MXPA98000500A - Inhibition of the expression of ge - Google Patents
Inhibition of the expression of geInfo
- Publication number
- MXPA98000500A MXPA98000500A MXPA/A/1998/000500A MX9800500A MXPA98000500A MX PA98000500 A MXPA98000500 A MX PA98000500A MX 9800500 A MX9800500 A MX 9800500A MX PA98000500 A MXPA98000500 A MX PA98000500A
- Authority
- MX
- Mexico
- Prior art keywords
- sequence
- nucleotide sequence
- intron
- antisense
- starch
- Prior art date
Links
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Abstract
A method for inhibiting the expression of genes is described, the method, which affects the enzymatic activity in a plant, comprises expressing in a plant (a cell, a tissue or an organ thereof) a sequence of nucleotides, further characterized by the sequence of nucleotides is encoded, partially or completely, for an intron in an antisense orientation, and because the nucleotide sequence does not contain a sequence that is antisense to a sequence of exons normally associated with the
Description
INHIBITION OF THE EXPRESSION OF GENES
DESCRIPTIVE MEMORY
The present invention relates to a method for inhibiting the expression of genes, particularly for inhibiting the expression of genes 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 the starch consists of amylose and amylopectin. Amylose consists essentially of straight chains of bound a-1-4 glycosyl residues. Amylopectin comprises chains of glycosyl residues a-1-4 linked to some branches a-1-6. The branched nature of amylopectin is achieved through the action of, among other things, an enzyme commonly known as the branched-chain enzyme starch ("SBE"). SBE catalyzes the formation of branching points in the amylopectin molecule by the addition of α-1,4 glucans through a-1, 6-glycosidic branch bonds. The biosynthesis of amylose and amylopectin is shown schematically in Figure 1, while the bonds a-1-4 and the bonds a-1-6 are shown in Figure 2. It is known that starch is an important raw material . Starch is widely used in the chemical, food and paper industries. However, a large fraction of the starches used in these industrial applications is modified, after harvest, by chemical, physical or enzymatic methods to obtain starches with certain required functional properties. In recent years, it has become desirable to obtain genetically modified plants that may be capable of producing modified starches that may be the same as modified post-harvest starches. It is also known that it may be possible to prepare such genetically modified plants by the expression of antisense nucleotide coding sequences. In this regard, June Bourque provides a detailed summary of antisense strategies for genetic manipulations in plants (Bourque 1995, Plant Science 105 pp 125-149). At this time, reference may be made to Figure 3, which is a schematic diagram of one of the proposed mechanisms of antisense RNA inhibition. In particular, WO 92/11375 reports a method for genetically modifying potato to form amylose type starch. The method involves the use of an antisense construct that can apparently inhibit, to a variable degree, the expression of the gene encoding the formation of the branching enzyme in potatoes. The antisense construct of WO 92/11375 consists of a tuber-specific promoter, a transcription initiation sequence and the first exon of the branching enzyme in the antisense direction. However, WO 92/11375 does not provide any data on the antisense sequence. In addition, WO 92/11375 only describes the use of the potato GBSS promoter. WO 92/14827 reports a plasmid which, after being inserted into the genome of a plant, can apparently cause changes in the concentration and composition of carbohydrates, such as changes in the concentration and composition of amylose and amylopectin, in the regenerated plant . The plasmid contains part of the coding sequence of a branching enzyme in an antisense orientation. EP-A-0647715 reports the use of DNA encoding endogenous antisense mRNA to alter the characteristics and metabolic pathways of ornamental plants. EP-A-0467349 reports the expression of sequences that are antisense to sequences towards the 5 'end of a promoter to control the expression of genes. EP-A-0458367 and US-A-5107065 report the expression of a nucleotide sequence to regulate the expression of genes in a plant. The nucleotide sequence is complementary to the sequence of the mRNA of a gene, and can cover the entire non-coding region of the gene, or a portion thereof. In other words, the nucleotide sequences of EP-A-0458367 and US-A-5107065 must comprise at least one sequence that is complementary to a coding region. EP-A-0458367 and US-A-5107065 contain minimal information on the sequence. Kuipers and others, in Mol. Gen. Genet. [1995] 246 745-755, reports the expression of a series of nucleotides that are antisense to part of the genomic sequences of the intron of the starch synthetase linked to potato granules. Here, the antisense sequences of the intron are linked to a portion of the antisense sequences of the exon, wherein the sequences of the intron and the exon sequences are naturally associated with each other. In addition, the expressed antisense sequences of the intron are, at most, 231 base pairs in length. Similarly, Kull and others, in J. Genet &; Breed. [1995] 49 69-76, reports the expression of a series of nucleotides that are antisense to part of the genomic sequences of the intron of the starch synthetase linked to potato granules. In the same way, here the intron antisense sequences are linked to a part of the exon antisense sequences, wherein the sequences of the intron and the exon sequences are naturally associated with each other. In addition, in the same way, the expressed antisense sequences of the intron are, at most, 231 base pairs in length. Shimada and others, in Theor. Appl. Genet [1993] 86 665-672, reports the expression of a series of nucleotides that are antisense to part of the genomic sequences of the intron of the starch synthetase linked to rice granules. Here, the antisense sequences of the intron are linked to a portion of the antisense sequences of the exon, wherein the sequences of the intron and the exon sequences are naturally associated with each other. In addition, the expressed antisense sequences of the intron are, at most, 350 base pairs in length. A review of how enzymatic activity can be affected by the expression of particular nucleotide sequences can be found in the teachings of Finnegan and
McElroy [1994] Biotechnology 12 883-888; and Matzke and Matzke
