CA2280210A1

CA2280210A1 - Sense intron inhibition of starch branching enzyme expression

Info

Publication number: CA2280210A1
Application number: CA002280210A
Authority: CA
Inventors: Peter Poulsen
Original assignee: Individual
Current assignee: DuPont Nutrition Biosciences ApS
Priority date: 1997-02-21
Filing date: 1998-02-23
Publication date: 1998-08-27
Also published as: WO1998037214A1; JP2001512972A; BR9808648A; AU738311B2; NZ336607A; EP1007708A1; AU6225998A; CN1248293A

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 a-1-4-linked glycosyl residues. Amylopectin comprises chains of a-1-4-linked glycosyl residues with some a-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 a-1,4 glucans through a-1,6-glucosidic branching linkages. The biosynthesis of amylose and amylopectin is schematically shown in Figure 1, whereas the a-1-4-links and the a-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 PCTlEP96103052 and PCT/EP96103053, class B potato SBE and a gene encoding it are discussed. In international patent application W096/34968, 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 ~5 pp 125-149).
W096/34968 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 1? 883-888; and Matzke and Matzke [1995] TIG ~ 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 I5 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 ,r 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 inter 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 andlor 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 1 ~

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.

5 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 W096/34968, 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 andlor 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. LD. 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 andlor 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 Agrobactericcm 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 Shl-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 6418, 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 Shl-intron or an 5 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
S' 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 a-Amy 1 promoter {otherwise known as the Amy 1 promoter, the Amy 637 promoter or the a-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 a-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the a-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 t . . r __. _ ._._.~._~.._. _ .. . _ _.....

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 bindine sites for regulatory proteins for example the CreA
protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the cataliolite 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 G01 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.
r ~

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 a-galactosidase. The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for a-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 a-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 a-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 a-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 mventton ~s 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 mtron.
5 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.~.
SEQ.I.D.No.

10 16).
The terms "cell", "tissue" and "organ" include cell, tissue and organ per se and when within an organism.
IS 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, andlor 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 Bacillces scebtilis. 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 ~enetic 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.
rt 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 turnefaciens or a Ri plasmid from Agrobacteritun 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 organism 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 turnefaciens Ti-plasmid or an Agrobacteritcrn rltizogenes 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.

WO 98/37214 PCT/IB98l00295 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 Agrobactericcm strain, e.~.
Agrobacterium tcemefaciens. The Ti-plasmid harbouring the nucleotide sequence or construct of the present invention is thus preferably transferred into a suitable Agrobacteritcm strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cel!
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 EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.B., Alblasserdam, 1985, Chapter V; Fraiey, et al., Crit.
Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985) 4:277-284.
Direct infection of plant tissues by Agrobacterictm 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 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 pan 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 5 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 ~ntron sequences.
Also, the present invention relates to a promoter useful for the expression of those sense mtron 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 ~.-SBE 3.2 as described herein), and NCIMB 40752 (which refers to i,.-SBE 3.4 as described herein).
2~ 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 a-1-4-links and the a-1-6 links of arnylopectin;
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 by in size;
Figure 5, which is a plasmid map of pABE7, which is 5106 by in size;
Figure 6, which is a plasmid map of pVictorIV Man, which is 7080 by in size;
Figure 7, which is a plasmid map of pBEAI l, 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 pSSlS; and Figure 14, which shows pSSl6.
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.LD.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:

1054) and the exon DNAs begin at 2083 by 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.
n ....

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 ?~-phages containing SBE DNA (~.SBE 3.2 -NCIMB 40751 - and ~.SBE-3.4 - NCIMB 40752) are identified by Southern analysis and then subcloned into pBluescript II vectors (Clontech Laboratories Inc., Palo Alto CA, USA). ?~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, pGBlS, pGBl6 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 by to 836 bp, pGBlS contains the sequences from 735 by to 2580 bp, pGBl6 contains the sequences from 2580 by to 5093 bp, pGBll contains the sequences from 3348 by to 7975 bp, and pGB3 contains the sequences from 7533 by to 11468 bp.
In more detail, pGB3 is constructed by insertion of a 4 kb EcoRI fragment isolated from ~.SBE 3.2 into the EcoRI site of pBluescript II SK (+). pGBll is constructed by insertion of a 4.7 kb XhoI fragment isolated from ~.SBE 3.4 into the XhoI site of pBluescript II SK (+). pGBIS is constructed by insertion of a 1.7 kb SpeI
fragment isolated from ~.SBE 3.4 into the SpeI site of pBluescript II SK (+). pGBl6 is constructed by insertion of a 2.5 kb SpeI fragment isolated from ~,SBE 3.4 into the SpeI
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 J~SBE 3.4 as a template.
The PCR fragment is digested with BamHI and EcoRI, 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 ?~SBE 3.4 phage containing the SBE gene as template.
The PCR product is digested with BamHI and inserted in a sense orientation in the BamHI site of plasmid pPATAl (described in WO 94/24292) between the patatin promoter and the 35S terminator. This construction, pABE7, is digested with KpnI, and the 2.4 kb "patatin promoter-SBE intron 1- 35S terminator" KpnI fragment is isolated and inserted in the KpnI site of the plant transformation vector pVictorIV Man yielding plasmid pBEAll.
CONSTRUCTION OF PLASMID pSSlS.

