WO1998037214A1 - Sense intron inhibition of starch branching enzyme expression - Google Patents

Sense intron inhibition of starch branching enzyme expression Download PDF

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Publication number
WO1998037214A1
WO1998037214A1 PCT/IB1998/000295 IB9800295W WO9837214A1 WO 1998037214 A1 WO1998037214 A1 WO 1998037214A1 IB 9800295 W IB9800295 W IB 9800295W WO 9837214 A1 WO9837214 A1 WO 9837214A1
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Prior art keywords
sequence
nucleotide sequence
intron
sense
seq
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PCT/IB1998/000295
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French (fr)
Inventor
Peter Poulsen
Original Assignee
Danisco A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9703672.7A external-priority patent/GB9703672D0/en
Priority claimed from GBGB9706075.0A external-priority patent/GB9706075D0/en
Application filed by Danisco A/S filed Critical Danisco A/S
Priority to BR9808648-0A priority Critical patent/BR9808648A/en
Priority to JP53643898A priority patent/JP2001512972A/en
Priority to CA002280210A priority patent/CA2280210A1/en
Priority to AU62259/98A priority patent/AU738311B2/en
Priority to EP98904329A priority patent/EP1007708A1/en
Priority to NZ336607A priority patent/NZ336607A/en
Publication of WO1998037214A1 publication Critical patent/WO1998037214A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/107—1,4-Alpha-glucan branching enzyme (2.4.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the present invention relates to a method of inhibiting gene expression, particularly inhibiting gene expression in a plant.
  • the present invention also relates to a nucleotide sequence useful in the method.
  • the present invention relates to a promoter that is useful for expressing the nucleotide sequence.
  • Starch is one of the main storage carbohydrates in plants, especially higher plants.
  • the structure of starch consists of amylose and amylopectin.
  • Amylose consists essentially of straight chains of ⁇ -l-4-linked glycosyl residues.
  • Amylopectin comprises chains of ⁇ -l-4-linked glycosyl residues with some ⁇ -1-6 branches.
  • the branched nature of amylopectin is accomplished by the action of inter alia an enzyme commonly known as the starch branching enzyme ("SBE").
  • SBE catalyses the formation of branch points in the amylopectin molecule by adding ⁇ -1,4 glucans through ⁇ -l,6-glucosidic branching linkages.
  • the biosynthesis of amylose and amylopectin is schematically shown in Figure 1, whereas the ⁇ -l-4-links and the ⁇ -1-6 links are shown in Figure 2.
  • starch is an important raw material. Starch is widely used in the food, paper, and chemical industries. However, a large fraction of the starches used in these industrial applications are post-harvest modified by chemical, physical or enzymatic methods in order to obtain starches with certain required functional properties.
  • WO96/34968 discusses the use of antisense sequences complementary to sequences which encode class A and class B potato SBE to downregulate SBE expression in potato plants.
  • the sequences used are complementary to SBE coding sequences.
  • a method of affecting enzymatic activity in a plant comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence partially or completely codes (is) an intron of the potato class A SBE gene in a sense orientation, optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; and wherein the nucleotide sequence does not contain a sequence that is a sense exon sequence normally associated with the intron.
  • a method of affecting enzymatic activity in a starch producing organism comprising expressing in the starch producing organism (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron of the potato class A SBE gene in a sense orientation optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
  • the class A SBE gene sense intron construct is used in combination with a potato class B SBE gene sense intron construct as defined in PCT/EP96/03053.
  • it may also be used independently thereof, to target class A SBE alone, or in combination with other transgenes such as other sense and/or antisense transgenes, for example antisense intron transgenes such as from SBE genes, to further manipulate starch quality in potato plants.
  • a sequence comprising the nucleotide sequence shown as SEQ. ID. No. 38 or a variant, derivative or homologue thereof.
  • a promoter comprising the sequence shown as SEQ.I.D. No. 14 or a variant, derivative or homologue thereof.
  • a construct capable of comprising or expressing the present invention.
  • a vector comprising or expressing the present invention.
  • a seventh aspect of the present invention there is provided a cell, tissue or organ comprising or expressing the present invention.
  • a transgenic starch producing organism comprising or expressing the present invention.
  • a starch obtained from the present invention is provided.
  • a key advantage of the present invention is that it provides a method for preparing modified starches that is not dependent on the need for post-harvest modification of starches. Thus the method of the present invention obviates the need for the use of hazardous chemicals that are normally used in the post-harvest modification of starches.
  • the present invention provides ter alia genetically modified plants which are capable of producing modified and/or novel and/or improved starches whose properties would satisfy various industrial requirements.
  • the present invention provides a method of preparing tailor-made starches in plants which could replace the post-harvest modified starches.
  • the present invention provides a method that enables modified starches to be prepared by a method that can have a more beneficial effect on the environment than the known post-harvest modification methods which are dependent on the use of hazardous chemicals and large quantities of energy.
  • An other key advantage of the present invention is that it provides a method that may more reliably and/or more efficiently and/or more specifically affect enzymatic activity when compared to the known methods of affecting enzymatic activity.
  • this advantage of the present invention it is to be noted that there is some degree of homology between coding regions of SBEs. However, there is little or no homology with the intron sequences of SBEs. Thus, sense intron expression provides a mechanism to affect selectively the expression of a particular SBE.
  • This advantageous aspect could be used, for example, to reduce or eliminate a particular SBE enzyme and replace that enzyme with another enzyme which can be another branching enzyme or even a recombinant version of the affected enzyme or even a hybrid enzyme which could for example comprise part of a SBE enzyme from one source and at least a part of another SBE enzyme from another source.
  • Another enzyme which can be another branching enzyme or even a recombinant version of the affected enzyme or even a hybrid enzyme which could for example comprise part of a SBE enzyme from one source and at least a part of another SBE enzyme from another source.
  • the present invention provides a mechanism for selectively affecting SBE activity. This is in contrast to the prior art methods which are dependent on the use of for example antisense exon expression whereby it would not be possible to introduce new SBE activity without affecting that activity as well.
  • class B SBE is synonymous with SBE I: class A SBE is synonymous with SBE II.
  • Class A SBE is as defined in WO96/34968, incorporated herein by reference.
  • the antisense intron construct used comprises intron 1 of class A SBE, which is 2.0 kb in length and is located starting at residue 45 of the coding sequence of class A SBE.
  • the boundaries of the intron may be calculated by searching for consensus intron boundary sequences, and are shown in attached figure 11.
  • the sequence of the intron is set forth in SEQ. ID. No. 38.
  • Class B SBE is substantially as defined in the sequences given herein and in PCT/EP96/03053.
  • starch branching enzyme activity is affected and/or wherein the levels of amylopectin are affected and/or the composition of starch is changed.
  • the nucleotide sequence does not contain a sequence that is sense to an exon sequence.
  • the promoter is in combination with a gene of interest ("GOI").
  • GOI gene of interest
  • the enzymatic activity is reduced or eliminated.
  • the nucleotide sequence codes for at least substantially all of at least one intron in a sense orientation.
  • the nucleotide sequence codes, partially or completely, for two or more introns and wherein each intron is in a sense orientation.
  • the nucleotide sequence comprises at least 350 nucleotides (e.g. 350 bp), more preferably at least 500 nucleotides (e.g. 500 bp).
  • nucleotide sequence comprises the sequence shown as SEQ. ID. No. 38, or a fragment thereof.
  • nucleotide sequence is expressed by a promoter having a sequence shown as SEQ. I.D. No. 14 or a variant, derivative or homologue thereof.
  • the transgenic starch producing organism is a plant.
  • a preferred aspect of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for a class A SBE intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
  • a more preferred aspect of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed; and wherein the nucleotide sequence comprises the sequence shown as SEQ. ID. No. 38, or fragments thereof.
  • nucleotide in relation to the present invention includes DNA and RNA.
  • DNA Preferably it means DNA, more preferably DNA prepared by use of recombinant DNA techniques.
  • intron is used in its normal sense as meaning a segment of nucleotides, usually DNA, that does not encode part or all of an expressed protein or enzyme.
  • exon is used in its normal sense as meaning a segment of nucleotides, usually DNA, encoding part or all of an expressed protein or enzyme.
  • the term “intron” refers to gene regions that are transcribed into RNA molecules, but which are spliced out of the RNA before the RNA is translated into a protein.
  • the term “exon” refers to gene regions that are transcribed into RNA and subsequently translated into proteins.
  • variant or “homologue” or “fragment” in relation to the nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the respective nucleotide sequence providing the resultant nucleotide sequence can affect enzyme activity in a plant, or cell or tissue thereof, preferably wherein the resultant nucleotide sequence has at least the same effect as the sequence shown in SEQ. ID. No. 38.
  • homologue covers homology with respect to similarity of structure and/or similarity of function providing the resultant nucleotide sequence has the ability to affect enzymatic activity in accordance with the present invention.
  • sequence homology i.e. similarity
  • sequence homology preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology.
  • variant or variant or fragment in relation to the promoter of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the respective promoter sequence providing the resultant promoter sequence allows expression of a GOI, preferably wherein the resultant promoter sequence has at least the same effect as SEQ. I.D. No. 14.
  • homologue covers homology with respect to similarity of structure and/or similarity of function providing the resultant promoter sequence has the ability to allow for expression of a GOI, such as a nucleotide sequence according to the present invention.
  • sequence homology i.e. similarity
  • sequence homology preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95 % homology, more preferably at least 98% homology.
  • sequence homology i.e. similarity
  • sequence homology preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95 % homology, more preferably at least 98% homology.
  • allelic variations of the sequences are also synonymous with allelic variations of the sequences.
  • the intron sequence of the present invention can be any one or all of the intron sequences of the present invention, including partial sequences thereof, provided that if partial sense sequences are used the partial sequences affect enzymatic activity. Suitable examples of partial sequences include sequences that are shorter than any one of the full sense sequences shown as SEQ. ID. No. 38 but which comprise nucleotides that are adjacent the respective exon or exons.
  • the nucleotide sequences of the present invention may comprise one or more sense or antisense exon sequences of the class A or class B SBE gene (but not sense exon sequences naturally associated with the intron sequence), including complete or partial sequences thereof, providing the nucleotide sequences can affect SBE activity, preferably wherein the nucleotide sequences reduce or eliminate SBE activity.
  • the nucleotide sequence of the second aspect of the present invention does not comprise sense exon sequences.
  • vector includes an expression vector and a transformation vector.
  • expression vector means a construct capable of in vivo or in vitro expression.
  • transformation vector means a construct capable of being transferred from one species to another - such as from an E.Coli plasmid to a fungus or a plant cell, or from an Agrobacterium to a plant cell.
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - in relation to the sense nucleotide sequence aspect of the present invention includes the nucleotide sequence according to the present invention directly or indirectly attached to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shi -intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention.
  • fused in relation to the present invention which includes direct or indirect attachment. The terms do not cover the natural combination of the wild type SBE gene when associated with the wild type SBE gene promoter in their natural environment.
  • the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred.
  • a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred.
  • Various markers exist which may be used in, for example, plants - such as mannose.
  • Other examples of markers include those that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • the construct of the present invention preferably comprises a promoter.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression.
  • suitable promoters are those that can direct efficient expression of the nucleotide sequence of the present invention and/or in a specific type of cell.
  • tissue specific promoters are disclosed in WO 92/11375.
  • the promoter could additionally include conserved regions such as a Pribnow Box or a TATA box.
  • the promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention.
  • sequences include the S/zi-intron or an ADH intron.
  • Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements.
  • suitable elements to enhance transcription or translation may be present.
  • An example of the latter element is the TMV 5' leader sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
  • the construct and/or the vector of the present invention may include a transcriptional initiation region which may provide for regulated or constitutive expression.
  • Any suitable promoter may be used for the transcriptional initiation region, such as a tissue specific promoter.
  • the promoter is the patatin promoter or the E35S promoter.
  • the promoter is the SBE promoter.
  • the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, tuber, stem, sprout, root and leaf tissues, preferably tuber.
  • the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the ⁇ -Amy 637 promoter) as described in our co-pending UK patent application No. 9421292.5 filed 21 October 1994.
  • the promoter for the nucleotide sequence of the present invention can be the ⁇ -Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the ⁇ -Amy 351 promoter) as described in our co-pending UK patent application No. 9421286.7 filed 21 October 1994.
  • the present invention also encompasses the use of a promoter to express a nucleotide sequence according to the present invention, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses the nucleotide sequence of the present invention in a more specific manner such as in just one specific tissue type or organ.
  • the term "inactivated” means partial inactivation in the sense that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter.
  • the modified promoter is capable of expressing a gene coding for the enzyme of the present invention in at least one (but not all) specific tissue of the original promoter.
  • partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase.
  • Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part.
  • Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter.
  • the construct and/or the vector of the present invention may include a transcriptional termination region.
  • the nucleotide according to the present invention can be expressed in combination (but not necessarily at the same time) with an additional construct.
  • the present invention also provides a combination of constructs comprising a first construct comprising the nucleotide sequence according to the present invention operatively linked to a first promoter; and a second construct comprising a GOI operatively linked to a second promoter (which need not be the same as the first promoter).
  • the combination of constructs may be present in the same vector, plasmid, cells, tissue, organ or organism.
  • This aspect of the present invention also covers methods of expressing the same, preferably in specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a plant.
  • the second construct does not cover the natural combination of the gene coding for an enzyme ordinarily associated with the wild type gene promoter when they are both in their natural environment.
  • An example of a suitable combination would be a first construct comprising the nucleotide sequence of the present invention and a promoter, such as the promoter of the present invention, and a second construct comprising a promoter, such as the promoter of the present invention, and a GOI wherein the GOI codes for another starch branching enzyme either in sense or antisense orientation.
  • GOI with reference to the promoter aspect of the present invention or the combination aspect of the present invention means any gene of interest, which need not necessarily code for a protein or an enzyme - as is explained later.
  • a GOI can be any nucleotide sequence that is either foreign or natural to the organism in question, for example a plant.
  • Typical examples of a GOI include genes encoding for other proteins or enzymes that modify metabolic and catabolic processes.
  • the GOI may code for an agent for introducing or increasing pathogen resistance.
  • the GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues.
  • An example of such a GOI is the nucleotide sequence according to the present invention.
  • the GOI may even code for a protein that is non-natural to the host organism - e.g. a plant.
  • the GOI may code for a compound that is of benefit to animals or humans.
  • the GOI could code for a pharmaceutically active protein or enzyme such as any one of the therapeutic compounds insulin, interferon, human serum albumin, human growth factor and blood clotting factors.
  • the GOI may even code for a protein giving additional nutritional value to a food or feed or crop.
  • Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non- transgenic plant).
  • the GOI may even code for an enzyme that can be used in food processing such as xylanases and ⁇ -galactosidase.
  • the GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for ⁇ -amylase, a protease or a glucanase.
  • the GOI can be a nucleotide sequence according to the present invention.
  • the GOI can be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of our co-pending UK patent application 9505479.7.
  • the GOI can be the nucleotide sequence coding for the glucanase enzyme which is the subject of our co-pending UK patent application 9505475.5.
  • the GOI can be the nucleotide sequence coding for the ⁇ -amylase enzyme which is the subject of our co-pending UK patent application 9413439.2.
  • the GOI can be the nucleotide sequence coding for the ⁇ - amylase enzyme which is the subject of our co-pending UK patent application 9421290.9.
  • the GOI can be any of the nucleotide sequences coding for the ⁇ -glucan lyase enzyme which are described in our co-pending PCT patent application PCT/EP94/03397.
  • the GOI can even be a nucleotide sequence according to the present invention but when operatively linked to a different promoter.
  • the GOI could include a sequence that codes for one or more of a xylanase, an arabinase, an acetyl esterase, a rhamnogalacturonase, a glucanase, a pectinase, a branching enzyme or another carbohydrate modifying enzyme or proteinase.
  • the GOI may be a sequence that is antisense to any of those sequences.
  • the present invention provides a mechanism for selectively affecting a particular enzymatic activity.
  • a genomic nucleotide sequence coding for a genomic protein or enzyme by expressing a sense intron construct for that particular genomic protein or enzyme and (e.g. at the same time) expressing a recombinant version of that enzyme or protein - in other words the GOI is a recombinant nucleotide sequence coding for the genomic enzyme or protein.
  • This application allows expression of desired recombinant enzymes and proteins in the absence of (or reduced levels of) respective genomic enzymes and proteins.
  • the desired recombinant enzymes and proteins can be easily separated and purified from the host organism.
  • This particular aspect of the present invention is very advantageous over the prior art methods which, for example, rely on the use of anti- sense exon expression which methods also affect expression of the recombinant enzyme.
