sequences. 15 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 the fda gene, fused to the Arabidopsis small subunit CTPl (pMON 17524) (SEQ ID NO:5, Figure 2) and the Arabidopsis EPSPS (CTP2) transit 20 peptide (pMON17542) (SEQ ID NO.6, Figure 3), 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 25 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-ex pressed fda. Subsequently, for pMON17524-expressing tobacco lines, leaf nonstructural carbohydrates were analyzed (sucrose, glucose, and hydrolyzed starch into 30 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.

Printed from Mimosa 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 NaH2P04 and 10 g/L Na2H2P04 in double de-ionized water) by homogenization with a Polytron. Test tubes were then placed in an 85°C water bath for 15 minutes. Tubes were centrifuged for 12 minutes at 5 3000 rpm and the supematants 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 p.L 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°C water bath and 4 mL of 200 mM acetate buffer (pH 4.5) were added followed by 0.1 mL 15 amyloglucosidase (20 U) 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 Table 1, expression of the fda gene in tobacco correlated with a 20 significant increase in leaf starch levels. However, referring to Figure 4, when a diurnal profile of starch levels was established in the ^fa-expressing leaves, this increase was apparent mainly early m 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 25 apparent increase in steady state levels of sucrose or glucose in tobacco leaves expressing E coh fda as compared to the control. 26 Printed from Mimosa WO 98/58069 PCT/US98/12447 Table 1 Leaf Carbohydrate Levels of Plants Expressing the fda Transgene1 (pMON17524) High Expressors Low Expressors Negative (>0.01% total protein) (<0.01%) Control (mg/g fresh weight) STARCH .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 ' Leaf samples were harvested at midday A second set of transgenic tobacco plants transformed with the construct 15 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 pMON17542-lines screened for expression by Western blot analysis, 18 were high expressors (>0 01% of the total cellular protein) and 15 lines were low expressors (<0.01%). Fifteen plants containing the null vector, pMON 17227, were used as control. 20 Fully expanded leaves from plants expressing the fda 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 25 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 the fda transgene.

This increase in sucrose export by jito-expressing leaves is an illustration of an increase in source capacity, very likely due to an increased carbon flow through the Calvin 30 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. 27 Printed from Mimosa Table 2 Levels of Sucrose in Phloem Exudate from Excised Leaves offda Transgenic Tobacco Plants (pMON17542) Water uptake sucrose in phloem exudate (|a.I/g F.Wt./h) (ng/leaf) (ng/gF.Wt.) fda high expressors 320 + 20 330 ± 60 fda low expressors 340+10 210+10 Control 390 + 30 160+ 10 Refernng to Table 3, preliminary analysis of plant growth and development revealed no significant differences in number of leaves or pods per plant, plant height, 15 stem diameter, or apparent seed weight per plant, between plants expressing the fda gene and the vector control under the specific growing and analysis conditions. However, as seen in Table 4, theyifa-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 20 illustration shows that the increase in root mass in the presence of the E coh fda 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 25 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 the fda transgene 28 108 ±22 77 ±3 56 + 3 Printed from Mimosa Table 3 Assessment of Certain Plant Growth and Development Parameters in Tobacco Expressing the fda Transgene1 (pMON 17542) #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 ' To achieve this analysis, 14 high-expressor lines were compared to 15 control plants 10 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 coli fda Transgene1 (pMON 17542) Root Dry Weight (g/plant) fda high expressors 64.0 + 39 fda low expressors 62 7 + 5 4 Control 31.7 + 16 ' Roots from 5 high and 7 low expressing lines and 6 control plants were excised and washed carefully then placed in a 65CC 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. 25 EXAMPLE 3 Plant transformation and fda expression in corn plants Targeting of FDA protein Vectors containing the fda gene with and without the plastid targeting peptide were made for transformation in corn and are also suitable for other monocots, including 30 rice, wheat, barley, sugarcane, triticale, etc. 29 Printed from Mimosa For the cytosolic expression of the fda gene in corn plants, a construct was made in which the fda 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' poiyadenylation sequence (Fraley et al., 1983). This created a NotI cassette [P-e35S/HSP70 intron//tfa/NOS3'] that was cloned into the NotI site of pMC)N30460, a monocot transformation vector, to form the plant transformation vector pMON13925, as shown in Figure 5 pMC)N30460 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 onentation 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 the fda gene in corn plants, a construct was made in which the fda 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' poiyadenylation sequences. This created a NotI cassette [P-e35S/HSP70 intronAnzSSuCTP//cfo/NOS3'] that was cloned into the NotI site (in the same onentation as the selectable marker cassette [P-35S/NPTII /NOS3']) of the monocot transformation vector pMC)N30460, to form the vector pMON 17590, as shown in Figure 6 From this vector a fragment containing the fda gene expression cassette and the selectable marker cassette could be excised from the bacterial selector (Kan) and on, gel punfied, 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 (Zheng et al, 1993), the HSP70 intron, and the NOS3' poiyadenylation 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//c/a/NOS3'] and the selectable marker cassette could be excised from the bacterial selector (Kan) and ori, gel purified, and used for plant transformation.

Printed from Mimosa Maize plant transformation Transgenic maize plants transformed with the vectors pMON13925 (described above) or pMC)N17590 (described above) were produced using microprojectile bombardment, a procedure well-known to the art (Fromm, 1990; Gordon-Kamm et al., 5 1990; Walters et al., 1992). Embryogenic callus initiated from immature maize embryos was used as a target tissue. Plasmid DNA at lmg/mL in TE buffer was precipitated onto M10 tungsten particles using a calcium chloride / spermidine procedure, essentially as descnbed by Klein et al. (1988). In addition to the gene of interest, the plasmids also contained the neomycin phosphotransferase II gene (nptll) dnven by the 35S promoter 10 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 15 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/nptII gene. Actively growing tissue sectors were transferred to fresh selective medium approximately every 3 weeks About 3 months after 20 bombardment, plants were regenerated from surviving embryogenic callus essentially as descnbed 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 25 grinding the leaf tissue at 4°C in 1 2 mL of the extraction buffer (100 mM Hepes, pH 8.0, 5 mM MgCl2, 5 mM MnCl2, 100 mM KC1, 10 mM DTT, 1% BSA, 1 mM PMSF, 10 Hg/mL leupeptin, 10 jig/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, 5 mM MgCl2, 4 units of alpha-glycerophosphate dehydrogenase, and 15 units of 31 Printed from Mimosa triosephosphate isomerase. The reaction was initiated by addition of 20 (J.L of leaf extract. The resulting data, generated from a single experiment, are presented in Table 5 Table 5 Aldolase Activity from Transgenic Maize Leaves Lines A340/min/20nL Activity % H99 (control) 0.113 100 pMON 17590 0.233 206 pMON 13925 0.251 222 A phenotype was visible m 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 R1 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 com 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 cell-specific promoter) has been used to construct vectors for cell-specific fda expression in maize. A class I aldolase (fdal), an fda without an iron sulfur cluster and with different properties from fdall, was utilized to improve carbon metabolism in C4 crops (e g. 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 and fdall) in a cell-specific manner in maize. The following cassettes have been made: pMON 13899: PmzSSU/hsp70/mzSSU CI?I fdal pMON13990PmzS SU/hsp70/mzS SU CTP!fdall 32 Printed from Mimosa pMONl 3988:P35S/hsp70//<fo/.

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 of aldolase gene overexpression that will lead to increase growth and 5 development and yield in maize.

Also, two vectors were used for transformation of corn which would target the expression of the E coh fda II gene in the maize endosperm. The vector pMON 13936 uses the rice gtl promoter to drive expression of aldolase m the cytoplasm of the endosperm cells Another vector (pMON 36416) uses the same promoter with the maize 10 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 15 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 20 Targeting of fda expression The plant expression vector, pMONl 7542 (described earlier), in which the fda gene is expressed behind the FMV promoter and the aldolase enzyme is fused to the chloroplast transit peptide CTP2, was used for Agrobacterium-mtdxaXed potato transformation A second potato transformation vector was constructed by cloning the NotI cassette [P-FMV/CTP2//£/a/NOS3'] (descnbed earlier) into the unique NotI site of pMON23616. pMON23616 is a potato transformation vector containing the nopaline-type T-DNA nght border region (Fraley et al., 1985), an expression cassette for the neomycin phosphotransferase typell gene [P-35S/NPTII /NOS3'] (selectable marker), a unique NotI 30 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/^/a/NOS3'] (described earlier) into the NotI site of pMON23616 results in the potato transformation vector 33 Printed from Mimosa 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 5 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 pMON17542 was used for potato transformation via Agrobacterium transformation of Russet Burbank potato callus, following the method descnbed in PCT Publication WO 96/03513 for glyphosate selection of transformed lines 10 After transformation with the vector pMON 17542, transgenic potato plantlets that came through selection on glyphosate were screened for expression of E coh aldolase by leaf Western blot analysis Out of 112 independent lines assayed. 50./t/a-expressing lines (45%) were identified, with fda expression levels ranging between 0 12% and 1.2 % of total extractable protein 15 The plant transformation vector PMON 17581 was used for Agrobacterium- mediated transformation of HS31-638 potato callus. HS31-638 is a Russet Burbank potato line previously transformed with the mutant ADPglucose pyrophosphorylase (glgC16) gene from E coh (U S Patent 5,498,830). The potato callus was transformed following the method described in PCT Publication WO 96/03513, substituting kanamycin 20 (administered at a concentration of 150-200 mg/L) for glyphosate as a selective agent The transgenic potato plants were screened for expression of the fda gene by assaying leaf punches from tissue culture plantlets. Western blot analysis (using antibodies raised against the E. colt aldolase) of leaf tissue from the pMONl 7581 -transformed lines identified 12 expressing lines out of 56 lines screened. Expression was detected of a 25 protein migrating at approximately 40 kDa, which is the molecular weight of the E coh (classll) aldolase subunit and the size of the protein observed after overexpression of the aldolase in E coh.