The 2122 by 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 5 PCR product is digested with BamHI and inserted in sense orientation after a patatin promoter in the BamHI site of a plant transformation vector in which the NPTII
gene is used as selectable marker (see figure 13).
CONSTRUCTION OF PLASMID pSSlG.
10 The 2122 by intron 1 sequence of the potato SBEII gene (SEQ. ID. No. 38) is amplified by PCR from a genomic SBEII subclone using the primers ~' - 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 BamHI and inserted in sense orientation after a patatin 15 promoter in the BamHI site of a plant transformation vector in which the manA gene is used as selectable marker {see figure 14).
PRODUCTION OF TRANSGENIC POTATO PLANTS
~xenic stock cultures 20 Shoot cultures of Solanum tLCberosum 'Bintje' and 'Dianelia' 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 pM 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 25 the shoots and cut into nodal segments (approximately 0.8 cm) each containing one node.
Inoculation of o~ tatO~issues 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 Agrobactericcm tccmefaciens 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/i) 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/l 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 mgll), gibberellic acid (U.1 mgll), carbenicillin (800 mg/1), and kanamycin sulphate (SO-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 mgll). The transgenic genotype of the regenerated shoot are verified by testing the rooting ability on the above mentioned substrates containing kanamycin sulphate (200 mg/I), 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 2~ 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 (1-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/m~/sec).
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-Hetmelink 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 ~,-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 CIaI and BamHI. The resultant 1.2 kb fragment is then inserted in pVictor5a (see Figure 9) linearised with CIaI and BgIII
yielding pBEP2 (see Figure 10).
STARCH BRANCHING ENZY1~IE MEASUREMENTS OF POTATO TUBERS
Potatoes from potato plants transformed with pBEAI l 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 yl composed of 0.1 M Na citrate buffer pH 7.0, 0.75 mglml amylose, 5 mg/ml bovine serum albumin and the potato extract. At 0, 15 30 and 60 minutes aliqouts of 50 lZl are removed from the reaction into 20 pl 3 N HC1. 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 transgenic Dianella potato plants transformed with plasmid pBEAl 1, pSS 15 and pSS 16.
The results show that the BEAT l , SS 15 and SS 16 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 pSSlS and pBEAI l 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 1? pp 883 - 887) and Matzke and Matzke (1995 TIG ~1_ 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.LD. No. 1 - SEQ.LD. No. 38. In brief, SEQ.LD.
No. 1 - SEQ.LD. No. 13 represent sense intron sequences (genomic DNA); SEQ.LD.

No. 14 represents the SBE promoter sequence (genomic sequence); SEQ.LD. 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
5 including the promoter, exons and introns is shown as SEQ. LD. 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.
...,..... .fi......

SEQUENCE LISTING
(1) GENERAL INFORMATION:
S
(i) APPLICANT:
(A) NAME: DANISCO A/S
(B) STREET: LANGEBROGADE 1 (C) CITY: COPENHAGEN K
lO (E) COUNTRY: DENMARK
(F) POSTAL CODE (ZIP): DK-1001 (ii) TITLE OF INVENTION: INHIBITION OF GENE EXPRESSION
IS (iii) NUMBER OF SEQUENCES: 38 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible ZO (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 3O (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

i ~i (xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 1:

CTTTTAAATGTACCAATA_~TAGATTCCGTATCGAACGAGT TACCTGGTCA900 ATGTTTTGAT

TTTTTTTGGT
GTTGAACTGC
AATTGAAAAT

TAGTTGAGAA
TGTGTTCTTT
GTATGGACCT
TGAGAAGCTC
AAACGCTACT

~ ......

(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 1S (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 2:

(2) INFORMATION EQ ID NO: 3:
FOR S

i ii (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 504 base pairs (B) TYPE: nucleic acid S (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) IO (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0: 3:

(2) INFORMATION
FOR SEQ
ID NO:
4:

(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 5 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
I~
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 3~ (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

i i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORNfATION FOR SEQ ID NO: 6:
IS (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
2S (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GTATTTTAAA TTTATTTCTA CAACTAAATA ATTCTCAGAA CA.~TTGTTAG ATAGAATCCA 60 ~.