  • a further aspect of the present invention relates to a method of expressing a recombinant protein or enzyme in a host organism comprising expressing a nucleotide sequence coding for the recombinant protein or enzyme; and expressing a further nucleotide sequence wherein the further nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the intron is an intron normally associated with the genomic gene encoding a protein or an enzyme corresponding to the recombinant protein or enzyme; and wherein the further nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron. Additional aspects cover the combination of those nucleotide sequences including their incorporation in constructs, vectors, cells, tissues and transgenic organisms.
  • the present invention also relates to a combination of nucleotide sequences comprising a first nucleotide sequence coding for a recombinant enzyme; and a second nucleotide sequence which corresponds to an intron in a sense orientation; wherein the intron is an intron that is associated with a genomic gene encoding the enzyme corresponding to the recombinant enzyme; and wherein the second nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron.
  • the GOI may even code for one or more introns but in an antisense orientation, such as any one or more of the antisense intron sequences presented in the attached sequence listings.
  • the present invention also covers the expression of for example a sense intron (e.g. SEQ. I.D. No. 38) in combination with for example an antisense intron which preferably is not complementary to the sense intron sequence (e.g. SEQ. I.D. No. 16).
  • cell include cell, tissue and organ per se and when within an organism.
  • organism in relation to the present invention includes any organism that could comprise the nucleotide sequence according to the present invention and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism.
  • the organism is a starch producing organism such as any one of a plant, algae, fungi, yeast and bacteria, as well as cell lines thereof.
  • the organism is a plant.
  • starch producing organism includes any organism that can biosynthesise starch.
  • the starch producing organism is a plant.
  • plant as used herein includes any suitable angiosperm, gymnosperm, monocotyledon and dicotyledon.
  • suitable plants include vegetables such as potatoes; cereals such as wheat, maize, and barley; fruit; trees; flowers; and other plant crops.
  • the term means "potato”.
  • transgenic organism in relation to the present invention includes any organism that comprises the nucleotide sequence according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism.
  • the nucleotide sequence of the present invention is incorporated in the genome of the organism.
  • the transgenic organism is a plant, more preferably a potato.
  • prokaryotic or eukaryotic organisms examples include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art. for example see Sambrook et al (Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press).
  • the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
  • the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
  • the vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system.
  • Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
  • Ti plasmids from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
  • Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
  • a non-limiting example of such a Ti plasmid is pGV3850.
  • the nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appears to be essential for insertion of modified T-DNA into the plant genome.
  • the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
  • the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof.
  • these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
  • the nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant.
  • An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used.
  • a vector of a vector system as defined above When a vector of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.
  • the Ti-plasmid harbouring the nucleotide sequence or construct of the present invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the present invention, which DNA is subsequently transferred into the plant cell to be modified.
  • T-DNA for the transformation of plant cells, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected.
  • T-DNA for the transformation of plant cells has been intensively studied and is described in ⁇ P-A-120516; Hoekema, in: The Binary Plant Vector System Offset- drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al., Cut. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985) 4:277-284.
  • a plant to be infected is wounded, e.g. by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive.
  • the wound is then inoculated with the Agrobacterium.
  • the inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
  • tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
  • Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
  • cloning vectors which contain a replication system in E. coli and a marker which allows a selection of the transformed cells.
  • the vectors contain for example pBR 322, pUC series, M13 mp series, pACYC 184 etc.
  • the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector.
  • the contained plasmid is then used for the transformation in E.coli.
  • the E.coli cells are cultivated in a suitable nutrient medium and then harvested and lysed.
  • the plasmid is then recovered.
  • sequence analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
  • nucleotide sequence or construct according to the present invention After the introduction of the nucleotide sequence or construct according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary - such as to create combination systems as outlined above (e.g. an organism comprising a combination of constructs).
  • the present invention relates to affecting enzyme activity by expressing sense intron sequences.
  • the present invention relates to a promoter useful for the expression of those sense intron sequences.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • NCIMB 40754 (which refers to pBEA 11 as described herein);
  • NCIMB 40751 (which refers to ⁇ -SBE 3.2 as described herein), and
  • NCIMB 40752 (which refers to ⁇ -SBE 3.4 as described herein).
  • a highly preferred embodiment of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed; and wherein the intron nucleotide sequence is the sequence of intron 1 of class A SBE as set forth in SEQ.
  • sequence of introns of class A SBE other than intron 1 may be obtained by sequencing of, for example, potato class A SBE genomic DNA, isolatable by hybridisation screening of a genomic DNA library with class A SBE cDNA obtainable according to W096/34968 according to methods well known in the art and set forth, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989.
  • Figure 1 is a schematic representation of the biosynthesis of amylose and amylopectin
  • Figure 2 which is a diagrammatic representation of the ⁇ -l-4-links and the ⁇ -1-6 links of amylopectin;
  • Figure 3 which is a diagrammatic representation of the exon-intron structure of a genomic SBE clone
  • Figure 4 which is a plasmid map of pPATAl, which is 3936 bp in size;
  • Figure 5 which is a plasmid map of pABE7, which is 5106 bp in size;
  • Figure 6 which is a plasmid map of pVictorlV Man, which is 7080 bp in size;
  • Figure 7 which is a plasmid map of pBEAll, which is 9.54 kb in size
  • Figure 8 which shows the full genomic nucleotide sequence for SBE including the promoter, exons and introns;
  • Figure 9 which is a plasmid map of pVictor5a, which is 9.12 kb in size;
  • Figure 10 which is a plasmid map of pBEP2, which is 10.32 kb in size;
  • Figure 11 which shows the positioning of intron 1 in the class A and class B SBE genes
  • Figures 1 and 2 were referred to above in the introductory description concerning starch in general.
  • Figure 3 is a diagrammatic representation of the exon-intron structure of a genomic SBE clone, the sequence of which is shown in Figure 8.
  • This clone which has about 11.5 k base pairs, comprises 14 exons and 13 introns.
  • the introns are numbered in increasing order from the 5 ' end to the 3 ' end and correspond to SEQ. I.D. No. s 1-13, respectively.
  • Their respective antisense intron sequences are shown as SEQ.I.D.No.s 15-27.
  • Figures 3 and 8 present information on the 11468 base pairs of a potato SBE gene.
  • the 5' region from nucleotides 1 to 2082 contain the promoter region of the SBE gene.
  • a TATA box candidate at nucleotide 2048 to 2051 is boxed.
  • the homology between a potato SBE cDNA clone (Poulsen & Kreiberg (1993) Plant Physiol 102: 1053- 1054) and the exon DNAs begin at 2083 bp and end at 9666 bp.
  • the homology between the cDNA and the exon DNA is indicated by nucleotides in upper case letters, while the translated amino acid sequences are shown in the single letter code below the exon DNA.
  • Figure 7 is a plasmid map of pBEA7, which is 9.54 k base pairs in size. Plasmid pBEA 11 comprises the first intron sequence of the potato SBE gene. This first intron sequence, which has 1177 base pairs, is shown in Figure 3 and lies between the first exon and the second exon.
  • ⁇ SBE 3.2 contains a 15 kb potato DNA insert and ⁇ SBE-3.4 contains a 13 kb potato DNA insert.
  • the resultant plasmids are called pGB3, pGBll, pGB15, pGB16 and pGB25 (see discussion below).
  • the respective inserts are then sequenced using the Pharmacia Autoread Sequencing Kit (Pharmacia, Uppsala) and a A.L.F. DNA sequencer (Pharmacia, Uppsala). In total, a stretch of 11.5 kb of the SBE gene is sequenced.
  • pGB25 contains the sequences from 1 bp to 836 bp
  • pGB15 contains the sequences from 735 bp to 2580 bp
  • pGB16 contains the sequences from 2580 bp to 5093 bp
  • pGBll contains the sequences from 3348 bp to 7975 bp
  • pGB3 contains the sequences from 7533 bp to 11468 bp.
  • pGB3 is constructed by insertion of a 4 kb EcoRl fragment isolated from ⁇ SBE 3.2 into the EcoRl site of pBluescript II SK (+).
  • pGBll is constructed by insertion of a 4.7 kb Xhol fragment isolated from ⁇ SBE 3.4 into the Xhol site of pBluescript II SK (+).
  • pGB15 is constructed by insertion of a 1.7 kb Spel fragment isolated from ⁇ SBE 3.4 into the Spel site of pBluescript II SK (+).
  • pGB16 is constructed by insertion of a 2.5 kb Spel fragment isolated from ⁇ SBE 3.4 into the Spel site of pBluescript II SK (+).
  • the PCR fragment is digested with BamHl and EcoRl, and inserted in pBluescript II SK (+) digested with the same restriction enzymes.
  • the SBE intron 1 is amplified by PCR using the oligonucleotides
  • the PCR product is digested with BamHl and inserted in a sense orientation in the BamHl site of plasmid pPATAl (described in WO 94/24292) between the patatin promoter and the 35S terminator.
  • This construction, pABE7 is digested with Kpnl, and the 2.4 kb "patatin promoter-SBE intron 1- 35S terminator" Kpnl fragment is isolated and inserted in the Kpnl site of the plant transformation vector pVictorlV Man yielding plasmid pBEAll .
  • the 2122 bp intron 1 sequence of the potato SBEII gene (see SEQ. ID. No. 38) is amplified by PCR from a genomic SBEII subclone using the primers 5' - CGG GAT CCC GTA TGT CTC ACT GTG TTT GTG GC - 3' (SEQ. ID. No. 34) and 5' - CGG GAT CCC CCT ACA TAC ATA TAT CAG ATT AG - 3' (SEQ. ID. No. 35).
  • the PCR product is digested with BamHl and inserted in sense orientation after a patatin promoter in the BamHl site of a plant transformation vector in which the NPTII gene is used as selectable marker (see figure 13).
  • the 2122 bp intron 1 sequence of the potato SBEII gene (SEQ. ID. No. 38) is amplified by PCR from a genomic SBEII subclone using the primers 5' - CGG GAT CCC GTA TGT CTC ACT GTG TTT GTG GC - 3' (SEQ. ID. No. 34) and 5' - CGG GAT CCC CCT ACA TAC ATA TAT CAG ATT AG - 3' (SEQ. ID. No. 35).
  • the PCR product is digested with BamHl and inserted in sense orientation after a patatin promoter in the BamHl site of a plant transformation vector in which the manA gene is used as selectable marker (see figure 14).
  • Axenic stock cultures Shoot cultures of Solanum tuberosum 'Bintje' and 'Dianella' are maintained on a substrate (LS) of a formula according to Linsmaier, E.U. and Skoog, F. (1965), Physiol.
  • Plant. 18 100-127, in addition containing 2 ⁇ M silver thiosulphate at 25°C and 16 h light/8 h dark.
  • the cultures are subcultured after approximately 40 days. Leaves are then cut off the shoots and cut into nodal segments (approximately 0.8 cm) each containing one node.
  • the Agrobacterium are grown overnight in YMB-substrate (di-potassium hydrogen phosphate, trihydrate (0.66 g/1); magnesium sulphate, heptahydrate (0.20 g/1); sodium chloride (0.10 g/1); mannitol (10.0 g/1); and yeast extract (0.40 g/1)) containing appropriate antibiotics (corresponding to the resistance gene of the Agrobacterium strain) to an optical density at 660 nm (OD-660) of approximately 0.8, centrifuged and resuspended in the LS-substrate to an OD-660 of 0.5.
  • YMB-substrate di-potassium hydrogen phosphate, trihydrate (0.66 g/1); magnesium sulphate, heptahydrate (0.20 g/1); sodium chloride (0.10 g/1); mannitol (10.0 g/1); and yeast extract (0.40 g/1)
  • appropriate antibiotics corresponding to the resistance gene of the Agrobacterium strain
  • the segments are left in the suspension of Agrobacterium for 30 minutes and then the excess of bacteria are removed by blotting the segments on sterile filter paper.
  • the shoot segments are co-cultured with bacteria for 48 hours directly on LS- substrate containing agar (8.0 g/1), 2,4-dichlorophenoxyacetic acid (2.0 mg/1) and trans- zeatin (0.5 mg/1).
  • the substrate and also the explants are covered with sterile filter papers, and the petri dishes are placed at 25°C and 16 h light/ 8 dark.
  • the segments are transferred to containers containing liquid LS-substrate containing 800 mg/1 carbenicillin.
  • the containers are gently shaken and by this procedure the major part of the Agrobacterium is either washed off the segments and/or killed.
  • the segments are transferred to plates containing the LS-substrate, agar (8 g/1), trans-zeatin (1-5 mg/1), gibberellic acid (0.1 mg/1), carbenicillin (800 mg/1), and kanamycin sulphate (50-100 mg/1) or phosphinotricin (1-5 mg/1) or mannose (5 g/1) depending on the vector construction used.
  • the segments are sub-cultured to fresh substrate each 3-4 weeks. In 3 to 4 weeks, shoots develop from the segments and the formation of new shoots continued for 3-4 months.
  • Rooting of regenerated shoots The regenerated shoots are transferred to rooting substrate composed of LS- substrate, agar (8 g/1) and carbenicillin (800 mg/1).
  • the transgenic genotype of the regenerated shoot are verified by testing the rooting ability on the above mentioned substrates containing kanamycin sulphate (200 mg/1), by performing NPTII assays (Radke, S. E. et al, Theor. Appl. Genet. (1988), 75: 685-694) or by performing PCR analysis according to Wang et al (1993, NAR 21 pp 4153-4154). Plants which are not positive in any of these assays are discarded or used as controls.
  • the transgenic plants could be verified by performing a GUS assay on the co-introduced ⁇ - glucuronidase gene according to Hodal, L. et al. (PI. Sci. (1992), 87: 115-122).
  • the newly rooted plants (height approx. 2-3 cms) are transplanted from rooting substrate to soil and placed in a growth chamber (21°C, 16 hour light 200-400uE/m7sec). When the plants are well established they are transferred to the greenhouse, where they are grown until tubers had developed and the upper part of the plants are senescing.
  • the potatoes are harvested after about 3 months and then analysed.
  • the SBE expression in the transgenic potato lines are measured using the SBE assays described by Blennow and Johansson (Phytochemistry (1991) 30:437-444) and by standard Western procedures using antibodies directed against class A and class B potato SBE.
  • STARCH ANALYSIS Starch is isolated from potato tubers and analysed for the amylose:amylopectin ratio (Hovenkamp-Hermelink et al. (1988) Potato Research 31:241-246). In addition, the chain length distribution of amylopectin is determined by analysis of isoamylase digested starch on a Dionex HPAEC. The number of reducing ends in isoamylase digested starch is determined by the method described by N. Nelson (1944) J. Biol. Chem. 153:375-380. The results revealed that there is a reduction in the level of synthesis of SBE and/or the level of activity of SBE and/or the composition of starch SBE in the transgenic plants.
  • An SBE promoter fragment is amplified from ⁇ -SBE 3.4 using primers: 5 ' CCA TCG ATA CTT TAA GTG ATT TGA TGG C 3'
  • Potatoes from potato plants transformed with pBEAl 1 are cut in small pieces and homogenised in extraction buffer (50 mM Tris-HCl pH 7.5, Sodium-dithionite (0.1 g/1), and 2 mM DTT) using a Ultra-Turax homogenizer; 1 g of Dowex xl. is added pr. 10 g of tuber.
  • the crude homogenate is filtered through a miracloth filter and centrifuged at 4°C for 10 minutes at 24.700 g. The supernatant is used for starch branching enzyme assays.
  • the starch branching enzyme assays are carried out at 25 °C in a volume of 400 ⁇ l composed of 0.1 M Na citrate buffer pH 7.0, 0.75 mg/ml amylose, 5 mg/ml bovine serum albumin and the potato extract. At 0, 15 30 and 60 minutes aliqouts of 50 ⁇ l are removed from the reaction into 20 ⁇ l 3 N HCl. 1 ml of iodine solution is added and the decrease in absorbance at 620 nm is measured with an ELISA spectrophotometer.
  • the starch branching enzyme (SBE) levels in tuber extracts are measured from 24 transgenic Dianella potato plants transformed with plasmid pBEAll, pSS15 and pSS16.
  • the above-mentioned examples relate to the isolation and sequencing of a gene for potato SBE.
  • the examples further demonstrate that it is possible to prepare SBE intron constructs.
  • These SBE intron constructs can be introduced into plants, such as potato plants. After introduction, a reduction in the level of synthesis of SBE and/or the level of activity of SBE and/or the composition of starch in plants can be achieved.
  • the expressed sense intron nucleotide sequence according to the present invention affects enzymatic activity via co-suppression and/or trans-activation. Reviews of these mechanisms has been published by Finnegan and McElroy (1994 Biotechnology 12 pp 883 - 887) and Matzke and Matzke (1995 TIG 11 No. 1 pp 1 - 3). By these mechanisms, it is believed that the sense introns of the present invention reduce the level of plant enzyme activity (in particular SBE activity), which in turn for SBE activity is believed to influence the amylose: amylopectin ratio and thus the branching pattern of amylopectin.
  • the present invention provides a method wherein it is possible to manipulate the starch composition in plants, or tissues or cells thereof, such as potato tubers, by reducing the level of SBE activity by using sense intron sequences.
  • the present invention therefore relates to the surprising use of SBE class A sense intron sequences in a method to affect class A SBE activity in plants.
  • SEQ.I.D. No. 13 represent sense intron sequences (genomic DNA); SEQ.I.D. No. 14 represents the SBE promoter sequence (genomic sequence); SEQ.I.D. No. 15 - SEQ.I.D. No. 27 represent antisense intron sequences; and SEQ. I.D. No. 28 represents the sequence complementary to the SBE promoter sequence - i.e. the SBE promoter sequence in antisense orientation.
  • SEQ. I.D. No. 29 see Figures 3 and 8 which highlight particular gene features.
  • SEQ. ID. No. 30 to 37 show primers used in the methods set forth above.
  • SEQ. ID. No. 38 represents the nucleotide sequence of intron 1 of the class A potato SBE gene.
  • MOLECULE TYPE DNA (genomic)
  • AGTGTATAAA ATTCAAAAAT TGAGAGATGG AGGGGGGGTG GGGGAAGACA ATATTTAGAA 240
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ATAGCTTCCT TAATATTAAA TTCATGATAC TTTTGTTGAG ATTTAGCAGT TTTTTCTTGT 480
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GTTTGTCTGT TTCTATTGCA TTTTAAGGTT CATATAGGTT AGCCACGGAA AATCTCACTC 60
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAAAGGATGC CAAAAAAATT CTTCTCTATC CTCTTTTTCC CTAAACCAGT GCATGTAGCT 240
  • AAAAAGTCCA AAAAGAAGTA ACAGAAGTGA AAGCAACATG TGTAGCTAAA GCGACCCAAC 540
  • MOLECULE TYPE DNA (genomic)
  • AAAATAAATT ATTTTTTGAC ATTCTAAAAC TTTAAGCAGA ATAAATAGTT TATCAATTAT 1440
  • TTTAGCCTAA CCAACGAATA TTTGTAAACT CACAACTTGA TTAAAAGGGA TTTACAACAA 1620
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTGCATTTCA TCATGAGGGG GAGGAAAGAC GGAGAAATAT AGATATCAGA TTTAGACCAT 60
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • TCTAATCCCA CTTGAAATGA GAGTTATTTT AATATCGACC GTTAATTACC ATTTTATTAT 1500 TAAATCTGCA ACTACAGTCA ACTACACCAA TGATTTTGCT GATGCCAACT CATAATATAA 1560
  • CTGTGTACTT GTCTTTTCCT TGTAATAATG TATGTATGAT AATAATAATA AGAGAACAAA 1860
  • MOLECULE TYPE DNA (genomic)
  • AAAATAAATT ATTTTTTGAC ATTCTAAAAC TTTAAGCAGA ATAAATAGTT TATCAATTAT 1440
  • TTTAGCCTAA CCAACGAATA TTTGTAAACT CACAACTTGA TTAAAAGGGA TTTACAACAA 1620
  • TTCTGTTCGA AATAGCTTCC TTAATATTAA ATTCATGATA CTTTTGTTGA GATTTAGCAG 4500
  • TCTTTTTCAA TATCCCTCTT ATTTGGAGGT AATTTTTCTC ATCTATTCAC TTTTAGCTTC 6660 TAACCACAGA TGATCCATTT TTTCACAATG GCCTTGGGAG GAGAGGGGTA CCTCAATTTC 6720
  • ACAAATGTAG ACGCCAGTGG AACCTCGCGG ATAGCGAACA CTTGAGATAC AAGGTTCAAG 7140
  • CTTTGTCTTC ATGCTGACAT ACAATCTGTT CTCATGACAG ACTGGTC TG ATGTTGACCA 8340
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "Synthetic DNA Primer"
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "Synthetic DNA Primer"
  • MOLECULE TYPE DNA (genomic)
  • AAAAGTTTTT TGATATACTT AAAAAATTGT CACACAGAAG AAAGAGTTTT TTACCATTAC 1860 TTAAGCTAGA TGGGACTGTT TGATTCTTAG ACCAAATAAT GAACCTTTTT GTTCTCTTAA 1920

Abstract

A method of inhibiting gene expression is described. The method, which affects enzymatic activity in a plant, comprises expressing in a plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for a class A SBE intron in a sense orientation; and wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron.