[1995] TIG 11 1-3. Even though it is known that enzymatic activity can be affected by the expression of particular nucleotide sequences, there is still a need for a method that can affect enzyme activity more reliably and / or more efficiently and / or more specifically. According to a first aspect of the present invention, a method is provided for affecting the enzymatic activity in a plant (or a cell, a tissue or an organ thereof), which comprises expressing in the plant (or a cell, a tissue or an organ thereof) a nucleotide sequence, wherein the nucleotide sequence encodes, partially or completely, an intron in an antisense orientation; and wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron. According to a second aspect of the present invention, a method is provided for affecting the enzymatic activity in a starch-producing organism (or a cell, a tissue or an organ thereof), which comprises expressing in the starch-producing organism ( or a cell, a tissue or an organ thereof) a nucleotide sequence, wherein the nucleotide sequence encodes, partially or completely, an intron in an antisense orientation; and wherein the activity of the starch branching enzyme is affected and / or the amylopectin levels are affected and / or the composition of the starch is modified. According to a third aspect of the present invention, there is provided an antisense sequence comprising the nucleotide sequence shown as any of I.D. FROM
SEC. No. 15 to I.D. SEC. No. 27, or a variant, derivative or homologous thereof. In accordance with a fourth aspect of the present invention, a promoter comprising the sequence shown as I.D. SEC. No. 14 or a variant, derivative or homologous thereof. In accordance with a fifth aspect of the present invention, a construct capable of understanding or expressing the present invention is provided. In accordance with a sixth aspect of the present invention, a vector comprising or expressing the present invention is provided. According to a seventh aspect of the present invention, a cell, tissue or organ comprising or expressing the present invention is provided. According to an eighth aspect of the present invention, a transgenic starch producing organism comprising or expressing the present invention is provided. According to a ninth aspect of the present invention, a starch obtained from the present invention is provided. In accordance with a tenth aspect of the present invention, pBEA8 (NCIMB 40753) or pBEA9 (NCIMB 40815) is provided. According to an eleventh aspect of the present invention, a nucleotide sequence is provided that is antisense to any one or more of the intron sequences obtained from \ -SBE 3.2 (NCIMB 40751) or \ -SBE 3.4 ( NCIMB 40752) or a variant, derivative or homologous thereof. A key advantage of the present invention is that it provides a method for preparing modified starches and does not depend on the need to modify postharvest starches. Thus, the method of the present invention avoids the need to use hazardous chemical compounds that are normally used in the modification of post-harvest starches. In addition, the present invention provides, among other things, genetically modified plants that 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 for preparing adapted starches in plants that can replace modified postharvest starches. In the same way, the present invention provides a method that allows to prepare modified starches by a method that can have a more beneficial effect on the environment, that the known methods of modification of postharvest, which depend on the use of dangerous chemical compounds and large amounts of energy. Another key advantage of the present invention is that it provides a method that can affect enzyme activity more reliably and / or more efficiently and / or more specifically as compared to methods known to affect enzyme activity. In relation to this advantage of the present invention, it should be noted that there is a certain degree of homology between the coding regions of the SBEs. However, there is little or no homology with the intron sequences of the SBEs. Thus, the antisense expression of the intron provides a mechanism that selectively affects the expression of a particular SBE. This advantageous aspect can be used, for example, to reduce or eliminate a particular enzyme of the SBE and replace this enzyme with another enzyme that could be another branching enzyme, or even a recombinant version of the affected enzyme, or a hybrid enzyme that can understand, for example, part of an SBE from one source and at least part of another SBE from another source. This particular feature of the present invention is covered by the combination aspect of the present invention which is discussed in more detail below. Thus, the present invention provides a mechanism to selectively affect the activity of SBE. This contrasts with the methods of the prior art, which depend on the use of, for example, the antisense expression of the exon, according to which it would not be possible to introduce a new activity of the SBE without also affecting that activity. Preferably with the first aspect of the present invention, the activity of the starch branching enzyme is affected and / or where amylopectin levels are affected and / or the composition of the starch is modified. Preferably with the second aspect of the present invention, the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron. Preferably with the fourth aspect of the present invention, the promoter is in combination with a gene of interest ("GOI"). Preferably, the enzymatic activity is reduced or eliminated. Preferably, the nucleotide sequence encodes at least substantially at least one intron in an antisense orientation. Preferably, the nucleotide sequence encodes, partially or completely, two or more intron, and wherein each intron is in an antisense orientation. Preferably, the nucleotide sequence comprises at least 350 nucleotides (e.g., at least 350 base pairs), more preferably at least 500 nucleotides (e.g., at least 500 base pairs). Preferably, the nucleotide sequence comprises the sequence shown as any of I.D. SEC. No. 15 to I.D. SEC. No. 27 or a variant, derivative or homologous thereof, including combinations thereof. Preferably, the nucleotide sequence is expressed by a promoter having a sequence shown as I.D. SEC. No. 14 or a variant, derivative or homologous thereof. Preferably, the transgenic organism that produces the starch is a plant. A preferred aspect of the present invention therefore relates to a method for affecting the enzymatic activity in a plant (or a cell, a tissue or an organ thereof), which comprises expressing in the plant (or a cell , a tissue or an organ thereof) a nucleotide sequence, wherein the nucleotide sequence encodes, partially or completely, an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; and wherein the activity of the starch branching enzyme is affected and / or the amylopectin levels are affected and / or the starch composition is modified. A more preferred aspect of the