AU738311B2 - Sense intron inhibition of starch branching enzyme expression - Google Patents

Description

WO 98/37214 PCT/IB98/00295 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 ca--4-linked glycosyl residues. Amylopectin comprises chains of ct-1-4-linked glycosyl residues with some-ct-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 catalyses the formation of branch points in the amylopectin molecule by adding a-1,4 glucans through ca-1,6-glucosidic branching linkages. The biosynthesis of amylose and amylopectin is schematically shown in Figure 1, whereas the ac-l-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 PCT/EP96/03052 and PCTIEP96/03053, 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 105 pp 125-149).

WO 96/34968 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 [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.

o* *#o i. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an 9. admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

990@* According to a first aspect of the present invention there is provided a 25 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 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 coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation does not contain a sequence that is sense to an exon sequence normally associated with the intron.

3 According to another aspect of the present invention there is provided a method of affecting starch branching 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 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 coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation 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 effected 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 So 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 *oo 'transgenes such as from SBE genes, to further manipulate starch quality in potato plants.

0 According to another aspect of the present invention there is provided a 25 sequence comprising the nucleotide sequence shown as SEQ. ID. No. 38 or a variant, derivative or homologue thereof.

According to another aspect of the present invention there is provided a promoter comprising the sequence shown as SEQ. ID. No. 14 or a variant, derivative or homologue thereof.

According to another aspect of the present invention there is provided a construct capable of comprising or expressing the nucleotide sequence shown as SEQ. ID. No. 38.

According to another aspect of the present invention there is provided a vector comprising or expressing the construct above or the nucleotide sequence shown as SEQ. ID. No. 38.

According to another aspect of the present invention there is provided a combination of first, second and third nucleotide sequences borne on one or more nucleic acid molecules, wherein the first nucleotide sequence codes for a recombinant class A SBE enzyme; the second nucleotide sequence corresponds to a class A SBE intron in a sense orientation; and the third nucleotide sequence corresponds to a class B SBE intron in a sense or antisense orientation; wherein the class A SBE 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.

According to another aspect of the present invention there is provided a cell, tissue or organ comprising or expressing the construct, vector or combination sequence previously described.

According to another aspect of the present invention there is provided a transgenic starch producing organism comprising or expressing the construct, vector or combination sequence previously described.

According to another aspect of the present invention there is provided starch obtained from the previously described novel methods or when obtained from the previously described novel organism.

WO 98/37214 PCT/IB98/00295 4 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 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 WO 98/37214 PCT/IB98/00295 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 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 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 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.

WO 98/37214 PCT/IB98/00295 6 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 350 bp), more preferably at least 500 nucleotides 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.

In another aspect the invention provides a method of expressing a recombinant class A SBE enzyme in a host organism comprising expressing a first nucleotide sequence coding for the recombinant enzyme; expressing a second nucleotide sequence, wherein the second nucleotide sequence codes, partially or completely, for a class A SBE intron in sense orientation; and expressing a third nucleotide sequence, wherein the third nucleotide sequence codes, partially or completely, for a class B SBE intron in sense or antisense orientation; wherein the class A SBE intron is an intron normally associated with the genomic gene encoding a protein or 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.

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.

25 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 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 homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences.

S

o• i WO 98/37214 PCT/IB98/00295 8 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 homology, more preferably at least 90% homology, even more preferably at least 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 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 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 no cover the natural combination of the wild type SBE gene when associated with the wild type SBE gene promoter in their natural environment.

o$ Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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.

9a 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.

*t go eo o *t WO 98/37214 PCT/IB98/00295 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 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 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 acc-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 ct-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 WO 98/37214 PCT/IB98/00295 11 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 WO 98/37214 PCT/IB98/00295 12 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.

WO 98/37214 PCT/IB98/00295 13 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 a higher lysine content than a nontransgenic plant). The GOI may even code for an enzyme that can be used in food processing such as xylanases and ca-galactosidase. The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for ca-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 copending UK patent application 9505475.5. The GOI can be the nucleotide sequence coding for the ca-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 aamylase 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 c-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.

WO 98/37214 PCT/IB98/00295 14 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 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 antisense 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 WO 98/37214 PCT/IB98/00295 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 SEQ.I.D.No. 38) in combination with for example an antisense intron which preferably is not complementary to the sense intron sequence 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 WO 98/37214 PCT/IB98/00295 16 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.

WO 98/37214 PCT/IB98/00295 17 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 rumefaciens 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 Methodsfor 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 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.

WO 98/37214 PCT/IB98/00295 18 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 EP-A-120516; Hoekema, in: The Binary Plant Vector System Offsetdrukkerij Kanters Alblasserdam, 1985, Chapter V; Fraley, 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 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 WO 98/37214 PCT/IB98/00295 19 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 WO 98/37214 PCT/IB98/00295 necessary such as to create combination systems as outlined above 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 -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 WO 98/37214 PCT/IB98/00295 22 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.

WO 98/37214 PCT/IB98/00295 21 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 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 pPATA1, 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 pBEA11, which is 9.54 kb in size; WO 98/37214 PCT/IB98/00295 23 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 X-phages containing SBE DNA (XSBE 3.2 NCIMB 40751 and XSBE-3.4 NCIMB 40752) are identified by Southern analysis and then subcloned into pBluescript II vectors (Clontech Laboratories Inc., Palo Alto CA, USA). XSBE 3.2 contains a 15 kb potato DNA insert and XSBE-3.4 contains a 13 kb potato DNA insert. The resultant plasmids are called pGB3, pGB11, pGB15, pGB16 and (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, pGB11 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 EcoRI fragment isolated from ASBE 3.2 into the EcoRI site of pBluescript II SK pGB11 is constructed by WO 98/37214 PCT/IB98/00295 24 insertion of a 4.7 kb XhoI fragment isolated from XSBE 3.4 into the XhoI site of pBluescript II SK pGB15 is constructed by insertion of a 1.7 kb SpeI fragment isolated from ASBE 3.4 into the SpeI site of pBluescript II SK pGB16 is constructed by insertion of a 2.5 kb SpeI fragment isolated from XSBE 3.4 into the SpeI site of pBluescript II SK For the construction of pGB25 a PCR fragment is produced with the primers GGA ATT CCA GTC GCA GTC TAC ATT AC 3' (SEQ. ID. No. and 5' CGG GAT CCA GAG GCA TTA AGA TTT CTG G 3' (SEQ. ID. No. 31) and XSBE 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 CGG GAT CCA AAG AAA TTC TCG AGG TTA CAT GG 3' (SEQ. ID. No. 32) and CGG GAT CCG GGG TAA TTT TTA CTA ATT TCA TG 3' (SEQ. ID. No. 33) and the XSBE 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 pPATA1 (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 pVictorlV Man yielding plasmid pBEA11.

CONSTRUCTION OF PLASMID WO 98/37214 PCT/IB98/00295 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 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 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 BamHI and inserted in sense orientation after a patatin 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 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 0 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 WO 98/37214 PCT/IB98/00295 26 (di-potassium hydrogen phosphate, trihydrate (0.66 magnesium sulphate, heptahydrate (0.20 sodium chloride (0.10 mannitol (10.0 and yeast extract (0.40 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 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 LSsubstrate containing agar (8.0 2,4-dichlorophenoxyacetic acid (2.0 mg/l) and transzeatin (0.5 mg/1). The substrate and also the explants are covered with sterile filter papers, and the petri dishes are placed at 25 0 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 trans-zeatin (1-5 mg/1), gibberellic acid (0.1 mg/1), carbenicillin (800 mg/l), and kanamycin sulphate (50-100 mg/1) or phosphinotricin mg/1) or mannose (5 g/l) 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 LSsubstrate, agar (8 g/1) and carbenicillin (800 mg/1). The transgenic genotype of the WO 98/37214 PCT/IB98/00295 27 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 Pglucuronidase gene according to Hodal, L. et al. (P1. 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 0 C, 16 hour light 200-400uE/m 2 /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-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.

WO 98/37214 PCT/IB98/00295 28 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 X-SBE 3.4 using primers: 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 ClaI and BamHI. The resultant 1.2 kb fragment is then inserted in pVictor5a (see Figure 9) linearised with ClaI and BglII yielding pBEP2 (see Figure STARCH BRANCHING ENZYME MEASUREMENTS OF POTATO TUBERS Potatoes from potato plants transformed with pBEA11 are cut in small pieces and homogenised in extraction buffer (50 mM Tris-HC1 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 0

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 OC 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 P1 are removed from the reaction into 20 p.1 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 24 transgenic Dianella potato plants transformed with plasmid pBEA11, pSS15 and pSS16.

The results show that the BEA11, 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.

WO 98/37214 PCT/IB98/00295 29 In a further experiment, plasmids pSS15 and pBEA1l 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 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.

WO 98/37214 PCT/IB98/00295 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.