Specific gravity measurements of transgenic potato plants From the 50fda-zx.pressing potato lines obtained after transformation with 30 pMONl 7542, 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" XA Metro 350 potting media, Va fine sand, lA Ready Earth 34 Printed from Mimosa 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 5 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 Vi 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 10 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 30g) RB 6609 4477 67.70% 1.087 40652 5138 neg 1307 neg 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 the fda lines show an increase in overall tuber yield, and that in four lines, there is a corresponding 20 increase in percentage of tubers that weigh more than 30 g. For combined tubers over 30 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 smallertubers. In other words, with the increase in overall yield, there is a corresponding increase in percentage of bigger tubers (over 30 g). However, there is no 25 increase in specific gravity of the tubers.

Printed from Mimosa In conclusion, it appears that expression offda m 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 5 carbohydrate metabolizing genes, such as glgC16, 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 10 were taken from 6 of the highest y#a-expressing potato lines, obtained after transformation with pMONl7542, 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°C in 1 2 mL of the extraction buffer: 100 mM Hepes, pH 8.0, 15 5 mM MgCl2, 5 mM MnCl2, 100 mM KC1, 10 mM DTT, 1% BSA, ImM PMSF, 10 |ig/mL leupeptm. 10 p.g/mL aprotimn The extract was assayed for aldolase activity as described earlier.

Six independent transgenic potato lines expressing ycfa were tested for aldolase activity The expression offda in leaves is an indicator of the expression in the whole 20 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. 36 Printed from Mimosa WO 98/58069 PCT/U S98/12447 Table 7 Aldolase Activity from Transgenic Russet Burbank Potato Leaves Exp. #1 Exp. #2 Average Lines Act (U/gFW) %Act Act (U/gFW) %Act % Activity Control 4.461 100 4.732 100 100 40608 6.969 156 8.055 170 163 40610 8.489 190 7.326 155 173 40652 812 130 6.367 135 132 40632 .257 118 4.244 90 104 40631 .764 129 4.968 105 117 40611 .715 128 836 123 126 Solids uniformity in transgenic potato 15 Twenty-five Russet Burbank lines expressing fda (potato lines designated "Maestro"), obtained after transformation with pMONl7542, and fifteen Russet Burbank Simple Solid lines, also containing glgC\6 (PCT Publication WO 91/19806 and US Patent 5,498,830), expressing(potato lines designated "Segal"), obtained after transformation with pMONl7581, were field tested at two different sites. For each field site, 36 plants 20 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 25 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 (penmedullary 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 30 and pooled cortex punches in air and then in water: Specific gravity = Air Wt /(Air Wt -Water Wt.) 37 Printed from Mimosa WO 98/58069 PCT/US98/12447 After calculating specific gravity, solids levels were determined by the following equation- -214.9206+ (218.1852* 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 5 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 10 and agricultural practices. Tables 8-11 provide the pith to cortex solids ratios by plant line number, with a higher pith to cortex sohds 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 15 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 20 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 coli fda, alone or with co-expression of g/gC16, increases tuber sohds uniformity even in a growing season when 25 tuber solids uniformity is already extremely high in nontransgenic Russet Burbank. That is, the fda gene continues to perform when agricultural conditions are already conducive to an abnormally high sohds uniformity level. 38 Printed from Mimosa Table 8. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio. Segal Russet Burbank Lines. Site 1 Line Ratio S-29 79.1 S-9 75.8 S-20 71.3 S-15 71.3 S-21 70.5 S-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-l 73 4 M-3 73 0 M-6 72.4 M-9 71.2 M-l 1 70.6 M-l 8 70.5 M-17 69.9 M-19 69 4 M-5 69.3 M-20 68 9 RB control 68.4 M-8 68.3 M-43 67.7 M-23 67.3 M-7 67.0 M-3 9 . 66.6 M-22 66.0 M-10 65.4 40 M-27 61 4 39 Printed from Mimosa 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 S-05 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-l 7 83 8 M-03 83.7 M-09 83.4 M-15 83.2 M-29 82 9 M-44 82 3 M-08 82 2 M-43 81 6 M-22 81 1 M-05 80 8 M-01 80 5 M-20 80 2 M-45 79 6 M-39 79.5 M-27 - 79.5 RB control 79.3 M-13 78.9 40 M-22 78.8 M-l 9 78.7 M-07 78.2 M-l 2 77.9 M-23 77.3 45 M-06 76.5 M-10 75.0 M-l 1 74.1 40 Printed from Mimosa 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 g/gC16 provides improved 5 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 10 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 5 Plant transformation and FDA expression in cotton plants 15 The E coli fda vectors pMONl7524 [FMV/CTPl//cfa] (Figure 2) and pMONl7542 [FMV/CTP2//cfa] (Figure 3) were transformed into cotton using Agrobacterium as descnbed by Umbeck et al. (1987) and in US Patent 5004863. The protein was targeted to the chloroplast using either the Arabidopsis SSU CTP 1 (pMON17524) or the Arabidopsis EPSPS (pMON17542) chloroplast transit peptide. 20 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 25 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 25 mM EDTA, which inhibits the E coli enzyme but not the plant enzyme. The results of the 30 assays are shown in Table 12. 41 Printed from Mimosa Table 12 Aldolase Activity in Cotton Calh and Cotton Leaf A A340 e"3/mg protein/5 min Colony# -EDTA +EDTA Fold Increase Controls Cotton Leaf (Coker) Uninoculated Calli Inoculated Calli (E35S/GUS) #1 #2 FDA calli pMON 17542 #1 #3 #5 #4 pMONl 7524 #2 #3 #5 #6 4.0 7.7 6.8 3.5 3 5 5 5 9.2 19.8 15.2 12.5 144 4 1 42 56 6 1 4.0 23 26 3 8 3.6 5 8 40 29 1.2 1.3X 1.5X 2.1X 2.4X 5.5X 2.6X 3.IX 4.9X 3.5X The results indicate that there is good expression of the fda 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 42 Printed from Mimosa REFERENCES CITED Alefounder et al. (1989) Biochem. J 257:529-534 Ammirato et al. (1984) Handbook of Plant Cell Culture - Crop Species. Macmillan Publ. 5 Co..

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Zheng et al. (1993) Plant J. 4: 3357-3366. 46 Printed from Mimosa WO 98/58069 PCT/US98/12447 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Gerard Barry NordmeCheikh Ganesh Kishore (ii) TITLE OF INVENTION Expression of Fructose 1,6 Bisphosphate Aldolase in Transgenic Plants (iii) NUMBER OF SEQUENCES- 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE- Arnold, White & Durkee (B) STREET. P O Box 443 3 (C) CITY. Houston (D) STATE Texas (E) COUNTRY: United States of America (F) ZIP: 77210-4433 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE Floppy disk (B) COMPUTER IBM PC compatible (C) OPERATING SYSTEM PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (VI) CURRENT APPLICATION DATA.