present invention therefore relates to a method for affecting the enzymatic activity in a plant (or a cell, a tissue or an organ thereof), which comprises expressing in the plant (or a cell, a tissue or an organ thereof) a sequence of nucleotides, wherein the nucleotide sequence encodes, partially or completely, an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; wherein the activity of the starch branching enzyme is affected and / or the amylopectin levels are affected and / or the starch composition is modified; and wherein the nucleotide sequence comprises the sequence shown as any of I.D. SEC. No. 15 to I.D. SEC. No. 27, or a variant, derivative or homologous thereof, including combinations thereof. The term "nucleotide", in relation to the present invention, includes DNA and RNA. Preferably it means DNA, more preferably DNA prepared by the use of recombinant DNA techniques. The term "intron" is used, in its normal sense, to indicate a segment of nucleotides, often DNA, that does not encode a whole protein or expressed enzyme, or part thereof. The term "exon" is used, in its normal sense, to indicate a segment of nucleotides, often DNA, that encodes an entire expressed protein or enzyme, or part thereof. Thus, the term "intron" refers to regions of genes that are transcribed into RNA molecules, but that bind outside the RNA before the RNA is translated into a protein. In contrast, the term "exon" refers to regions of genes that are transcribed into RNA and that are subsequently translated into proteins. The terms "variant" or "homologous" or "fragment" in relation to the nucleotide sequence of the present invention, include any substitution of, variation of, modification of, replacement of, deletion (loss) of, or addition of, one (or more) nucleic acids for the respective nucleotide sequence, provided that the resulting nucleotide sequence can affect the enzymatic activity in a plant, or cell or tissue thereof, preferably wherein the resulting nucleotide sequence has at least same effect as any of the antisense sequences shown as ID SEC. Nos. 15-27. In particular, the term "homologous" covers homology with respect to the similarity of structure and / or similarity of function, provided that the resulting nucleotide sequence has the ability to affect the enzymatic activity in accordance with the present invention. In relation to the homology (i.e., similarity) of the sequence, preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences. The terms "variant" or "homologous" or "fragment" in relation to the nucleotide sequence of the present invention, include any substitution of, variation of, modification of, replacement of, deletion (loss) of, or addition of, one (or more) nucleic acids for the respective nucleotide sequence, provided that the resulting sequence of promoters allows the expression of a GOI, preferably wherein the resulting nucleotide sequence has at least the same effect as ID SEC. Nos. 14. In particular, the term "homologous" covers homology with respect to similarity of structure and / or similarity of function, provided that the resulting nucleotide sequence has the ability to allow the expression of a GOI, such as a nucleotide sequence in accordance with the present invention. In relation to the homology (i.e., similarity) of the sequence, preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology. The above terms are also synonymous with allelic variations of the sequences. The term "antisense" means a sequence of nucleotides that is complementary to, and can therefore hybridize to, any or all of the sequences of the intron of the present invention, including partial sequences thereof. With the present invention, the antisense intron can be complementary to a complete intron of the gene to be inhibited. However, in some circumstances, they may use their partial antisense sequences (i.e., sequences that are not or do not comprise, the complete complementary sequence) provided that the partial sequences affect the enzymatic activity. Suitable examples of partial sequences include sequences that are shorter than any of the complete antisense sequences shown as SEQ. I.D. Nos. 15 to 27 but comprising nucleotides that are at least antisense to the intron sense sequences adjacent to the respective exon or exons.
With respect to the second aspect of the present invention (ie, specifically affecting the activity of SBEs), the nucleotide subsequences of the present invention may comprise one or more sense or antisense sequences of the exon of the SBE gene, including complete or partial thereof, provided that the nucleotide sequences can affect the activity of the SBE, preferably wherein the nucleotide sequences are reduced or eliminate the activity of the SBE. Preferably, the nucleotide sequence of the second aspect of the present invention does not comprise an antisense sequence of the exon. The term "vector" includes an expression vector and a transformation vector. The term "expression vector" means a construct capable of expression in vivo or in vitro. 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 Agrobacterium to a plant cell. The term "construct", which is synonymous with terms such as "conjugate", "cassette" and "hybrid", in relation to the antisense nucleotide sequence aspect of the present invention, includes the nucleotide sequence according to the invention. present invention linked directly or indirectly to a promoter. An example of an indirect linkage is the provision of a suitable spacer group such as an intron sequence, such as the Shl intron or the ADH intron, intermediate to the promoter and the nucleotide sequence of the present invention. The same is true for the term "merged" in relation to the present invention, which includes direct or indirect linkage. The terms do not cover the natural combination of the wild-type SBE gene when associated with the wild-type SBE gene promoter in its natural environment. The construct can contain or even express a marker that allows the selection of the direct genetic construct, for example, in a plant cell in which it has been transferred. There are several markers that can be used, for example, in plants - such as handy. Other examples of markers include those that provide resistance to antibiotics - for example, resistance to G418, hygromycin, bleomycin, kanamycin and gentamicin. The construct of the present invention preferably comprises a promoter. The term "promoter" is used in the ordinary sense of the art, for example, an RNA polymerase binding site in Jacob's and Monod's theory of gene expression. Examples of suitable promoters are those that can direct the 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 described in WO 92/11375. The promoter may additionally include conserved regions such as a