WO 98/37214 PCT/IB98/00295 31 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: DANISCO A/S STREET: LANGEBROGADE 1 CITY: COPENHAGEN K COUNTRY: DENMARK POSTAL CODE (ZIP): DK-1001 (ii) TITLE OF INVENTION: INHIBITION OF GENE EXPRESSION (iii) NUMBER OF SEQUENCES: 38 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1165 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 98/37214 PCT/1B98/00295 32 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTAATTTTTA CTAATTTCAT GTTAATTTCA ATTATTTTTA GCCTTTGCAT TTCATTTTCC

AATATATCTG

GAAAAATGAC

AGTGTATAA

AGAGTGTTCT

GAGTGTTGTC

GTTTTGTTAT

ATATATCTTG

TTTTATCATG

CTATTAGAAA

TGTGGGATTA

TTTACAACAC

GTCCAGGGAT

TTGCTGGAAA

CTTTTAAATG

TGATGTTTCT

GATCATCTCC

ACTTGTAGAG

ATTCAAAAAT

AGGAGGTTAT

TGGCTTATCC

TTGATCTTTG

TCGTAGTTAT

CCTTCTTTAT

CAATCTCTCT

CATTGTGTTT

AAATACATGG

ATGATAAA~A

CATAATAAGT

TACCAATAAIT

ATTTTTTACA

TTAGTTTTTT

CCATATGTAA

TGAGAGATGG

GGAGGACACG

TTTCATACTA

TTATTCTATT

TGTTCCTCGG

ATTCGCGTTG

ATCTCGTAAG

GTTGTTGTAA

TCAAGGGCAA

TTGTTTCTTT

TATAATGCTG

AGATTCCGTA

TTTTTTTGGT

ATTTTATTTT

GTATCATGTG

AGGGGGGGTG

GATGAGGGGT

GTAGTCGTGG

TTCTGTTTCT

TAAGAATGCT

CTTTGAAATG

GTAGGGGTAA

ATCAATTATG

AGTTCTGAAC

GTGAAAGTTA

AGATAGCTAC

TCGAACGAGT

GTTGA.ACTGC

TTATAATATC

ACAAATTTGC

GGGGAAGACA

AGAAGGTTAG

AATTATTTGG

TGTACTTCGA

CTAGCATGCT

CTTTTACTTT

AGTCCTCACC

TATACATAAT

ACATAAAGGG

TATAAGATTT

TGAAGTTTGT

ATGTTTTGAT

AATTGAAAAT

AAATATGGAA

AAGGTGGTTG

ATATTTAGAA

TTAGGTATTT

GTAGTTTCTT

TTATTGTATT

TCCTTTAGTG

AGCCGAGGGT

ACACTCCACT

AAGTGGATTT

TTCATTATAT

GTTATGGCTT

TTTTTCTAGC

TACCTGGTCA

GTTGTATCCT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 ATGAGACGGA TAGTTGAGAA TGTGTTCTTT GTATGGACCT TGAGAAGCTC AAACGCTACT 0 WO 98/37214 PCT/IB98/00295 CCAATAATTT CTATGAATTC AAATTCAGTT TATGGCTACC AGTCAGTCCA GAAATTAGGA TATGCTGCAT ATACTTGTTC AATTATACTG TAAAATTTCT TAAGTTCTCA AGATATCCAT GTAACCTCGA GAATTTCTTT GACAG INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 317 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 1080 1140 1165 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTATGTTTGA TAATTTATAT GGTTGCATGG ATAGTATATA AATAGTTGGA ACTGGTGCTC ATGGCATATT TGATCTGTGC ACCGTGTGGA GATGTCAAAC TCGTTCCGCC AATTTATAAT ACCTTAACTT GGGAAAGACA GCTCTTTACT TTTGTTATTT GAATTACAAT CTTTATGAGC ATGGTGTTTT CACATTATCA TGTGGTATAT AACAGTTTTT AGCTCCGTTA ATACCTTTCT TCTTTTTGAT TGTGGTGCAT TGCTTGC INFORMATION FOR SEQ ID NO: 3:

AAACTTCTGG

ATGTGTTACT

CCTGTGGGCA

ACTTCTTTCA

ATAAACTAAC

120 180 240 300 WO 98/37214 PCT/IB98/00295 SEQUENCE CHARACTERISTICS: LENGTH: 504 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 TACCTACTTT GACTTTGCTA GAGAATTTTG CATACCGGGG AGTAAGTAGT AGGTGGCACC TGGCCATTTT TTTGATCTTT TAAAAAGCTG TTTGATTGGG AGTAGACAAG GTTTTTGGAG AAGTGACACA CCCCCGGAGT GTCAGTGGCA TTTCACTAAG GAGATTCAAA ATATAAAAAA AGTATAGACA TAAAGAAGCT AACATGTACT ATACAAGCAT CAAATATAGT CTTAAAGCAA TTTTGTAGAA TCTTCCTTCT GTTGCTTCAC AATTTCCTTC TATTATCATG AGTTACTCTT ATAGCTTCCT TAATATTAAA TTCATGATAC TTTTGTTGAG ATTTAGCAGT GTAAACTGCT CTCTTTTTTT GCAG INFORMATION FOR SEQ ID NO: 4:

GAATTGTTTA

GGCTCCATTT

TCTTCAAAAA

AAGCAAAGAT

GAGGGGATTC

ATAAAGAAAG

TCTGTTCGAA

TTTTTCTTGT

120 180 240 300 360 420 480 504 WO 98/37214 PCT/IB98/00295 SEQUENCE CHARACTERISTICS: LENGTH: 146 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 AGCATGATGT TGCAGCATCA TTGGCTTTCT TACATGTTCT AATTGCTATT AAGGTTATGC 120 TTCTAATTAA CTCATCCACA ATGCAG 146 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 218 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 98/37214 PCT/IB98/00295 36 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTTTGTTAT TCATACCTTG AAGCTGAATT TTGAACACCA TCATCACAGG CATTTCGATT CATGTTCTTA CTAGTCTTGT TATGTAAGAC ATTTTGAAAT GCAAAAGTTA AAATAATTGT GTCTTTACTA ATTTGGACTT GATCCCATAC TCTTTCCCTT AACAAAATGA GTCAATTCTA TAAGTGCTTG AGAACTTACT ACTTCAGCAA TTAAACAG INFORMATION FOR SEQ ID NO: 6: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 198 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 AATATATACG TCCTGAAAGT ATAAAAGTAC TTATTTTCGC CATGGGCCTT CAGAATATTG GTAGCCGCTG AATATCATGA TAAGTTATTT ATCCAGTGAC ATTTTTATGT TCACTCCTAT TATGTCTGCT GGATACAG 120 180 218 120 180 WO 98/37214 PCT/IB98/00295 37 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 208 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 TTTGTGAGGT AACCAGGGTT CTGATGGATT ATTCAATTTT CTCGTTTATC ATTTGTTTAT 120 TCTTTTCATG CATTGTGTTT CTTTTTCAAT ATCCCTCTTA TTTGGAGGTA ATTTTTCTCA 180 TCTATTCACT TTTAGCTTCT AACCACAG 208 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 293 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 98/37214 PCT/IB98/00295 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GTATGTCTTA CATCTTTAGA TATTTTGTGA TAATTACAAT AGATTCATTC CTCAAAATGA CCTGAACTGT TGAACATCAA TAGTTTGGCT TACTTGAACA AAACAACATG ATGAATGTTT CCATTGTCTA GGGA7 GTCATCTTAA AAAAAACATT GTTTACTTTT TTGT$ TGCAAGTGTG TCTGTTTTGG AGTAATTGTG AAATC INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 376 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO rTTCTA

AGTATA

3TTTGA

AGGGGTTGAA

TTATGTTGCT

GAAGATTACT

TGAACTTGTA

ACATAGAGGA

GAGAACAAAT

GTATAGAGTT

CAG

120 180 240 293 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: WO 98/37214 PTI9/09 PCT/IB98/00295

GTTCAAGTAT

GGAAGTCTAC

CCGAATTTCT

CTCATTTCTA

TCAGGCTACC

TTTGAATCGC

TTGGTTCTGG

GATTTTTGTT

CCACTAAGGC

AATCCACAGC

AGCTTGTTAA

GGATGATAGC

TCGAGATCCA

CTTGATGAGC

ATAATCTAGT

TCATTTCATC

AGTATTAGAT

AGCTTAAGTT

AATTTTTAGA

TTGTTCTACT

TCATTTACAC

GATTCTTTGA

TTGCTTACTT

TATTTTCCAA

TTATTACCGC

AGCTATAGTT

TTTACAATGA

TTCCTCCCCC

CTGCTATATT TGTTGGATAC TTACCTTTTC AGTGATACTA ATTGAAATGG TCTAAATCTG ATATCTATAT TTCTCCGTCT TCATGATGAA ATGCAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 172 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTAAAATCAT CTAAAGTTGA AAGTGTTGGG TTTATGAAGT GCTTTAATTC TATCCAAGGA CAAGTAGAAA CCTTTTTACC TTCCATTTCT TGATGATGGA TTTCATATTA TTTAATCCAA TAGCTGGTCA AATTCGGTAA TAGCTGTACT GATTAGTTAC TTCACTTTGC AG SWO 98/37214 PCT/IB98/00295 INFORMATION FOR SEQ ID NO: 1i: SEQUENCE CHARACTERISTICS: LENGTH: 145 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 AGTTTTATGG AGAAGTAACT GAAACAAATC ATTTTCACAT TGTCTAATTT AACTCTTTTT 120 TCTGATCCTC GCATGACGAA AACAG 145 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 242 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO WO 98/37214 PCT/IB98/00295 41 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GTAAGGATTT GCTTGAATAA CTTTTGATAA TAAGATAACA GATGTAGGGT ACAGTTCTCT CACCAAAAAG AACTGTAATT GTCTCATCCA TCTTTAGTTG TATAAGATAT CCGACTGTCT 120 GAGTTCGGAA GTGTTTGAGC CTCCTGCCCT CCCCCTGCGT TGTTTAGCTA ATTCAAAAAG 180 GAGAAAACTG TTTATTGATG ATCTTTGTCT TCATGCTGAC ATACAATCTG TTCTCATGAC 240 AG 242 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 797 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 WO 98/37214 WO 9837214PCTIIB98/00295

GCGATAGAAG

ATTTGAAAGG

AAAAGGATGC

TGCACCTGCA

CCTGAAGTAA

ACAACAACAT

AAAACTTACT

AAAAAGTCCA

TTGTTTGGGA

GAAAATGGAC

CAGCTTTATG

TTAACTATTG

TAGGAATAGC

CAAAAAAATT

TAAACTTAGG

AGCTAGGAAT

ACCTCGTGTA.