(A) APPLICATION NUMBER. US Unknown (B) FILING DATE: Concurrently Herewith (C) CLASSIFICATION Unknown (vi) PRIOR APPLICATION DATA 35 (A) APPLICATION NUMBER: US Prov App. Serial No. 60/049,995 (B) FILING DATE: June 17, 1997 (Vlli) ATTORNEY/AGENT INFORMATION 40 (A) NAME: Patricia A. Kammerer (B) REGISTRATION NUMBER: 29,775 (C) REFERENCE/DOCKET NUMBER. MOBTO86 (ix) TELECOMMUNICATION INFORMATION: 45 (A) TELEPHONE: (713) 787-1400 (B) TELEFAX: (713) 787-1440 50 (2) INFORMATION FOR SEQ ID NO.l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1080 base pairs (B) TYPE: nucleic acid 55 (C) STRANDEDNESS: single 47 Printed from Mimosa PCTAJS98/12447 (D) TOPOLOGY: linear (XI) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGTCTAAGA TTTTTGATTT CGTAAAACCT GGCGTAATCA CTGGTGATGA CGTACAGAAA 10 60 GTTTTCCAGG TAGCAAAAGA AAACAACTTC GCACTGCCAG CAGTAAACTG CGTCGGTACT 120 GACTCCATCA ACGCCGTACT GGAAACCGCT GCTAAAGTTA AAGCGCCGGT TATCGTTCAG 180 TTCTCCAACG GTGGTGCTTC CTTTATCGCT GGTAAAGGCG TGAAATCTGA CGTTCCGCAG 240 GGTGCTGCTA TCCTGGGCGC GATCTCTGGT GCGCATCACG TTCACCAGAT GGCTGAACAT 300 TATGGTGTTC CGGTTATCCT GCACACTGAC CACTGCGCGA AGAAACTGCT GCCGTGGATC 25 360 GACGGTCTGT TGGACGCGGG TGAAAAACAC TTCGCAGCTA CCGGTAAGCC GCTGTTCTCT 420 TCTCACATGA TCGACCTGTC TGAAGAATCT CTGCAAGAGA ACATCGAAAT CTGCTCTAAA 480 TACCTGGAGC GCATGTCCAA AATCGGCATG ACTCTGGAAA TCGAACTGGG TTGCACCGGT 540 GGTGAAGAAG ACGGCGTGGA CAACAGCCAC ATGGACGCTT CTGCACTGTA CACCCAGCCG 600 GAAGACGTTG ATTACGCATA CACCGAACTG AGCAAAATCA GCCCGCGTTT CACCATCGCA 40 660 GCGTCCTTCG GTAACGTACA CGGTGTTTAC AAGCCGGGTA ACGTGGTTCT GACTCCGACC 720 45 ATCCTGCGTG ATTCTCAGGA ATATGTTTCC AAGAAACACA ACCTGCCGCA CAACAGCCTG 780 AACTTCGTAT TCCACGGTGG TTCCGGTTCT ACTGCTCAGG AAATCAAAGA CTCCGTAAGC 840 50 TACGGCGTAG TAAAAATGAA CATCGATACC GATACCCAAT GGGCAACCTG GGAAGGCGTT 900 CTGAACTACT ACAAAGCGAA CGAAGCTTAT CTGCAGGGTC AGCTGGGTAA CCCGAAAGGC 55 960 GAAGATCAGC CGAACAAGAA ATACTACGAT CCGCGCGTAT GGCTGCGTGC CGGTCAGACT 1020 48 Printed from Mimosa WO 98/58069 PCT/US98/12447 TCGATGATCG CTCGTCTGGA GAAAGCATTC CAGGAACTGA ACGCGATCGA CGTTCTGTAA 1080 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 359ammo acids 10 (B) TYPE • ammo (C) STRANDEDNES S• (D) TOPOLOGY. Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 Met Ser Lys lie Phe Asp Phe Val Lys Pro Gly Val lie Thr Gly 5 10 15 Asp Asp Val Gin Lys Val Phe Gin Val Ala Lys Glu Asn Asn Phe 25 30 Ala Leu Pro Ala Val Asn Cys Val Gly Thr Asp Ser lie Asn Ala 35 40 45 Val Leu Glu Thr Ala Ala Lys Val Lys Ala Pro Val lie Val Gin 50 55 60 Phe Ser Asn Gly Gly Ala Ser Phe lie Ala Gly Lys Gly Val Lys 30 65 70 75 Ser Asp Val Pro Gin Gly Ala Ala lie Leu Gly Ala lie Ser Gly 80 85 90 Ala His His Val His Gin Met Ala Glu His Tyr Gly Val Pro Val 95 100 105 lie Leu His Thr Asp His Cys Ala Lys Lys Leu Leu Pro Trp He 110 115 120 40 Asp Gly Leu Leu Asp Ala Gly Glu Lys His Phe Ala Ala Thr Gly 125 120 135 Lys Pro Leu Phe Ser Ser His Met lie Asp Leu Ser Glu Glu Ser 45 140 145 150 Leu Gin Glu Asn lie Glu lie Cys Ser Lys Tyr Leu Glu Arg Met 155 160 165 50 Ser Lys lie Gly Met Thr Leu Glu lie Glu Leu Gly Cys Thr Gly 170 175 180 Gly Glu Glu Asp Gly Val Asp Asn Ser His Met Asp Ala Ser Ala 185 190 195 55 Leu Tyr Thr Gin Pro Glu Asp Val Asp Tyr Ala Tyr Thr Glu Leu 200 205 210 49 Printed from Mimosa 40 45 WO 98/58069 PCT/US98/12447 Ser Lys lie Ser Pro Arg Phe Thr lie Ala Ala Ser Phe Gly Asn 215 220 225 Val His Gly Val Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr 230 235 240 lie Leu Arg Asp Ser Gin Glu Tyr Val Ser Lys Lys His Asn Leu 245 250 255 Pro His Asn Ser Leu Asn Phe Val Phe His Gly Gly Ser Gly Ser 260 265 270 Thr Ala Gin Glu lie Lys Asp Ser Val Ser Tyr Gly Val Val Lys 275 280 285 Met Asn lie Asp Thr Asp Thr Gin Trp Ala Thr Trp Glu Gly Val 290 295 300 Leu Asn Tyr Tyr Lys Ala Asn Glu Ala Tyr Leu Gin Gly Gin Leu 305 310 315 Gly Asn Pro Lys Gly Glu Asp Gin Pro Asn Lys Lys Tyr Tyr Asp 320 325 330 Pro Arg Val Trp Leu Arg Ala Gly Gin Thr Ser Met lie Ala Arg 335 340 345 Leu Glu Lys Ala Phe Gin Glu Leu Asn Ala lie Asp Val Leu 350 355 (2) INFORMATION FOR SEQ ID NO 3 (l) sequence characteristics: (A) LENGTH 3 0 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY Linear (xi) SEQUENCE DESCRIPTION SEQ ID NO.3 GGGGCCATGG CTAAGATTTT TGATTTCGTA (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4. CCCCGAGCTC TTACAGAACG TCGATCGCGT TCAG (2) INFORMATION FOR SEQ ID NO:5: (l) SEQUENCE CHARACTERISTICS: 50 Printed from Mimosa 40 45 (A) LENGTH: 10847 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION- SEQ ID NO:5: 1 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA ATCCCCATTC 51 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT AGCAGAATCT 101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC GAAATCCAGT 151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC AGCATCCACG 201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT GGGATGACGT 251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC TGTTTCCACG 301 GCGTGCATGC TTCACGGTGC AAGCAGCCGT CCAGCAACTG CTCGTAAGTC 351 CTCTGGTCTT TCTGGAACCG TCCGTATTCC AGGTGACAAG TCTATCTCCC 401 ACAGGTCCTT CATGTTTGGA GGTCTCGCTA GCGGTGAAAC TCGTATCACC 451 GGTCTTTTGG AAGGTGAAGA TGTTATCAAC ACTGGTAAGG CTATGCAAGC 501 TATGGGTGCC AGAATCCGTA AGGAAGGTGA TACTTGGATC ATTGATGGTG 551 TTGGTAACGG TGGACTCCTT GCTCCTGAGG CTCCTCTCGA TTTCGGTAAC 601 GCTGCAACTG GTTGCCGTTT GACTATGGGT CTTGTTGGTG TTTACGATTT 651 CGATAGCACT TTCATTGGTG ACGCTTCTCT CACTAAGCGT CCAATGGGTC 701 GTGTGTTGAA CCCACTTCGC GAAATGGGTG TGCAGGTGAA GTCTGAAGAC 751 GGTGATCGTC TTCCAGTTAC CTTGCGTGGA CCAAAGACTC CAACGCCAAT 801 CACCTACAGG GTACCTATGG CTTCCGCTCA AGTGAAGTCC GCTGTTCTGC 851 TTGCTGGTCT CAACACCCCA GGTATCACCA CTGTTATCGA GCCAATCATG 901 ACTCGTGACC ACACTGAAAA GATGCTTCAA GGTTTTGGTG CTAACCTTAC 951 CGTTGAGACT GATGCTGACG GTGTGCGTAC CATCCGTCTT GAAGGTCGTG 1001 GTAAGCTCAC CGGTCAAGTG ATTGATGTTC CAGGTGATCC ATCCTCTACT 1051 GCTTTCCCAT TGGTTGCTGC CTTGCTTGTT CCAGGTTCCG ACGTCACCAT 1101 CCTTAACGTT TTGATGAACC CAACCCGTAC TGGTCTCATC TTGACTCTGC 1151 AGGAAATGGG TGCCGACATC GAAGTGATCA