Description

SENSE INTRON INHIBITION OF STARCH BRANCHING ENZYME EXPRESSION
The present invention relates to a method of inhibiting gene expression, particularly inhibiting gene expression in a plant. The present invention also relates to a nucleotide sequence useful in the method. In addition, the present invention relates to a promoter that is useful for expressing the nucleotide sequence.
Starch is one of the main storage carbohydrates in plants, especially higher plants. The structure of starch consists of amylose and amylopectin. Amylose consists essentially of straight chains of α-l-4-linked glycosyl residues. Amylopectin comprises chains of α-l-4-linked glycosyl residues with some α-1-6 branches. The branched nature of amylopectin is accomplished by the action of inter alia an enzyme commonly known as the starch branching enzyme ("SBE"). SBE catalyses the formation of branch points in the amylopectin molecule by adding α-1,4 glucans through α-l,6-glucosidic branching linkages. The biosynthesis of amylose and amylopectin is schematically shown in Figure 1, whereas the α-l-4-links and the α-1-6 links are shown in Figure 2.
In Potato, it is known that two classes of SBE exist. In our copending international patent applications PCT/EP96/03052 and PCT/EP96/03053, class B potato SBE and a gene encoding it are discussed. In international patent application WO96/34968, class A potato SBE and a cDNA encoding it are disclosed.
It is known that starch is an important raw material. Starch is widely used in the food, paper, and chemical industries. However, a large fraction of the starches used in these industrial applications are post-harvest modified by chemical, physical or enzymatic methods in order to obtain starches with certain required functional properties.
Within the past few years it has become desirable to make genetically modified plants which could be capable of producing modified starches which could be the same as the post-harvest modified starches. It is also known that it may be possible to prepare such genetically modified plants by expression of antisense nucleotide coding sequences. In this regard, June Bourque provides a detailed summary of antisense strategies for the genetic manipulations in plants (Bourque 1995 Plant Science 1Q5 pp 125-149).
WO96/34968 discusses the use of antisense sequences complementary to sequences which encode class A and class B potato SBE to downregulate SBE expression in potato plants. The sequences used are complementary to SBE coding sequences.
Whilst it is known that enzymatic activity can be affected by expression of particular nucleotide sequences (for example see the teachings of Finnegan and McElroy [1994] Biotechnology 12 883-888; and Matzke and Matzke [1995] TIG 11 1-3) there is still a need for a method that can more reliably and/or more efficiently and/or more specifically affect enzymatic activity.
According to a first aspect of the present invention there is provided a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence partially or completely codes (is) an intron of the potato class A SBE gene in a sense orientation, optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; and wherein the nucleotide sequence does not contain a sequence that is a sense exon sequence normally associated with the intron.
According to a second aspect of the present invention there is provided a method of affecting enzymatic activity in a starch producing organism (or a cell, a tissue or an organ thereof) comprising expressing in the starch producing organism (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron of the potato class A SBE gene in a sense orientation optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
Preferably, the class A SBE gene sense intron construct is used in combination with a potato class B SBE gene sense intron construct as defined in PCT/EP96/03053. However, it may also be used independently thereof, to target class A SBE alone, or in combination with other transgenes such as other sense and/or antisense transgenes, for example antisense intron transgenes such as from SBE genes, to further manipulate starch quality in potato plants.
According to a third aspect of the present invention there is provided a sequence comprising the nucleotide sequence shown as SEQ. ID. No. 38 or a variant, derivative or homologue thereof.
According to a fourth aspect of the present invention there is provided a promoter comprising the sequence shown as SEQ.I.D. No. 14 or a variant, derivative or homologue thereof.
According to a fifth aspect of the present invention there is provided a construct capable of comprising or expressing the present invention.
According to a sixth aspect of the present invention there is provided a vector comprising or expressing the present invention.
According to a seventh aspect of the present invention there is provided a cell, tissue or organ comprising or expressing the present invention.
According to an eighth aspect of the present invention there is provided a transgenic starch producing organism comprising or expressing the present invention. According to a ninth aspect of the present invention there is provided a starch obtained from the present invention. A key advantage of the present invention is that it provides a method for preparing modified starches that is not dependent on the need for post-harvest modification of starches. Thus the method of the present invention obviates the need for the use of hazardous chemicals that are normally used in the post-harvest modification of starches.
In addition, the present invention provides ter alia genetically modified plants which are capable of producing modified and/or novel and/or improved starches whose properties would satisfy various industrial requirements.
Thus, the present invention provides a method of preparing tailor-made starches in plants which could replace the post-harvest modified starches.
Also, the present invention provides a method that enables modified starches to be prepared by a method that can have a more beneficial effect on the environment than the known post-harvest modification methods which are dependent on the use of hazardous chemicals and large quantities of energy.
An other key advantage of the present invention is that it provides a method that may more reliably and/or more efficiently and/or more specifically affect enzymatic activity when compared to the known methods of affecting enzymatic activity. With regard to this advantage of the present invention it is to be noted that there is some degree of homology between coding regions of SBEs. However, there is little or no homology with the intron sequences of SBEs. Thus, sense intron expression provides a mechanism to affect selectively the expression of a particular SBE. This advantageous aspect could be used, for example, to reduce or eliminate a particular SBE enzyme and replace that enzyme with another enzyme which can be another branching enzyme or even a recombinant version of the affected enzyme or even a hybrid enzyme which could for example comprise part of a SBE enzyme from one source and at least a part of another SBE enzyme from another source. This particular feature of the present invention is covered by the combination aspect of the present invention which is discussed in more detail later.
Thus the present invention provides a mechanism for selectively affecting SBE activity. This is in contrast to the prior art methods which are dependent on the use of for example antisense exon expression whereby it would not be possible to introduce new SBE activity without affecting that activity as well.
In the context of the present invention, class B SBE is synonymous with SBE I: class A SBE is synonymous with SBE II. Class A SBE is as defined in WO96/34968, incorporated herein by reference. Preferably, the antisense intron construct used comprises intron 1 of class A SBE, which is 2.0 kb in length and is located starting at residue 45 of the coding sequence of class A SBE. The boundaries of the intron may be calculated by searching for consensus intron boundary sequences, and are shown in attached figure 11. The sequence of the intron is set forth in SEQ. ID. No. 38. Class B SBE is substantially as defined in the sequences given herein and in PCT/EP96/03053.
Preferably with the first aspect of the present invention starch branching enzyme activity is affected and/or wherein the levels of amylopectin are affected and/or the composition of starch is changed.
Preferably with the first or second aspect of the present invention the nucleotide sequence does not contain a sequence that is sense to an exon sequence.
Preferably with the fourth aspect of the present invention the promoter is in combination with a gene of interest ("GOI").
Preferably the enzymatic activity is reduced or eliminated.
Preferably the nucleotide sequence codes for at least substantially all of at least one intron in a sense orientation. Preferably the nucleotide sequence codes, partially or completely, for two or more introns and wherein each intron is in a sense orientation.
Preferably the nucleotide sequence comprises at least 350 nucleotides (e.g. 350 bp), more preferably at least 500 nucleotides (e.g. 500 bp).
Preferably the nucleotide sequence comprises the sequence shown as SEQ. ID. No. 38, or a fragment thereof.
Preferably the nucleotide sequence is expressed by a promoter having a sequence shown as SEQ. I.D. No. 14 or a variant, derivative or homologue thereof.
Preferably the transgenic starch producing organism is a plant.
A preferred aspect of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for a class A SBE intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
A more preferred aspect of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed; and wherein the nucleotide sequence comprises the sequence shown as SEQ. ID. No. 38, or fragments thereof.
The term "nucleotide" in relation to the present invention includes DNA and RNA. Preferably it means DNA, more preferably DNA prepared by use of recombinant DNA techniques.
The term "intron" is used in its normal sense as meaning a segment of nucleotides, usually DNA, that does not encode part or all of an expressed protein or enzyme.
The term "exon" is used in its normal sense as meaning a segment of nucleotides, usually DNA, encoding part or all of an expressed protein or enzyme.
Thus, the term "intron" refers to gene regions that are transcribed into RNA molecules, but which are spliced out of the RNA before the RNA is translated into a protein. In contrast, the term "exon" refers to gene regions that are transcribed into RNA and subsequently translated into proteins.
The terms "variant" or "homologue" or "fragment" in relation to the nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the respective nucleotide sequence providing the resultant nucleotide sequence can affect enzyme activity in a plant, or cell or tissue thereof, preferably wherein the resultant nucleotide sequence has at least the same effect as the sequence shown in SEQ. ID. No. 38. In particular, the term "homologue" covers homology with respect to similarity of structure and/or similarity of function providing the resultant nucleotide sequence has the ability to affect enzymatic activity in accordance with the present invention. With respect to sequence homology (i.e. similarity), preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences. Likewise, the terms "variant" or "homologue" or "fragment" in relation to the promoter of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the respective promoter sequence providing the resultant promoter sequence allows expression of a GOI, preferably wherein the resultant promoter sequence has at least the same effect as SEQ. I.D. No. 14. In particular, the term "homologue" covers homology with respect to similarity of structure and/or similarity of function providing the resultant promoter sequence has the ability to allow for expression of a GOI, such as a nucleotide sequence according to the present invention. With respect to sequence homology (i.e. similarity), preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95 % homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences.
The intron sequence of the present invention can be any one or all of the intron sequences of the present invention, including partial sequences thereof, provided that if partial sense sequences are used the partial sequences affect enzymatic activity. Suitable examples of partial sequences include sequences that are shorter than any one of the full sense sequences shown as SEQ. ID. No. 38 but which comprise nucleotides that are adjacent the respective exon or exons.
With regard to the second aspect of the present invention (i.e. specifically affecting SBE activity), the nucleotide sequences of the present invention may comprise one or more sense or antisense exon sequences of the class A or class B SBE gene (but not sense exon sequences naturally associated with the intron sequence), including complete or partial sequences thereof, providing the nucleotide sequences can affect SBE activity, preferably wherein the nucleotide sequences reduce or eliminate SBE activity. Preferably, the nucleotide sequence of the second aspect of the present invention does not comprise sense exon sequences. The term "vector" includes an expression vector and a transformation vector. The term "expression vector" means a construct capable of in vivo or in vitro expression. The term "transformation vector" means a construct capable of being transferred from one species to another - such as from an E.Coli plasmid to a fungus or a plant cell, or from an Agrobacterium to a plant cell.
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - in relation to the sense nucleotide sequence aspect of the present invention includes the nucleotide sequence according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shi -intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. The terms do not cover the natural combination of the wild type SBE gene when associated with the wild type SBE gene promoter in their natural environment.
The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a plant cell into which it has been transferred. Various markers exist which may be used in, for example, plants - such as mannose. Other examples of markers include those that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
The construct of the present invention preferably comprises a promoter. The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression. Examples of suitable promoters are those that can direct efficient expression of the nucleotide sequence of the present invention and/or in a specific type of cell. Some examples of tissue specific promoters are disclosed in WO 92/11375. The promoter could additionally include conserved regions such as a Pribnow Box or a TATA box. The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. Suitable examples of such sequences include the S/zi-intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' leader sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
As mentioned, the construct and/or the vector of the present invention may include a transcriptional initiation region which may provide for regulated or constitutive expression. Any suitable promoter may be used for the transcriptional initiation region, such as a tissue specific promoter. In one aspect, preferably the promoter is the patatin promoter or the E35S promoter. In another aspect, preferably the promoter is the SBE promoter.
If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, tuber, stem, sprout, root and leaf tissues, preferably tuber. By way of example, the promoter for the nucleotide sequence of the present invention can be the α-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the α-Amy 637 promoter) as described in our co-pending UK patent application No. 9421292.5 filed 21 October 1994. Alternatively, the promoter for the nucleotide sequence of the present invention can be the α-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the α-Amy 351 promoter) as described in our co-pending UK patent application No. 9421286.7 filed 21 October 1994.
The present invention also encompasses the use of a promoter to express a nucleotide sequence according to the present invention, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses the nucleotide sequence of the present invention in a more specific manner such as in just one specific tissue type or organ. The term "inactivated" means partial inactivation in the sense that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter. However, as mentioned above, the modified promoter is capable of expressing a gene coding for the enzyme of the present invention in at least one (but not all) specific tissue of the original promoter. Examples of partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase. Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part. Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter.