CCTATCTCAG

AAAAGAAGTA

CTGAAGTAGT

AACACAGTTA

TATAGAAAAG

ATTACCGCCA

CGTAATAAGG

CTTCTCTATC

TAAATGATCA

AGTCATATAA

GTCCCACAA-A

AGGTAGAGAG

ACAGAAGTGA

TGTTGTTGTT

TTTTGTGCAA

TTAAATAACT

CAATCGCCAG

TCTACTTTTG

CTCTTTTTCC

AAAATGAAGT

TGTCCACCTT

GTGGTTTCAG

GATTTTTTCA

AAGCAACATG

GA.AACAGTGC

GTCAAAAAAA

AATGAATTTT

TTAAGTCCTC

GCATCTTACT

CTAAACCAGT

TGATGGGAAC

TGGTGTCTGC

GGGGAGGGTA

ATAGACCCTT

TGTAGCTAAA

ATGTAGATGA

TGTACTACTA

GCTAGCAGAA

TGAACTACTA

GTTACAAAAC

GCATGTAGCT

TTAAAACCGC

GCTAACATCA

GAGTGTATGC

GGCTCAAGAA

GCGACCCAAC

ACACATGTCA

TTTCTTTGTG

AAATAGCTTG

120 180 240 300 360 420 480 540 600 660 720 GAGAGAAATT TTTTATATTG AACTAAGCTA ACTATATTCA TCTTTCTTTT TGCTTCTTCT TCTCCTTGTT TGTGAAG INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 2169 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO WO 98/37214 PTI9/09 PCT/IB98/00295 (iv) ANTI-SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ATCATGGCCA ATTACTGGTT GACCGGTCCT ACTACAGACG CTATGGCTAT TTTCGTTAGC CATTTACAGT CAAAATGTTG TTGTCATCTG TACTTTTGCC TTGCCATATT TTGTTCTCTT AGTACACAGA TCTTAACGTT AATTTTGAGT TTATGATTAA CATAAAAATA GCTAATGATA AACGTTAATT TAGTAATTTT CGGTGGATAT TATATTATGA GCAGATTTAA TAATAAAATG GGGATTAGAA CTAGTTATTA AAAGTTTTTC ATTTCATGCT ATTATAGTGT AA.ATTTATGC CAAATGCATT ACTTCCTTTC

ATACTAACCC

TTGGCGTCGG

TATGGTTTTT

TATTACTTGT

ATTATTATTA

TATGTTCAAT

GTTCAATCTT

GAACATTGAC

TGTTACGTAC

GTTGGCATCA

GTAATTAACG

AAAAAATGTA

AAAATTGTTA

ATTCAGTGTA

GTGGAACTGT

TTTGAACATA

GTTTTCCTCA

TTTGAGTTAC

TCATACATAC

CAACTTTTGG

AGAATATGAA

ATTTGGCAGA

GTATATGAAA

GCAAAATCAT

GTCGATATTA

TACTTTAAGT

*ATTATTGTAP

AAATTAAAGI

AGATTCTTTC

TGCATCTGCT

GTTTTTGTTT

ATGATGTTTA

ATGTTAAAAA

ATTATTACAA

AGGCATTGAC

TTTAACATCT

GCTTAGGGTA

TATTGAATTA

TGGTGTAGTT

AAATAACTCT

GATTTGATGG

TGTAGACTGC

ATTGAACTTG

GAGTTCTCAT

TCTTAGAACT

TCAAACTCTT

CAGTGTTGTG

AGTGTTTATT

GGAAAAGACA

AGGTACCACA

ATTATAGATG

TGGTATATCC

ATCACATGAA

GACTGTAGTT

CATTTCAAGT

CATATAATTT

GACTGGAATT

TCTGTTTTAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 AAAATACTTT ATACTTTAAT ATAGGATTTT GTCATGCGAA TTTAAATTAA. TCGATATTGA WO 98/37214 PTI9/09 PCT/IB98/00295 ACACGGAATA CCAAkAATTAA ATAACGTGGA AGTTCAAAGA ATTTGGCCCA CTACTAAATT GAATGATATT CATTTTTCAT CTGAAAAATG CTTGGCCATT TTTAATTTGA CATGTTATTT TTAATATTGT AACTTTGTAA AAAATAAATT ATTTTTTGAC TAAAAACAAA AAACGACTTA TTTTCCAAAT TAAATGCAGA TTTAGCCTAA CCAACGAATA GATATATATA AGTAGTGACA TACCATAATC ATTTGTATTT AACTTTTAAA TATACGTATA AATCCTTAAC TATATCTGCC CATGTATTAT GTATACAAAA AGTAAGTAGA AATATAAAAA AAACTAATGG GGTCTGAGTG GTTTTTTTAT AAAAAGCCAC

AAAGGATACA

AGGTAAAGTT

TGCTTTACTT

CCCATAAGTT

CACAAAGTTT

ATATTAGATG

TTGAGTGTGT

ATTCTAAA-AC

TTTATAAATC

AAATGCATAA

TTTGTAAACT

AATCTTGATT

ATAATTAAAT

TACAAAATAT

TTACCACTAG

AGTCGATTAG

AACTACAATA

AAATATTCAG-

TAAAATGAGC

CATGGCCTTC

TAAGAATAAA

TCTAACATGT

CAATTTGATT

ATCTTAGTTC

ATATAATCCA

CTACATCTTA

ATATGAACCG

CTGACAAATT

CAAGTTGTGC

GTCATACCAC

CTATGAACTT

TGACCCAATA

TTCAATCATT

TTTAAGCAGA ATAAATAGTT

AACAAACAAT

TTTTATACTT

CACAACTTGA

TTAAATATTT

TTTAAATATC

AAAATTATTG

GAGAAAGTAA

ATTACCTAAA

CTAAAAAAAA

AAAGGGGAGG

AAATCA.AGAA

TTTAGATTGC

GATCTTTATA

TTAAAAGGGA

TAATTTGGAG

TTATTTATAC

GCGTTCATAT

AAAACTCTTT

TAGAAATTGT

TATGTTTTAC

ACTAACAAAA

TCAGAACATA

rGAACCTTTG

AATTTCTTTT

CCTCTTAGTT

CCATGATGTT

IATAAGAAGC

GACAAGTGTA

TAAGGTCATT

TATCAATTAT

TCCAACATAT

GCTTATTTTT

TTTACAACAA

GTCAAAATTT

ATATCTAGTA

TAGGTCAATA

ACCAAAAATA

ATAACGAGTA

TTCAATTTCG

GGGTCATAAT

CAAGAAGGCA

1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 WO 98/37214 PCT/1B98/00295 GCAGCTGAAG CAAAGTACCA TAATTTAATC AATGGAAATT AATTTCAAAG TTTTATCAAA

ACCCATTCG

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1165 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES 2160 2169 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTGTCAAAGA AATTCTCGAG GTTACATGGA TATCTTGAGA A TAATTGAACA AGTATATGCA GCATATCCTA ATTTCTGGAC T AATTTGAATT CATAGAAATT ATTGGAGTAG CGTTTGAGCT TI ACACATTCTC AACTATCCGT CTCATAGGAT ACAACATTTT C AAAAATGTAA AAAATAGAAA CATCATGACC AGGTAATCAA AATCTATTAT TGGTACATTT AAAAGGCTAG AAAAAACAAA C TTATAACTTA TTATGTTTCC AGCAAAAGCC ATAACAAATC

CTTAAGAAA

'GACTGGTAG

'CTCAAGGTC

:AATTGCAGT

ACATACTCG

TTCAGTAGC

TTTTACAGTA

CCATAAACTG

CATACAAAGA

TCAACACCAA

TTCGATACGG

TATCTCAGCA

120 180 240 300 360 420 E'TATATAACT TTCACAAAGA WO 98/37214 PTI9/09 PCT/IB98/00295

AACAATTTTT

CTTGACCATG

ACAACAAACA

GAGATAGAGA

CGAATATAAA

GAACAATAAC

AATAACAAAG

TGAAAGGATA

CCTCCATAAC

TCTCAATTTT

TATGGCTCTA

ATCATATCCC

TATTTGTGTT

CAATGTAATC

GATTGTTTCT

GAAGGCATGA

TACGACAAGA

ATCAAATAAC

AGCCAGACAA

CTCCTAGAAC

TGAATTTTAT

CAAGTGTCAT

TGGACATATA

GTAAAAAATC

CCACAAGTGG

AATAGACCCT

TAAAACACTA

TATATAATAC

AAAACAAGAA

CACTCAAATA

ACTCTTTCTA

ACACTCAACC

TTTTCTTCCA

ATGAACCCTT

CACTTATTAT

AGTGTGGTGA

CGGCTAAAGT

AAGGAAGCAT

AATAATCGAA

ACTACCCAAA

CCTAACTAAC

AATATTGTCT

TATGTGTTCA

GTATACATAA

GGACTTTACC

AAAAGCATTT

GCTAGAGCAT

GTACAAGAAA

TAATTCCACG

CTTCTACCCC

TCCCCCACCC

GAACTTTGCC

TTGATTTACA

CCTACCTTAC

CAAAGCAACG

TCTTACCGAG

CAGAAAATAG

ACTACTAGTA

TCATCCGTGT

CCCCTCCATC

480 540 600 660 720 780 840 900 960 1020 1080 1140 1165 ACCTTGCAAA TTTGTCACAT GATACTTACA TATTTGATAT TATAAAAAAT AAAATAAAAA ACTAAGGAGA TGATCCAGAT ATATTGGAAA ATGAAATGCA AAGGCTAAAA ATAATTGAAA TTAACATGAA ATTAGTAAAA ATTAC INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 317 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO WO 98/37214 PTI9/09 PCT/IB98/00295 (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GCAAGCAATG CACCACAGTT AGTTTATATC AAAAAGAAGAA AACTGTTATA TACCACATGA AAGAAGTTGA TAATGTGAAA A GTAATTCAAA TAACAAATGC CCACAGGAGT AAAG ATAAATTGGC GGAACGAAGT AACACATGTT TGAC2 ATGCCATGAG CACCAGTCCA GAAGTTTTCC AACT TAAATTATCA AACATAC INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 504 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES kGCXT(T

C

AGGTATTAA

CACCATGCT

TTTCCCAAG

CACGGTGCA

.TACTATCCA

CGGAGCTAAA

CATAAAGATT

TTAAGGTATT

CAGATCAAAT

TGCAACCATA

120 180 240 300 kCTC C

%TTTAT

A

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: WO 98/37214 PCTIIB98/00295