Primbow block or a TATA block. The promoters may even contain other sequences that affect (so that they maintain, increase, decrease) the expression levels 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 agents, light or voltage-inducible elements. Also, suitable elements may be present that increase transcription or translation. An example of the last element is the TMV 5 'master sequence (see Sleat Gene 217 [1987] 217-225 and Dawson Plant Mol. Biol. 23 [1993] 97). As mentioned above, the construct and / or vector of the present invention may include a region of early transcription that can provide regulated or constitutive expression. Any suitable promoter can be used for the region of the start of transcription, such as a tissue-specific promoter. In one aspect, preferably the promoter is a 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 may be one that affects the expression of the nucleotide sequence in any one or more tissues of the seed, tuber, stem, shoot, root and leaf. Preferably the tuber. By way of example, the promoter for the nucleotide sequence of the present invention may be the α-Amy 1 promoter (also known as the Amy 1 promoter, the Amy 637 promoter or the α-Amy 637 promoter) as described in UK co-pending patent application No. 9421292.5 filed October 21, 1994. Alternatively, the promoter for the nucleotide sequence of the present invention may be the α-Amy 3 promoter (also known as the Amy 3 promoter, the promoter Amy 351 or promoter a-Amy 351) as described in co-pending UK patent application No. 9421286.7 filed October 21, 1994. The present invention also encompasses the use of a promoter that expresses a nucleotide sequence of according to the present invention, wherein a part of the promoter is inactivated, but wherein the promoter can still function as a promoter. The partial inactivation of the promoter is in some cases advantageous. In particular, in the case of the Amy 351 promoter mentioned above, it is possible to inactivate a part thereof so that the partially inactivated promoter expresses the nucleotide sequence of the present invention in a more specific manner such as in a tissue or organ type. specific. The term "inactivated" means partial inactivation in the sense that the expression pattern of the promoter is modified, but where the partially inactivated promoter still functions as a promoter. However, as mentioned above, the promoter is modified capable of expressing a gene encoding the enzyme of the present invention in at least one specific tissue (but not all) of the original promoter. Examples of partial inactivation include altering the folding pattern of the promoter sequence, or joining species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognized by, for example, RNA polymerase. Another (preferable) way to partially inactivate 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 such as, for example, the CreA protein of filamentous fungi known to exert repression of the catabolite by carbon, and thus suppress the repression of the catabolite of the native promoter. The construct and / or vector of the present invention may include a region of transcription terms. 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 can be present in the same vector, plasmid, cells, tissue, organ or organism. This aspect of the present invention also covers methods of expression thereof, preferably in specific cells or tissues, such as expression in a specific cell or tissue of an organism, typically a plant. With this aspect of the present invention, the second construct does not cover the natural combination of the gene encoding an enzyme ordinarily associated with the wild-type gene promoter when both are 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 the second construct comprising a promoter, such as the promoter of the present invention. , and a GOI in which the GOI codes for another starch branching enzyme in either sense or antisense orientation. The above comments related to the term "construct" for the antisense 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 GOI.
The term "GOI" with reference to the promoter aspect of the present invention or to the combination aspect of the present invention means any gene of interest, which does not need a code for a protein or an enzyme - as explained below. A GOI can be any nucleotide sequence that is foreign or natural to the organism, in question, for example, a plant. Typical examples of a GOI include genes that code for other proteins or enzymes that modify metabolic and catabolic processes. The GOI can code for an agent to introduce or increase resistance to pathogens. The GOI can even be an antisense construct to modify the expression of natural transcripts present in the relevant tissues. An example of said GOI is the nucleotide sequence according to the present invention. The GOI can even code for a protein that is unnatural to the host organism and / or, for example, a plant. The GOI can code for a compound that is of benefit to animals or humans. For example, GOI could code for a pharmaceutically active protein or enzyme such as any of the therapeutic compounds insulin, interferon, human serum alum, human growth factor and blood agglutination factors. The GOI can even code for a protein that gives additional nutritional value to a food or feed for livestock or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and vegetable proteins having a more desirable amino acid composition (e.g., a higher lysine content than a non-transgenic plant). The GOI can even code for an enzyme that can be used in food processing such as xylanases and α-galactosidase. The GOI can be a gene that codes for either a pest toxin, an antisense transcript such as for α-amylase, a protease or a glucanase. Alternatively, the GOI may be a nucleotide sequence according to the present invention. The GOI may be a nucleotide sequence encoding the arabinofuranosidase enzyme which is the subject of our co-pending United Kingdom Patent Application No. 9505479.7. The GOI may be the nucleotide sequence encoding the glucanase enzyme which is the subject of copending United Kingdom Patent Application No. 9505475.5 of the present inventors. The GOI may be the nucleotide sequence encoding the α-amylase enzyme which is the subject of copending United Kingdom Patent Application No. 9413439.2 of the present inventors. The GOI may be the nucleotide sequence encoding the α-amylase enzyme which is the subject of copending United Kingdom Patent Application No. 9421290.9 of the present inventors. The GOI may be any of the nucleotide sequences encoding the a-glucanliase enzyme that are described in copending PCT Patent Application No. PCT / EP94 / 03397 of the present inventors. In one aspect, the GOI can be even a nucleotide sequence