The construct and/or the vector of the present invention may include a transcriptional termination region.
The nucleotide according to the present invention can be expressed in combination (but not necessarily at the same time) with an additional construct. Thus the present invention also provides a combination of constructs comprising a first construct comprising the nucleotide sequence according to the present invention operatively linked to a first promoter; and a second construct comprising a GOI operatively linked to a second promoter (which need not be the same as the first promoter). With this aspect of the present invention the combination of constructs may be present in the same vector, plasmid, cells, tissue, organ or organism. This aspect of the present invention also covers methods of expressing the same, preferably in specific cells or tissues, such as expression in just a specific cell or tissue, of an organism, typically a plant. With this aspect of the present invention the second construct does not cover the natural combination of the gene coding for an enzyme ordinarily associated with the wild type gene promoter when they are both in their natural environment.
An example of a suitable combination would be a first construct comprising the nucleotide sequence of the present invention and a promoter, such as the promoter of the present invention, and a second construct comprising a promoter, such as the promoter of the present invention, and a GOI wherein the GOI codes for another starch branching enzyme either in sense or antisense orientation.
The above comments relating to the term "construct" for the sense nucleotide aspect of the present invention are equally applicable to the term "construct" for the promoter aspect of the present invention. In this regard, the term includes the promoter according to the present invention directly or indirectly attached to a GOI.
The term "GOI" with reference to the promoter aspect of the present invention or the combination aspect of the present invention means any gene of interest, which need not necessarily code for a protein or an enzyme - as is explained later. A GOI can be any nucleotide sequence that is either foreign or natural to the organism in question, for example a plant.
Typical examples of a GOI include genes encoding for other proteins or enzymes that modify metabolic and catabolic processes. The GOI may code for an agent for introducing or increasing pathogen resistance.
The GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. An example of such a GOI is the nucleotide sequence according to the present invention. The GOI may even code for a protein that is non-natural to the host organism - e.g. a plant. The GOI may code for a compound that is of benefit to animals or humans. For example, the GOI could code for a pharmaceutically active protein or enzyme such as any one of the therapeutic compounds insulin, interferon, human serum albumin, human growth factor and blood clotting factors. The GOI may even code for a protein giving additional nutritional value to a food or feed or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non- transgenic plant). The GOI may even code for an enzyme that can be used in food processing such as xylanases and α-galactosidase. The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for α-amylase, a protease or a glucanase. Alternatively, the GOI can be a nucleotide sequence according to the present invention.
The GOI can be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of our co-pending UK patent application 9505479.7. The GOI can be the nucleotide sequence coding for the glucanase enzyme which is the subject of our co- pending UK patent application 9505475.5. The GOI can be the nucleotide sequence coding for the α-amylase enzyme which is the subject of our co-pending UK patent application 9413439.2. The GOI can be the nucleotide sequence coding for the α- amylase enzyme which is the subject of our co-pending UK patent application 9421290.9. The GOI can be any of the nucleotide sequences coding for the α-glucan lyase enzyme which are described in our co-pending PCT patent application PCT/EP94/03397.
In one aspect the GOI can even be a nucleotide sequence according to the present invention but when operatively linked to a different promoter.
The GOI could include a sequence that codes for one or more of a xylanase, an arabinase, an acetyl esterase, a rhamnogalacturonase, a glucanase, a pectinase, a branching enzyme or another carbohydrate modifying enzyme or proteinase. Alternatively, the GOI may be a sequence that is antisense to any of those sequences. As mentioned above, the present invention provides a mechanism for selectively affecting a particular enzymatic activity.
In an important application of the present invention it is now possible to reduce or eliminate expression of a genomic nucleotide sequence coding for a genomic protein or enzyme by expressing a sense intron construct for that particular genomic protein or enzyme and (e.g. at the same time) expressing a recombinant version of that enzyme or protein - in other words the GOI is a recombinant nucleotide sequence coding for the genomic enzyme or protein. This application allows expression of desired recombinant enzymes and proteins in the absence of (or reduced levels of) respective genomic enzymes and proteins. Thus the desired recombinant enzymes and proteins can be easily separated and purified from the host organism. This particular aspect of the present invention is very advantageous over the prior art methods which, for example, rely on the use of anti- sense exon expression which methods also affect expression of the recombinant enzyme.
Thus, a further aspect of the present invention relates to a method of expressing a recombinant protein or enzyme in a host organism comprising expressing a nucleotide sequence coding for the recombinant protein or enzyme; and expressing a further nucleotide sequence wherein the further nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the intron is an intron normally associated with the genomic gene encoding a protein or an enzyme corresponding to the recombinant protein or enzyme; and wherein the further nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron. Additional aspects cover the combination of those nucleotide sequences including their incorporation in constructs, vectors, cells, tissues and transgenic organisms.
Therefore the present invention also relates to a combination of nucleotide sequences comprising a first nucleotide sequence coding for a recombinant enzyme; and a second nucleotide sequence which corresponds to an intron in a sense orientation; wherein the intron is an intron that is associated with a genomic gene encoding the enzyme corresponding to the recombinant enzyme; and wherein the second nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron.
The GOI may even code for one or more introns but in an antisense orientation, such as any one or more of the antisense intron sequences presented in the attached sequence listings. For example, the present invention also covers the expression of for example a sense intron (e.g. SEQ. I.D. No. 38) in combination with for example an antisense intron which preferably is not complementary to the sense intron sequence (e.g. SEQ. I.D. No. 16).
The terms "cell", "tissue" and "organ" include cell, tissue and organ per se and when within an organism.
The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence according to the present invention and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism. Preferably the organism is a starch producing organism such as any one of a plant, algae, fungi, yeast and bacteria, as well as cell lines thereof. Preferably the organism is a plant.
The term "starch producing organism" includes any organism that can biosynthesise starch. Preferably, the starch producing organism is a plant.
The term "plant" as used herein includes any suitable angiosperm, gymnosperm, monocotyledon and dicotyledon. Typical examples of suitable plants include vegetables such as potatoes; cereals such as wheat, maize, and barley; fruit; trees; flowers; and other plant crops. Preferably, the term means "potato".
The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the nucleotide sequence of the present invention is incorporated in the genome of the organism. Preferably the transgenic organism is a plant, more preferably a potato.
To prepare the host organism one can use prokaryotic or eukaryotic organisms. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art. for example see Sambrook et al (Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press).
Even though the enzyme according to the present invention and the nucleotide sequence coding for same are not disclosed in EP-B-0470145 and CA-A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention. Some of these background teachings are now included in the following commentary.
The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/ April 1994 17-27).
Thus, in one aspect, the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant. The vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208. Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.
The nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appears to be essential for insertion of modified T-DNA into the plant genome.
As will be understood from the above explanation, if the orgamsm is a plant the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct.
Furthermore, the vector system is preferably an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof. As these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof. In the construction of a transgenic plant the nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant. An example of a useful microorganism is E. coli, but other microorganisms having the above properties may be used. When a vector of a vector system as defined above has been constructed in E. coli, it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the nucleotide sequence or construct of the present invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the present invention, which DNA is subsequently transferred into the plant cell to be modified.
If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in ΕP-A-120516; Hoekema, in: The Binary Plant Vector System Offset- drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraley, et al., Cut. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985) 4:277-284.
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17- 27). With this technique, infection of a plant may be performed in or on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
Typically, with direct infection of plant tissues by Agrobacterium carrying the GOI (such as the nucleotide sequence according to the present invention) and, optionally, a promoter, a plant to be infected is wounded, e.g. by cutting the plant with a razor blade or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
Further teachings on plant transformation may be found in EP-A-0449375.
As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 322, pUC series, M13 mp series, pACYC 184 etc. In this way, the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is then used for the transformation in E.coli. The E.coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
After the introduction of the nucleotide sequence or construct according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary - such as to create combination systems as outlined above (e.g. an organism comprising a combination of constructs).
The above commentary for the transformation of prokaryotic organisms and plants with the nucleotide sequence of the present invention is equally applicable for the transformation of those organisms with the promoter of the present invention.
In summation, the present invention relates to affecting enzyme activity by expressing sense intron sequences.
Also, the present invention relates to a promoter useful for the expression of those sense intron sequences.
The following samples have been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on 13 July 1995:
NCIMB 40754 (which refers to pBEA 11 as described herein);
NCIMB 40751 (which refers to λ-SBE 3.2 as described herein), and
NCIMB 40752 (which refers to λ-SBE 3.4 as described herein).
A highly preferred embodiment of the present invention therefore relates to a method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron in a sense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed; and wherein the intron nucleotide sequence is the sequence of intron 1 of class A SBE as set forth in SEQ. ID. No. 38, or any other intron of class A SBE, including fragments thereof, and including combinations of class A sense intron sequences and class B sense or antisense intron sequences. The sequence of introns of class A SBE other than intron 1 may be obtained by sequencing of, for example, potato class A SBE genomic DNA, isolatable by hybridisation screening of a genomic DNA library with class A SBE cDNA obtainable according to W096/34968 according to methods well known in the art and set forth, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989.
The present invention will now be described only by way of example, in which reference is made to the following attached Figures:
Figure 1, which is a schematic representation of the biosynthesis of amylose and amylopectin;
Figure 2, which is a diagrammatic representation of the α-l-4-links and the α-1-6 links of amylopectin;
Figure 3, which is a diagrammatic representation of the exon-intron structure of a genomic SBE clone;
Figure 4, which is a plasmid map of pPATAl, which is 3936 bp in size;
Figure 5, which is a plasmid map of pABE7, which is 5106 bp in size;
Figure 6, which is a plasmid map of pVictorlV Man, which is 7080 bp in size;
Figure 7, which is a plasmid map of pBEAll, which is 9.54 kb in size; Figure 8, which shows the full genomic nucleotide sequence for SBE including the promoter, exons and introns;
Figure 9, which is a plasmid map of pVictor5a, which is 9.12 kb in size;
Figure 10, which is a plasmid map of pBEP2, which is 10.32 kb in size;
Figure 11 , which shows the positioning of intron 1 in the class A and class B SBE genes;
Figure 12, which shows the sequence of intron 1 of the potato class A SBE;
Figure 13, which shows pSS15; and
Figure 14, which shows pSS16.
Figures 1 and 2 were referred to above in the introductory description concerning starch in general. As mentioned, Figure 3 is a diagrammatic representation of the exon-intron structure of a genomic SBE clone, the sequence of which is shown in Figure 8. This clone, which has about 11.5 k base pairs, comprises 14 exons and 13 introns. The introns are numbered in increasing order from the 5 ' end to the 3 ' end and correspond to SEQ. I.D. No. s 1-13, respectively. Their respective antisense intron sequences are shown as SEQ.I.D.No.s 15-27.
In more detail, Figures 3 and 8 present information on the 11468 base pairs of a potato SBE gene. The 5' region from nucleotides 1 to 2082 contain the promoter region of the SBE gene. A TATA box candidate at nucleotide 2048 to 2051 is boxed. The homology between a potato SBE cDNA clone (Poulsen & Kreiberg (1993) Plant Physiol 102: 1053- 1054) and the exon DNAs begin at 2083 bp and end at 9666 bp. The homology between the cDNA and the exon DNA is indicated by nucleotides in upper case letters, while the translated amino acid sequences are shown in the single letter code below the exon DNA. Intron sequences are indicated by lower case letters. Figure 7 is a plasmid map of pBEA7, which is 9.54 k base pairs in size. Plasmid pBEA 11 comprises the first intron sequence of the potato SBE gene. This first intron sequence, which has 1177 base pairs, is shown in Figure 3 and lies between the first exon and the second exon.
These experiments and aspects of the present invention are now discussed in more detail.
EXPERIMENTAL PROTOCOL
ISOLATION, SUBCLONING IN PLASMIDS, AND SEQUENCING OF GENOMIC SBE CLONES