CTGCAAAAAA

TGAATTTAAT

AATTGTGAAG

TTTGATGCTT

ATATTTTGAA

AGAGAGCAGT

ATTAAGGAAG

CAACAGAAGG

GTATAGTACA

TCTCCTTAGT

TTACACAAGA

CTATTTCGAA

AAGACTTTCT

TGTTGAATCC

GAAAATCTTT

AAAAACTGCT

CAGAAAGAGT

TTATTTCTAC

CCTCAGCTTC

GCTTTGCCAC

AAATCTCAAC

AACTCATGAT

AAAATTGCTT

TTTATGTCTA

TGACACTCCG

CAAACAGCTT

TACTCCCCGG

CAAAATAAGG

AAAAGTATCA

AATAGA-AGGA

TAAGACTATA

TACTTTTTTT

GGGGTGTGTC

TTTAAAAGAT

TATGCAAAAT

TCAAACTGCC

ACTTC1'CA- AAACCTTGTC TACTTTTTTG AAGACCCAAT CAAAAAA.ATG GCCAGGTGCC ACCTAAATGG AGCCACTACT TCTCTAGCAA AGTCAAAGTA GGTATAAACA ATTCATCTTC TAAAGCACAA CTTTTGGCTG TTAC INFORMATION FOR SEQ ID NO: 2.8: SEQUENCE CHARACTERISTICS: LENGTH: 146 base pairs TYPE: nucleic acid STRAN~DEDNESS: single 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 WO 98/37214 PCT/IB98/00295 49 AGCCAATGAT GCTGCAACAT CATGCTTTAA TAGGAAAATC TGTTATGATG ATGGAAACTA 120 CTATTTTGTA GTAGACGAGG ACCTAC 146 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 218 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 GGGAAAGAGT ATGGGATCAA GTCCAAATTA GTAAAGACAC AATTATTTTA ACTTTTGCAT 120 TTCAAAATGT CTTACATAAC AAGACTAGTA AGAACATGAA TCGAAATGCC TGTGATGATG 180 GTGTTCAAAA TTCAGCTTCA AGGTATGAAT AACAAAAC 218 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 198 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/37214 PCT/IB98/00295 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTGTATCCAG CAGACATAAT AGGAGTGAAC ATAAAAATGT CACTGGATAA ATAACTTATC ATGATATTCA GCGGCTACCA ATATTCTGAA GGCCCATGGC GAAAATAAGT ACTTTTATAC 120 TTTCAGGACG TATATATTTG GATTCTATCT AACAATTGTT CTGAGAATTA TTTAGTTGTA 180 GAAATAAATT TAAAATAC 198 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 208 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES WO 98/37214 PTI9/09 PCT/IB98/00295 51 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CTGTGGTTAG AAGCTAAAAG TGAATAGATG AGAAAAATTA CCTCCAAATA AGAGGGATAT TGAAAAAGAA ACACAATGCA TGAAAAGAAT AAACAAATGA TAAACGAGAA AATTGAATAA TCCATCAGAA CCCTGGTTAC CTCAC.AAAGA GTGAGATTTT CCGTGGCTAA CCTATATGAA CCTTAAAATG CAATAGAAAC AGACAAAC INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 293 base pairs TYPE: nucleic acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CTGTACAAGT TCATCAAACA TTTCACAATT ACTCCAAA.AC AGACACACTT GCAAACTCTA TACAGTAATC TTCTATACTA CAA.AAAAGTA AACAATGTTT TTTTTAAGAT GACATTTGTT CTCAGCARCA TAATAGAAAT CCCTAGACAA TGGAAACATT CATCATGTTG TTTTCCTCTA TGTTTCAACC CCTTTGATGT TCAACAGTTC AGGTCATTTT GAGGAATGAA TCTTGTTCAA GTAAGCCAAA CTAATTGTAA TTATCACAAA ATATCTAAAG ATGTAAGACA TAC 120 180 240 293 WO 98/37214 PCT/IB98/00295 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 376 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 TTCAATTAGT ATCACTTCAT TGTAAAGAAA AGGTAAGTAT CCAACAAATA TAGCAGGCTG TGGATTGGTA GCCTGAAACT ATAGCTTCAA AGAATCAACT TAAGCTGCTC ATCAAGGCCT TAGTGGTAGA AATGAGGCGG TAATAAGTGT AAATGAATCT AATACTTGGA TCTCGAAACA AAAATCAGAA ATTCGGTTGG AAAATAAGTA GAACAAGATG AAATGAGCTA TCATCCCCAG AACCAAGTAG ACTTCCAAGT AAGCAATCTA AAAATTACTA GATTATTTAA CAAGCTGCGA TTCAAAATAC TTGAAC INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 172 base pairs 120 180 240 300 360 376 WO 98/37214 PCT/IB98/00295 53 TYPE: nucleic acid STRANDEDNESS: single 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 AATAATATGA AATCCATCAT CAAGAAATGG AAGGTAAAAA GGTTTCTACT TGTCCTTGGA 120 TAGAATTAAA GCACTTCATA AACCCAACAC TTTCAACTTT AGATGATTTT AC 172 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 145 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES WO 98/37214 PCT/IB98/00295 54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTGTTTTCGT CATGCGAGGA TCAGAAAAAA GAGTTAAATT AGACAATGTG AAAATGATTT GTTTCAGTTA CTTCTCCATA AAACTTGTTC AGTACATTAA AAACAAGCAG AGCAATAATT 120 TCATGGATAA GTAAAACATA TATAC 145 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 242 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 TCCTTTTTGA ATTAGCTAAA CAACGCAGGG GGAGGGCAGG AGGCTCAAAC ACTTCCGAAC 120 TCAGACAGTC GGATATCTTA TACAACTAAA GATGGATGAG ACAATTACAG TTCTTTTTGG 180 TGAGAGAACT GTACCCTACA TCTGTTATCT TATTATCAAA AGTTATTCAA GCAAATCCTT 240 AC 242 INFORMATION FOR SEQ ID NO: 27: WO 98/37214 PCTIIB98/00295 SEQUENCE CHARACTERISTICS: LENGTH: 797 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genornic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CTTCACAAAC AAGGAGAAGA

TATAAAAAAT

TTCTATACAT

CTGTGTTGTC

ACTTCAGTCC

TTCTTTTTGG

AGATAGGAGT

ACGAGGTATG

CCTAGCTTTA

AAGTTTATGC

TTCTCTCCAA

AAAGCTGCAC

CATTTTCTGA

CAAACAAGTT

ACTTTTTTTC

AAGTTTTGCA

TTGTTGTTGA

CTTCAGGGCG

AGGTGCAAGC

AGAAGCAAAA

GCTATTTTTC

AAAGAAATAG

CATGTGTTCA

GGGTCGCTTT

TTGAGCCAAG

TACACTCTAC

TGTTAGCGCA

GTTTTAAGTT

TACATGCACT

AGAAAGATGA

TGCTAGCAAA

TAGTACATTT

TCTACATGCA

AGCTACACAT

GGTCTATTGA

CCTCCCCCTG

GACACCAAAG

CCCATCAACT

GGTTTAGGGA

ATATAGTTAG

ATTCATTAGT

CTTAGTTCAA

TATTTAACTT

TTTTGACTTG CACAAAATAA

CTGTTTCA.AC

GTTGCTTTCA

AAAAATCCTC

AAACCACTTT

GTGGACATTA

TCATTTTTGA

AAAAGAGGAT

AACAACAACT

CTTCTGTTAC

TCTACCTCTG

GTGGGACTAC

TATGACTATT

TCATTTACCT

AGAGAAGAAT

120 180 240 300 360 420 480 540 600 WO 98/37214 PCT/IB98/00295 TTTTTTGGCA TCCTTTTGTT TTGTAACAGT AAGATGCCAA AAGTAGACCT TATTACGGCT ATTCCTACCT TTCAAATTAG TAGTTCAGAG GACTTAACTG GCGATTGTGG CGGTAATCAA TAGTTAACTT CTATCGCATT CAAATAACTA TGAACAAAAC CACAATAAAA AGGGAGGTCA CACGGCAAGA ACTGTAC INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 2169 base pairs TYPE: nucleic acid STRANDEDNESS: single 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 TTCAGCTGCT GCCTTCTTGT ATGTTCTGAT TCTTGATTTC CTCATTTTAG TAAAAAAACA TTATGACCCT TTTGTTAGTC CTCCCCTTTC TGAATATTTC CATTAGTTTC GAAATTGAAG TAAAACATAT TTTTTTTAGT ATTGTAGTTT CTACTTACTT ACTCGTTATA CAATTTCTAT TTAGGTAATC TAATCGACTT TAATACATGT ATTTTTGGTA AAGAGTTTTT TACTTTCTCC TAGTGGTAAG