according to the present invention but when it is operatively linked to a different promoter. The GOI could include a sequence encoding one or more than one xylanase, an arabinase, an acetyl esterase, a rhamnogalactinase, a glucanase, a pectinase, a branching enzyme or another carbohydrate or protein modifying enzyme. Alternatively, the GOI may be a sequence that is antisense to any of those sequences. As mentioned above, the present invention provides a mechanism to selectively affect a particular enzymatic activity. In an important application of the present invention, it is now possible to reduce or eliminate the expression of a genomic nucleotide sequence encoding a genomic protein or enzyme by expressing an antisense intron construct for that particular protein or genomic enzyme and (for example, same time) expressing a recombinant version of that enzyme or protein - in other words, GOI is a recombinant nucleotide sequence that codes for the enzyme or genomic protein. This application allows the expression of enzymes and recombinant proteins desired in the absence (or reduced levels) of enzymes and respective genomic proteins. Therefore, 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 methods of the prior art which, for example, are based on the use of antisense exon expression, said methods also affect the expression of the recombinant enzyme. Therefore, 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 encoding the protein or recombinant enzyme; and which expresses a sequence of nucleotides wherein the nucleotide sequence encodes, partially or completely, for an intron in an antisense orientation; wherein the intron is an intron normally associated with the genomic gene that encodes a protein or an enzyme corresponding to the protein or recombinant enzyme; and wherein the additional nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron. Additional aspects cover the combination of nucleotide sequences including their incorporation into transgenic constructs, vectors, cells, tissues and organisms. Therefore, the present invention also relates to a combination of nucleotide sequences comprising a first nucleotide sequence encoding a recombinant enzyme; and a second nucleotide sequence corresponding to an intron in antisense orientation; wherein the intron is an intron that is associated with the genomic gene encoding an enzyme corresponding to the recombinant enzyme; and wherein the second nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron. The GOI can even code for one or more introns, such as any or more of the intron sequences presented in the attached sequence listings. For example, the present invention also covers the expression, for example, of an antisense intron (e.g., SEQ ID NO: 15) in combination, for example, with a sense intron that is preferably not complementary to the sequence of the antisense intron (e.g., SEQ ID No. 2). The term "cell", "tissue" and "organ" includes cell, tissue and organ per se and when it is within an organism. The term "organism" in relation to the present invention includes any organism that can comprise the nucleotide sequence according to the present invention and / or wherein the nucleotide sequence according to the present invention can be expressed when it is present in the organism. Preferably, the organism is an organism starch products such as any of a plant, algae, fungus, 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 biosynthesize starch. Preferably, the starch producing organism is a plant. The term "plant", as used herein, includes any suitable angiosperm, gymnosperm, monocot and dicot. Typical examples of suitable plants include vegetables such as potatoes; cereals such as wheat, corn and barley; fruits; Trees flowers and other plant crops. Preferably, the term means "potato." The term "transgenic organism" in relation to the present invention includes any organism comprising the nucleotide sequence according to the present invention and / or products obtained therefrom, and / or wherein the nucleotide sequence according to the present invention invention can be expressed within the organism. Preferably, the nucleotide sequence of the present invention is incorporated into the genome of the organism. Preferably, the transgenic organism is a plant, most preferably a potato. Prokaryotic or eukaryotic organisms can be used to prepare the host organism. Examples of suitable prokaryotic hosts include E coli and Bacillus subtilis.
Teachings on the transformation of prokaryotic hosts are 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). Although the enzyme according to the present invention and the nucleotide sequence coding for it are not described in EP-B-0470145 and CA-A-2006454, these two documents provide useful supporting comments on the types of techniques that they can be used to prepare transgenic plants in accordance with the present invention. Some of these back-up teachings are now included in the following comment. The basic principle in the construction of genetically modified plants is to insert genetic information into the genome of the plant to obtain a stable maintenance of the inserted genetic material. There are several techniques for inserting genetic information, the two most important principles being the direct introduction of genetic information and the introduction of genetic information through the use of a vector system. A review of the general techniques can be found in Potrykus articles (Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Christo (Agro-food-Industry Hi-Tech March / April 1994 17-27).
Therefore, 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 or construct sequence into the genome of an organism , such as a plant. The vector system may comprise a vector, but may also comprise two vectors. In the case of two vectors, the vector system is usually referred to as a binary vector system. The binary vector systems are described in greater detail in Gynheung An et al., (1980), binary Vectors, Plant Molecular Biology Manual A3, 1-19. A system extensively employed for the transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al. (1986), Plant Physiol, 81, 301-305 and Butcher DN and others (1980), Tissue Culture Methods for Plant Pathologists, eds .: D.S. Ingrams and J.P. Helgeson, 203-208. Several different Ti and Ri plasmids have been constructed which are suitable for the construction of previously described plant or plant cell constructs. A non-limiting example of said Ti plasmid is pGV3850. A 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 a T-DNA sequence to avoid altering the sequences immediately surrounding the boundaries of the T-DNA, at least one of these regions appears to be essential for insertion of modified T-DNA into the genome of the plant. As will be