Various clones containing the potato SBE gene are isolated from a Desiree potato genomic library (Clontech Laboratories Inc., Palo Alto CA, USA) using radioactively labelled potato SBE cDNA (Poulsen & Kreiberg (1993) Plant Physiol. 102:1053-1054) as probe. The fragments of the isolated λ-phages containing SBE DNA (λSBE 3.2 - NCIMB 40751 - and λSBE-3.4 - NCIMB 40752) are identified by Southern analysis and then subcloned into pBluescript II vectors (Clontech Laboratories Inc., Palo Alto CA, USA). λSBE 3.2 contains a 15 kb potato DNA insert and λSBE-3.4 contains a 13 kb potato DNA insert. The resultant plasmids are called pGB3, pGBll, pGB15, pGB16 and pGB25 (see discussion below). The respective inserts are then sequenced using the Pharmacia Autoread Sequencing Kit (Pharmacia, Uppsala) and a A.L.F. DNA sequencer (Pharmacia, Uppsala). In total, a stretch of 11.5 kb of the SBE gene is sequenced. The sequence is deduced from the above-mentioned plasmids, wherein: pGB25 contains the sequences from 1 bp to 836 bp, pGB15 contains the sequences from 735 bp to 2580 bp, pGB16 contains the sequences from 2580 bp to 5093 bp, pGBll contains the sequences from 3348 bp to 7975 bp, and pGB3 contains the sequences from 7533 bp to 11468 bp. In more detail, pGB3 is constructed by insertion of a 4 kb EcoRl fragment isolated from λSBE 3.2 into the EcoRl site of pBluescript II SK (+). pGBll is constructed by insertion of a 4.7 kb Xhol fragment isolated from λSBE 3.4 into the Xhol site of pBluescript II SK (+). pGB15 is constructed by insertion of a 1.7 kb Spel fragment isolated from λSBE 3.4 into the Spel site of pBluescript II SK (+). pGB16 is constructed by insertion of a 2.5 kb Spel fragment isolated from λSBE 3.4 into the Spel site of pBluescript II SK (+). For the construction of pGB25 a PCR fragment is produced with the primers
5' GGA ATT CCA GTC GCA GTC TAC ATT AC 3'
(SEQ. ID. No.30) and 5' CGG GAT CCA GAG GCA TTA AGA TTT CTG G 3'
(SEQ. ID. No. 31) and λSBE 3.4 as a template.
The PCR fragment is digested with BamHl and EcoRl, and inserted in pBluescript II SK (+) digested with the same restriction enzymes.
CONSTRUCTION OF PLASMID pBEAll
The SBE intron 1 is amplified by PCR using the oligonucleotides
5' CGG GAT CCA AAG AAA TTC TCG AGG TTA CAT GG 3'
(SEQ. ID. No.32) and
5' CGG GAT CCG GGG TAA TTT TTA CTA ATT TCA TG 3'
(SEQ. ID. No. 33) and the λSBE 3.4 phage containing the SBE gene as template.
The PCR product is digested with BamHl and inserted in a sense orientation in the BamHl site of plasmid pPATAl (described in WO 94/24292) between the patatin promoter and the 35S terminator. This construction, pABE7, is digested with Kpnl, and the 2.4 kb "patatin promoter-SBE intron 1- 35S terminator" Kpnl fragment is isolated and inserted in the Kpnl site of the plant transformation vector pVictorlV Man yielding plasmid pBEAll .
CONSTRUCTION OF PLASMID pSS15. The 2122 bp intron 1 sequence of the potato SBEII gene (see SEQ. ID. No. 38) is amplified by PCR from a genomic SBEII subclone using the primers 5' - CGG GAT CCC GTA TGT CTC ACT GTG TTT GTG GC - 3' (SEQ. ID. No. 34) and 5' - CGG GAT CCC CCT ACA TAC ATA TAT CAG ATT AG - 3' (SEQ. ID. No. 35). The PCR product is digested with BamHl and inserted in sense orientation after a patatin promoter in the BamHl site of a plant transformation vector in which the NPTII gene is used as selectable marker (see figure 13).
CONSTRUCTION OF PLASMID pSS16. The 2122 bp intron 1 sequence of the potato SBEII gene (SEQ. ID. No. 38) is amplified by PCR from a genomic SBEII subclone using the primers 5' - CGG GAT CCC GTA TGT CTC ACT GTG TTT GTG GC - 3' (SEQ. ID. No. 34) and 5' - CGG GAT CCC CCT ACA TAC ATA TAT CAG ATT AG - 3' (SEQ. ID. No. 35). The PCR product is digested with BamHl and inserted in sense orientation after a patatin promoter in the BamHl site of a plant transformation vector in which the manA gene is used as selectable marker (see figure 14).
PRODUCTION OF TRANSGENIC POTATO PLANTS
Axenic stock cultures Shoot cultures of Solanum tuberosum 'Bintje' and 'Dianella' are maintained on a substrate (LS) of a formula according to Linsmaier, E.U. and Skoog, F. (1965), Physiol.
Plant. 18: 100-127, in addition containing 2 μM silver thiosulphate at 25°C and 16 h light/8 h dark.
The cultures are subcultured after approximately 40 days. Leaves are then cut off the shoots and cut into nodal segments (approximately 0.8 cm) each containing one node.
Inoculation of potato tissues
Shoots from approximately 40 days old shoot cultures (height approximately 5-6 cms) are cut into internodal segments (approximately 0.8 cm). The segments are placed into liquid LS-substrate containing the transformed Agrobacterium tumefaciens containing the binary vector of interest. The Agrobacterium are grown overnight in YMB-substrate (di-potassium hydrogen phosphate, trihydrate (0.66 g/1); magnesium sulphate, heptahydrate (0.20 g/1); sodium chloride (0.10 g/1); mannitol (10.0 g/1); and yeast extract (0.40 g/1)) containing appropriate antibiotics (corresponding to the resistance gene of the Agrobacterium strain) to an optical density at 660 nm (OD-660) of approximately 0.8, centrifuged and resuspended in the LS-substrate to an OD-660 of 0.5.
The segments are left in the suspension of Agrobacterium for 30 minutes and then the excess of bacteria are removed by blotting the segments on sterile filter paper.
Co-cultivation The shoot segments are co-cultured with bacteria for 48 hours directly on LS- substrate containing agar (8.0 g/1), 2,4-dichlorophenoxyacetic acid (2.0 mg/1) and trans- zeatin (0.5 mg/1). The substrate and also the explants are covered with sterile filter papers, and the petri dishes are placed at 25°C and 16 h light/ 8 dark.
"Washing" procedure
After the 48 h on the co-cultivation substrate the segments are transferred to containers containing liquid LS-substrate containing 800 mg/1 carbenicillin. The containers are gently shaken and by this procedure the major part of the Agrobacterium is either washed off the segments and/or killed.
Selection
After the washing procedure the segments are transferred to plates containing the LS-substrate, agar (8 g/1), trans-zeatin (1-5 mg/1), gibberellic acid (0.1 mg/1), carbenicillin (800 mg/1), and kanamycin sulphate (50-100 mg/1) or phosphinotricin (1-5 mg/1) or mannose (5 g/1) depending on the vector construction used. The segments are sub-cultured to fresh substrate each 3-4 weeks. In 3 to 4 weeks, shoots develop from the segments and the formation of new shoots continued for 3-4 months.
Rooting of regenerated shoots The regenerated shoots are transferred to rooting substrate composed of LS- substrate, agar (8 g/1) and carbenicillin (800 mg/1). The transgenic genotype of the regenerated shoot are verified by testing the rooting ability on the above mentioned substrates containing kanamycin sulphate (200 mg/1), by performing NPTII assays (Radke, S. E. et al, Theor. Appl. Genet. (1988), 75: 685-694) or by performing PCR analysis according to Wang et al (1993, NAR 21 pp 4153-4154). Plants which are not positive in any of these assays are discarded or used as controls. Alternatively, the transgenic plants could be verified by performing a GUS assay on the co-introduced β- glucuronidase gene according to Hodal, L. et al. (PI. Sci. (1992), 87: 115-122).
Transfer to soil The newly rooted plants (height approx. 2-3 cms) are transplanted from rooting substrate to soil and placed in a growth chamber (21°C, 16 hour light 200-400uE/m7sec). When the plants are well established they are transferred to the greenhouse, where they are grown until tubers had developed and the upper part of the plants are senescing.
Harvesting
The potatoes are harvested after about 3 months and then analysed.
BRANCHING ENZYME ANALYSIS
The SBE expression in the transgenic potato lines are measured using the SBE assays described by Blennow and Johansson (Phytochemistry (1991) 30:437-444) and by standard Western procedures using antibodies directed against class A and class B potato SBE.
STARCH ANALYSIS Starch is isolated from potato tubers and analysed for the amylose:amylopectin ratio (Hovenkamp-Hermelink et al. (1988) Potato Research 31:241-246). In addition, the chain length distribution of amylopectin is determined by analysis of isoamylase digested starch on a Dionex HPAEC. The number of reducing ends in isoamylase digested starch is determined by the method described by N. Nelson (1944) J. Biol. Chem. 153:375-380. The results revealed that there is a reduction in the level of synthesis of SBE and/or the level of activity of SBE and/or the composition of starch SBE in the transgenic plants.
CONSTRUCTION OF SBE PROMOTER CONSTRUCT
An SBE promoter fragment is amplified from λ-SBE 3.4 using primers: 5 ' CCA TCG ATA CTT TAA GTG ATT TGA TGG C 3'
(SEQ. ID. No.36) and 5' CGG GAT CCT GTT CTG ATT CTT GAT TTC C 3'.
(SEQ. ID. No. 37) The PCR product is digested with Clal and BamHl. The resultant 1.2 kb fragment is then inserted in pVictor5a (see Figure 9) linearised with Clal and BgUl yielding pBEP2 (see Figure 10).
STARCH BRANCHING ENZYME MEASUREMENTS OF POTATO TUBERS
Potatoes from potato plants transformed with pBEAl 1 are cut in small pieces and homogenised in extraction buffer (50 mM Tris-HCl pH 7.5, Sodium-dithionite (0.1 g/1), and 2 mM DTT) using a Ultra-Turax homogenizer; 1 g of Dowex xl. is added pr. 10 g of tuber. The crude homogenate is filtered through a miracloth filter and centrifuged at 4°C for 10 minutes at 24.700 g. The supernatant is used for starch branching enzyme assays. The starch branching enzyme assays are carried out at 25 °C in a volume of 400 μl composed of 0.1 M Na citrate buffer pH 7.0, 0.75 mg/ml amylose, 5 mg/ml bovine serum albumin and the potato extract. At 0, 15 30 and 60 minutes aliqouts of 50 μl are removed from the reaction into 20 μl 3 N HCl. 1 ml of iodine solution is added and the decrease in absorbance at 620 nm is measured with an ELISA spectrophotometer.
The starch branching enzyme (SBE) levels in tuber extracts are measured from 24 transgenic Dianella potato plants transformed with plasmid pBEAll, pSS15 and pSS16.
The results show that the BEAU, SS15 and SS16 transgenic lines produce tubers which have class B and class A SBE levels, respectively, that are only 10 % to 15 % of the SBE levels found in non transformed Dianella plants. In a further experiment, plasmids pSS15 and pBEAll are cotransfected into potato plants, as described above. In the cotransfectants, when analysed as set forth above, simultaneous reduction of class A and class B SBE levels are observed.
SUMMATION
The above-mentioned examples relate to the isolation and sequencing of a gene for potato SBE. The examples further demonstrate that it is possible to prepare SBE intron constructs. These SBE intron constructs can be introduced into plants, such as potato plants. After introduction, a reduction in the level of synthesis of SBE and/or the level of activity of SBE and/or the composition of starch in plants can be achieved.
Without wishing to be bound by theory it is believed that the expressed sense intron nucleotide sequence according to the present invention affects enzymatic activity via co-suppression and/or trans-activation. Reviews of these mechanisms has been published by Finnegan and McElroy (1994 Biotechnology 12 pp 883 - 887) and Matzke and Matzke (1995 TIG 11 No. 1 pp 1 - 3). By these mechanisms, it is believed that the sense introns of the present invention reduce the level of plant enzyme activity (in particular SBE activity), which in turn for SBE activity is believed to influence the amylose: amylopectin ratio and thus the branching pattern of amylopectin.
Thus, the present invention provides a method wherein it is possible to manipulate the starch composition in plants, or tissues or cells thereof, such as potato tubers, by reducing the level of SBE activity by using sense intron sequences.
The simultaneous reduction or elimination of class A and class B SBE sequences from the doubly transformed potato plants, moreover, offers the possibility to transform such plants with different SBE genes at will, thus allowing the manipulation of branching in starch according to the desired result.
In summation the present invention therefore relates to the surprising use of SBE class A sense intron sequences in a method to affect class A SBE activity in plants.
Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. The following pages present a number of sequence listings which have been consecutively numbered from SEQ.I.D. No. 1 - SEQ.I.D. No. 38. In brief, SEQ.I.D.
No. 1 - SEQ.I.D. No. 13 represent sense intron sequences (genomic DNA); SEQ.I.D. No. 14 represents the SBE promoter sequence (genomic sequence); SEQ.I.D. No. 15 - SEQ.I.D. No. 27 represent antisense intron sequences; and SEQ. I.D. No. 28 represents the sequence complementary to the SBE promoter sequence - i.e. the SBE promoter sequence in antisense orientation. The full genomic nucleotide sequence for SBE including the promoter, exons and introns is shown as SEQ. I.D. No. 29 (see Figures 3 and 8 which highlight particular gene features). SEQ. ID. No. 30 to 37 show primers used in the methods set forth above. SEQ. ID. No. 38 represents the nucleotide sequence of intron 1 of the class A potato SBE gene.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: DANISCO A/S
(B) STREET: LANGEBROGADE 1
(C) CITY: COPENHAGEN K (E) COUNTRY: DENMARK
(F) POSTAL CODE (ZIP) : DK-1001
(ii) TITLE OF INVENTION: INHIBITION OF GENE EXPRESSION
(iii) NUMBER OF SEQUENCES: 38
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO : 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1165 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GTAATTTTTA CTAATTTCAT GTTAATTTCA ATTATTTTTA GCCTTTGCAT TTCATTTTCC 60
AATATATCTG GATCATCTCC TTAGTTTTTT ATTTTATTTT TTATAATATC AAATATGGAA 120
GAAAAATGAC ACTTGTAGAG CCATATGTAA GTATCATGTG ACAAATTTGC AAGGTGGTTG 180
AGTGTATAAA ATTCAAAAAT TGAGAGATGG AGGGGGGGTG GGGGAAGACA ATATTTAGAA 240
AGAGTGTTCT AGGAGGTTAT GGAGGACACG GATGAGGGGT AGAAGGTTAG TTAGGTATTT 300
GAGTGTTGTC TGGCTTATCC TTTCATACTA GTAGTCGTGG AATTATTTGG GTAGTTTCTT 360
GTTTTGTTAT TTGATCTTTG TTATTCTATT TTCTGTTTCT TGTACTTCGA TTATTGTATT 420
ATATATCTTG TCGTAGTTAT TGTTCCTCGG TAAGAATGCT CTAGCATGCT TCCTTTAGTG 480
TTTTATCATG CCTTCTTTAT ATTCGCGTTG CTTTGAAATG CTTTTACTTT AGCCGAGGGT 540
CTATTAGAAA CAATCTCTCT ATCTCGTAAG GTAGGGGTAA AGTCCTCACC ACACTCCACT 600
TGTGGGATTA CATTGTGTTT GTTGTTGTAA ATCAATTATG TATACATAAT AAGTGGATTT 660
TTTACAACAC AAATACATGG TCAAGGGCAA AGTTCTGAAC ACATAAAGGG TTCATTATAT 720
GTCCAGGGAT ATGATAAAAA TTGTTTCTTT GTGAAAGTTA TATAAGATTT GTTATGGCTT 780
TTGCTGGAAA CATAATAAGT TATAATGCTG AGATAGCTAC TGAAGTTTGT TTTTTCTAGC 840
CTTTTAAATG TACCAATAAT AGATTCCGTA TCGAACGAGT ATGTTTTGAT TACCTGGTCA 900
TGATGTTTCT ATTTTTTACA TTTTTTTGGT GTTGAACTGC AATTGAAAAT GTTGTATCCT 960
ATGAGACGGA TAGTTGAGAA TGTGTTCTTT GTATGGACCT TGAGAAGCTC AAACGCTACT 1020 CCAATAATTT CTATGAATTC AAATTCAGTT TATGGCTACC AGTCAGTCCA GAAATTAGGA 1080
TATGCTGCAT ATACTTGTTC AATTATACTG TAAAATTTCT TAAGTTCTCA AGATATCCAT 1140
GTAACCTCGA GAATTTCTTT GACAG 1165
(2) INFORMATION FOR SEQ ID NO : 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GTATGTTTGA TAATTTATAT GGTTGCATGG ATAGTATATA AATAGTTGGA AAACTTCTGG 60
ACTGGTGCTC ATGGCATATT TGATCTGTGC ACCGTGTGGA GATGTCAAAC ATGTGTTACT 120
TCGTTCCGCC AATTTATAAT ACCTTAACTT GGGAAAGACA GCTCTTTACT CCTGTGGGCA 180
TTTGTTATTT GAATTACAAT CTTTATGAGC ATGGTGTTTT CACATTATCA ACTTCTTTCA 240
TGTGGTATAT AACAGTTTTT AGCTCCGTTA ATACCTTTCT TCTTTTTGAT ATAAACTAAC 300
TGTGGTGCAT TGCTTGC 317
(2) INFORMATION FOR SEQ ID NO : 3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 504 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GTAACAGCCA AAAGTTGTGC TTTAGGCAGT TTGACCTTAT TTTGGAAGAT GAATTGTTTA 60
TACCTACTTT GACTTTGCTA GAGAATTTTG CATACCGGGG AGTAAGTAGT GGCTCCATTT 120
AGGTGGCACC TGGCCATTTT TTTGATCTTT TAAAAAGCTG TTTGATTGGG TCTTCAAAAA 180
AGTAGACAAG GTTTTTGGAG AAGTGACACA CCCCCGGAGT GTCAGTGGCA AAGCAAAGAT 240
TTTCACTAAG GAGATTCAAA ATATAAAAAA AGTATAGACA TAAAGAAGCT GAGGGGATTC 300
AACATGTACT ATACAAGCAT CAAATATAGT CTTAAAGCAA TTTTGTAGAA ATAAAGAAAG 360
TCTTCCTTCT GTTGCTTCAC AATTTCCTTC TATTATCATG AGTTACTCTT TCTGTTCGAA 420
ATAGCTTCCT TAATATTAAA TTCATGATAC TTTTGTTGAG ATTTAGCAGT TTTTTCTTGT 480
GTAAACTGCT CTCTTTTTTT GCAG 504
( 2 ) INFORMATION FOR SEQ ID NO : 4 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GTAGGTCCTC GTCTACTACA AAATAGTAGT TTCCATCATC ATAACAGATT TTCCTATTAA 60
AGCATGATGT TGCAGCATCA TTGGCTTTCT TACATGTTCT AATTGCTATT AAGGTTATGC 120
TTCTAATTAA CTCATCCACA ATGCAG 146
(2) INFORMATION FOR SEQ ID NO : 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5:
GTTTTGTTAT TCATACCTTG AAGCTGAATT TTGAACACCA TCATCACAGG CATTTCGATT 60
CATGTTCTTA CTAGTCTTGT TATGTAAGAC ATTTTGAAAT GCAAAAGTTA AAATAATTGT 120
GTCTTTACTA ATTTGGACTT GATCCCATAC TCTTTCCCTT AACAAAATGA GTCAATTCTA 180
TAAGTGCTTG AGAACTTACT ACTTCAGCAA TTAAACAG 218