ACCCACGTCT TGCTGGTGGA 1201 GAAGACGTGG CTGACTTGCG TGTTCGTTCT TCTACTTTGA AGGGTGTTAC 1251 TGTTCCAGAA GACCGTGCTC CTTCTATGAT CGACGAGTAT CCAATTCTCG 1301 CTGTTGCAGC TGCATTCGCT GAAGGTGCTA CCGTTATGAA CGGTTTGGAA 1351 GAACTCCGTG TTAAGGAAAG CGACCGTCTT TCTGCTGTCG CAAACGGTCT 1401 CAAGCTCAAC GGTGTTGATT GCGATGAAGG TGAGACTTCT CTCGTCGTGC 1451 GTGGTCGTCC TGACGGTAAG GGTCTCGGTA ACGCTTCTGG AGCAGCTGTC 1501 GCTACCCACC TCGATCACCG TATCGCTATG AGCTTCCTCG TTATGGGTCT 1551 CGTTTCTGAA AACCCTGTTA CTGTTGATGA TGCTACTATG ATCGCTACTA 1601 GCTTCCCAGA GTTCATGGAT TTGATGGCTG GTCTTGGAGC TAAGATCGAA 1651 CTCTCCGACA CTAAGGCTGC TTGATGAGCT CAAGAATTCG AGCTCGGTAC 1701 CGGATCCAGC TTTCGTTCGT ATCATCGGTT TCGACAACGT TCGTCAAGTT 1751 CAATGCATCA GTTTCATTGC GCACACACCA GAATCCTACT GAGTTCGAGT 1801 ATTATGGCAT TGGGAAAACT GTTTTTCTTG TACCATTTGT TGTGCTTGTA 1851 ATTTACTGTG TTTTTTATTC GGTTTTCGCT ATCGAACTGT GAAATGGAAA 1901 TGGATGGAGA AGAGTTAATG AATGATATGG TCCTTTTGTT CATTCTCAAA 1951 TTAATATTAT TTGTTTTTTC TCTTATTTGT TGTGTGTTGA ATTTGAAATT 2001 ATAAGAGATA TGCAAACATT TTGTTTTGAG TAAAAATGTG TCAAATCGTG 2051 GCCTCTAATG ACCGAAGTTA ATATGAGGAG TAAAACACTT GTAGTTGTAC 2101 CATTATGCTT ATTCACTAGG CAACAAATAT ATTTTCAGAC CTAGAAAAGC 2151 TGCAAATGTT ACTGAATACA AGTATGTCCT CTTGTGTTTT AGACATTTAT 2201 GAACTTTCCT TTATGTAATT TTCCAGAATC CTTGTCAGAT TCTAATCATT 2251 GCTTTATAAT TATAGTTATA CTCATGGATT TGTAGTTGAG TATGAAAATA 2301 TTTTTTAATG CATTTTATGA CTTGCCAATT GATTGACAAC ATGCATCAAT 2351 CGACCTGCAG CCACTCGAAG CGGCCGCGTT CAAGCTTGAG CTCAGGATTT 2401 AGCAGCATTC CAGATTGGGT TCAATCAACA AGGTACGAGC CATATCACTT 2451 TATTCAAATT GGTATCGCCA AAACCAAGAA GGAACTCCCA TCCTCAAAGG 2501 TTTGTAAGGA AGAATTCTCA GTCCAAAGCC TCAACAAGGT CAGGGTACAG 51 Printed from Mimosa 40 45 2551 AGTCTCCAAA CCATTAGCCA AAAGCTACAG GAGATCAATG AAGAATCTTC 2601 AATCAAAGTA AACTACTGTT CCAGCACATG CATCATGGTC AGTAAGTTTC 2651 AGAAAAAGAC ATCCACCGAA GACTTAAAGT TAGTGGGCAT CTTTGAAAGT 2701 AATCTTGTCA ACATCGAGCA GCTGGCTTGT GGGGACCAGA CAAAAAAGGA 2751 ATGGTGCAGA ATTGTTAGGC GCACCTACCA AAAGCATCTT TGCCTTTATT 2801 GCAAAGATAA AGCAGATTCC TCTAGTACAA GTGGGGAACA AAATAACGTG 2851 GAAAAGAGCT GTCCTGACAG CCCACTCACT AATGCGTATG ACGAACGCAG 2901 TGACGACCAC AAAAGAATTC CCTCTATATA AGAAGGCATT CATTCCCATT 2951 TGAAGGATCA TCAGATACTG AACCAATCCT TCTAGAAGAT CTCCACAATG 3 001 GCTTCCTCTA TGCTCTCTTC CGCTACTATG GTTGCCTCTC CGGCTCAGGC 3051 CACTATGGTC GCTCCTTTCA ACGGACTTAA GTCCTCCGCT GCCTTCCCAG 3101 CCACCCGCAA GGCTAACAAC GACATTACTT CCATCACAAG CAACGGCGGA 3151 AGAGTTAACT GCATGCAGGT GTGGCCTCCG ATTGGAAAGA AGAAGTTTGA 3201 GACTCTCTCT TACCTTCCTG ACCTTACCGA TTCCGGTGGT CGCGTCAACT 3251 GCATGCAGGC CATGGCTAAG ATTTTTGATT TCGTAAAACC TGGCGTAATC 33 01 ACTGGTGATG ACGTACAGAA AGTTTTCCAG GTAGCAAAAG AAAACAACTT 33 51 CGCACTGCCA GCAGTAAACT GCGTCGGTAC TGACTCCATC AACGCCGTAC 3401 TGGAAACCGC TGCTAAAGTT AAAGCGCCGG TTATCGTTCA GTTCTCCAAC 3451 GGTGGTGCTT CCTTTATCGC TGGTAAAGGC GTGAAATCTG ACGTTCCGCA 3501 GGGTGCTGCT ATCCTGGGCG CGATCTCTGG TGCGCATCAC GTTCACCAGA 3 551 TGGCTGAACA TTATGGTGTT CCGGTTATCC TGCACACTGA CCACTGCGCG 3601 AAGAAACTGC TGCCGTGGAT CGACGGTCTG TTGGACGCGG GTGAAAAACA 3651 CTTCGCAGCT ACCGGTAAGC CGCTGTTCTC TTCTCACATG ATCGACCTGT 3701 CTGAAGAATC TCTGCAAGAG AACATCGAAA TCTGCTCTAA ATACCTGGAG 3751 CGCATGTCCA AAATCGGCAT GACTCTGGAA ATCGAACTGG GTTGCACCGG 3801 TGGTGAAGAA GACGGCGTGG ACAACAGCCA CATGGACGCT TCTGCACTGT 3851 ACACCCAGCC GGAAGACGTT GATTACGCAT ACACCGAACT GAGCAAAATC 3901 AGCCCGCGTT TCACCATCGC AGCGTCCTTC GGTAACGTAC ACGGTGTTTA 3 951 CAAGCCGGGT AACGTGGTTC TGACTCCGAC CATCCTGCGT GATTCTCAGG 4001 AATATGTTTC CAAGAAACAC AACCTGCCGC ACAACAGCCT GAACTTCGTA 4051 TTCCACGGTG GTTCCGGTTC TACTGCTCAG GAAATCAAAG ACTCCGTAAG 4101 CTACGGCGTA GTAAAAATGA ACATCGATAC CGATACCCAA TGGGCAACCT 4151 GGGAAGGCGT TCTGAACTAC TACAAAGCGA ACGAAGCTTA TCTGCAGGGT 42 01 CAGCTGGGTA ACCCGAAAGG CGAAGATCAG CCGAACAAGA AATACTACGA 4251 TCCGCGCGTA TGGCTGCGTG CCGGTCAGAC TTCGATGATC GCTCGTCTGG 43 01 AGAAAGCATT CCAGGAACTG AACGCGATCG ACGTTCTGTA AGAGCTCGGT 4 351 ACCGGATCCA ATTCCCGATC GTTCAAACAT TTGGCAATAA AGTTTCTTAA 4401 GATTGAATCC TGTTGCCGGT CTTGCGATGA TTATCATATA ATTTCTGTTG 4451 AATTACGTTA AGCATGTAAT AATTAACATG TAATGCATGA CGTTATTTAT 4501 GAGATGGGTT TTTATGATTA GAGTCCCGCA ATTATACATT TAATACGCGA 4551 TAGAAAACAA AATATAGCGC GCAAACTAGG ATAAATTATC GCGCGCGGTG 4601 TCATCTATGT TACTAGATCG GGGATCGATC CCCGGGCGGC CGCCACTCGA 4651 GTGGTGGCCG CATCGATCGT GAAGTTTCTC ATCTAAGCCC CCATTTGGAC 4701 GTGAATGTAG ACACGTCGAA ATAAAGATTT CCGAATTAGA ATAATTTGTT 4751 TATTGCTTTC GCCTATAAAT ACGACGGATC GTAATTTGTC GTTTTATCAA 4801 AATGTACTTT CATTTTATAA TAACGCTGCG GACATCTACA TTTTTGAATT 4851 GAAAAAAAAT TGGTAATTAC TCTTTCTTTT TCTCCATATT GACCATCATA 4901 CTCATTGCTG ATCCATGTAG ATTTCCCGGA CATGAAGCCA TTTACAATTG 4951 AATATATCCT GCCGCCGCTG CCGCTTTGCA CCCGGTGGAG CTTGCATGTT 5001 GGTTTCTACG CAGAACTGAG CCGGTTAGGC AGATAATTTC CATTGAGAAC 5051 TGAGCCATGT GCACCTTCCC CCCAACACGG TGAGCGACGG GGCAACGGAG 5101 TGATCCACAT GGGACTTTTc CTAGCTTGGC TGCCATTTTT GGGGTGAGGC 5151 CGTTCGCGCG GGGCGCCAGC TGGGGGGATG GGAGGCCCGC GTTACCGGGA 5201 GGGTTCGAGA AGGGGGGGCA CCCCCCTTCG GCGTGCGCGG TCACGCGCCA 5251 GGGCGCAGCC CTGGTTAAAA ACAAGGTTTA TAAATATTGG TTTAAAAGCA 5301 GGTTAAAAGA CAGGTTAGCG GTGGCCGAAA AACGGGCGGA AACCCTTGCA 5351 AATGCTGGAT TTTCTGCCTG TGGACAGCCC CTCAAATGTC AATAGGTGCG 5401 CCCCTCATCT GTCATCACTC TGCCCCTCAA GTGTCAAGGA TCGCGCCCCT 52 Printed from Mimosa 40 45 5451 CATCTGTCAG TAGTCGCGCC CCTCAAGTGT CAATACCGCA GGGCACTTAT 5501 CCCCAGGCTT GTCCACATCA TCTGTGGGAA ACTCGCGTAA AATCAGGCGT 5551 TTTCGCCGAT TTGCGAGGCT GGCCAGCTCC ACGTCGCCGG CCGAAATCGA 5601 GCCTGCCCCT CATCTGTCAA CGCCGCGCCG GGTGAGTCGG CCCCTCAAGT 5651 GTCAACGTCC GCCCCTCATC TGTCAGTGAG GGCCAAGTTT TCCGCGTGGT 5701 ATCCACAACG CCGGCGGCCG GCCGCGGTGT CTCGCACACG GCTTCGACGG 5751 CGTTTCTGGC GCGTTTGCAG GGCCATAGAC GGCCGCCAGC CCAGCGGCGA 5B01 GGGCAACCAG CCCGGTGAGC GTCGGAAAGG GTCGATCGAC CGATGCCCTT 5851 GAGAGCCTTC AACCCAGTCA GCTCCTTCCG GTGGGCGCGG GGCATGACTA 5901 TCGTCGCCGC ACTTATGACT GTCTTCTTTA TCATGCAACT CGTAGGACAG 5951 GTGCCGGCAG CGCTCTGGGT CATTTTCGGC GAGGACCGCT TTCGCTGGAG 6001 CGCGACGATG ATCGGCCTGT CGCTTGCGGT ATTCGGAATC TTGCACGCCC 6051 TCGCTCAAGC CTTCGTCACT GGTCCCGCCA