GGTACTTTGC

TGGCTTTTTA

ACTCAGACCC

TTTTATATTT

TTTGTATACA

GCAGATATAG

120 180 240 300 360 WO 98/37214 PT19/09 PCT/IB98/00295 TTAAGGATTT ATTGACCTAA TATGAACGCC AATAATTTTA

TTAAAAGTTT

ATTATGGTAA

ATATATATCT

TAGGCTAAAA

TTTGGAAAAA

TTGTTTTTAA

ATTTATTTTA

CAATATTAAT

CAAATTAAAG

ATTTTTCAGA

ATATCATTCA

GGGCCAAATA

CCACGTTATC

ATTCCGTGTT

AAGTATTTTC

CACTATAATA

ACTAGATATG

AATTTTGACC

TGTTGTAAAT

AAAATAAGCT

TATGTTGGAG

TAATTGATAA

ATGACCTTAA

ACACTTGTCT

CTTCTTATAA

ACATCATGGG

ACTAAGAGGG

AAAGAAATTA

AAAGGTTCAC

CAATATCGAT

TAAAACAGAC

ATTCCAGTCG

AATTATATGC

TATAAATAAG

TCCAAATTAA

CCCTTTTAAT

ATAAAGATCA

CAATCTAAAA

ACTATTTATT

ATGATTGAAT

ATTGGGTCAT

AGTTCATAGG

TGGTATGACA

CACAACTTGA

ATTTGTCAGT

GGTTCATATG

TAATTTAAAT

AAGTTCAATA

CAGTCTACAT

CATCAAATCA

ATATTTAAAA

AATATTTAAA

CAAGTTGTGA

AGTATAAAAT

TTGTTTGTTG

CTGCTTAAAG

AAGATGTAGA

GGATTATATC

AACTAAGATA

ATCAAATTGA

CATGTTAGAA

TTATTCTTAA

AAGGCCATGT

TCGCATGACA

CTTTAATTTT

TACAATAATT

CTTAAAGTAT

TATTTTGTAT

TTTAATTATA

ATCAAGATTT

GTTTACAAAT

TATGCATTTT

ATTTATAA-AT

TTTTAGAATG

CACACTCAAT

ATCTAATATA

AACTTTGTGA

ACTTATGGGA

AGTAAAGCAA

ACTTTACCTT

GTATCCTTTT

AAATCCTATA

ACACTGAATG

AACAATTTTA

ACATTTTTTT

ATACGTATAT

AATACAAATG

GTCACTACTT

ATTCGTTGGT

CTGCATTTA.A

AAGTCGTTTT

TCAAAAAATA

TACAAAGTTA

AATAACATGT

ATGGCCAAGC

TGAAAA-ATGA

ATTTAGTAGT

CTTTGAACTT

TAATTTTGGT

TTAAAGTATA

CATAAATTTA

GCATGAAATG

AATAACTAGT

420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500

AAAAACTTTA

TCTAATCCCA CTTGAAATGA GAGTTATTTT AATATCGACC GTTAATTACC ATTTTATTAT WO 98/37214 PTT9/09 PCT/IB98/00295

TAAATCTGCA

TATCCACCGT

ATTAACGTTG

ATTTTTATGC

CTCAAAATTT

CTGTGTACTT

ATATGGCAAA

AGATGACAAC

CTGTAAATGA

TAG CCATAGA

ACTACAGTCA

TCATGTGATT

GATATACCAT

ATCTATAATA

GTGGTACCTG

GTCTTTTCCT

ATAAACACTT

ACAACACTGT

AGAGTTTGAA

GTTCTAAGAA

ACTACACCAA

AATTCAATAT

ACCCTAAGCT

GATGTTAAAT

TCAATGCCTC

TGTAATAATG

TTTTAACATG

AAACATCATT

AACAAAAACT

GCAGATGCAA

TGATTTTGCT

TTCATATACG

CTGCCAAATG

TCATATTCTA

CAAAAGTTGA

TATGTATGAT

TAACTCAAAA

GAGGAAAACA

ATGTTCAAAC

CAGTTCCACG

GATGCCAACT

TACGTAACAA

TCAATGTTCT

AGATTGAACT

TTGAACATAA

AATAATAATA

CAAGTAATAG

AAAACCATAC

CGACGCCAAG

GGTTAGTATC

CATAATATAA

AA.ATTACTAA

ATCATTAGCT

TAATCATAAA

ACGTTAAGAT

AGAGAACAAA

GCAAAAGTAC

AACATTTTGA

CTAACGAAAA

GTCTGTAGTA

1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2169 GGACCGGTCA TGAGAACTCG AAAGAATCTG AAAGGAAGTA ATGCATTTGA ACCAGTAATT

GGCCATGAT

INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 11469 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 98/37214 PT19/09 PCT/IB98/00295 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ATCATGGCCA ATTACTGGTT CAAATGCATT ACTTCCTTTC AGATTCTTTC GAGTTCTCAT GACCGGTCCT ACTACAGACG CTATGGCTAT TTTCGTTAGC CATTTACAGT CAAAATGTTG TTGTCATCTG TACTTTTGCC TTGCCATATT TTGTTCTCTT AGTACACAGA TCTTAACGTT AATTTTGAGT TTATGATTAA CATAAAAATA GCTAATGATA AACGTTAATT TAGTAATTTT CGGTGGATAT TATATTATGA GCAGATTTAA TAATAAAATG GGGATTAGAA CTAGTTATTA AAAGTTTTTC ATTTCATGCT ATTATAGTGT AAATTTATGC

ATACTAACCC

TTGGCGTCGG

TATGGTTTTT

TATTACTTGT

ATTATTATTA

TATGTTCAAT

GTTCAATCTT

GAACATTGAC

TGTTACGTAC

GTTGGCATCA

GTAATTAACG

AAAAAATGTA

AAAATTGTTA

GTGGAACTGT

TTTGAACATA

GTTTTCCTCA

TTTGAGTTAC

TCATACATAC

CAACTTTTGG

AGAATATGALA

ATTTGGCAGA

GTATATGAAA

GCAAAAT CAT

GTCGATATTA

TACTTTAAGT

*ATTATTGTAP

TGCATCTGCT

GTTTTTGTTT

ATGATGTTTA

ATGTTAAAAA

ATTATTACAA

AGGCATTGAC

TTTAACATCT

GCTTAGGGTA

TATTGAATTA

TGGTGTAGTT

AAATAACTCT

GATTTGATGG

TGTAGACTGC

TCTTAGAACT

TCAAACTCTT

CAGTGTTGTG

AGTGTTTATT

GGAAAAGACA

AGGTACCACA

ATTATAGATG

TGGTATATCC

ATCACATGAA

GACTGTAGTT

CATTTCAAGT

CATATAATTT

GACTGGAATT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 ATTCAGTGTA AATTAAAGT ATTGAACTTG TCTGTTTTAG AAAATACTTT ATACTTTAAT ATAGGATTTT GTCATGCGAA TTTAAATTAA TCGATATTGA WO098/37214 PTI9/09 PCT/IB98/00295 ACACGGAATA CCAAAATTAA ATAACGTGGA AGTTCAAAGA ATTTGGCCCA CTACTAAATT GAATGATATT CATTTTTCAT CTGAAAAATG CTTGGCCATT TTTAATTTGA CATGTTATTT TTAATATTGT AACTTTGTAA AAAATAAATT ATTTTTTGAC TAAAAACAAA AAACGACTTA TTTTCCAAAT TAAATGCAGA TTTAGCCTAA CCAACGAATA GATATATATA AGTAGTGACA TACCATAATC ATTTGTATTT AACTTTTAAA TATACGTATA AATCCTTAAC TATATCTGCC CATGTATTAT GTATACAAA.A AGTAAGTAGA AATATAAAAA

AAAGGATACA

AGGTAAAGTT

TGCTTTACTT

CCCATAAGTT

CACAAAGTTT

ATATTAGATG

TTGAGTGTGT

ATTCTAAAAC

TTTATAAATC

AAATGCATAA

TTTGTAAACT

AATCTTGATT

ATAATTAAAT

TACAAAATAT

TTACCACTAG

AGTCGATTAG

AACTACAATA

CATGGCCTTC

TAAGAATAAA

TCTAACATGT

CAATTTGATT

ATCTTAGTTC

ATATAAT CCA

CTACATCTTA

TTTAAGCAGA

AACAAACAAT

TTTTATACTT

CACAACTTGA

TTAAATATTT

TTTAAATATC

AAAATTATTG

GAGAAAGTA-A

ATTACCTAAA

*CTAAAAAAAA

*AAAGGGGAGG

ATATGAACCG

CTGACAAATT

CAAGTTGTGC

GTCATACCAC

CTATGAACTT

TGACCCAATA

TTCAATCATT

ATAAATAGTT

TTTAGATTGC

GATCTTTATA

TTAAAAGGGA

TAATTTGGAG

TTATTTATAC

GCGTTCATAT

AAAACTCTTT

TAGAAATTGT

TATGTTTTAC

ACTAACAAAA

TGAACCTTTG

AATTTCTTTT

CCTCTTAGTT

CCATGATGTT

TATAAGAAGC

GACAAGTGTA

TA7AGGTCATT

TATCAATTAT

TCCAACATAT

GCTTATTTTT

TTTACAACAA

GTCAAAATTT

ATATCTAGTA

TAGGTCAATA

ACCAAAAATA

ATAACGAGTA

TTCAATTTCG

GGGTCATAAT

1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 AALACTAATGG GGTCTGAGTG AALATATTCAG GTTTTTTTAT AAAAAGCCAC TAAAATGAGG AALATCAAGAA TCAGAACATA CAAGAAGGCA WO 98/37214 WO 9837214PCTJIB98/00295 GCAGCTGAAG CAAAGTACCA TAATTTAATC ACCCATTCGA GGATCTTTTC CATCTTTCTC D TAATTTCATG TTAATTTCAA ATCATCTCCT TAGTTTTTTA CTTGTAGAGC CATATGTAAG TTCAAAAATT GAGAGATGGA GGAGGTTATG GAGGACACGG GGCTTATCCT TTCATACTAG TGATCTTTGT TATTCTATTT CGTAGTTATT GTTCCTCGGT CTTCTTTATA TTCGCGTTGC AATCTCTCTA TCTCGTAAGG ATTGTGTTTG TTGTTGTAAA AATACATGGT CAAGGGCAAA TGATAAAAAT TGTTTCTTTG

TTATTTTTAG

TTTTATTTTT

TATCATGTGA

GGGGGGGTGG

ATGAGGGGTA

TAGTCGTGGA

TCTGTTTCTT

AAGAATGCTC

TTTGAAATGC

TAGGGGTAAA

TCAATTATGT

GTTCTGAACA

TGAAAGTTAT

P.ATGGAAATT

ACCTAAAGTT

CCTTTGCATT

TATAATATCA

CAAATTTGCA

GGGAAGACAA

GAAGGTTAGT

ATTATTTGGG

GTACTTCGAT

TAGCATGCTT

TTTTACTTTA

GTCCTCACCA

ATACATAATA

CATAAAGGGT

ATAAGATTTG

GAAGTTTGTIl

TGTTTTGATI

AATTTCAAAG

TCTTCAGGGG

TCATTTTCCA

AATATGGAAG

AGGTGGTTGA

TATTTAGAA.