(2) INFORMATION FOR SEQ ID NO : 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GTATTTTAAA TTTATTTCTA CAACTAAATA ATTCTCAGAA CAATTGTTAG ATAGAATCCA 60
AATATATACG TCCTGAAAGT ATAAAAGTAC TTATTTTCGC CATGGGCCTT CAGAATATTG 120
GTAGCCGCTG AATATCATGA TAAGTTATTT ATCCAGTGAC ATTTTTATGT TCACTCCTAT 180
TATGTCTGCT GGATACAG 198 (2) INFORMATION FOR SEQ ID NO : 7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 208 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GTTTGTCTGT TTCTATTGCA TTTTAAGGTT CATATAGGTT AGCCACGGAA AATCTCACTC 60
TTTGTGAGGT AACCAGGGTT CTGATGGATT ATTCAATTTT CTCGTTTATC ATTTGTTTAT 120
TCTTTTCATG CATTGTGTTT CTTTTTCAAT ATCCCTCTTA TTTGGAGGTA ATTTTTCTCA 180
TCTATTCACT TTTAGCTTCT AACCACAG 208
(2) INFORMATION FOR SEQ ID NO : 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 293 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GTATGTCTTA CATCTTTAGA TATTTTGTGA TAATTACAAT TAGTTTGGCT TACTTGAACA 60
AGATTCATTC CTCAAAATGA CCTGAACTGT TGAACATCAA AGGGGTTGAA ACATAGAGGA 120
AAACAACATG ATGAATGTTT CCATTGTCTA GGGATTTCTA TTATGTTGCT GAGAACAAAT 180
GTCATCTTAA AAAAAACATT GTTTACTTTT TTGTAGTATA GAAGATTACT GTATAGAGTT 240
TGCAAGTGTG TCTGTTTTGG AGTAATTGTG AAATGTTTGA TGAACTTGTA CAG 293
(2) INFORMATION FOR SEQ ID NO : 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 376 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9: GTTCAAGTAT TTTGAATCGC AGCTTGTTAA ATAATCTAGT AATTTTTAGA TTGCTTACTT 60
GGAAGTCTAC TTGGTTCTGG GGATGATAGC TCATTTCATC TTGTTCTACT TATTTTCCAA 120
CCGAATTTCT GATTTTTGTT TCGAGATCCA AGTATTAGAT TCATTTACAC TTATTACCGC 180
CTCATTTCTA CCACTAAGGC CTTGATGAGC AGCTTAAGTT GATTCTTTGA AGCTATAGTT 240
TCAGGCTACC AATCCACAGC CTGCTATATT TGTTGGATAC TTACCTTTTC TTTACAATGA 300
AGTGATACTA ATTGAAATGG TCTAAATCTG ATATCTATAT TTCTCCGTCT TTCCTCCCCC 360
TCATGATGAA ATGCAG 376
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 172 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GTAAAATCAT CTAAAGTTGA AAGTGTTGGG TTTATGAAGT GCTTTAATTC TATCCAAGGA 60
CAAGTAGAAA CCTTTTTACC TTCCATTTCT TGATGATGGA TTTCATATTA TTTAATCCAA 120
TAGCTGGTCA AATTCGGTAA TAGCTGTACT GATTAGTTAC TTCACTTTGC AG 172 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 145 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GTATATATGT TTTACTTATC CATGAAATTA TTGCTCTGCT TGTTTTTAAT GTACTGAACA 60
AGTTTTATGG AGAAGTAACT GAAACAAATC ATTTTCACAT TGTCTAATTT AACTCTTTTT 120
TCTGATCCTC GCATGACGAA AACAG 145
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 242 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GTAAGGATTT GCTTGAATAA CTTTTGATAA TAAGATAACA GATGTAGGGT ACAGTTCTCT 60
CACCAAAAAG AACTGTAATT GTCTCATCCA TCTTTAGTTG TATAAGATAT CCGACTGTCT 120
GAGTTCGGAA GTGTTTGAGC CTCCTGCCCT CCCCCTGCGT TGTTTAGCTA ATTCAAAAAG 180
GAGAAAACTG TTTATTGATG ATCTTTGTCT TCATGCTGAC ATACAATCTG TTCTCATGAC 240
AG 242
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
GTACAGTTCT TGCCGTGTGA CCTCCCTTTT TATTGTGGTT TTGTTCATAG TTATTTGAAT 60 GCGATAGAAG TTAACTATTG ATTACCGCCA CAATCGCCAG TTAAGTCCTC TGAACTACTA 120
ATTTGAAAGG TAGGAATAGC CGTAATAAGG TCTACTTTTG GCATCTTACT GTTACAAAAC 180
AAAAGGATGC CAAAAAAATT CTTCTCTATC CTCTTTTTCC CTAAACCAGT GCATGTAGCT 240
TGCACCTGCA TAAACTTAGG TAAATGATCA AAAATGAAGT TGATGGGAAC TTAAAACCGC 300
CCTGAAGTAA AGCTAGGAAT AGTCATATAA TGTCCACCTT TGGTGTCTGC GCTAACATCA 360
ACAACAACAT ACCTCGTGTA GTCCCACAAA GTGGTTTCAG GGGGAGGGTA GAGTGTATGC 420
AAAACTTACT CCTATCTCAG AGGTAGAGAG GATTTTTTCA ATAGACCCTT GGCTCAAGAA 480
AAAAAGTCCA AAAAGAAGTA ACAGAAGTGA AAGCAACATG TGTAGCTAAA GCGACCCAAC 540
TTGTTTGGGA CTGAAGTAGT TGTTGTTGTT GAAACAGTGC ATGTAGATGA ACACATGTCA 600
GAAAATGGAC AACACAGTTA TTTTGTGCAA GTCAAAAAAA TGTACTACTA TTTCTTTGTG 660
CAGCTTTATG TATAGAAAAG TTAAATAACT AATGAATTTT GCTAGCAGAA AAATAGCTTG 720
GAGAGAAATT TTTTATATTG AACTAAGCTA ACTATATTCA TCTTTCTTTT TGCTTCTTCT 780
TCTCCTTGTT TGTGAAG 797
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2169 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
ATCATGGCCA ATTACTGGTT CAAATGCATT ACTTCCTTTC AGATTCTTTC GAGTTCTCAT 60
GACCGGTCCT ACTACAGACG ATACTAACCC GTGGAACTGT TGCATCTGCT TCTTAGAACT 120
CTATGGCTAT TTTCGTTAGC TTGGCGTCGG TTTGAACATA GTTTTTGTTT TCAAACTCTT 180
CATTTACAGT CAAAATGTTG TATGGTTTTT GTTTTCCTCA ATGATGTTTA CAGTGTTGTG 240
TTGTCATCTG TACTTTTGCC TATTACTTGT TTTGAGTTAC ATGTTAAAAA AGTGTTTATT 300
TTGCCATATT TTGTTCTCTT ATTATTATTA TCATACATAC ATTATTACAA GGAAAAGACA 360
AGTACACAGA TCTTAACGTT TATGTTCAAT CAACTTTTGG AGGCATTGAC AGGTACCACA 420
AATTTTGAGT TTATGATTAA GTTCAATCTT AGAATATGAA TTTAACATCT ATTATAGATG 480
CATAAAAATA GCTAATGATA GAACATTGAC ATTTGGCAGA GCTTAGGGTA TGGTATATCC 540
AACGTTAATT TAGTAATTTT TGTTACGTAC GTATATGAAA TATTGAATTA ATCACATGAA 600
CGGTGGATAT TATATTATGA GTTGGCATCA GCAAAATCAT TGGTGTAGTT GACTGTAGTT 660
GCAGATTTAA TAATAAAATG GTAATTAACG GTCGATATTA AAATAACTCT CATTTCAAGT 720
GGGATTAGAA CTAGTTATTA AAAAAATGTA TACTTTAAGT GATTTGATGG CATATAATTT 780
AAAGTTTTTC ATTTCATGCT AAAATTGTTA ATTATTGTAA TGTAGACTGC GACTGGAATT 840
ATTATAGTGT AAATTTATGC ATTCAGTGTA AAATTAAAGT ATTGAACTTG TCTGTTTTAG 900
AAAATACTTT ATACTTTAAT ATAGGATTTT GTCATGCGAA TTTAAATTAA TCGATATTGA 960 ACACGGAATA CCAAAATTAA AAAGGATACA CATGGCCTTC ATATGAACCG TGAACCTTTG 1020
ATAACGTGGA AGTTCAAAGA AGGTAAAGTT TAAGAATAAA CTGACAAATT AATTTCTTTT 1080
ATTTGGCCCA CTACTAAATT TGCTTTACTT TCTAACATGT CAAGTTGTGC CCTCTTAGTT 1140
GAATGATATT CATTTTTCAT CCCATAAGTT CAATTTGATT GTCATACCAC CCATGATGTT 1200
CTGAAAAATG CTTGGCCATT CACAAAGTTT ATCTTAGTTC CTATGAACTT TATAAGAAGC 1260
TTTAATTTGA CATGTTATTT ATATTAGATG ATATAATCCA TGACCCAATA GACAAGTGTA 1320
TTAATATTGT AACTTTGTAA TTGAGTGTGT CTACATCTTA TTCAATCATT TAAGGTCATT 1380
AAAATAAATT ATTTTTTGAC ATTCTAAAAC TTTAAGCAGA ATAAATAGTT TATCAATTAT 1440
TAAAAACAAA AAACGACTTA TTTATAAATC AACAAACAAT TTTAGATTGC TCCAACATAT 1500
TTTTCCAAAT TAAATGCAGA AAATGCATAA TTTTATACTT GATCTTTATA GCTTATTTTT 1560
TTTAGCCTAA CCAACGAATA TTTGTAAACT CACAACTTGA TTAAAAGGGA TTTACAACAA 1620
GATATATATA AGTAGTGACA AATCTTGATT TTAAATATTT TAATTTGGAG GTCAAAATTT 1680
TACCATAATC ATTTGTATTT ATAATTAAAT TTTAAATATC TTATTTATAC ATATCTAGTA 1740
AACTTTTAAA TATACGTATA TACAAAATAT AAAATTATTG GCGTTCATAT TAGGTCAATA 1800
AATCCTTAAC TATATCTGCC TTACCACTAG GAGAAAGTAA AAAACTCTTT ACCAAAAATA 1860
CATGTATTAT GTATACAAAA AGTCGATTAG ATTACCTAAA TAGAAATTGT ATAACGAGTA 1920
AGTAAGTAGA AATATAAAAA AACTACAATA CTAAAAAAAA TATGTTTTAC TTCAATTTCG 1980
AAACTAATGG GGTCTGAGTG AAATATTCAG AAAGGGGAGG ACTAACAAAA GGGTCATAAT 2040
GTTTTTTTAT AAAAAGCCAC TAAAATGAGG AAATCAAGAA TCAGAACATA CAAGAAGGCA 2100 GCAGCTGAAG CAAAGTACCA TAATTTAATC AATGGAAATT AATTTCAAAG TTTTATCAAA 2160
ACCCATTCG 2169
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1165 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTGTCAAAGA AATTCTCGAG GTTACATGGA TATCTTGAGA ACTTAAGAAA TTTTACAGTA 60
TAATTGAACA AGTATATGCA GCATATCCTA ATTTCTGGAC TGACTGGTAG CCATAAACTG 120
AATTTGAATT CATAGAAATT ATTGGAGTAG CGTTTGAGCT TCTCAAGGTC CATACAAAGA 180
ACACATTCTC AACTATCCGT CTCATAGGAT ACAACATTTT CAATTGCAGT TCAACACCAA 240
AAAAATGTAA AAAATAGAAA CATCATGACC AGGTAATCAA AACATACTCG TTCGATACGG 300
AATCTATTAT TGGTACATTT AAAAGGCTAG AAAAAACAAA CTTCAGTAGC TATCTCAGCA 360
TTATAACTTA TTATGTTTCC AGCAAAAGCC ATAACAAATC TTATATAACT TTCACAAAGA 420 AACAATTTTT ATCATATCCC TGGACATATA ATGAACCCTT TATGTGTTCA GAACTTTGCC 480
CTTGACCATG TATTTGTGTT GTAAAAAATC CACTTATTAT GTATACATAA TTGATTTACA 540
ACAACAAACA CAATGTAATC CCACAAGTGG AGTGTGGTGA GGACTTTACC CCTACCTTAC 600
GAGATAGAGA GATTGTTTCT AATAGACCCT CGGCTAAAGT AAAAGCATTT CAAAGCAACG 660
CGAATATAAA GAAGGCATGA TAAAACACTA AAGGAAGCAT GCTAGAGCAT TCTTACCGAG 720
GAACAATAAC TACGACAAGA TATATAATAC AATAATCGAA GTACAAGAAA CAGAAAATAG 780
AATAACAAAG ATCAAATAAC AAAACAAGAA ACTACCCAAA TAATTCCACG ACTACTAGTA 840
TGAAAGGATA AGCCAGACAA CACTCAAATA CCTAACTAAC CTTCTACCCC TCATCCGTGT 900
CCTCCATAAC CTCCTAGAAC ACTCTTTCTA AATATTGTCT TCCCCCACCC CCCCTCCATC 960
TCTCAATTTT TGAATTTTAT ACACTCAACC ACCTTGCAAA TTTGTCACAT GATACTTACA 1020
TATGGCTCTA CAAGTGTCAT TTTTCTTCCA TATTTGATAT TATAAAAAAT AAAATAAAAA 1080
ACTAAGGAGA TGATCCAGAT ATATTGGAAA ATGAAATGCA AAGGCTAAAA ATAATTGAAA 1140
TTAACATGAA ATTAGTAAAA ATTAC 1165
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 317 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : YES
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GCAAGCAATG CACCACAGTT AGTTTATATC AAAAAGAAGA AAGGTATTAA CGGAGCTAAA 60
AACTGTTATA TACCACATGA AAGAAGTTGA TAATGTGAAA ACACCATGCT CATAAAGATT 120
GTAATTCAAA TAACAAATGC CCACAGGAGT AAAGAGCTGT CTTTCCCAAG TTAAGGTATT 180
ATAAATTGGC GGAACGAAGT AACACATGTT TGACATCTCC ACACGGTGCA CAGATCAAAT 240
ATGCCATGAG CACCAGTCCA GAAGTTTTCC AACTATTTAT ATACTATCCA TGCAACCATA 300
TAAATTATCA AACATAC 317
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 504 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CTGCAAAAAA AGAGAGCAGT TTACACAAGA AAAAACTGCT AAATCTCAAC AAAAGTATCA 60
TGAATTTAAT ATTAAGGAAG CTATTTCGAA CAGAAAGAGT AACTCATGAT AATAGAAGGA 120
AATTGTGAAG CAACAGAAGG AAGACTTTCT TTATTTCTAC AAAATTGCTT TAAGACTATA 180
TTTGATGCTT GTATAGTACA TGTTGAATCC CCTCAGCTTC TTTATGTCTA TACTTTTTTT 240
ATATTTTGAA TCTCCTTAGT GAAAATCTTT GCTTTGCCAC TGACACTCCG GGGGTGTGTC 300
ACTTCTCCAA AAACCTTGTC TACTTTTTTG AAGACCCAAT CAAACAGCTT TTTAAAAGAT 360
CAAAAAAATG GCCAGGTGCC ACCTAAATGG AGCCACTACT TACTCCCCGG TATGCAAAAT 420
TCTCTAGCAA AGTCAAAGTA GGTATAAACA ATTCATCTTC CAAAATAAGG TCAAACTGCC 480
TAAAGCACAA CTTTTGGCTG TTAC 504
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CTGCATTGTG GATGAGTTAA TTAGAAGCAT AACCTTAATA GCAATTAGAA CATGTAAGAA 60 AGCCAATGAT GCTGCAACAT CATGCTTTAA TAGGAAAATC TGTTATGATG ATGGAAACTA 120
CTATTTTGTA GTAGACGAGG ACCTAC 146
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CTGTTTAATT GCTGAAGTAG TAAGTTCTCA AGCACTTATA GAATTGACTC ATTTTGTTAA 60
GGGAAAGAGT ATGGGATCAA GTCCAAATTA GTAAAGACAC AATTATTTTA ACTTTTGCAT 120
TTCAAAATGT CTTACATAAC AAGACTAGTA AGAACATGAA TCGAAATGCC TGTGATGATG 180
GTGTTCAAAA TTCAGCTTCA AGGTATGAAT AACAAAAC 218
(2) INFORMATION FOR SEQ ID NO : 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CTGTATCCAG CAGACATAAT AGGAGTGAAC ATAAAAATGT CACTGGATAA ATAACTTATC 60
ATGATATTCA GCGGCTACCA ATATTCTGAA GGCCCATGGC GAAAATAAGT ACTTTTATAC 120
TTTCAGGACG TATATATTTG GATTCTATCT AACAATTGTT CTGAGAATTA TTTAGTTGTA 180
GAAATAAATT TAAAATAC I98
(2) INFORMATION FOR SEQ ID NO : 21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 208 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
;ϋi) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CTGTGGTTAG AAGCTAAAAG TGAATAGATG AGAAAAATTA CCTCCAAATA AGAGGGATAT 60
TGAAAAAGAA ACACAATGCA TGAAAAGAAT AAACAAATGA TAAACGAGAA AATTGAATAA 120
TCCATCAGAA CCCTGGTTAC CTCACAAAGA GTGAGATTTT CCGTGGCTAA CCTATATGAA 180
CCTTAAAATG CAATAGAAAC AGACAAAC 208
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 293 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CTGTACAAGT TCATCAAACA TTTCACAATT ACTCCAAAAC AGACACACTT GCAAACTCTA 60
TACAGTAATC TTCTATACTA CAAAAAAGTA AACAATGTTT TTTTTAAGAT GACATTTGTT 120
CTCAGCAACA TAATAGAAAT CCCTAGACAA TGGAAACATT CATCATGTTG TTTTCCTCTA 180
TGTTTCAACC CCTTTGATGT TCAACAGTTC AGGTCATTTT GAGGAATGAA TCTTGTTCAA 240
GTAAGCCAAA CTAATTGTAA TTATCACAAA ATATCTAAAG ATGTAAGACA TAC 293 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 376 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CTGCATTTCA TCATGAGGGG GAGGAAAGAC GGAGAAATAT AGATATCAGA TTTAGACCAT 60
TTCAATTAGT ATCACTTCAT TGTAAAGAAA AGGTAAGTAT CCAACAAATA TAGCAGGCTG 120
TGGATTGGTA GCCTGAAACT ATAGCTTCAA AGAATCAACT TAAGCTGCTC ATCAAGGCCT 180
TAGTGGTAGA AATGAGGCGG TAATAAGTGT AAATGAATCT AATACTTGGA TCTCGAAACA 240
AAAATCAGAA ATTCGGTTGG AAAATAAGTA GAACAAGATG AAATGAGCTA TCATCCCCAG 300
AACCAAGTAG ACTTCCAAGT AAGCAATCTA AAAATTACTA GATTATTTAA CAAGCTGCGA 360
TTCAAAATAC TTGAAC 376
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 172 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CTGCAAAGTG AAGTAACTAA TCAGTACAGC TATTACCGAA TTTGACCAGC TATTGGATTA 60
AATAATATGA AATCCATCAT CAAGAAATGG AAGGTAAAAA GGTTTCTACT TGTCCTTGGA 120
TAGAATTAAA GCACTTCATA AACCCAACAC TTTCAACTTT AGATGATTTT AC 172
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 145 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
CTGTTTTCGT CATGCGAGGA TCAGAAAAAA GAGTTAAATT AGACAATGTG AAAATGATTT 60
GTTTCAGTTA CTTCTCCATA AAACTTGTTC AGTACATTAA AAACAAGCAG AGCAATAATT 120
TCATGGATAA GTAAAACATA TATAC 145
(2) INFORMATION FOR SEQ ID NO : 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
CTGTCATGAG AACAGATTGT ATGTCAGCAT GAAGACAAAG ATCATCAATA AACAGTTTTC 60
TCCTTTTTGA ATTAGCTAAA CAACGCAGGG GGAGGGCAGG AGGCTCAAAC ACTTCCGAAC 120
TCAGACAGTC GGATATCTTA TACAACTAAA GATGGATGAG ACAATTACAG TTCTTTTTGG 180
TGAGAGAACT GTACCCTACA TCTGTTATCT TATTATCAAA AGTTATTCAA GCAAATCCTT 240
AC 242
(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
CTTCACAAAC AAGGAGAAGA AGAAGCAAAA AGAAAGATGA ATATAGTTAG CTTAGTTCAA 60
TATAAAAAAT TTCTCTCCAA GCTATTTTTC TGCTAGCAAA ATTCATTAGT TATTTAACTT 120
TTCTATACAT AAAGCTGCAC AAAGAAATAG TAGTACATTT TTTTGACTTG CACAAAATAA 180
CTGTGTTGTC CATTTTCTGA CATGTGTTCA TCTACATGCA CTGTTTCAAC AACAACAACT 240
ACTTCAGTCC CAAACAAGTT GGGTCGCTTT AGCTACACAT GTTGCTTTCA CTTCTGTTAC 300
TTCTTTTTGG ACTTTTTTTC TTGAGCCAAG GGTCTATTGA AAAAATCCTC TCTACCTCTG 360
AGATAGGAGT AAGTTTTGCA TACACTCTAC CCTCCCCCTG AAACCACTTT GTGGGACTAC 420
ACGAGGTATG TTGTTGTTGA TGTTAGCGCA GACACCAAAG GTGGACATTA TATGACTATT 480
CCTAGCTTTA CTTCAGGGCG GTTTTAAGTT CCCATCAACT TCATTTTTGA TCATTTACCT 540
AAGTTTATGC AGGTGCAAGC TACATGCACT GGTTTAGGGA AAAAGAGGAT AGAGAAGAAT 600 TTTTTTGGCA TCCTTTTGTT TTGTAACAGT AAGATGCCAA AAGTAGACCT TATTACGGCT 660
ATTCCTACCT TTCAAATTAG TAGTTCAGAG GACTTAACTG GCGATTGTGG CGGTAATCAA 720
TAGTTAACTT CTATCGCATT CAAATAACTA TGAACAAAAC CACAATAAAA AGGGAGGTCA 780
CACGGCAAGA ACTGTAC 797
(2) INFORMATION FOR SEQ ID NO : 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2169 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
[iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
CGAATGGGTT TTGATAAAAC TTTGAAATTA ATTTCCATTG ATTAAATTAT GGTACTTTGC 60
TTCAGCTGCT GCCTTCTTGT ATGTTCTGAT TCTTGATTTC CTCATTTTAG TGGCTTTTTA 120
TAAAAAAACA TTATGACCCT TTTGTTAGTC CTCCCCTTTC TGAATATTTC ACTCAGACCC 180
CATTAGTTTC GAAATTGAAG TAAAACATAT TTTTTTTAGT ATTGTAGTTT TTTTATATTT 240
CTACTTACTT ACTCGTTATA CAATTTCTAT TTAGGTAATC TAATCGACTT TTTGTATACA 300
TAATACATGT ATTTTTGGTA AAGAGTTTTT TACTTTCTCC TAGTGGTAAG GCAGATATAG 360 TTAAGGATTT ATTGACCTAA TATGAACGCC AATAATTTTA TATTTTGTAT ATACGTATAT 420
TTAAAAGTTT ACTAGATATG TATAAATAAG ATATTTAAAA TTTAATTATA AATACAAATG 480
ATTATGGTAA AATTTTGACC TCCAAATTAA AATATTTAAA ATCAAGATTT GTCACTACTT 540
ATATATATCT TGTTGTAAAT CCCTTTTAAT CAAGTTGTGA GTTTACAAAT ATTCGTTGGT 600
TAGGCTAAAA AAAATAAGCT ATAAAGATCA AGTATAAAAT TATGCATTTT CTGCATTTAA 660
TTTGGAAAAA TATGTTGGAG CAATCTAAAA TTGTTTGTTG ATTTATAAAT AAGTCGTTTT 720
TTGTTTTTAA TAATTGATAA ACTATTTATT CTGCTTAAAG TTTTAGAATG TCAAAAAATA 780
ATTTATTTTA ATGACCTTAA ATGATTGAAT AAGATGTAGA CACACTCAAT TACAAAGTTA 840
CAATATTAAT ACACTTGTCT ATTGGGTCAT GGATTATATC ATCTAATATA AATAACATGT 900
CAAATTAAAG CTTCTTATAA AGTTCATAGG AACTAAGATA AACTTTGTGA ATGGCCAAGC 960
ATTTTTCAGA ACATCATGGG TGGTATGACA ATCAAATTGA ACTTATGGGA TGAAAAATGA 1020
ATATCATTCA ACTAAGAGGG CACAACTTGA CATGTTAGAA AGTAAAGCAA ATTTAGTAGT 1080
GGGCCAAATA AAAGAAATTA ATTTGTCAGT TTATTCTTAA ACTTTACCTT CTTTGAACTT 1140
CCACGTTATC AAAGGTTCAC GGTTCATATG AAGGCCATGT GTATCCTTTT TAATTTTGGT 1200
ATTCCGTGTT CAATATCGAT TAATTTAAAT TCGCATGACA AAATCCTATA TTAAAGTATA 1260
AAGTATTTTC TAAAACAGAC AAGTTCAATA CTTTAATTTT ACACTGAATG CATAAATTTA 1320
CACTATAATA ATTCCAGTCG CAGTCTACAT TACAATAATT AACAATTTTA GCATGAAATG 1380
AAAAACTTTA AATTATATGC CATCAAATCA CTTAAAGTAT ACATTTTTTT AATAACTAGT 1440
TCTAATCCCA CTTGAAATGA GAGTTATTTT AATATCGACC GTTAATTACC ATTTTATTAT 1500 TAAATCTGCA ACTACAGTCA ACTACACCAA TGATTTTGCT GATGCCAACT CATAATATAA 1560
TATCCACCGT TCATGTGATT AATTCAATAT TTCATATACG TACGTAACAA AAATTACTAA 1620
ATTAACGTTG GATATACCAT ACCCTAAGCT CTGCCAAATG TCAATGTTCT ATCATTAGCT 1680
ATTTTTATGC ATCTATAATA GATGTTAAAT TCATATTCTA AGATTGAACT TAATCATAAA 1740
CTCAAAATTT GTGGTACCTG TCAATGCCTC CAAAAGTTGA TTGAACATAA ACGTTAAGAT 1800
CTGTGTACTT GTCTTTTCCT TGTAATAATG TATGTATGAT AATAATAATA AGAGAACAAA 1860
ATATGGCAAA ATAAACACTT TTTTAACATG TAACTCAAAA CAAGTAATAG GCAAAAGTAC 1920
AGATGACAAC ACAACACTGT AAACATCATT GAGGAAAACA AAAACCATAC AACATTTTGA 1980
CTGTAAATGA AGAGTTTGAA AACAAAAACT ATGTTCAAAC CGACGCCAAG CTAACGAAAA 2040
TAGCCATAGA GTTCTAAGAA GCAGATGCAA CAGTTCCACG GGTTAGTATC GTCTGTAGTA 2100
GGACCGGTCA TGAGAACTCG AAAGAATCTG AAAGGAAGTA ATGCATTTGA ACCAGTAATT 2160
GGCCATGAT 2169
(2) INFORMATION FOR SEQ ID NO : 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11469 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 29:
ATCATGGCCA ATTACTGGTT CAAATGCATT ACTTCCTTTC AGATTCTTTC GAGTTCTCAT 60
GACCGGTCCT ACTACAGACG ATACTAACCC GTGGAACTGT TGCATCTGCT TCTTAGAACT 120
CTATGGCTAT TTTCGTTAGC TTGGCGTCGG TTTGAACATA GTTTTTGTTT TCAAACTCTT 180
CATTTACAGT CAAAATGTTG TATGGTTTTT GTTTTCCTCA ATGATGTTTA CAGTGTTGTG 240
TTGTCATCTG TACTTTTGCC TATTACTTGT TTTGAGTTAC ATGTTAAAAA AGTGTTTATT 300
TTGCCATATT TTGTTCTCTT ATTATTATTA TCATACATAC ATTATTACAA GGAAAAGACA 360
AGTACACAGA TCTTAACGTT TATGTTCAAT CAACTTTTGG AGGCATTGAC AGGTACCACA 420