understood from the above explanation, if the organism is a plant, the vector system of the present invention is preferably one that contains the sequences necessary to infect the plant (e.g., the vir region) and so minus a borderline part of a T-DNA sequence, the borderline part being located on the same vector as the genetic construct. In addition, the vector system is preferably a Ti plasmid of Agrsbacterium trumefaciens or a Ri plasmid of Agrobacterium rhizogenes or a derivative thereof. Since these plasmids are well known and widely used in the construction of transgenic plants, there are many vector systems that rely on these plasmids or derivatives thereof. In the construction of a transgenic plant, the nucleotide sequence or contruct of the present invention can first be constructed in a microorganism in which the vector can be replicated and which is easy to manipulate before being inserted into the plant. An example of a useful organism is E ^. coli but other organisms having the above properties can be used. When a vector or a vector system as defined above has been constructed in E. coli. is transferred, if necessary, in a suitable strain of Agrobacterium, e.g., Agrobacterium tumefaciens. The Ti plasmid carrying the nucleotide sequence or construct of the present invention is thus preferably transferred to a suitable Agrobacterium separator e.g., A. tumefacies. to obtain an A robacterium cell carrying the promoter or nucleotide sequence or construct of the present invention, said DNA being subsequently tansferid to the cell of the plant to be modified. If, for example, for the transformation the Ti or Ri plasmid of the plant cells is used, at least the right border and often the right border and the left border of the T-DNA of the Ti plasmid and the Ri plasmid, as areas of flanking the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been studied intensively and is described in EP-A-120516; Hoekema in: The Binary Plant Vector System Offset-drukkerij Kanters B.B. , Alblasserdam, 1985, Chapter V; Fraley et al., Crit. Rev. Plant Sci., R: 1-46; and An et al., EMBO J. (1985) 4: 277-284. Direct infection of plant tissues by A robacterium is a simple technique that has been widely used and described in Butcher D.N. and others (1980), Tissue Culture Methods .for Plant Pathologists, eds .: D.S. Ingrams and J.P. Helgeson, 203-208. For additional teachings on this topic, see Potrykus (Annu Rev Plant Physiol Plant MolBiol [1991] 42: 205-225) and Christou (Agro-Food-Industry HiTech March / April 1994 17-27). With this technique, the infection of a plant can be done inside or on a certain part or tissue of the plant, such as 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 injured, e.g., by cutting the Plant with a blade by shaving or pricking the plant with a needle or by rubbing the plant with an abrasive. The lesion is then inoculated with Agrobacterium. The plant or part of the inoculated plant is then grown on an appropriate culture medium to allow development in mature plants. When plant cells are constructed, these cells can be cultured and maintained according to well-known tissue culture methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamin , etc. Regeneration of the transformed cells in genetically modified plants can be achieved using known methods for the regeneration of plants from cell or tissue cultures, for example, by selecting transformed offspring using an antibiotic and subculturing the rods in a medium containing the nutrients appropriate, vegetative hormones, etc. Further teachings about the transformation of plants can be found in EP-A-0449375. As reported in CA-A-2006454, a large number of cloning vectors are available that contain a replication system in E. coli and a marker that 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 contruct of the present invention can be introduced into a suitable restriction position in the vector. The plasmid contained is then used for transformation into E. coli. The E. coli cells are grown in an appropriate nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis, a sequence analysis, restriction analysis, electrophoresis, and biochemical and molecular biology methods are generally used. After each manipulation, the DNA sequence used can be restricted and connected to the next DNA sequence. Each sequence can be cloned in the same plasmid or in a different one. After introduction of the nucleotide sequence or construct according to the present invention into the plants, the presence and / or insertion of additional DNA sequence may be necessary - such as to create combination systems as indicated above (e.g. , an organism that comprises a combination of constructs). The above comment 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 summary, the present invention relates to affecting the enzymatic activity by expressing antisense intron sequences. Also, the present invention relates to a promoter useful for the expression of those antisense intron sequences. The following samples have been deposited in accordance with the Budapest Treaty in the recognized warehouse The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on July 13, 1995: NCIMB 40753 (which refers to pBEA 8 as described here); NCIMB 40751 (which refers to \ -SBE 3.2 as described here); and NCIMB 40752 (which refers to \ -SBE 3.4 as described here). The following sample has been deposited according to the Budapest Treaty in the recognized warehouse The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, UK, on July 9, 1996: NCIMB (which refers to pBEA 9 as described here). A highly preferred embodiment of the present invention, therefore, relates to a method for affecting the enzymatic activity in a plant (or a cell, or a tissue or an organ thereof) comprising expressing in the plant (or a cell, or a tissue or an organ thereof) a nucleotide sequence in which the nucleotide sequence encodes partially or completely for an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; wherein the enzymatic starch branching activity is affected and / or the amylopectin levels are affected and / or the starch composition is changed; and wherein the nucleotide sequence can be obtained from NCIMB 40753 or NCIMB 40815, or is antisense to any one or more of the sequences of the intron obtainable either from [-SBE 3.2 (NCIMB 40751) or [SBE 3.4] (NCIMB 40752 ) or a variant, derivative or homologous thereof. A more highly preferred aspect of the present invention, therefore, relates to a method for affecting the 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 sequence of nucleotides wherein the nucleotide sequence encodes, partially or completely, for an intron in an antisense orientation; wherein the nucleotide sequence does not contain a sequence that is antisense to