CCAAACGTTT CGGCGAGAAG 6101 CAGGCCATTA TCGCCGGCAT GGCGGCCGAC GCGCTGGGCT ACGTCTTGCT 6151 GGCGTTCGCG ACGCGAGGCT GGATGGCCTT CCCCATTATG ATTCTTCTCG 6201 CTTCCGGCGG CATCGGGATG CCCGCGTTGC AGGCCATGCT GTCCAGGCAG 6251 GTAGATGACG ACCATCAGGG ACAGCTTCAA GGATCGCTCG CGGCTCTTAC 6301 CAGCCTAACT TCGATCACTG GACCGCTGAT CGTCACGGCG ATTTATGCCG 6351 CCTCGGCGAG CACATGGAAC GGGTTGGCAT GGATTGTAGG CGCCGCCCTA 6401 TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA GCCGGGCCAC 6451 CTCGACCTGA ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCACTCC 6501 AAGAATTGGA GCCAATCAAT TCTTGCGGAG AACTGTGAAT GCGCAAACCA 6551 ACCCTTGGCA GAACATATCC ATCGCGTCCG CCATCTCCAG CAGCCGCACG 6601 CGGCGCATCT CGGGCAGCGT TGGGTCCTGG CCACGGGTGC GCATGATCGT 6651 GCTCCTGTCG TTGAGGACCC GGCTAGGCTG GCGGGGTTGC CTTACTGGTT 6701 AGCAGAATGA ATCACCGATA CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC 6751 AAAACGTCTG CGACCTGAGC AACAACATGA ATGGTCTTCG GTTTCCGTGT 6801 TTCGTAAAGT CTGGAAACGC GGAAGTCAGC GCCCTGCACC ATTATGTTCC 6851 GGATCTGCAT CGCAGGATGC TGCTGGCTAC CCTGTGGAAC ACCTACATCT 6901 GTATTAACGA AGCGCTGGCA TTGACCCTGA GTGATTTTTC TCTGGTCCCG 6951 CCGCATCCAT ACCGCCAGTT GTTTACCCTC ACAACGTTCC AGTAACCGGG 7001 CATGTTCATC ATCAGTAACC CGTATCGTGA GCATCCTCTC TCGTTTCATC 7051 GGTATCATTA CCCCCATGAA CAGAAATTCC CCCTTACACG GAGGCATCAA 7101 GTGACCAAAC AGGAAAAAAC CGCCCTTAAC ATGGCCCGCT TTATCAGAAG 7151 CCAGACATTA ACGCTTCTGG AGAAACTCAA CGAGCTGGAC GCGGATGAAC 7201 AGGCAGACAT CTGTGAATCG CTTCACGACC ACGCTGATGA GCTTTACCGC 7251 AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA 7301 GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC 7351 AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC 7401 ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT TAACTATGCG 7451 GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATGCGGT GTGAAATACC 7501 GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCTCT TCCGCTTCCT 7551 CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA 7601 GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC 7651 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA 7701 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT 7751 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA 7801 AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC 7851 CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC 7901 GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 7951 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC 8001 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC 8051 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG 8101 GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT 8151 ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC 8201 TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG 8251 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG 8301 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG 53 Printed from Mimosa 40 45 8351 aacgaaaact cacgttaagg 8401 cttcacctag atccttttaa 8451 gtatatatga gtaaacttgg 8501 gcacctatct cagcgatctg 8551 ccccgtcgtg tagataacta 8601 gtgctgcaat gataccgcga 8651 gcaataaacc agccagccgg 8701 tttatccgcc tccatccagt 8751 gtagttcgcc agttaatagt 8801 cgggagcaca ggatgacgcc 8851 cggcgcggct taattcagga 8901 cgaagtatcg actcaactat 8951 tcgaaccgac gttgctggcc 9001 ggcctgaagc cacacagtga 9051 gcttgatgaa acaacgcggc 9101 cggcttcccc tggagagagc 9151 gttgtgcacg acgacatcat 9201 gcaatttgga gaatggcagc 9251 cagccacgat cgacattgat 9301 catagcgttg ccttggtagg 9351 tcctgaacag gatctatttg 9401 actcgccgcc cgactgggct 9451 tcccgcattt ggtacagcgc 9501 cgctgccgac tgggcaatgg 9551 tacttgaagc taggcaggct 9601 cgcgcagatc agttggaaga 9651 caaggtagtc ggcaaataat 9701 tcgcggcgcg gcttaactca 9751 acgctcgtcg tttggtatgg 9801 ggcgagttac atgatccccc 9851 ggtcctccga tcgaggattt 9901 tgagggatca agccacagca 9951 gccaagggat ctttttggaa 10001 tgggtggttg aacagaagtc 10051 ggggaaccct gtggttggca 10101 ttttcacgcc cttttaaata 10151 taggtttacc cgccaatata 10201 ggcgggaaac gacaatctga 10251 agcattccag attgggttca 10301 tcaaattggt atcgccaaaa 10351 gtaaggaaga attctcagtc 10401 ctccaaacca ttagccaaaa 10451 caaagtaaac tactgttcca 10501 aaaagacatc caccgaagac 10551 cttgtcaaca tcgagcagct 10601 gtgcagaatt gttaggcgca 10651 aagataaagc agattcctct 10701 aagagctgtc ctgacagccc 10751 cgaccacaaa agaattccct 10801 aggatcatca gatactgaac GATTTTGGTC ATGAGATTAT CAAAAAGGAT ATTAAAAATG AAGTTTTAAA TCAATCTAAA TCTGACAGTT ACCAATGCTT AATCAGTGAG TCTATTTCGT TCATCCATAG TTGCCTGACT CGATACGGGA GGGCTTACCA TCTGGCCCCA GACCCACGCT CACCGGCTCC AGATTTATCA AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC CTATTAATTG TTGCCGGGAA GCTAGAGTAA TTGCGCAACG TTGTTGCCAT TGCTGCAGGT TAACAATTCA TTCAAGCCGA CACCGCTTCG GTTAAACATC ATGAGGGAAG CGGTGATCGC CAGAGGTAGT TGGCGTCATC GAGCGCCATC GTACATTTGT ACGGCTCCGC AGTGGATGGC TATTGATTTG CTGGTTACGG TGACCGTAAG GAGCTTTGAT CAACGACCTT TTGGAAACTT GAGATTCTCC GCGCTGTAGA AGTCACCATT TCCGTGGCGT TATCCAGCTA AGCGCGAACT GCAATGACAT TCTTGCAGGT ATCTTCGAGC CTGGCTATCT TGCTGACAAA AGCAAGAGAA TCCAGCGGCG GAGGAACTCT TTGATCCGGT AGGCGCTAAA TGAAACCTTA ACGCTATGGA GGCGATGAGC GAAATGTAGT GCTTACGTTG AGTAACCGGC AAAATCGCGC CGAAGGATGT AGCGCCTGCC GGCCCAGTAT CAGCCCGTCA TATCTTGGAC AAGAAGATCG CTTGGCCTCG ATTTGTTCAC TACGTGAAAG GCGAGATCAC GTCTAACAAT TCGTTCAAGC CGACGCCGCT AGCGTTAGAT GCTGCAGGCA TCGTGGTGTC CTTCATTCAG CTCCGGTTCC CAACGATCAA ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC TTCGGCGCTG CGCTACGTCC GCKACCGCGT GCCCACTCGA CCTCTAGCCG ACCCAGACGA TGCTGCTCCG TCGTCAGGCT TTCCGACGTT ATTATCGTAC GGAATGCCAA GCACTCCCGA TGCACATACA AATGGACGAA CGGATAAACC TCCGTTATTC TAATAAACGC TCTTTTCTCT TCCTGTCAAA CACTGATAGT TTAAACTGAA TCCCCATCAA GCTTGAGCTC AGGATTTAGC ATCAACAAGG TACGAGCCAT ATCACTTTAT CCAAGAAGGA ACTCCCATCC TCAAAGGTTT CAAAGCCTCA ACAAGGTCAG GGTACAGAGT GCTACAGGAG ATCAATGAAG AATCTTCAAT GCACATGCAT CATGGTCAGT AAGTTTCAGA TTAAAGTTAG TGGGCATCTT TGAAAGTAAT GGCTTGTGGG GACCAGACAA AAAAGGAATG CCTACCAAAA GCATCTTTGC CTTTATTGCA AGTACAAGTG GGGAACAAAA TAACGTGGAA ACTCACTAAT GCGTATGACG AACGCAGTGA CTATATAAGA AGGCATTCAT TCCCATTTGA CAATCCTTCT AGAAGATCTA AGCTTAT (2) INFORMATION FOR SEQ ID NO. 6 .