AATTTTGAGT TTATGATTAA GTTCAATCTT AGAATATGAA TTTAACATCT ATTATAGATG 480
CATAAAAATA GCTAATGATA GAACATTGAC ATTTGGCAGA GCTTAGGGTA TGGTATATCC 540
AACGTTAATT TAGTAATTTT TGTTACGTAC GTATATGAAA TATTGAATTA ATCACATGAA 600
CGGTGGATAT TATATTATGA GTTGGCATCA GCAAAATCAT TGGTGTAGTT GACTGTAGTT 660
GCAGATTTAA TAATAAAATG GTAATTAACG GTCGATATTA AAATAACTCT CATTTCAAGT 720
GGGATTAGAA CTAGTTATTA AAAAAATGTA TACTTTAAGT GATTTGATGG CATATAATTT 780
AAAGTTTTTC ATTTCATGCT AAAATTGTTA ATTATTGTAA TGTAGACTGC GACTGGAATT 840
ATTATAGTGT AAATTTATGC ATTCAGTGTA AAATTAAAGT ATTGAACTTG TCTGTTTTAG 900
AAAATACTTT ATACTTTAAT ATAGGATTTT GTCATGCGAA TTTAAATTAA TCGATATTGA 960 ACACGGAATA CCAAAATTAA AAAGGATACA CATGGCCTTC ATATGAACCG TGAACCTTTG 1020
ATAACGTGGA AGTTCAAAGA AGGTAAAGTT TAAGAATAAA CTGACAAATT AATTTCTTTT 1080
ATTTGGCCCA CTACTAAATT TGCTTTACTT TCTAACATGT CAAGTTGTGC CCTCTTAGTT 1140
GAATGATATT CATTTTTCAT CCCATAAGTT CAATTTGATT GTCATACCAC CCATGATGTT 1200
CTGAAAAATG CTTGGCCATT CACAAAGTTT ATCTTAGTTC CTATGAACTT TATAAGAAGC 1260
TTTAATTTGA CATGTTATTT ATATTAGATG ATATAATCCA TGACCCAATA GACAAGTGTA 1320
TTAATATTGT AACTTTGTAA TTGAGTGTGT CTACATCTTA TTCAATCATT TAAGGTCATT 1380
AAAATAAATT ATTTTTTGAC ATTCTAAAAC TTTAAGCAGA ATAAATAGTT TATCAATTAT 1440
TAAAAACAAA AAACGACTTA TTTATAAATC AACAAACAAT TTTAGATTGC TCCAACATAT 1500
TTTTCCAAAT TAAATGCAGA AAATGCATAA TTTTATACTT GATCTTTATA GCTTATTTTT 1560
TTTAGCCTAA CCAACGAATA TTTGTAAACT CACAACTTGA TTAAAAGGGA TTTACAACAA 1620
GATATATATA AGTAGTGACA AATCTTGATT TTAAATATTT TAATTTGGAG GTCAAAATTT 1680
TACCATAATC ATTTGTATTT ATAATTAAAT TTTAAATATC TTATTTATAC ATATCTAGTA 1740
AACTTTTAAA TATACGTATA TACAAAATAT AAAATTATTG GCGTTCATAT TAGGTCAATA 1800
AATCCTTAAC TATATCTGCC TTACCACTAG GAGAAAGTAA AAAACTCTTT ACCAAAAATA 1860
CATGTATTAT GTATACAAAA AGTCGATTAG ATTACCTAAA TAGAAATTGT ATAACGAGTA 1920
AGTAAGTAGA AATATAAAAA AACTACAATA CTAAAAAAAA TATGTTTTAC TTCAATTTCG 1980
AAACTAATGG GGTCTGAGTG AAATATTCAG AAAGGGGAGG ACTAACAAAA GGGTCATAAT 2040
GTTTTTTTAT AAAAAGCCAC TAAAATGAGG AAATCAAGAA TCAGAACATA CAAGAAGGCA 2100 GCAGCTGAAG CAAAGTACCA TAATTTAATC AATGGAAATT AATTTCAAAG TTTTATCAAA 2160
ACCCATTCGA GGATCTTTTC CATCTTTCTC ACCTAAAGTT TCTTCAGGGG TAATTTTTAC 2220
TAATTTCATG TTAATTTCAA TTATTTTTAG CCTTTGCATT TCATTTTCCA ATATATCTGG 2280
ATCATCTCCT TAGTTTTTTA TTTTATTTTT TATAATATCA AATATGGAAG AAAAATGACA 2340
CTTGTAGAGC CATATGTAAG TATCATGTGA CAAATTTGCA AGGTGGTTGA GTGTATAAAA 2400
TTCAAAAATT GAGAGATGGA GGGGGGGTGG GGGAAGACAA TATTTAGAAA GAGTGTTCTA 2460
GGAGGTTATG GAGGACACGG ATGAGGGGTA GAAGGTTAGT TAGGTATTTG AGTGTTGTCT 2520
GGCTTATCCT TTCATACTAG TAGTCGTGGA ATTATTTGGG TAGTTTCTTG TTTTGTTATT 2580
TGATCTTTGT TATTCTATTT TCTGTTTCTT GTACTTCGAT TATTGTATTA TATATCTTGT 2640
CGTAGTTATT GTTCCTCGGT AAGAATGCTC TAGCATGCTT CCTTTAGTGT TTTATCATGC 2700
CTTCTTTATA TTCGCGTTGC TTTGAAATGC TTTTACTTTA GCCGAGGGTC TATTAGAAAC 2760
AATCTCTCTA TCTCGTAAGG TAGGGGTAAA GTCCTCACCA CACTCCACTT GTGGGATTAC 2820
ATTGTGTTTG TTGTTGTAAA TCAATTATGT ATACATAATA AGTGGATTTT TTACAACACA 2880
AATACATGGT CAAGGGCAAA GTTCTGAACA CATAAAGGGT TCATTATATG TCCAGGGATA 2940
TGATAAAAAT TGTTTCTTTG TGAAAGTTAT ATAAGATTTG TTATGGCTTT TGCTGGAAAC 3000
ATAATAAGTT ATAATGCTGA GATAGCTACT GAAGTTTGTT TTTTCTAGCC TTTTAAATGT 3060
ACCAATAATA GATTCCGTAT CGAACGAGTA TGTTTTGATT ACCTGGTCAT GATGTTTCTA 3120
TTTTTTACAT TTTTTTGGTG TTGAACTGCA ATTGAAAATG TTGTATCCTA TGAGACGGAT 3180
AGTTGAGAAT GTGTTCTTTG TATGGACCTT GAGAAGCTCA AACGCTACTC CAATAATTTC 3240 TATGAATTCA AATTCAGTTT ATGGCTACCA GTCAGTCCAG AAATTAGGAT ATGCTGCATA 3300
TACTTGTTCA ATTATACTGT AAAATTTCTT AAGTTCTCAA GATATCCATG TAACCTCGAG 3360
AATTTCTTTG ACAGGCTTCT AGAAATAAGA TATGTTTTCC TTCTCAACAT AGTACTGGAC 3420
TGAAGTTTGG ATCTCAGGAA CGGTCTTGGG ATATTTCTTC CACCCCAAAA TCAAGAGTTA 3480
GAAAAGATGA AAGGGTATGT TTGATAATTT ATATGGTTGC ATGGATAGTA TATAAATAGT 3540
TGGAAAACTT CTGGACTGGT GCTCATGGCA TATTTGATCT GTGCACCGTG TGGAGATGTC 3600
AAACATGTGT TACTTCGTTC CGCCAATTTA TAATACCTTA ACTTGGGAAA GACAGCTCTT 3660
TACTCCTGTG GGCATTTGTT ATTTGAATTA CAATCTTTAT GAGCATGGTG TTTTCACATT 3720
ATCAACTTCT TTCATGTGGT ATATAACAGT TTTTAGCTCC GTTAATACCT TTCTTCTTTT 3780
TGATATAAAC TAACTGTGGT GCATTGCTTG CATGAAGCAC AGTTCAGCTA TTTCCGCTGT 3840
TTTGACCGAT GACGACAATT CGACAATGGC ACCCCTAGAG GAAGATGTCA AGACTGAAAA 3900
TATTGGCCTC CTAAATTTGG ATCCAACTTT GGAACCTTAT CTAGATCACT TCAGACACAG 3960
AATGAAGAGA TATGTGGATC AGAAAATGCT CATTGAAAAA TATGAGGGAC CCCTTGAGGA 4020
ATTTGCTCAA GGTAACAGCC AAAAGTTGTG CTTTAGGCAG TTTGACCTTA TTTTGGAAGA 4080
TGAATTGTTT ATACCTACTT TGACTTTGCT AGAGAATTTT GCATACCGGG GAGTAAGTAG 4140
TGGCTCCATT TAGGTGGCAC CTGGCCATTT TTTTGATCTT TTAAAAAGCT GTTTGATTGG 4200
GTCTTCAAAA AAGTAGACAA GGTTTTTGGA GAAGTGACAC ACCCCCGGAG TGTCAGTGGC 4260
AAAGCAAAGA TTTTCACTAA GGAGATTCAA AATATAAAAA AAGTATAGAC ATAAAGAAGC 4320
TGAGGGGATT CAACATGTAC TATACAAGCA TCAAATATAG TCTTAAAGCA ATTTTGTAGA 4380 AATAAAGAAA GTCTTCCTTC TGTTGCTTCA CAATTTCCTT CTATTATCAT GAGTTACTCT 4440
TTCTGTTCGA AATAGCTTCC TTAATATTAA ATTCATGATA CTTTTGTTGA GATTTAGCAG 4500
TTTTTTCTTG TGTAAACTGC TCTCTTTTTT TGCAGGTTAT. TTAAAATTTG GATTCAACAG 4560
GGAAGATGGT TGCATAGTCT ATCGTGAATG GGCTCCTGCT GCTCAGTAGG TCCTCGTCTA 4620
CTACAAAATA GTAGTTTCCA TCATCATAAC AGATTTTCCT ATTAAAGCAT GATGTTGCAG 4680
CATCATTGGC TTTCTTACAT GTTCTAATTG CTATTAAGGT TATGCTTCTA ATTAACTCAT 4740
CCACAATGCA GGGAAGCAGA AGTTATTGGC GATTTCAATG GATGGAACGG TTCTAACCAC 4800
ATGATGGAGA AGGACCAGTT TGGTGTTTGG AGTATTAGAA TTCCTGATGT TGACAGTAAG 4860
CCAGTCATTC CACACAACTC CAGAGTTAAG TTTCGTTTCA AACATGGTAA TGGAGTGTGG 4920
GTAGATCGTA TCCCTGCTTG GATAAAGTAT GCCACTGCAG ACGCCACAAA GTTTGCAGCA 4980
CCATATGATG GTGTCTACTG GGACCCACCA CCTTCAGAAA GGTTTTGTTA TTCATACCTT 5040
GAAGCTGAAT TTTGAACACC ATCATCACAG GCATTTCGAT TCATGTTCTT ACTAGTCTTG 5100
TTATGTAAGA CATTTTGAAA TGCAAAAGTT AAAATAATTG TGTCTTTACT AATTTGGACT 5160
TGATCCCATA CTCTTTCCCT TAACAAAATG AGTCAATTCT ATAAGTGCTT GAGAACTTAC 5220
TACTTCAGCA ATTAAACAGG TACCACTTCA AATACCCTCG CCCTCCCAAA CCCCGAGCCC 5280
CACGAATCTA TGAAGCACAT GTCGGCATGA GCAGCTCTGA GCCACGTGTA AATTCGTATC 5340
GTGAGTTTGC AGATGATGTT TTACCTCGGA TTAAGGCAAA TAACTATAAT ACTGTCCAGT 5400
TGATGGCCAT AATGGAACAT TCTTACTATG GATCATTTGG ATATCATGTT ACAAACTTTT 5460
TTGCTGTGAG CAGTAGATAT GGAAACCCGG AGGACCTAAA GTATCTGATA GATAAAGCAC 5520 ATAGCTTGGG TTTACAGGTT CTGGTGGATG TAGTTCACAG TCATGCAAGC AATAATGTCA 5580
CTGATGGCCT CAATGGCTTT GATATTGGCC AAGGTTCTCA AGAATCCTAC TTTCATGCTG 5640
GAGAGCGAGG GTACCATAAG TTGTGGGATA GCAGGCTGTT CAACTATGCC AATTGGGAGG 5700
TTCTTCGTTT CCTTCTTTCC AACTTGAGGT GGTGGCTAGA AGAGTATAAC TTTGACGGAT 5760
TTCGATTTGA TGGAATAACT TCTATGCTGT ATGTTCATCA TGGAATCAAT ATGGGATTTA 5820
CAGGAAACTA TAATGAGTAT TTCAGCGAGG CTACAGATGT TGATGCTGTG GTCTATTTAA 5880
TGTTGGCCAA TAATCTGATT CACAAGATTT TCCCAGATGC AACTGTTATT GCCGAAGATG 5940
TTTCTGGTAT GCCGGGCCTT GGCCGGCCTG TTTCTGAGGG AGGAATTGGT TTTGTTTACC 6000
GCCTGGCAAT GGCAATCCCA GATAAGTGGA TAGATTATTT AAAGAATAAG AATGATGAAG 6060
ATTGGTCCAT GAAGGAAGTA ACATCGAGTT TGACAAATAG GAGATATACA GAGAAGTGTA 6120
TAGCATATGC GGAGACCCAT GATCAGGTAT TTTAAATTTA TTTCTACAAC TAAATAATTC 6180
TCAGAACAAT TGTTAGATAG AATCCAAATA TATACGTCCT GAAAGTATAA AAGTACTTAT 6240
TTTCGCCATG GGCCTTCAGA ATATTGGTAG CCGCTGAATA TCATGATAAG TTATTTATCC 6300
AGTGACATTT TTATGTTCAC TCCTATTATG TCTGCTGGAT ACAGTCTATT GTTGGTGACA 6360
AGACCATTGC ATTTCTCCTA ATGGACAAAG AGATGTATTC TGGCATGTCT TGCTTGACAG 6420
ATGCTTCTCC TGTTGTTGAT CGAGGAATTG CGCTTCACAA GGTTTGTCTG TTTCTATTGC 6480
ATTTTAAGGT TCATATAGGT TAGCCACGGA AAATCTCACT CTTTGTGAGG TAACCAGGGT 6540
TCTGATGGAT TATTCAATTT TCTCGTTTAT CATTTGTTTA TTCTTTTCAT GCATTGTGTT 6600
TCTTTTTCAA TATCCCTCTT ATTTGGAGGT AATTTTTCTC ATCTATTCAC TTTTAGCTTC 6660 TAACCACAGA TGATCCATTT TTTCACAATG GCCTTGGGAG GAGAGGGGTA CCTCAATTTC 6720
ATGGGTAACG AGGTATGTCT TACATCTTTA GATATTTTGT GATAATTACA ATTAGTTTGG 6780
CTTACTTGAA CAAGATTCAT TCCTCAAAAT GACCTGAACT GTTGAACATC AAAGGGGTTG 6840
AAACATAGAG GAAAACAACA TGATGAATGT TTCCATTGTC TAGGGATTTC TATTATGTTG 6900
CTGAGAACAA ATGTCATCTT AAAAAAAACA TTGTTTACTT TTTTGTAGTA TAGAAGATTA 6960
CTGTATAGAG TTTGCAAGTG TGTCTGTTTT GGAGTAATTG TGAAATGTTT GATGAACTTG 7020
TACAGTTTGG CCATCCTGAG TGGATTGACT TCCCTAGAGA GGGCAATAAT TGGAGTTATG 7080
ACAAATGTAG ACGCCAGTGG AACCTCGCGG ATAGCGAACA CTTGAGATAC AAGGTTCAAG 7140
TATTTTGAAT CGCAGCTTGT TAAATAATCT AGTAATTTTT AGATTGCTTA CTTGGAAGTC 7200
TACTTGGTTC TGGGGATGAT AGCTCATTTC ATCTTGTTCT ACTTATTTTC CAACCGAATT 7260
TCTGATTTTT GTTTCGAGAT CCAAGTATTA GATTCATTTA CACTTATTAC CGCCTCATTT 7320
CTACCACTAA GGCCTTGATG AGCAGCTTAA GTTGATTCTT TGAAGCTATA GTTTCAGGCT 7380
ACCAATCCAC AGCCTGCTAT ATTTGTTGGA TACTTACCTT TTCTTTACAA TGAAGTGATA 7440
CTAATTGAAA TGGTCTAAAT CTGATATCTA TATTTCTCCG TCTTTCCTCC CCCTCATGAT 7500
GAAATGCAGT TTATGAATGC ATTTGATAGA GCTATGAATT CGCTCGATGA AAAGTTCTCA 7560
TTCCTCGCAT CAGGAAAACA GATAGTAAGC AGCATGGATG ATGATAATAA GGTAAAATCA 7620
TCTAAAGTTG AAAGTGTTGG GTTTATGAAG TGCTTTAATT CTATCCAAGG ACAAGTAGAA 7680
ACCTTTTTAC CTTCCATTTC TTGATGATGG ATTTCATATT ATTTAATCCA ATAGCTGGTC 7740
AAATTCGGTA ATAGCTGTAC TGATTAGTTA CTTCACTTTG CAGGTTGTTG TGTTTGAACG 7800 TGGTGACCTG GTATTTGTAT TCAACTTCCA CCCAAAGAAC ACATACGAAG GGTATATATG 7860
TTTTACTTAT CCATGAAATT ATTGCTCTGC TTGTTTTTAA TGTACTGAAC AAGTTTTATG 7920
GAGAAGTAAC TGAAACAAAT CATTTTCACA TTGTCTAATT TAACTCTTTT TTCTGATCCT 7980
CGCATGACGA AAACAGGTAT AAAGTTGGAT GTGACTTGCC AGGGAAGTAC AGAGTTGCAC 8040
TGGACAGTGA TGCTTGGGAA TTTGGTGGCC ATGGAAGAGT AAGGATTTGC TTGAATAACT 8100
TTTGATAATA AGATAACAGA TGTAGGGTAC AGTTCTCTCA CCAAAAAGAA CTGTAATTGT 8160
CTCATCCATC TTTAGTTGTA TAAGATATCC GACTGTCTGA GTTCGGAAGT GTTTGAGCCT 8220
CCTGCCCTCC CCCTGCGTTG TTTAGCTAAT TCAAAAAGGA GAAAACTGTT TATTGATGAT 8280
CTTTGTCTTC ATGCTGACAT ACAATCTGTT CTCATGACAG ACTGGTC TG ATGTTGACCA 8340
TTTCACATCA CCAGAAGGAA TACCTGGAGT TCCAGAAACA AATTTCAATG GTCGTCCAAA . 8400
TTCCTTCAAA GTGCTGTCTC CTGCGCGAAC ATGTGTGGTA CAGTTCTTGC CGTGTGACCT 8460
CCCTTTTTAT TGTGGTTTTG TTCATAGTTA TTTGAATGCG ATAGAAGTTA ACTATTGATT 8520
ACCGCCACAA TCGCCAGTTA AGTCCTCTGA ACTACTAATT TGAAAGGTAG GAATAGCCGT 8580
AATAAGGTCT ACTTTTGGCA TCTTACTGTT ACAAAACAAA AGGATGCCAA AAAAATTCTT 8640
CTCTATCCTC TTTTTCCCTA AACCAGTGCA TGTAGCTTGC ACCTGCATAA ACTTAGGTAA 8700
ATGATCAAAA ATGAAGTTGA TGGGAACTTA AAACCGCCCT GAAGTAAAGC TAGGAATAGT 8760
CATATAATGT CCACCTTTGG TGTCTGCGCT AACATCAACA ACAACATACC TCGTGTAGTC 8820
CCACAAAGTG GTTTCAGGGG GAGGGTAGAG TGTATGCAAA ACTTACTCCT ATCTCAGAGG 8880
TAGAGAGGAT TTTTTCAATA GACCCTTGGC TCAAGAAAAA AAGTCCAAAA AGAAGTAACA 8940 GAAGTGAAAG CAACATGTGT AGCTAAAGCG ACCCAACTTG TTTGGGACTG AAGTAGTTGT 9000
TGTTGTTGAA ACAGTGCATG TAGATGAACA CATGTCAGAA AATGGACAAC ACAGTTATTT 9060
TGTGCAAGTC AAAAAAATGT ACTACTATTT CTTTGTGCAG CTTTATGTAT AGAAAAGTTA 9120
AATAACTAAT GAATTTTGCT AGCAGAAAAA TAGCTTGGAG AGAAATTTTT TATATTGAAC 9180
TAAGCTAACT ATATTCATCT TTCTTTTTGC TTCTTCTTCT CCTTGTTTGT GAAGGCTTAT 9240
TACAGAGTTG ATGAACGCAT GTCAGAAACT GAAGATTACC AGACAGACAT TTGTAGTGAG 9300
CTACTACCAA CAGCCAATAT CGAGGAGAGT GACGAGAAAC TTAAAGATTC GTTATCTACA 9360
AATATCAGTA ACATTGACGA ACGCATGTCA GAAACTGAAG TTTACCAGAC AGACATTTCT 9420
AGTGAGCTAC TACCAACAGC CAATATTGAG GAGAGTGACG AGAAACTTAA AGATTCGTTA 9480
TCTACAAATA TCAGTAACAT TGATCAGACT GTTGTAGTTT CTGTTGAGGA GAGAGACAAG 9540
GAACTTAAAG ATTCACCGTC TGTAAGCATC ATTAGTGATG TTGTTCCAGC TGAATGGGAT 9600
GATTCAGATG CAAACGTCTG GGGTGAGGAC TAGTCAGATG ATTGATCGAC CCTTCTACCG 9660
ATTGGTGATC GCTATCCTTG CTCTCTGAGA AATAGGTGAG GCGAAACAAA AAATAATTTG 9720
CATGATAAAA AGTCTGATTT TATGATCGCT ATCCTCGCTC TCTGAGAAAG AAGCGAAACA 9780
AAGGCGACTC CTGGACTCGA ATCTATAAGA TAACAAAGGC GACTCCTGGG ACTCGAATCT 9840
ATAAGATAAC AAAGGCAATT CCAAGACTTG AATCTATAAA AAATTTAGTT AAGAATGATT 9900
AACGTCCGAT CCTAATTCGA ATCGAGGCAT CTTACCACTC CATTGATAAT TATATAAGTC 9960
AATAAGTCAT ATAAAGTATT AAAAACTAAA TTGACTTGAT CGGTCTATCA AAAATAGATA 10020
AATTGTGTTC ATATGTAACA TTTTTGTTGT CACAATTAGC TTAATTACAT CTTTCATGTG 10080 CAATAACAAA GAAATGATAG GAATTTAGAG ATTCCAATTT TTTTGTTGCC ACAATTAACT 10140
TAATTACATC TTTCATTTGC AATAACAAAG AAATGATAGG AATTTAGAGA TCCAGTGTCA 10200
ATACACAACC TAGGCCAACA TCGAAAGCAT AACTGTAAAC TCATGCATGA AGAAATCAGT 10260
CGTAAAAATG AATAAATGCG ACATAAAAAC AAATTGCATG TATCATTAAT GTGACTTAAC 10320
TACAAGTAAA AATAAATTTA ACAAATGTAA CTTAACTACA AGTAAAAATA AATTGCTTCT 10380
ATCATTAACA AACAAACAGA ATTAAAAAGA AAAAAACATA CTAAATCTTA CCGTCATTCG 10440
ATAAAAAAAA ATACCAAATT CATAATGCAA GGAAAACGAA ACGCGTCCTG ATCGGGTATC 10500
AACGATGAAA TGGACCAGTT GGATCGACTG CCTGCACAAC GTTAGGTATG CCAAAAAAAA 10560
GAACACGATC CTTTGCACCC GTTCGATGAT TATCAGTATG TTCACAAAAA AAACTTAAGT 10620
TCATCCCAGT GTACAACAGC CCCAACATCT GCCCCAAGTA ACAAAAAACA ACCAATTTAT 10680
CTTATTCTTA TCTGCCACAA AATAATCGGT TTCACACTAT TCTCTTGTTA TACAAAATTG 10740
ACAAGTAGGA AGGAGAGGAG TCATCCAAAT AAACGGTGCA CGTTCTTTGA GAAAAGTCTT 10800
ATTTTTCGTA AGATCCAATT TCAACAAACT TTTCTTCAAG TCAAAATTCC TGATAGTGTA 10860
TCTCCTCTCG ACGACCTCTT GCATTGAACG ATCTCCGCTT ATCATGAAAA GTTGCTTGGA 10920
TAACAAGTAT TGCAAGGGGG GGACAGTAGC TATTAAGTTA GTCGGCCCAA GGAAATGGAG 10980
GAGTGATAGT CTCGAATATT ATTCACCTCT TTAGCATTAC CCGGTCTGGC TTTAAGGAGT 11040
TACGTCTTTT ACGCTCGCCA ATTTCTTTTT TTAGAATGGT TGGTGTCAAA ATCGCGAGTT 11100
GTGGAAGGTT CAAGTTACTC GATTCGTGAT TTTCAAGTAT GAGTGGTGAG AGAGATTCGA 11160
TATTTTCACG AGGTGTATTC GAGGTCTAGT AGAACGAAGG GTGTCACTAA TGAAAGTTTC 11220 AAGAGTTCAT CATCATCTTC TTCTAGTAGA TTTTCGCTTT CAAATGAGTA TGAAAATTCT 11280
TCCTCTTTTC TATTGATTTT CTTCATTGTT TTCTTCATTG TTGTGGTTGT TATTGAAAAG 11340
AAAGAAAATT TATAACAGAA AAAGATGTCA AAAAAAAGGT AAAATGAAAG AGTATCATAT 11400
ACTTAAAGAG TTGCGTAGAG ATAAGTCAAA AGAAACAGAA TTATAGTAAT TTCAGCTAAG 11460
TTAGAATTC 11469
(2) INFORMATION FOR SEQ ID NO : 30:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 30:
GGAATTCCAG TCGCAGTCTA CATTAC 6
(2) INFORMATION FOR SEQ ID NO : 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 31:
CGGGATCCAG AGGCATTAAG ATTTCTGG
(2) INFORMATION FOR SEQ ID NO : 32:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
CGGGATCCAA AGAAATTCTC GAGGTTACAT GG 32 (2) INFORMATION FOR SEQ ID NO : 33:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CGGGATCCGG GGTAATTTTT ACTAATTTCA TG 32
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 34:
CGGGATCCCG TATGTCTCAC TGTGTTTGTG GC 32
(2) INFORMATION FOR SEQ ID NO : 35:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CGGGATCCCC CTACATACAT ATATCAGATT AG 32
(2) INFORMATION FOR SEQ ID NO : 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
CCATCGATAC TTTAAGTGAT TTGATGGC 28
(2) INFORMATION FOR SEQ ID NO : 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
CGGGATCCTG TTCTGATTCT TGATTTCC 28
(2) INFORMATION FOR SEQ ID NO : 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2122 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 38:
GTATGTCTCA CTGTGTTTGT GGCTGTGTGT GTTTTTTTCT CTGTCTTTTT GTGTTTTGTG 60
TAATTGGGGC TCTTTAAAGT TGGTATTGTG TATACCCTTT TGAGTATAGT CTTTGAGGAA 120
GCAAAATGAT GAATCTTGAT TGACATTAGT AAGGGTTGTA ACTTTTTGAA GTTTGGTTAG 180
GTGTAATTGA GTTTGGCTTG TGTGTCTGTG TGTCGAGGTT ATTTTTTTGG TTTGTGTTAT 240
TGGGGATTCT TAAAAGTTGG TATTGTGTAT ACCCTTTTGA GTATAGTCTT TGAGGAAGCA 300
AAAATGATGA ATCTTGATTG GCATTAGTAA AGGTTGTAGC TTTTTGAAGT GTGGTTAGGT 360
GTAATTGAGT TTGGCTTGTG TGTCTGTGTG TTTTGGAATC CTGATGTGTG TCAAGTCCTG 420
ATATGGGTCG AGGTTCTTTC TTTGGTTTGT GTAATTGGGG GTTCTTAAAA GTTGGTATTA 480
TGTACCTTTT TAAGAATAGT GTCTGAGAAA GCAAAATCGA TGAATTTTGA TTGACAGCAT 540
ATTCTTTGAG AAAGCAAAAA ATGGTGAGTT TTCATGGAGA AACTTGATTG ACATTACTAA 600
AGGTAGCAAC TTTTTCAACT CCTGATATGG GTCAAGGTTC TTTGTTTGGT TTGTGTAATT 660
TGGGGTTCTT TGAAGTTTTG AGAAAGAAAA ATTATGATTT TTCATGGAGA AATTTGATTT 720 ACATTAATAA AGGTAGTAGC TTTTTAAAGT GTGGTCAGCT GTAATGAGTT CAGCTTGGTT 780
TAAAGGGGCC CTACATATGG TGCTTTCTGG TGAGATATTT GTTGCTCCAC CATACGAGTT 840
ATAAGAATCA TAGTGTTAGG ATCTTTTTTC ττττττττττ CATTTTTCAC TTGACTAGCT 900
ACTAGAGGAG TGATCTTGAC GGCGGAAAAT CTTAGAAAGG GGAAGGTTGT TTGCATCAAC 960
TGGTGTTATA TGTGCAAGGA GACGGGAGAT GATGTAGATC ATCTTCTTCT TCATTGTGGT 1020
CTTTCCATGA GGTTATGATG TGATATGTTT GAATGGTTTG GTACTTCTTG GCTATGCCAA 1080
GAACTGTGAA AGAATTGATA TTCAGTTGGA AGTGTGGAGT TGGAAGAGTG GAAGAATTGA 1140
CACTTGGTTC CATTAGCTTT AATGTGGGTG GTGTGGAGAG AGAGAGAAAT AGGAGAGCTT 1200
TTGAGGGGGT AGAGTTGAGC TTTCCTCAGT TGAGAAGTAG CCTTTGATAT CTTTTTTTTT 1260
TTTTTTTGTA CACCCATAGA ATTCCCAATT GTATAGAAGA TTGGGTGGAG TTTGTAGAGA 1320
ATCATCTTTT GTAGTAGATT CTTTACCTTT TGGTATATCC ATTGTATACA GCCAGGCCTT 1380
TGACTATGTT TATGAATGAA TATACATTAC TTGAAAAAAA AAGAAGTGAA GCCAGTCTGT 1440
TGTACCTTTG TAGACAATGT TGTTGCAGCA TCTTGATAAT TCCCTGAAAA TTGTCTCCCT 1500
GAAGGAATAG TTTGGTTGAT ATTGATTATT TCTTGGTTTG TTTAATTCGG TGTTCTTGAA 1560
GGCCATTTTA AATCCTTTGA CATTGTTAAA GGTGTTTACA AGTGTTGGTC TGGGTTTAAA 1620
AGCACCTCTT GTATGGTGCT TTCTGGAGTG ATCTTTCTTC CTCCAAAAGA GAAGTTGCAA 1680
GAATCAGTGT GTGTACTTTT TTCTCTTGTA TGATCAGATC TTTTTTCAAT TTTTCCGTTT 1740
TAGTTGATTT ATCCATATAG TGAAAGTTGG TGTCATAGTT GCTGTTTGTG GACTTCCTGT 1800
AAAAGTTTTT TGATATACTT AAAAAATTGT CACACAGAAG AAAGAGTTTT TTACCATTAC 1860 TTAAGCTAGA TGGGACTGTT TGATTCTTAG ACCAAATAAT GAACCTTTTT GTTCTCTTAA 1920
CGTGTACTTG AAATAGTTTG GTAAAATTGT GATAGGAAAA AAGATAATTC TTGATTGCTT 1980
TTGGAGCATC ACTTCTAATC ATAAAAGTCT TTGCTCTCTT CAACCATGAA TGATAAATTG 2040
GACACTTATG TGGCCCTAAG TTGCTCTCAG TAGTGGTCTT TAATTGTGGA GATATAACTA 2100
ATCTGATATA TGTATGTAGG GA 2122