an exon sequence normally associated with the intron; wherein the activity of the starch branching enzyme is affected and / or the levels of amylopectin are affected and / or the starch composition is changed; wherein the nucleotide sequence comprises the sequence shown as any of SEQ ID No. 15 to SEQ ID No. 27 or a variant; derivative or homolog thereof, including combinations thereof; and wherein the nucleotide sequence is obtainable from NCIMB 40753 or NCIMB 40815, or is antisense to any or more of the sequences of the intron obtainable either from SBE 3.2 (NCIMB 40751) or SBE 3.4 (NCIMB 40752) or a variant , derivative or homologous thereof. The present invention will now be described only by way of example, in which reference is made to the following appended figures: Figure 1 is a schematic representation of the biosynthesis of amylose and amylopectin; Figure 2 is a diagrammatic representation of the a-1-4 bonds and the α-1-6 amylopectin bonds; Figure 3 is a diagrammatic representation of a possible mechanism of inhibition of antisense RNA;
Figure 4 is a diagrammatic representation of the exon-intron structure of a genomic SBE clone; Figure 5 is a plasmid map of pPATAl, which is 3936 base pairs in size; Figure 6 is a plasmid map of pABE6, which is 5106 base pairs in size; Figure 7 is a plasmid map of pVictorlV Man, which is 7080 base pairs in size; Figure 8 is a plasmid map of pBEA8, which is 9.54 kb in size; Figure 9 is a plasmid map of pBEA9, which is 9.54 kb in size; Figure 10 is a plasmid map of pBEP2, which is 10.32 kb in size; Figure 11 is a plasmid map of pVictor5a, which is 9.12 kb in size; and Figure 12 shows the complete genomic nucleotide sequence for SBE including the promoter, exons and introñes. Figures 1 and 2 were previously mentioned in the introductory description referring to starch in general. Figure 3 was mentioned earlier in the introductory description referring to antisense expression. As mentioned, Figure 4 is a diagrammatic representation of the exon-intron structure of a genomic SBE clone, the sequence of which is shown in Figure 12. This clone, which is approximately 11.5 kb, 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 ID Nos. 1-13, respectively. Their respective antisense intron sequences are shown as SEQ ID NOs 15-27. In more detail, Figures 4 and 12 present information about 11478 base pairs of the potato SBE gene. The 5 'region of nucleotides 1 to 2082 contains the promoter region of the SBE gene. A TATA box candidate in nucleotide 2048 through 2051 is enclosed in a box. The homology between a potato SBE cDNA clone (Poulsen &Kreiberg (1993) Plant Physiol 102: 1053-1054) and the DNAs of exons being at 2083 bp and ending at 9666 bp. The homology between the cDNA and the DNA of the exons is indicated by the nucleotides with capital letters, while the translated sequences of the amino acids in the code are shown with unique letters under the DNA of the exons. The sequence of the introns is indicated with lowercase letters. FIGS. 5 to 7 are considered below. As mentioned, FIG. 8 is a plasmid map of pBEA8, which is 9.54 kb of base size; and Figure 9 is a plasmid map of pBEA9, which is 9.54 kbps in size. From pBEA 8 and pBEA 9 each one comprises an antisense sequence to the first sequence of the introns of the SBE gene of the potato. Figure 4 shows this first sequence of introns, which has 1177 base pairs, and is between the first exon and the second exon. These experiments and aspects of the present invention are now considered in more detail.
EXPERIMENTAL PROTOCOL
ISOLATION. SUBCLONATION IN PLASMIDS. AND SEQUENCING OF GENETIC SBE CLONES
Several clones containing the potato SBE gene were isolated from a genomic library of potato Desiree (Clontech Laboratories Inc., Palo Alto CA, USA) using radiolabelled potato SBE cDNA as a probe (Poulsen &Kreiberg (Poulsen &Kreiberg)). 1993) Plant Physiol. 102: 1053-1054). The fragments of the isolated β-bodies containing SBE DNA (\ SBE 3.2-NCIMB 40751- and \ SBE-3.4-NCIMB 40752) were identified by Southern analysis and then subcloned into pBluescript II vectors (Clontech Laboratories Inc., Palo Alto CA, USA., \ SBE 3.2 contains a 15 kb potato DNA insert and \ SBE-3.4 contains a 13 kb potato DNA insert called the resulting plasmids pGB3, pGBll, pGB15, pGB16 and pGB25 (see further discussion) The respective inserts were then sequenced using the Pharmacia Autoread Sequencing kit (Pharmacia, Uppsala) and an ALF DNA sequencer (Pharmacia, Uppsala) In total, an 11.5 kb stretch of the gene was sequenced. SBE The sequence of the aforementioned plasmids was derived, where: 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 to 5093 bp, pGBll contains the sequences from 3348 bp to 7975 bp and pGB3 contains the sequences from 7533 bp to 11468 bp. In more detail, a pGB3 was constructed by inserting a 4 kb EcoRI fragment isolated from \ SBE 3.2 into the EcoRI site of pBluescript II SK (+). A pGBll was constructed by inserting a 4.7 kb Xhol fragment isolated from \ SBE 3.4 to the Xhol site of pBluescript II SK (+). A pGB15 was constructed by inserting a 1.7 kb Spel fragment isolated from \ SBE 3.4 into the Spel site of pBluescript II SK (+). PGB16 was constructed by inserting a 2.5 kb Spel fragment isolated from \ SBE 3.4 into the Spel site of pBluescript II SK (+). For the construction of pGB25 a PCR fragment was produced with the primers
'GGA ATT CCA GTC GTC GTC TAC ATT AC 3 'and 5' CGG GAT CCA GAG GCA TTA AGA TTT CTG G 3 '
and \ SBE 3.4 as a template. A PCR fragment was digested with BamHI and EcoRI, and inserted into pBluescript II SK (+) digested by the same restriction enzymes.
CONSTRUCTION OF PLASMIDS PBEA8 V PBEA9 OF THE INTRONES IN ANTICIPATION OF SBE
Intron 1 of SBE was amplified by PCR using the oligonucleotides:
'CGG GAT CCA AAG AAA TTC TCG AGG TTA CAT GG 3' and 5 'CGG GAT CCG GGG TAA TTT TTA CTA ATT TCA TG 3'
and the phage \ SBE 3.4 that contained the SBE gene as a template. The PCR product was digested with BamHI and inserted in an antisense orientation at the BamHI site of plasmid pPATAI (described in WO 94/24292) between the patatin promoter and the 35S terminator. This construction was digested pABE6 with Kpnl, and the Kpnl fragment of "promoter of patatin-intron of SBE-terminator 35S" of 2.4 kb was isolated and inserted into the Kpnl site of the plant transformation vector pVictorlV Man. The fragment was inserted Kpnl in two orientations producing plasmids pBEA8 and pBEA9. PVictorlV Man is shown in Figure 7 and is formed by insertion of an included Xbal fragment containing an E35S-manA-terminator 35S promoter cassette isolated from plasmid pVictorlV SGiN Man (WO 94/24292) to the included Xhol site of pVictorIV. The pVictor regions of pVictor IV Man contained between the coordinates of 2.52 bp to 0.32 bp (see Figure 7).