TAGGTATTTG

TAGTTTCTTG

TATTGTATTA

CCTTTAGTGT

GCCGAGGGTC

CACTCCACTT

AGTGGATTTT

TCATTATATG

TTATGGCTTT

TTTTCTAGCC

ACCTGGTCAT

TTTTATCAAA

TAATTTTTAC

ATATATCTGG

AAAATGACA

GTGTATAAAA

GAGTGTTCTA

AGTGTTGTCT

TTTTGTTATT

TATATCTTGT

TTTATCATGC

TATTAGAAAC

GTGGGATTAC

TTACAACACA

TCCAGGGATA

TGCTGGAAAC

TTTTAA-ATGT

GATGTTTCTA

2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 ATAATAAGTT ATAATGCTGA GATAGCTACT

ACCAATAATA

TTTTTTACAT

GATTCCGTAT

TTTTTTGGTG

CGAACGAGTA

TTGAACTGCA ATTGAAAATG TTGTATCCTA TGAGACGGAT AGTTGAGAAT GTGTTCTTTG TATGGACCTT GAGAAGCTCA AACGCTACTC CAATAATTTC 34 3240 WO 98/37214 PT19/09 PCT/IB98/00295 62 TATGAATTCA AATTCAGTTT ATGGCTACCA GTCAGTCCAG AAATTAGGAT ATGCTGCATA TACTTGTTCA ATTATACTGT AATTTCTTTG ACAGGCTTCT TGAAGTTTGG ATCTCAGGAA GAAAAGATGA AAGGGTATGT TGGAAAACTT CTGGACTGGT AAACATGTGT TACTTCGTTC TACTCCTGTG GGCATTTGTT ATCAACTTCT TTCATGTGGT TGATATAAAC TAACTGTGGT TTTGACCGAT GACGACAATT TATTGGCCTC CTAAATTTGG AATGAAGAGA TATGTGGATC ATTTGCTCAA GGTAACAGCC TGAATTGTTT ATACCTACTT TGGCTCCATT TAGGTGGCAC GTCTTCAAAA AAGTAGACAA AAAGCAAAGA TTTTCACTAA TGAGGGGATT CAACATGTAC

AAAATTTCTT

A.GAAATAAGA

CGGTCTTGGG

TTGATA.ATTT

GCTCATGGCA

CGCCAATTTA

ATTTGAATTA

ATATAACAGT

GCATTGCTTG

CGACAATGGC

ATCCAACTTT

AGAAAATGCT

AAAAGTTGTG

TGACTTTGCT

CTGGCCATTT

GGTTTTTGGA

GGAGAT'rCAA

TATACAAGCA

ALAGTTCTCAA

TATGTTTTCC

PTATTTCTTC

ATATGGTTGC

TATTTGATCT

TAATACCTTA

CAATCTTTAT

TTTTAGCTCC

CATGAAGCAC

ACCCCTAGAG

GGAACCTTAT

CATTGAAAAA

CTTTAGGCAG

AGAGAATTTT

TTTTGATCTT

GAAGTGACAC

GATATCCATG

TTCTCAACAT

CACCCCAAAA

ATGGATAGTA

GTGCACCGTG

ACTTGGGAAA

GAGCATGGTG

GTTAATACCT

AGTTCAGCTA

GAAGATGTCA

CTAGATCACT

TATGAGGGAC

TTTGACCTTA

GCATACCGGG

TTAAAAAGCT

ACCCCCGGAG

TAACCTCGAG

AGTACTGGAC

TCAAGAGTTA

TATAAATAGT

TGGAGATGTC

GACAGCTCTT

TTTTCACATT

TTCTTCTTTT

TTTCCGCTGT

AGACTGAAAA

TCAGACACAG

CCCTTGAGGA

TTTTGGAAGA

GAGTAAGTAG

GTTTGATTGG

TGTCAGTGGC

ATAAAGAAGC

ATTTTGTAGA

3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 AATATAAAAA AAGTATAGAC TCAAATATAG TCTTAPAGCA WO 98/37214 PT19/09 PCT/IB98/00295 AATAAAGAAA GTCTTCCTTC TTCTGTTCGA AATAGCTTCC TTTTTTCTTG TGTAAACTGC GGAAGATGGT TGCATAGTCT CTACAAAkATA GTAGTTTCCA CATCATTGGC TTTCTTACAT CCACAATGCA GGGAAGCAGA ATGATGGAGA AGGACCAGTT CCAGTCATTC CACACAACTC GTAGATCGTA TCCCTGCTTG CCATATGATG GTGTCTACTG GAAGCTGAAT TTTGAACACC TTATGTAAGA CATTTTGAAA TGATCCCATA CTCTTTCCCT TACTTCAGCA ATTAAACAGG CACGAATCTA TGAAGCACAT GTGAGTTTGC AGATGATGTT TGATGGCCAT AATGGAACAT

TGTTGCTTCA

TTAATATTAA

TCTCTTTTTT

ATCGTGAATG

TCATCATAAC

GTTCTAATTG

AGTTATTGGC

TGGTGTTTGG

CAGAGTTAAG

GATAAAGTAT

GGACCCACCA

ATCATCACAG

TGCAAAAGTT

TAACAAAATG

TACCACTTCA

GTCGGCATGA

TTACCTCGGA

TCTTACTATG

CAATTTCCTT CTATTATCAT

ATTCATGATA

TGCAGGTTAT.

GGCTCCTGCT

AGATTTTCCT

CTATTAAGGT

GATTTCAATG

AGTATTAGAA

TTTCGTTTCA

GCCACTGCAG

CCTTCAGAAA

GCATTTCGAT

AAAATAATTG

AGTCAATTCT

AATACCCTCG

GCAGCTCTGA

TTAAGGCAAA

CTTTTGTTGA

TTAAAATTTG

GCTCAGTAGG

ATTAAAGCAT

TATGCTTCTA

GATGGAACGG

TTCCTGATGT

AACATGGTAA

ACGCCACAAA

GGTTTTGTTA

TCATGTTCTT

TGTCTTTACT

ATAAGTGCTT

CCCTCCCAAA

GCCACGTGTA

TAACTATAAT

GAGTTACTCT

GATTTAGCAG

GATTCA-ACAG

TCCTCGTCTA

GATGTTGCAG

ATTAACTCAT

TTCTAACCAC

TGACAGTAAG

TGGAGTGTGG

GTTTGCAGCA

TTCATACCTT

ACTAGTCTTG

AATTTGGACT

GAGAACTTAC

CCCCGAGCCC

AATTCGTATC

ACTGTCCAGT

ACAAACTTTT

4440 4500 4560 4620 4680 4740.

4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 GATCATTTGG ATATCATGTT TTGCTGTGAG CAGTAGATAT GGAAACCCGG AGGACCTAAA GTATCTGATA GATAAAGCAC WO 98/37214 PT19/09 PCT/IB98/00295 64 ATAGCTTGGG TTTACAGGTT CTGGTGGATG TAGTTCACAG TCATGCAAGC AATAATGTCA CTGATGGCCT CAATGGCTTT GAGAGCGAGG GTACCATAAG TTCTTCGTTT CCTTCTTTCC TTCGATTTGA TGGAATAACT CAGGAAACTA TAATGAGTAT TGTTGGCCAA TAATCTGATT TTTCTGGTAT GCCGGGCCTT GCCTGGCAAT GGCAATCCCA ATTGGTCCAT GAAGGAAGTA 3ATATTGGCC rTGTGGGATA C

AACTTGAGGTC

LCTATGCTGT2

TTCAGCGAGG

CACAAGATTT

GGCCGGCCTG

GATAAGTGGA

ACATCGAGTT

GATCAGGTAT

AATCCAAATA

ATATTGGTAG

TCCTATTATG

ATGGACAAAG

CGAGGAATTG

TAGCCACGGA

AGGTTCTCA2

CAGGCTGTT

3GTGGCTAGA

%TGTTCATCA

CTACAGATGT

TCCCAGATGC

TTTCTGAGGG

TAGATTATTT

TGACAAATAG

TTTAAATTTA

TATACGTCCT

CCGCTGAATA

TCTGCTGGAT

AGATGTATTC

CGCTTCACAA

AAATCTCACT

kGAATCCTAC

CAACTATGCC

%GAGTATAAC

TGGAATCAAT

TGATGCTGTG

AACTGTTATT

AGGAATTGGT

AAAGAATAAG

GAGATATACA

TTTCTACAAC

GAAAGTATAA

TCATGATAAG

ACAGTCTATT

TGGCATGTCT

TTTCATGCTG

AATTGGGAGG

TTTGACGGAT

1TGGGATTTA

GTCTATTTAA

GCCGAAGATG

TTTGTTTACC

AATGATGAAG

GAGAAGTGTA

TAAATAATTC

AAGTACTTAT

TTATTTATCC

GTTGGTGACA

TGCTTGACAG

5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660

TAGCATATGC

TCAGAACAAT

TTTCGCCATG

AGTGACATTT

AGACCATTGC

GGAGACCCAT

TGTTAGATAG

GGCCTTCAGA

TTATGTTCAC

ATTTCTCCTA

ATGCTTCTCC

ATTTTAAGGT

TCTGATGGAT

TGTTGTTGAT

TCATATAGGT

TATTCAATTT

GGTTTGTCTG TTTCTATTGC CTTTGTGAGG TAACCAGGGT TCTCGTTTAT CATTTGTTTA TTCTTTTCAT GCATTGTGTT TCTTTTTCAA TATCCCTCTT ATTTGGAGGT AATTTTTCTC ATCTATTCAC TTTTAGCTTC WO098/37214 PCT/IB98/00295 TAACCACAGA TGATCCATTT TTTCACAATG GCCTTGGGAG GAGAGGGGTA CCTCAATTTC 6720 ATGGGTAACG AGGTATGTCT TACATCTTTA 3 CTTACTTGAA CAAGATTCAT AAACATAGAG GAAAACAACA CTGAGAACAA ATGTCATCTT CTGTATAGAG TTTGCAAGTG TACAGTTTGG CCATCCTGAG ACAAATGTAG ACGCCAGTGG TATTTTGAAT CGCAGCTTGT TACTTGGTTC TGGGGATGAT TCTGATTTTT GTTTCGAGAT CTACCACTAA GGCCTTGATG ACCAATCCAC AGCCTGCTAT CTAATTGAAA TGGTCTAAAT GAAATGCAGT TTATGAATGC TTCCTCGCAT CAGGAAAACT