Claims

1. A method of affecting enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron of a class A potato starch branching enzyme in a sense orientation, optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; and wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron.
2. A method according to claim 1 wherein starch branching enzyme activity is affected and/or wherein the levels of amylopectin are affected and/or the composition of starch is changed.
3. A method of affecting enzymatic activity in a starch producing organism (or a cell, a tissue or an organ thereof) comprising expressing in the starch producing organism (or a cell, a tissue or an organ thereof) a nucleotide sequence wherein the nucleotide sequence codes, partially or completely, for an intron of a class A starch branching enzyme in a sense orientation, optionally together with a nucleotide sequence which codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron; and wherein starch branching enzyme activity is affected and/or the levels of amylopectin are affected and/or the composition of starch is changed.
4. A method according to any one of claims 1 to 3 wherein the nucleotide sequence does not contain a sequence that is sense to an exon sequence.
5. A method according to any one of the preceding claims wherein the enzymatic activity is reduced or eliminated.
6. A method according to any one of the preceding claims wherein the nucleotide sequence codes for at least substantially all of at least one intron in a sense orientation.
7. A method according to any one of the preceding claims wherein the nucleotide sequence codes for all of at least one intron in a sense orientation.
8. A method according to any one of the preceding claims wherein the nucleotide sequence comprises the sequence shown as SEQ. ID. No. 38, or a variant, derivative or homologue thereof.
9. A method according to any one of the preceding claims wherein the nucleotide sequence is expressed by a promoter having a sequence shown as SEQ.I.D. No. 14 or a variant, derivative or homologue thereof.
10. A promoter having a sequence shown as SEQ.I.D. No. 14, or a variant, derivative or homologue thereof.
11. A promoter according to claim 10 in combination with a gene of interest ("GOI").
12. A construct capable of comprising or expressing the invention according to any one of claims 10 and 11.
13. A vector comprising or expressing the invention according to any one of claims 10 to 12.
14. A combination of nucleotide sequences comprising a first nucleotide sequence coding for a recombinant enzyme; and a second nucleotide sequence which corresponds to a class A SBE intron in a sense orientation; wherein the intron is an intron that is associated with a genomic gene encoding an enzyme corresponding to the recombinant enzyme; and wherein the second nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron.
15. A cell, tissue or organ comprising or expressing the invention according to any one of claims 10 to 14.
16. A transgenic starch producing organism comprising or expressing the invention according to any one of claims 10 to 15.
17. A transgenic starch producing organism according to claim 16 wherein the organism is a plant.
18. A starch obtained from the invention according to any one of the preceding claims.
19. A method of expressing a recombinant protein or enzyme in a host organism comprising expressing a nucleotide sequence coding for the recombinant protein or enzyme; and expressing a further nucleotide sequence; wherein the further nucleotide sequence codes, partially or completely, for a class A SBE intron in a sense orientation; wherein the intron is an intron normally associated with the genomic gene encoding a protein or an enzyme corresponding to the recombinant protein or enzyme; and wherein the further nucleotide sequence does not contain a sequence that is sense to an exon sequence normally associated with the intron.
PCT/IB1998/000295 1997-02-21 1998-02-23 Sense intron inhibition of starch branching enzyme expression WO1998037214A1 (en)

Priority Applications (6)

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BR9808648-0A BR9808648A (en) 1997-02-21 1998-02-23 Intron sense inhibition of starch branching enzyme expression
JP53643898A JP2001512972A (en) 1997-02-21 1998-02-23 Sense intron inhibition of starch branching enzyme expression
CA002280210A CA2280210A1 (en) 1997-02-21 1998-02-23 Sense intron inhibition of starch branching enzyme expression
AU62259/98A AU738311B2 (en) 1997-02-21 1998-02-23 Sense intron inhibition of starch branching enzyme expression
EP98904329A EP1007708A1 (en) 1997-02-21 1998-02-23 Sense intron inhibition of starch branching enzyme expression
NZ336607A NZ336607A (en) 1997-02-21 1998-02-23 Method of inhibiting gene expression where the nucleotide codes for a Class A SBE intron in the sense direction

Applications Claiming Priority (4)

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GBGB9703672.7A GB9703672D0 (en) 1997-02-21 1997-02-21 Inhibition of gene expression
GB9703672.7 1997-02-21
GBGB9706075.0A GB9706075D0 (en) 1997-03-24 1997-03-24 Inhibition of gene expession
GB9706075.0 1997-03-24

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BR (1) BR9808648A (en)
CA (1) CA2280210A1 (en)
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WO2002101091A2 (en) * 2001-06-13 2002-12-19 Centre National De La Recherche Scientifique Method for detecting and identifying the presence of biological materials derived from fish, and oligonucleotides therefor
FR2858631A1 (en) * 2004-09-24 2005-02-11 Centre Nat Rech Scient Method for detecting material from gadiform fish, useful for detecting adulteration of foods, based on amplification of mitochondrial DNA, also new oligonucleotides and amplicons
US7714186B2 (en) 2002-12-19 2010-05-11 Bayer Cropscience Ag Plant cells and plants which synthesize a starch with an increased final viscosity
US8148517B2 (en) 2005-04-01 2012-04-03 Bayer Cropscience Ag Phosphorylated waxy potato starch
WO2013053729A1 (en) 2011-10-12 2013-04-18 Bayer Cropscience Ag Plants with decreased activity of a starch dephosphorylating enzyme
WO2013053730A1 (en) 2011-10-12 2013-04-18 Bayer Cropscience Ag Plants with decreased activity of a starch dephosphorylating enzyme
WO2019238832A1 (en) 2018-06-15 2019-12-19 Nunhems B.V. Seedless watermelon plants comprising modifications in an abc transporter gene
US11174493B2 (en) 2016-05-26 2021-11-16 Nunhems B.V. Seedless fruit producing plants

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CN104017829A (en) * 2011-12-06 2014-09-03 中国科学院上海生命科学研究院 Method for increasing amylose content of plants

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002101091A2 (en) * 2001-06-13 2002-12-19 Centre National De La Recherche Scientifique Method for detecting and identifying the presence of biological materials derived from fish, and oligonucleotides therefor
FR2826021A1 (en) * 2001-06-13 2002-12-20 Centre Nat Rech Scient Detecting material from gadiform fish, useful for determining adulteration of foods, by amplification and sequencing of mitochondrial DNA
WO2002101091A3 (en) * 2001-06-13 2004-05-06 Centre Nat Rech Scient Method for detecting and identifying the presence of biological materials derived from fish, and oligonucleotides therefor
US7714186B2 (en) 2002-12-19 2010-05-11 Bayer Cropscience Ag Plant cells and plants which synthesize a starch with an increased final viscosity
US8017832B2 (en) 2002-12-19 2011-09-13 Bayer Cropscience Ag Plant cells and plants which synthesize a starch with an increased final viscosity
FR2858631A1 (en) * 2004-09-24 2005-02-11 Centre Nat Rech Scient Method for detecting material from gadiform fish, useful for detecting adulteration of foods, based on amplification of mitochondrial DNA, also new oligonucleotides and amplicons
US8148517B2 (en) 2005-04-01 2012-04-03 Bayer Cropscience Ag Phosphorylated waxy potato starch
WO2013053729A1 (en) 2011-10-12 2013-04-18 Bayer Cropscience Ag Plants with decreased activity of a starch dephosphorylating enzyme
WO2013053730A1 (en) 2011-10-12 2013-04-18 Bayer Cropscience Ag Plants with decreased activity of a starch dephosphorylating enzyme
US11174493B2 (en) 2016-05-26 2021-11-16 Nunhems B.V. Seedless fruit producing plants
US11761018B2 (en) 2016-05-26 2023-09-19 Nunhems B.V. Seedless fruit producing plants
WO2019238832A1 (en) 2018-06-15 2019-12-19 Nunhems B.V. Seedless watermelon plants comprising modifications in an abc transporter gene

Also Published As

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JP2001512972A (en) 2001-08-28
EP1007708A1 (en) 2000-06-14
AU738311B2 (en) 2001-09-13
CA2280210A1 (en) 1998-08-27
NZ336607A (en) 2001-06-29
AU6225998A (en) 1998-09-09
BR9808648A (en) 2000-05-23
CN1248293A (en) 2000-03-22

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