(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
1$
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

~S TCTTTTCATG CATTGTGTTT CTTTTTCAAT ATCCCTCTTA TTTGGAGGTA ATTTTTCTCA 180 (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) i i (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
lO GTATGTCTTA CATCTTTAGA TATTTTGTGA TAATTACAAT TAGTTTGGCT TACTTGAACA 60 ZO (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) 3O (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATAATCTAGT

TCATTTCATC

AGTATTAGAT

AGCTTAAGTT

TGTTGGATAC

ATATCTATAT

IS (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) ZS (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
S (A) LENGTH: 145 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(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 3S (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO
S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GATGTAGGGT

lO CACCAAAAAG AACTGTAATT GTCTCATCCA TCTTTAGTTG CCGACTGTCT 120 TATAAGATAT

TGTTTAGCTA

ATACAATCTG

(2) INFORMATION FOR SEQ ID NO: 13:

2O (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

3O (iv) ANTI-SENSE: NO

3S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GAAAATGGAC AACACAGTTA TTTTGTGCAA GTCAAAAA.AATGTACTACTA TTTCTTTGTG660 (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 3S (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

t n (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION:
SEQ ID NO: 14:

AAAAAATGTA TACTTTAAGT
GATTTGATGG CATATAATTT

ATTCAGTGTA AAATTAAAGT
ATTGAACTTG TCTGTTTTAG

ATAGGATTTT GTCATGCGAA
TTTAAATTAA TCGATATTGA

i ii TGAACCTTTG

AATTTCTTTT

CCTCTTAGTT

CCATGATGTT

TATAAGAAGC

GACAAGTGTA

TAAGGTCATT

TATCAATTAT

TCCAACATAT

GCTTATTTTT

TTTACAACAA

GTCAAAATTT

ATATCTAGTA

TAGGTCAATA

ACCAAAAATA

ATAACGAGTA

AGTAAGTAGA AATATAAAAA AACTACAATA CTP.AAAAAAA TATGTTTTAC 1980 TTCAATTTCG

GGGTCATAAT

CAAGAAGGCA

ACCCATTCG
S
(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:

m AAGGCTAAAA ATAATTGAAA

~S 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 3S (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

n (iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

AAGGTATTAA

ACACCATGCT

CTTTCCCAAG

ACACGGTGCA

1$

ATACTATCCA

ZO (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 504 base pairs (B) TYPE: nucleic acid 2$ (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 3O (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

I II

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 146 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 2S (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

S
(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:

(2) INFORMATION FOR SEQ ID NO: 20:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single i ii (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) S (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

(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) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 293 base pairs 1S (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(ivy ANTI-SENSE: YES

(xi) SEQUENCE
DESCRIPTION:
SEQ ID NO:
22:

TACAGTAATC TTCTATACTACAA.A.AAAGTA AACAATGTTTTTTTTAAGATGACATTTGTT120 i ii (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:

(2) INFORMATION EQ ID NO: 24:
FOR S

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH:172 base pairs r rt (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear S (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
1~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

(2) INFORMATION FOR SEQ ID N0: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 3~ (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

i a (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
CTGTTTTCGT CATGCGAGGA TCAGAAAP.AA GAGTTAAATT AGACAATGTG AAAATGATTT 60 (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single IS (D) TOPOLOGY. linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

(2) INFORMATION FOR SEQ ID NO: 27:
i ~, SS
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 797 base pairs (B) TYPE: nucleic acid S (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iiy MOLECULE TYPE: DNA (genomic) lO (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 27:

i ii TATTACGGCT

CGGTAATCAA

AGGGAGGTCA

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2169 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 15(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(xi) SEQUENCE
DESCRIPTION:
SEQ ID NO:
28:

1 1f $7 ATACGTATAT

AATACAAATG

GTCACTACTT

ATTCGTTGGT

CTGCATTTAA

AAGTCGTTTT

TCF~AAAAATA

1$

TACAAAGTTA

AATAACATGT

ATGGCCAAGC

TGAAAAATGA

ATTTAGTAGT

CTTTGAACTT

TAATTTTGGT

TTAAAGTATA

CATAAATTTA

GCATGAAATG

3$

AATAACTAGT

ATTTTATTAT

i ii (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11469 base pairs 3O (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

1 if S (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 29:

AGATTCTTTC

TGCATCTGCT

GTTTTTGTTT

ATGATGTTTA

ATGTTAAAAA

ATTATTACAA

AGGCATTGAC

TTTAACATCT

GCTTAGGGTA

TATTGAATTA

TGGTGTAGTT

AAATAACTCT

GATTTGATGG

TGTAGACTGC GACTGGAATT

ATTGAACTTG TCTGTTTTAG

TTTAAATTAA TCGATATTGA

i ii WO 98/37214 PCT/IB98/0!0295 i ii S TTTTTTCTTG TGTAAACTGC TCTCTTTTTT TGCAGGTTAT.TTAAAATTTG GATTCAACAG 4560 f II

AATAATGTCA

TTTCATGCTG

AATTGGGAGG

TTTGACGGAT

ATGGGATTTA

GTCTATTTAA

GCCGAAGATG

TTTGTTTACC

AATGATGAAG

GAGAAGTGTA

TAAATAATTC

AAGTACTTAT

TTATTTATCC

GTTGGTGACA

TGCTTGACAG

TTTCTATTGC

TAACCAGGGT

GCATTGTGTT

TTTTAGCTTC

~.

CCTCAATTTC

ATTAGTTTGG

AAAGGGGTTG

TATTATGTTG

CTGAGAACAA ATGTCATCTT AA.AAAP.AACA TTGTTTACTT TTTTGTAGTA6960 TAGAAGATTA

GATGAACTTG

TGGAGTTATG

AAGGTTCAAG

CTTGGAAGTC

CAACCGAATT

CGCCTCATTT

GTTTCAGGCT

TGAAGTGATA

CCCTCATGAT

AAAGTTCTCA

GGTAAAATCA

ACAAGTAGAA

ATAGCTGGTC

TGTTTGAACG

i ii GGTATATATG

AAGTTTTATG

TTCTGATCCT

AGAGTTGCAC

TTGAATAACT

CTGTAATTGT

GTTTGAGCCT

TATTGATGAT

ATGTTGACCA

GTCGTCCAAA

CGTGTGACCT

ACTATTGATT

GAATAGCCGT

AAAAATTCTT

ACTTAGGTAA

TAGGAATAGT

TCGTGTAGTC

ATCTCAGAGG

AGAAGTAACA

AAGTAGTTGT

ACAGTTATTT

AGAAAAGTTA

TATATTGAAC

GAAGGCTTAT

TTGTAGTGAG

GTTATCTACA

AGACATTTCT

AGATTCGTTA

GAGAGACAAG

TGAATGGGAT

CCTTCTACCG

AAATAATTTG

AAGCGAAACA

ACTCGAATCT

AAGAATGATT

TATATAAGTC

AAAATAGATA

CTTTCATGTG

i ii ATCATTAACA AACAAACAGA ATTAAAAAGA AAAAA.ACATA CTAAATCTTA CCGTCATTCG 10440 ATAA.AA.AAAA ATACCAAATT CATAATGCAA GGAAAACGAA ACGCGTCCTG ATCGGGTATC 10500 IS P.ACGATGAAA TGGACCAGTT GGATCGACTG CCTGCACAAC GTTAGGTATG CCAAAAAAA.A 10560 TCATCCCAGT GTACAACAGC CCCAACATCT GCCCCAAGTA ACAF1A.A.AACA ACCAATTTAT 10680 (2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs 1S (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:

(2) INFORMATION FOR SEQ ID NO: 31:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single i ii (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: 3I:

(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 ZS (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
3O (iv) ANTI-SENSE: YES
3S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

t (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:

(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) MOLECUhE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

(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 IS (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Synthetic DNA Primer"
(iii) HYPOTHETICAL: NO
2O (iv) ANTI-SENSE: YES
2S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 3S (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:

(2) INFORMATION FOR SEQ ID NO: 37:
IS (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:

3S (2) INFORMATION FOR SEQ ID NO: 3B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2122 base pairs i ii (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
1~
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

GTGTTTTGTG

CTTTGAGGAA

GTTTGGTTAG

TTTGTGTTAT

TGAGGAAGCA

GTGGTTAGGT

TCAAGTCCTG

GTTGGTATTA

TTGACAGCAT

ACATTACTAA

TTGTGTAATT

AATTTGATTT

i ~

CAGCTTGGTT

CATACGAGTT

TTGACTAGCT

TTGCATCAAC

TCATTGTGGT

GCTATGCCAA

GAAGAATTGA

AGGAGAGCTT

CTTTTTTTTT

TTTGTAGAGA

GCCAGGCCTT

GCCAGTCTGT

TTGTCTCCCT

TGTTCTTGAA

TGGGTTTAAA

GAAGTTGCAA

TTTTCCGTTT

GACTTCCTGT

TTACCATTAC

I II

Claims

77

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.