(X) SEQUENCE CHARACTERISTICS: (A) LENGTH. 10901 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: Linear 54 Printed from Mimosa 40 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 1 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA ATCCCCATTC 51 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT AGCAGAATCT 101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC GAAATCCAGT 151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC AGCATCCACG 201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT GGGATGACGT 251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC TGTTTCCACG 301 GCGTGCATGC TTCACGGTGC AAGCAGCCGT CCAGCAACTG CTCGTAAGTC 351 CTCTGGTCTT TCTGGAACCG TCCGTATTCC AGGTGACAAG TCTATCTCCC 401 ACAGGTCCTT CATGTTTGGA GGTCTCGCTA GCGGTGAAAC TCGTATCACC 451 GGTCTTTTGG AAGGTGAAGA TGTTATCAAC ACTGGTAAGG CTATGCAAGC 501 TATGGGTGCC AGAATCCGTA AGGAAGGTGA TACTTGGATC ATTGATGGTG 551 TTGGTAACGG TGGACTCCTT GCTCCTGAGG CTCCTCTCGA TTTCGGTAAC 601 GCTGCAACTG GTTGCCGTTT GACTATGGGT CTTGTTGGTG TTTACGATTT 651 CGATAGCACT TTCATTGGTG ACGCTTCTCT CACTAAGCGT CCAATGGGTC 701 GTGTGTTGAA CCCACTTCGC GAAATGGGTG TGCAGGTGAA GTCTGAAGAC 751 GGTGATCGTC TTCCAGTTAC CTTGCGTGGA CCAAAGACTC CAACGCCAAT 801 CACCTACAGG GTACCTATGG CTTCCGCTCA AGTGAAGTCC GCTGTTCTGC 851 TTGCTGGTCT CAACACCCCA GGTATCACCA CTGTTATCGA GCCAATCATG 901 ACTCGTGACC ACACTGAAAA GATGCTTCAA GGTTTTGGTG CTAACCTTAC 951 CGTTGAGACT GATGCTGACG GTGTGCGTAC CATCCGTCTT GAAGGTCGTG 1001 GTAAGCTCAC CGGTCAAGTG ATTGATGTTC CAGGTGATCC ATCCTCTACT 1051 GCTTTCCCAT TGGTTGCTGC CTTGCTTGTT CCAGGTTCCG ACGTCACCAT 1101 CCTTAACGTT TTGATGAACC CAACCCGTAC TGGTCTCATC TTGACTCTGC 1151 AGGAAATGGG TGCCGACATC GAAGTGATCA ACCCACGTCT TGCTGGTGGA 1201 GAAGACGTGG CTGACTTGCG TGTTCGTTCT TCTACTTTGA AGGGTGTTAC 1251 TGTTCCAGAA GACCGTGCTC CTTCTATGAT CGACGAGTAT CCAATTCTCG 1301 CTGTTGCAGC TGCATTCGCT GAAGGTGCTA CCGTTATGAA CGGTTTGGAA 1351 GAACTCCGTG TTAAGGAAAG CGACCGTCTT TCTGCTGTCG CAAACGGTCT 14 01 CAAGCTCAAC GGTGTTGATT GCGATGAAGG TGAGACTTCT CTCGTCGTGC 1451 GTGGTCGTCC TGACGGTAAG GGTCTCGGTA ACGCTTCTGG AGCAGCTGTC 1501 GCTACCCACC TCGATCACCG TATCGCTATG AGCTTCCTCG TTATGGGTCT 1551 CGTTTCTGAA AACCCTGTTA CTGTTGATGA TGCTACTATG ATCGCTACTA 1601 GCTTCCCAGA GTTCATGGAT TTGATGGCTG GTCTTGGAGC TAAGATCGAA 1651 CTCTCCGACA CTAAGGCTGC TTGATGAGCT CAAGAATTCG AGCTCGGTAC 1701 CGGATCCAGC TTTCGTTCGT ATCATCGGTT TCGACAACGT TCGTCAAGTT 1751 CAATGCATCA GTTTCATTGC GCACACACCA GAATCCTACT GAGTTCGAGT 1801 ATTATGGCAT TGGGAAAACT GTTTTTCTTG TACCATTTGT TGTGCTTGTA 1851 ATTTACTGTG TTTTTTATTC GGTTTTCGCT ATCGAACTGT GAAATGGAAA 1901 TGGATGGAGA AGAGTTAATG AATGATATGG TCCTTTTGTT CATTCTCAAA 1951 TTAATATTAT TTGTTTTTTC TCTTATTTGT TGTGTGTTGA ATTTGAAATT 2001 ATAAGAGATA TGCAAACATT TTGTTTTGAG TAAAAATGTG TCAAATCGTG 2051 GCCTCTAATG ACCGAAGTTA ATATGAGGAG TAAAACACTT GTAGTTGTAC 2101 CATTATGCTT ATTCACTAGG CAACAAATAT ATTTTCAGAC CTAGAAAAGC 2151 TGCAAATGTT ACTGAATACA AGTATGTCCT CTTGTGTTTT AGACATTTAT 2201 GAACTTTCCT TTATGTAATT TTCCAGAATC CTTGTCAGAT TCTAATCATT 2251 GCTTTATAAT TATAGTTATA CTCATGGATT TGTAGTTGAG TATGAAAATA 2301 TTTTTTAATG CATTTTATGA CTTGCCAATT GATTGACAAC ATGCATCAAT 2351 CGACCTGCAG CCACTCGAAG CGGCCGCGTT CAAGCTTGAG CTCAGGATTT 2401 AGCAGCATTC CAGATTGGGT TCAATCAACA AGGTACGAGC CATATCACTT 2451 TATTCAAATT GGTATCGCCA AAACCAAGAA GGAACTCCCA TCCTCAAAGG 2501 TTTGTAAGGA AGAATTCTCA GTCCAAAGCC TCAACAAGGT CAGGGTACAG 2551 AGTCTCCAAA CCATTAGCCA AAAGCTACAG GAGATCAATG AAGAATCTTC 2601 AATCAAAGTA AACTACTGTT CCAGCACATG CATCATGGTC AGTAAGTTTC 2651 AGAAAAAGAC ATCCACCGAA GACTTAAAGT TAGTGGGCAT CTTTGAAAGT 2701 AATCTTGTCA ACATCGAGCA GCTGGCTTGT GGGGACCAGA CAAAAAAGGA 55 Printed from Mimosa 40 45 2751 ATGGTGCAGA ATTGTTAGGC GCACCTACCA AAAGCATCTT TGCCTTTATT 28 01 GCAAAGATAA AGCAGATTCC TCTAGTACAA GTGGGGAACA AAATAACGTG 28 51 GAAAAGAGCT GTCCTGACAG CCCACTCACT AATGCGTATG ACGAACGCAG 2901 TGACGACCAC AAAAGAATTC CCTCTATATA AGAAGGCATT CATTCCCATT 2951 TGAAGGATCA TCAGATACTG AACCAATCCT TCTAGAAGAT CTAAGCTTAT 3001 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA ATCCCCATTC 3051 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT AGCAGAATCT 3101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC GAAATCCAGT 3151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC AGCATCCACG 3201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT GGGATGACGT 3251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC TGTTTCCACG 33 01 GCGTGCATGC AGGCcatggC TAAGATTTTT GATTTCGTAA AACCTGGCGT 33 51 AATCACTGGT GATGACGTAC AGAAAGTTTT CCAGGTAGCA AAAGAAAACA 3401 ACTTCGCACT GCCAGCAGTA AACTGCGTCG GTACTGACTC CATCAACGCC 3451 GTACTGGAAA CCGCTGCTAA AGTTAAAGCG CCGGTTATCG TTCAGTTCTC 3501 CAACGGTGGT GCTTCCTTTA TCGCTGGTAA AGGCGTGAAA TCTGACGTTC 51 CGCAGGGTGC TGCTATCCTG GGCGCGATCT CTGGTGCGCA TCACGTTCAC 36 01 CAGATGGCTG AACATTATGG TGTTCCGGTT ATCCTGCACA CTGACCACTG 3651 CGCGAAGAAA CTGCTGCCGT GGATCGACGG TCTGTTGGAC GCGGGTGAAA 37 01 AACACTTCGC AGCTACCGGT AAGCCGCTGT TCTCTTCTCA CATGATCGAC 3751 CTGTCTGAAG AATCTCTGCA AGAGAACATC GAAATCTGCT CTAAATACCT 38 01 GGAGCGCATG TCCAAAATCG GCATGACTCT GGAAATCGAA CTGGGTTGCA 38 51 CCGGTGGTGA AGAAGACGGC GTGGACAACA GCCACATGGA CGCTTCTGCA 3901 CTGTACACCC AGCCGGAAGA CGTTGATTAC GCATACACCG AACTGAGCAA 3951 AATCAGCCCG CGTTTCACCA TCGCAGCGTC CTTCGGTAAC GTACACGGTG 4001 TTTACAAGCC GGGTAACGTG GTTCTGACTC CGACCATCCT GCGTGATTCT 4051 CAGGAATATG TTTCCAAGAA ACACAACCTG CCGCACAACA GCCTGAACTT 4101 CGTATTCCAC GGTGGTTCCG GTTCTACTGC TCAGGAAATC AAAGACTCCG 4151 TAAGCTACGG CGTAGTAAAA ATGAACATCG ATACCGATAC CCAATGGGCA 4201 ACCTGGGAAG GCGTTCTGAA CTACTACAAA GCGAACGAAG