TCCTCAAAAT

TGATGAATGT

AAAAAAAACA

TGTCTGTTTT

TGGATTGACT

AACCTCGCGG

TAAATAATCT

AGCTCATTTC

CCAAGTATTA

GATATTTTGT

GACCTGAACT

TTCCATTGTC

TTGTTTACTT

GGAGTAATTG

TCCCTAGAGA

ATAGCGAACA

AGTAATTTTT

ATCTTGTTCT

GATTCATTTA

GATAATTACA ATTAGTTTGG

"TTGAACATC

rAGGGATTTC

"TTTGTAGTA

TGAAATGTTT

GGGCAATAAT

CTTGAGATAC

AGATTGCTTA

ACTTATTTTC

CACTTATTAC

TGAAGCTATA

TTCTTTACAA

TCTTTCCTCC

CGCTCGATGA

'AAGGGGTTG

TATTATGTTG

TAGAAGATTA

GATGAACTTG

TGGAGTTATG

AAGGTTCAAG

CTTGGAAGTC

CAACCGAATT

CGCCTCATTT

GTTTCAGGCT

TGAAGTGATA

CCCTCATGAT

AAAGTTCTCA

GGTAAAATCA

ACAAGTAGAA

6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 AGCAGCTTAA GTTGATTCTT

ATTTGTTGGA

CTGATATCTA

ATTTGATAGA

GATAGTAAGC

TACTTACCTT

TATTTCTCCG

GCTATGAATT

AGCATGGATG ATGATAATAA TCTAAAGTTG AAAGTGTTGG GTTTATGAAG TGCTTTAATT CTATCCAAGG ACCTTTTTAC CTTCCATTTC TTGATGATGG ATTTCATATT ATTTAATCCA ATAGCTGGTC AAATTCGGTA ATAGCTGTAC TGATTAGTTA CTTCACTTTG CAGGTTGTTG TGTTTGAACG 7800 WO 98/37214 PT19/09 PCT/IB98/00295 TGGTGACCTG GTATTTGTAT TCAACTTCCA CCCAAPAGAAC ACATACGAAG GGTATATATG TTTTACTTAT CCATGAAATT ATTGCTCTGC D GAGAAGTAAC TGAAACAAAT CGCATGACGA AAACAGGTAT TGGACAGTGA TGCTTGGGAJ4 TTTGATAATA AGATAACAGA CTCATCCATC TTTAGTTGTA CCTGCCCTCC CCCTGCGTTG CTTTGTCTTC ATGCTGACAT TTTCACATCA CCAGAAGGAA TTCCTTCAAA GTGCTGTCTC CCCTTTTTAT TGTGGTTTTG ACCGCCACAA TCGCCAGTTA AATAAGGTCT ACTTTTGGCA CTCTATCCTC TTTTTCCCTP ATGATCAAAA ATGAAGTTG; CATATAATGT CCACCTTTGC CCACAA.AGTG GTTTCAGGG(

CATTTTCACA

AAAGTTGGAT

TTTGGTGGCC

TGTAGGGTAC

TAAGATATCC

TTTAGCTAAT

ACAATCTGTT

TACCTGGAGT

CTGCGCGAAC

TTCATAGTTA

AGTCCTCTGA

TCTTACTGTT

LAACCAGTGCA.

STGGGAACTTz

'TGTCTGCGCJ

3GAGGGTAGAC

TTGTTTTTAA

TTGTCTAATT

GTGACTTGCC

ATGGAAGAGT

AGTTCTCTCA

GACTGTCTGA

TCAAAAAGGA

CTCATGACAG

TCCAGAAACA

ATGTGTGGTA

TTTGAATGCG

ACTACTAATT

ACAAAACAAA

TGTAGCTTGC

AAACCGCCCI

AACATCAACI

;TGTATGCAA-i

TGTACTGAAC

TAACTCTTTT

AGGGAAGTAC

AAGGATTTGC

AAGTTTTATG

TTCTGATCCT

AGAGTTGCAC

TTGA.ATAACT

CCAAAAAGAA CTGTAATTGT

GTTCGGAAGT

GAAAACTGTT

ACTGGTCATG

AATTTCAATG

CAGTTCTTGC

ATAGAAGTTA

TGAAAGGTAG

AGGATGCCAA

ACCTGCATAA

GAAGTAAAGC

ACAACATACC

GTTTGAGCCT

TATTGATGAT

ATGTTGACCA

GTCGTCCAAA

CGTGTGACCT

ACTATTGATT

GAATAGCCGT

AAAAATTCTT

ACTTAGGTAA

TAGGAATAGT

TCGTGTAGTC

7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 ACTTACTCCT ATCTCAGAGG TAGAGAGGAT TTTTTCAATA GACCCTTGGC TCAAGAAAAA AAGTCCAAAA AGAAGTAACA 84 8940 WO 98/37214 PTI9/09 PCT/IB98/00295 GAAGTGAAAG CAACATGTGT AGCTAAAGCG ACCCAACTTG TTTGGGACTG AAGTAGTTGT TGTTGTTGAA ACAGTGCATG TGTGCAAGTC AAAAAAATGT AATAACTAAT GAATTTTGCT TAAGCTAACT ATATTCATCT TACAGAGTTG ATGAACGCAT CTACTACCAA CAGCCAATAT AATATCAGTA ACATTGACGA AGTGAGCTAC TACCAACAGC TCTACAAATA TCAGTAACAT GAACTTAAAG ATTCACCGTC GATTCAGATG CAAACGTCTG ATTGGTGATC GCTATCCTTG CATGATAAAA AGTCTGATTT AAGGCGACTC CTGGACTCGA ATAAGATAAC AAAGGCAATI TAGATGAACA CATGTCAGAA

ACTACTATTT

AGCAGAAAAA

TTCTTTTTGC

GTCAGAAACT

CGAGGAGAGT

ACGCATGTCA

CAATATTGAG

TGATCAGACT

TGTAAGCATC

GGGTGAGGAC

CTCTCTGAGA

TATGATCGCT

ATCTATAAGA

CTTTGTGCAG

TAGCTTGGAG

TTCTTCTTCT

GAAGATTACC

GACGAGAAAC

GAAACTGAAG

GAGAGTGACG

GTTGTAGTTT

ATTAGTGATG

TAGTCAGATG

AATAGGTGAG

ATCCTCGCTC

TAACAAAGGC

A.ATGGACAAC

CTTTATGTAT

A.GAAATTTTT

CCTTGTTTGT

AGACAGACAT

TTAAAGATTC

TTTACCAGAC

AGAAACTTAA

CTGTTGAGGA

TTGTTCCAGC

ATTGATCGAC

GCGAAACAAA

TCTGAGAAAG

GACTCCTGGG

A.CAGTTATTT

PGAAAAGTTA

TATATTGAAC

GAAGGCTTAT

TTGTAGTGAG

GTTATCTACA

AGACATTTCT

AGATTCGTTA

GAGAGACAAG

TGAATGGGAT

CCTTCTACCG

AAATAATTTG

AAGCGAAACA

ACTCGAATCT

AAGAATGATT

TATATAAGTC

9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 CCAAGACTTG AATCTATAAA AAATTTAGTT AACGTCCGAT CCTAATTCGA ATCGAGGCAT CTTACCACTC CATTGATAAT

AATAAGTCAT

AATTGTGTTC

ATAAAGTATT

ATATGTAACA

AAAAACTAAA

TTTTTGTTGT

TTGACTTGAT

CACAATTAGC

CGGTCTATCA

TTAATTACAT

AAAATAGATA

CTTTCATGTG

WO 98/3721 4 PCT/IB98/00295

CAATAACAAA

TAATTACATC

ATACACAACC

CGTAAAAATG

TACAAGTAAA

ATCATTAACA

ATAAAAAAA

AACGATGAA

GAAATGATAG

TTTCATTTGC

TAGGCCAACA

AATAAATGCG

AATAAATTTA

AACAAACAGA

ATACCAAATT

TGGACCAGTT

GAACACGATC CTTTGCACCC TCATCCCAGT GTACAACAGC CTTATTCTTA TCTGCCACAA ACAA.GTAGGA AGGAGAGGAG ATTTTTCGTA AGATCCAATT TCTCCTCTCG ACGACCTCTT TAACAAGTAT TGCAAGGGGG GAGTGATAGT CTCGAATATI TACGTCTTTT ACGCTCGCCT GTGGAAGGTT CAAGTTACTC

GAATTTAGAG

AATAACAAAG

TCGAAAGCAT

ACATAAAAAC

ACAAATGTAA

ATTAAAAAGA

CATAATGCAA

GGATCGACTG

GTTCGATGAT

CCCAACATCT

AATAATCGGT

TCATCCAAAT

TCAACAAACT

GCATTGAACG

GGACAGTAGC

ATTCACCTCT

ATTTCTTTT7

GATTCGTGAI

ATTCCAATTT

AAATGATAGG

AACTGTAAAC TCATGCATGA

AAATTGCATG

CTTAACTACA

AAAAAACATA

GGAAAACGAA

CCTGCACAAC

TATCAGTATG

GCCCCAAGTA

TTCACACTAT

AAACGGTGCA

TTTCTTCAAG

ATCTCCGCTT

TATTAAGTTA

TTAGCATTAC

TATCATTAAT

AGTAAAAATA

CTAAATCTTA

ACGCGTCCTG

GTTAGGTATG

TTCACAAAAA

ACAAAAAACA

TCTCTTGTTA

CGTTCTTTGA

TCAAA.ATTCC

ATCATGAAAA

GTCGGCCCAA

CCGGTCTGGC

TTTTGTTGCC

AATTTAGAGA

A.CAATTAACT

I'CCAGTGTCA

AGAAATCAGT

GTGACTTAAC

AATTGCTTCT

CCGTCATTCG

ATCGGGTATC

CCAAAAAAAA

AAACTTAAGT

ACCAATTTAT

TACAAAATTG

GAAAAGTCTT

TGATAGTGTA

GTTGCTTGGA

GGAAATGGAG

TTTAAGGAGT

ATCGCGAGTT

AGAGATTCGA

10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 TTAGAATGGT TGGTGTCAAA TTTCAAGTAT GAGTGGTGAG TATTTTCACG AGGTGTATTC GAGGTCTAGT AGAACGAAGG GTGTCACTAA TGAAAGTTTC WO 98/37214 PCT/IB98/00295 69 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 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGAATTCCAG TCGCAGTCTA CATTAC 26 INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/37214 PCT/IB98/00295 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: CGGGATCCAG AGGCATTAAG ATTTCTGG 28 INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CGGGATCCAA AGAAATTCTC GAGGTTACAT GG 3: WO 98/37214 PCT/IB98/00295 71 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CGGGATCCGG GGTAATTTTT ACTAATTTCA TG 32 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES WO 98/37214 PCT/IB98/00295 72 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CGGGATCCCG TATGTCTCAC TGTGTTTGTG GC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGGGATCCCC CTACATACAT ATATCAGATT AG INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" WO 98/37214 PCT/IB98/00295 73 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: CCATCGATAC TTTAAGTGAT TTGATGGC 28 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Synthetic DNA Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: CGGGATCCTG TTCTGATTCT TGATTTCC 28 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: 2122 base pairs WO 98/37214 WO 9837214PCT/1B98/00295 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

GTATGTCTCA.

TAATTGGGGC

GCAAAATGAT

GTGTAATTGA

TGGGGATTCT

AAAATGATGA

GTAATTGAGT

CTGTGTTTGT

TCTTTAAAGT

GAATCTTGAT

GTTTGGCTTG

GGCTGTGTGT

TGGTATTGTG

TGACATTAGT

TGTGTCTGTG

3TTTTTTTCT

TATACCCTTT

AAGGGTTGTA

TGTCGAGGTT

ACCCTTTTGA

AGGTTGTAGC

TTTTGGAATC

CTGTCTTTTT

TGAGTATAGT

ACTTTTTGAA

ATTTTTTTGG

GTATAGTCTT

TTTTTGAAGT

CTGATGTGTG

GTGTTTTGTG

CTTTGAGGAA

GTTTGGTTAG

TTTGTGTTAT

TGAGGAAGCA

GTGGTTAGGT

TCAAGTCCTG

GTTGGTATTA

TAAAAGTTGG TATTGTGTAT

ATCTTGATTG

TTGGCTTGTG

GCATTAGTAA

TGTCTGTGTG

120 180 240 300 360 420 480 540 600 660 720 ATATGGGTCG AGGTTCTTTC TTTGGTTTGT GTAATTGGGG GTTCTTAAAA

TGTACCTTTT

ATTCTTTGAG

AGGTAGCAAC

TGGGGTTCTT

TAAGAATAGT

AAAGCAAAAA

TTTTTCAACT

TGAAGTTTTG

GTCTGAGAAA GCAAAATCGA TGAATTTTGA ATGGTGAGTT TTCATGGAGA AACTTGATTG CCTGATATGG GTCAAGGTTC TTTGTTTGGT AGAAAGAAAA ATTATGATTT TTCATGGAGA

TTGACAGCAT

ACATTACTAA

TTGTGTAATT

AATTTGATTT

WO 98/37214 PT19/09 PCT/IB98/00295 ACATTAATAA AGGTAGTAGC TAAAGGGGCC CTACATATGG ATAAGAATCA TAGTGTTAGG ACTAGAGGAG TGATCTTGAC TGGTGTTATA TGTGCAAGGA CTTTCCATGA GGTTATGATG GAACTGTGAA AGAATTGATA CACTTGGTTC CATTAGCTTT TTGAGGGGGT AGAGTTGAGC TTTTTTTGTA CACCCATAGA ATCATCTTTT GTAGTAGATT TGACTATGTT TATGAATGAA TGTACCTTTG TAGACAATGT GAAGGAATAG TTTGGTTGAT GGCCATTTTA AATCCTTTGA AGCACCTCTT GTATGGTGCT GAATCAGTGT GTGTACTTTI

TTTTTAAAGT

TGCTTTCTGG

ATCTTTTTTC

GGCGGAAAAT

GACGGGAGAT

TGATATGTTT

TTCAGTTGGA

AATGTGGGTG

TTTCCTCAGT

ATTCCCAATT

CTTTACCTTT

TATACATTAC

TGTTGCAGCA

ATTGATTATT

.CATTGTTAAA

ITTCTGGAGTG

GTGGTCAGCT

TGAGATATTT

~TTTTTTTTTT

CTTAGAAAGG

GATGTAGATC

GAATGGTTTG

AGTGTGGAGT

GTGTGGAGAG

TGAGAAGTAG

GTATAGAAGA

TGGTATATCC

TTGAAAAAAA

TCTTGATAAT

TCTTGGTTTG

GGTGTTTACA

ATCTTTCTTC

GTAATGAGTT

GTTGCTCCAC

CATTTTTCAC

GGAAGGTTGT

ATCTTCTTCT

GTACTTCTTG

TGGAAGAGTG

CAGCTTGGTT

CATACGAGTT

TTGACTAGCT

TTGCATCAAC

TCATTGTGGT

GCTATGCCAA

GAAGAATTGA

AGAGAGAAAT AGGAGAGCTT

CCTTTGATAT

TTGGGTGGAG

ATTGTATACA

AAGAAGTGAA

TCCCTGAAAA

TTTAATTCGG

AGTGTTGGTC

CTCCAAAAGA

CTTTTTTTTT

TTTGTAGAGA

GCCAGGCCTT

GCCAGTCTGT

TTGTCTCCCT

TGTTCTTGAA

TGGGTTTAAA

GAAGTTGCAA

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 TTCTCTTGTA TGATCAGATC TTTTTTCAAT TTTTCCGTTT TAGTTGATTT ATCCATATAG TGAAAGTTGG TGTCATAGTT GCTGTTTGTG GACTTCCTGT AAAAGTTTTT TGATATACTT AAAAATTGT CACACAGAAG AAAGAGTTTT TTACCATTAC WO 98/37214 PCT/1B98/00295 76 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 (8)

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 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 coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation does not contain a sequence that is sense to an exon sequence normally associated with the intron. 0. 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. o: So: A method of affecting starch branching enzymatic activity in a starch producing organism (or a cell, a tissue or an organ thereof) comprising expressing in the starch o 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 o o* 20 branching enzyme in a sense orientation together with a nucleotide sequence which o codes, partially or completely, for an intron of a class B starch branching enzyme in a sense or antisense orientation; wherein the nucleotide sequence coding, partially or o completely, for an intron of class A potato starch branching enzyme in sense orientation 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 coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation does not contain a sequence that is sense to an exon sequence. 78 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 coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation 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 coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation 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 coding, partially or completely, for an intron of class A potato starch branching 15 enzyme in sense orientation comprises the sequence shown as SEQ. ID. No. 38, or a variant, derivative or hcmologue thereof.

9. A method according to any one of the preceding claims wherein the nucleotide sequence coding, partially or completely, for an intron of class A potato starch branching enzyme in sense orientation is expressed by a promoter having a sequence shown as SEQ. ID. No. 14 or a variant, derivative or homologue thereof. 0 A construct comprising or expressing the nucleotide sequence shown as SEQ ID No. 38.

11. A vector comprising or expressing the nucleotide sequence shown as SEQ ID No.

38. or the construct according to claim 12. A combination of first, second and third nucleotide sequences borne on one or more nucleic acid molecules, wherein the first nucleotide sequence codes for a recombinant class A SBE enzyme; the second nucleotide sequence corresponds to a class A SBE intron in a sense orientation; and the third nucleotide sequence corresponds to a class B SBE intron in a sense or antisense orientation; wherein the class A SBE 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. 13. A cell, tissue or organ comprising or expressing the construct, vector or combination according to any one of claims 10 to 12. 14. A transgenic starch-producing organism comprising or expressing the construct, vector or combination according to any one of claims 10 to 12. 15. A transgenic starch-producing organism according to claim 14 wherein the organism is a plant. 16. A starch obtained by carrying out the method according to any one of claims 1 to 9. 17. A method of expressing a recombinant class A SBE enzyme in a host organism comprising expressing a first nucleotide sequence coding for the :1 recombinant enzyme; expressing a second nucleotide sequence, wherein the second nucleotide sequence codes, partially or completely, for a class A SBE intron in sense orientation; and expressing a third nucleotide sequence, wherein the third nucleotide sequence codes, partially or completely, for a A 25 class B SBE intron in sense or antisense orientation; wherein the class A SEE intron is an intron normally associated with the genomic gene encoding a protein or 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. I 18. A method according to any one of claims 1 to 9 and 17 substantially as hereinbefore described with particular reference to the examples. 19. A construct according to claim 10 substantially as hereinbefore described with particular reference to the examples. A vector according to claim 11 substantially as hereinbefore described with particular reference to the examples. 21. A sequence combination according to claim 12 substantially as hereinbefore described with particular reference to the examples. 22. A cell, tissue or organ according to claim 13 substantially as hereinbefore described with particular reference to the examples. 23. An organism according to claims 14 or 15 substantially as hereinbefore described with particular reference to the examples. 24. Starch according to claim 16 substantially as hereinbefore described with particular reference to the examples. Dated this 18 t day of January 2001 o Danisco A/S Patent Attorneys for the Applicant: F B RICE CO