CTTATCTGCA 42 51 GGGTCAGCTG GGTAACCCGA AAGGCGAAGA TCAGCCGAAC AAGAAATACT 4301 ACGATCCGCG CGTATGGCTG CGTGCCGGTC AGACTTCGAT GATCGCTCGT 43 51 CTGGAGAAAG CATTCCAGGA ACTGAACGCG ATCGACGTTC TGTAAGAGCT 44 01 CGGTACCGGA TCCAATTccc GATCGTTCAA ACATTTGGCA ATAAAGTTTC 4451 TTAAGATTGA ATCCTGTTGC CGGTCTTGCG ATGATTATCA TATAATTTCT 4501 GTTGAATTAC GTTAAGCATG TAATAATTAA CATGTAATGC ATGACGTTAT 4551 TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA CATTTAATAC 4601 GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC 4651 GGTGTCATCT ATGTTACTAG ATCGGGGATC GATCCCCGGG CGGCCGCCAC 4701 TCGAGTGGTG GCCGCATCGA TCGTGAAGTT TCTCATCTAA GCCCCCATTT 4751 GGACGTGAAT GTAGACACGT CGAAATAAAG ATTTCCGAAT TAGAATAATT 4801 TGTTTATTGC TTTCGCCTAT AAATACGACG GATCGTAATT TGTCGTTTTA 4851 TCAAAATGTA CTTTCATTTT ATAATAACGC TGCGGACATC TACATTTTTG 4901 AATTGAAAAA AAATTGGTAA TTACTCTTTC TTTTTCTCCA TATTGACCAT 4951 CATACTCATT GCTGATCCAT GTAGATTTCC CGGACATGAA GCCATTTACA 5001 ATTGAATATA TCCTGCCGCC GCTGCCGCTT TGCACCCGGT GGAGCTTGCA 5051 TGTTGGTTTC TACGCAGAAC TGAGCCGGTT AGGCAGATAA TTTCCATTGA 5101 GAACTGAGCC ATGTGCACCT TCCCCCCAAC ACGGTGAGCG ACGGGGCAAC 5151 GGAGTGATCC ACATGGGACT TTTCCTAGCT TGGCTGCCAT TTTTGGGGTG 5201 AGGCCGTTCG CGCGGGGCGC CAGCTGGGGG GATGGGAGGC CCGCGTTACC 5251 GGGAGGGTTC GAGAAGGGGG GGCACCCCCC TTCGGCGTGC GCGGTCACGC 5301 GCCAGGGCGC AGCCCTGGTT AAAAACAAGG TTTATAAATA TTGGTTTAAA 5351 AGCAGGTTAA AAGACAGGTT AGCGGTGGCC GAAAAACGGG CGGAAACCCT 5401 TGCAAATGCT GGATTTTCTG CCTGTGGACA GCCCCTCAAA TGTCAATAGG 5451 TGCGCCCCTC ATCTGTCATC ACTCTGCCCC TCAAGTGTCA AGGATCGCGC 5501 CCCTCATCTG TCAGTAGTCG CGCCCCTCAA GTGTCAATAC CGCAGGGCAC 5551 TTATCCCCAG GCTTGTCCAC ATCATCTGTG GGAAACTCGC GTAAAATCAG 5601 GCGTTTTCGC CGATTTGCGA GGCTGGCCAG CTCCACGTCG CCGGCCGAAA 56 Printed from Mimosa 40 45 PCT AJS98/12447 5651 TCGAGCCTGC CCCTCATCTG TCAACGCCGC GCCGGGTGAG TCGGCCCCTC 5701 AAGTGTCAAC GTCCGCCCCT CATCTGTCAG TGAGGGCCAA GTTTTCCGCG 5751 TGGTATCCAC AACGCCGGCG GCCGGCCGCG GTGTCTCGCA CACGGCTTCG 5801 ACGGCGTTTC TGGCGCGTTT GCAGGGCCAT AGACGGCCGC CAGCCCAGCG 5851 GCGAGGGCAA CCAGCCCGGT GAGCGTCGGA AAGGGTCGAT CGACCGATGC 5901 CCTTGAGAGC CTTCAACCCA GTCAGCTCCT TCCGGTGGGC GCGGGGCATG 5951 ACTATCGTCG CCGCACTTAT GACTGTCTTC TTTATCATGC AACTCGTAGG 6001 ACAGGTGCCG GCAGCGCTCT GGGTCATTTT CGGCGAGGAC CGCTTTCGCT 6051 GGAGCGCGAC GATGATCGGC CTGTCGCTTG CGGTATTCGG AATCTTGCAC 6101 GCCCTCGCTC AAGCCTTCGT CACTGGTCCC GCCACCAAAC GTTTCGGCGA 6151 GAAGCAGGCC ATTATCGCCG GCATGGCGGC CGACGCGCTG GGCTACGTCT 6201 TGCTGGCGTT CGCGACGCGA GGCTGGATGG CCTTCCCCAT TATGATTCTT 6251 CTCGCTTCCG GCGGCATCGG GATGCCCGCG TTGCAGGCCA TGCTGTCCAG 6301 GCAGGTAGAT GACGACCATC AGGGACAGCT TCAAGGATCG CTCGCGGCTC 6351 TTACCAGCCT AACTTCGATC ACTGGACCGC TGATCGTCAC GGCGATTTAT 6401 GCCGCCTCGG CGAGCACATG GAACGGGTTG GCATGGATTG TAGGCGCCGC 6451 CCTATACCTT GTCTGCCTCC CCGCGTTGCG TCGCGGTGCA TGGAGCCGGG 6501 CCACCTCGAC CTGAATGGAA GCCGGCGGCA CCTCGCTAAC GGATTCACCA 6551 CTCCAAGAAT TGGAGCCAAT CAATTCTTGC GGAGAACTGT GAATGCGCAA 6601 ACCAACCCTT GGCAGAACAT ATCCATCGCG TCCGCCATCT CCAGCAGCCG 6651 CACGCGGCGC ATCTCGGGCA GCGTTGGGTC CTGGCCACGG GTGCGCATGA 6701 TCGTGCTCCT GTCGTTGAGG ACCCGGCTAG GCTGGCGGGG TTGCCTTACT 6751 GGTTAGCAGA ATGAATCACC GATACGCGAG CGAACGTGAA GCGACTGCTG 6801 CTGCAAAACG TCTGCGACCT GAGCAACAAC ATGAATGGTC TTCGGTTTCC 6851 GTGTTTCGTA AAGTCTGGAA ACGCGGAAGT CAGCGCCCTG CACCATTATG 6901 TTCCGGATCT GCATCGCAGG ATGCTGCTGG CTACCCTGTG GAACACCTAC 6951 ATCTGTATTA ACGAAGCGCT GGCATTGACC CTGAGTGATT TTTCTCTGGT 7001 CCCGCCGCAT CCATACCGCC AGTTGTTTAC CCTCACAACG TTCCAGTAAC 7051 CGGGCATGTT CATCATCAGT AACCCGTATC GTGAGCATCC TCTCTCGTTT 7101 CATCGGTATC ATTACCCCCA TGAACAGAAA TTCCCCCTTA CACGGAGGCA 7151 TCAAGTGACC AAACAGGAAA AAACCGCCCT TAACATGGCC CGCTTTATCA 72 01 GAAGCCAGAC ATTAACGCTT CTGGAGAAAC TCAACGAGCT GGACGCGGAT 7251 GAACAGGCAG ACATCTGTGA ATCGCTTCAC GACCACGCTG ATGAGCTTTA 7301 CCGCAGCTGC CTCGCGCGTT TCGGTGATGA CGGTGAAAAC CTCTGACACA 7351 TGCAGCTCCC GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC 7401 AGACAAGCCC GTCAGGGCGC GTCAGCGGGT GTTGGCGGGT GTCGGGGCGC 7451 AGCCATGACC CAGTCACGTA GCGATAGCGG AGTGTATACT GGCTTAACTA 7501 TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCATATG CGGTGTGAAA 7551 TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG CTCTTCCGCT 7601 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT 7651 ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA 7701 ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT 7751 AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA 7801 GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 7851 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT 7901 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG 7951 AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT 8001 AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 8051 GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 8101 ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 8151 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 8201 GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT 8251 TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG 8301 CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 8351 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 8401 GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA 8451 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 8501 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG 57 Printed from Mimosa 8551 TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT 8601 GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 8651 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 8701 ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG 8751 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA B801 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC 8851 AGGTCGGGAG CACAGGATGA CGCCTAACAA TTCATTCAAG CCGACACCGC 8901 TTCGCGGCGC GGCTTAATTC AGGAGTTAAA CATCATGAGG GAAGCGGTGA 8951 TCGCCGAAGT ATCGACTCAA CTATCAGAGG TAGTTGGCGT CATCGAGCGC 9001 CATCTCGAAC CGACGTTGCT GGCCGTACAT TTGTACGGCT CCGCAGTGGA 9051 TGGCGGCCTG AAGCCACACA GTGATATTGA TTTGCTGGTT ACGGTGACCG 9101 TAAGGCTTGA TGAAACAACG CGGCGAGCTT TGATCAACGA CCTTTTGGAA 9151 ACTTCGGCTT CCCCTGGAGA GAGCGAGATT CTCCGCGCTG TAGAAGTCAC 9201 CATTGTTGTG CACGACGACA TCATTCCGTG GCGTTATCCA GCTAAGCGCG 9251 AACTGCAATT TGGAGAATGG CAGCGCAATG ACATTCTTGC AGGTATCTTC 9301 GAGCCAGCCA CGATCGACAT TGATCTGGCT ATCTTGCTGA CAAAAGCAAG 9351 AGAACATAGC GTTGCCTTGG TAGGTCCAGC GGCGGAGGAA CTCTTTGATC 9401 CGGTTCCTGA ACAGGATCTA TTTGAGGCGC TAAATGAAAC CTTAACGCTA 9451 TGGAACTCGC CGCCCGACTG GGCTGGCGAT GAGCGAAATG TAGTGCTTAC 9501 GTTGTCCCGC ATTTGGTACA GCGCAGTAAC CGGCAAAATC GCGCCGAAGG 9551 ATGTCGCTGC CGACTGGGCA ATGGAGCGCC TGCCGGCCCA GTATCAGCCC 9601 GTCATACTTG AAGCTAGGCA GGCTTATCTT GGACAAGAAG ATCGCTTGGC 9651 CTCGCGCGCA GATCAGTTGG AAGAATTTGT TCACTACGTG AAAGGCGAGA 9701 TCACCAAGGT AGTCGGCAAA TAATGTCTAA CAATTCGTTC AAGCCGACGC 9751 CGCTTCGCGG CGCGGCTTAA CTCAAGCGTT AGATGCTGCA GGCATCGTGG 9801 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA 9851 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC 9901 CTTCGGTCCT CCGATCGAGG ATTTTTCGGC GCTGCGCTAC GTCCGCKACC 9951 GCGTTGAGGG ATCAAGCCAC AGCAGCCCAC TCGACCTCTA GCCGACCCAG 10001 ACGAGCCAAG GGATCTTTTT GGAATGCTGC TCCGTCGTCA GGCTTTCCGA 10051 CGTTTGGGTG GTTGAACAGA AGTCATTATC GTACGGAATG CCAAGCACTC 10101 CCGAGGGGAA CCCTGTGGTT GGCATGCACA TACAAATGGA CGAACGGATA 10151 AACCTTTTCA CGCCCTTTTA AATATCCGTT ATTCTAATAA ACGCTCTTTT 10201 CTCTTAGGTT TACCCGCCAA TATATCCTGT CAAACACTGA TAGTTTAAAC 10251 TGAAGGCGGG AAACGACAAT CTGATCCCCA TCAAGCTTGA GCTCAGGATT 10301 TAGCAGCATT CCAGATTGGG TTCAATCAAC AAGGTACGAG CCATATCACT 10351 TTATTCAAAT TGGTATCGCC AAAACCAAGA AGGAACTCCC ATCCTCAAAG 10401 GTTTGTAAGG AAGAATTCTC AGTCCAAAGC CTCAACAAGG TCAGGGTACA 10451 GAGTCTCCAA ACCATTAGCC AAAAGCTACA GGAGATCAAT GAAGAATCTT 40 10501 CAATCAAAGT AAACTACTGT TCCAGCACAT GCATCATGGT CAGTAAGTTT 10551 CAGAAAAAGA CATCCACCGA AGACTTAAAG TTAGTGGGCA TCTTTGAAAG 10601 TAATCTTGTC AACATCGAGC AGCTGGCTTG TGGGGACCAG ACAAAAAAGG 10651 AATGGTGCAG AATTGTTAGG CGCACCTACC AAAAGCATCT TTGCCTTTAT 10701 TGCAAAGATA AAGCAGATTC CTCTAGTACA AGTGGGGAAC AAAATAACGT 45 10751 GGAAAAGAGC TGTCCTGACA GCCCACTCAC TAATGCGTAT GACGAACGCA 10801 GTGACGACCA CAAAAGAATT CCCTCTATAT AAGAAGGCAT TCATTCCCAT 10851 TTGAAGGATC ATCAGATACT GAACCAATCC TTCTAGAAGA TCTAAGCTTA 10901 T 50 58 Printed from Mimosa

Claims (13)

1. A recombinant, double-stranded DNA molecule containing [a] a promoter functional in plant cells, and [b] a DNA sequence coding for a polypeptide having the enzymatic activitv 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 prokarvotic organism
3. The DNA molecule according to claim 2. wherein the prokarvotic organism is Escherichia coh
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 prokarvotic DNA sequence coding for fructose-1,6-bisphosphate aldolase class II
5. 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 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"C to 55°C
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 PONDED 9 60
8. 8
9. 9
10. 10
11. 11
12. 12. 13 14 15 16 17 the coding region depicted as SEQ TD NO 1, or encodes the same peptide as SEQ ID NO 1 in accordance with the dcgeneracv of the genetic code A transgenic plant cell containing in its genome a recombinant DNA molecule according to any one of claims 1 to 7 A transgenic plant containing plant cells according to claim 8 The transgenic plant of claim 9, 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 The transgenic plant according to claim 9 or claim 10 which is a crop plant 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, piamtain, tobacco, lettuce, cassava, cruciferous vegetables, forestry species and horticultural species The transgenic plant of claim 11, wherein the plant is a potato A food product derived from the potato of claim 12. The food product of claim
13. 13. which is a french fry or a potato chip Propagation material derived from the transgenic plant of any one of claims 9 to 12. 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 7, and regenerating the transformed cells to produce a transgenic plant 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 7, and regenerating the transformed cells to produce a transgenic plant INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 3 MAY 2001 RECEIVED 18 A process for increasing the growth rate in plants which comprises transforming plant cells with a DNA molecule according to any one of claims I to 7. and regenerating the transformed cells to produce a transgenic plant 19 A process for improving the solids uniformity in plants which comprises transforming plant cells with a DNA molecule according to any one of claims 1 to 7, and regenerating the transformed cells to produce a transgenic plant 20 A method for the processing of potatoes into french fries or potato chips, comprising producing the french fries or potato chips from the potato of claim 12. INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 3 MAY 2001 RECEIVED