PRODUCTION OF TRANSGENIC PLANTS OF POTATO
Axénica Class Crops Cultivation of shoots of Solanum tuberosum 'Bintje' and 'Dianella' on a substrate (LS) of a formula according to Linsmaier, E.U. and Skoog, F. (1965), Physiol. Plant.
18: 100-127, which also contains 2 μM of silver thiosulfate a
° C and 16 hours of light / 8 hours of darkness. The cultures were subcultured after approximately 40 days. Leaves were then cut from the rods and cut into nodal segments (approximately 0.8 cm) each containing a node.
Inoculation of Potato Tissues Crop stems were cut with stems of approximately 40 days (approximate height of 5-6 cm) internodal instruments (approximately 0.8 cm). The segments were placed on liquid LS substrate containing transformed Agrobacterium tumefaciens containing the binary vector of interest. Agrobacterium was grown overnight on YMB substrate (dipotassium acid phosphate, trihydrate (0.66 g / 1), magnesium sulfate, heptahydrate (0.20 g / 1), sodium chloride (0.10 g / 1), mannitol (10.0 g / 1), and yeast extract (0.40 g / 1)) containing appropriate antibiotics (corresponding to the resistance gene of the A robacterium strain) at an optical density at 660 nm (OD-660) of approximately 0.8, is centrifuged and resuspend in the LS substrate to a 0D-660 of 0.5. The segments were left in Agrobacterium suspension for 30 minutes and then the excess bacteria were removed by staining the segments on sterile filter paper.
Coculture The segments of the rod were cocultivated for 48 hours directly on the LS substrate containing 2,4-dichlorophenoxyacetic acid (2.0 mg / l) and trans-zeatin (0.5 mg / l). The substrate and also the explants were covered with sterile filter papers and the Petri dishes were placed at 25 ° C and 16 hours light / 8 dark.
"Washing" Procedure After 48 hours on the coculture substrate, the segments were transferred to containers containing liquid LS substrate containing 800 mg / l of carbenicillin. The containers were shaken gently and by this procedure either they were separated by washing the segments and / or most of the A robacterium was killed.
Selection After the washing procedure, the segments were transferred to plates containing the LS substrate, agar (8 g / 1), trans-zeatin (1-5 mg / l), gibberellic acid (0.1 mg / l), carbenicillin (800 mg / l) and kanamycin sulphate (50-100 mg / l) or phosphonothricin (1-5 mg / l) or mannose (5 g / 1) depending on the construction of the vector used. The segments were subcultured to fresh substrate every 3-4 weeks. In 3 to 4 weeks the rods were developed from the segments and the formation of new rods continues for 3-4 months.
Rooting of the Regenerated Vases Regenerated rods were transferred to substratum substrates composed of LS substrate, agar (8 g / 1) and carbenicillin (800 mg / l). The transgenic genotype of the regenerated stem was verified by examining the rooting capacity on the aforementioned substrate containing kanamycin sulphate (200 mg / l), performing the NPTII S.E. and others, Theor. Appl. Genet (1988), 7: 68-694) and performing the PCR analysis according to Wang et al. (1993, NAR 21 pp4153-4154). Plants that were not positive in any of these trials were discarded or used as controls. Alternatively, the transgenic plants could be verified by performing a GUS assay on the ü-glucuronidase gene or introduced according to Hodal, L. et al. (Pl.Sci. (1992), 87: 11-122).
Transfer to Soil Freshly rooted plants (approximate height 2-3 cm) of the rooting substrate were transplanted to the soil and placed in a culture chamber (21 ° C, 16 hours light 200-400uE / m2sec). When the plants were well established, they were transferred to the greenhouse, where they were grown until the tubers had developed and the upper part of the plants was aging.
Harvest The potatoes were harvested after approximately 3 months and then analyzed.
ANALYSIS OF THE BRANCH ENZYMES
The expression of SBE in the transgenic potato lines was measured using the SBE assays described by
Blennow and Johansson (Phytochemistry (1991) 30437-444) and by normal Western procedures using antibodies directed against potato SBE.
STARCH ANALYSIS
Starch was isolated from potato tubers and altered for the amine ratio: amylopectin (Hovenka p- Hermelink et al. (1988) Potato Research 31: 241-246). In addition, the chain length distribution of amylopectin was determined by analyzing the starch digested by the isoalase on a Dionex HPAEC. The number of reducing ends was determined by the starch digested by the isoamylase by the method described by N. Nelson (1994) J. Biol. Chem. 13: 37-380. The results revealed that there was a reduction in the level of synthesis of the SBE and / or the activity level of the SBE and / or the composition of the SBE of the starch in the transgenic plants.
TRAINING OF THE SBE PROMOTER CONSTRUCTION
An SBE promoter fragment of the amine \ -SBE 3.4 was amplified using the primers:
paste sequence 1 and paste sequence 2
The PCR product was digested with Clal and BamHI. The resulting 1.2 kb fragment was then inserted into pVictor5a (see Figure 11) linearized with Clal and Bg / II producing pBEP2 (see Figure 10).
MEASUREMENTS OF THE STARCH BRANCH ENZYMES OF POTATO TUBERS
Potatoes were cut from potato plants transformed with either pBEA8 or pBEA9 into small pieces and homogenized in extraction regulators (0 mM Tris-HCl pH 7., Sodium dithionite (0.1 g / 1) and 2 mM DTT) Ultra-Turax; 1 g of Dowex xl was added per 10 g of tuber. The crude homogenate was filtered through a mirate filter and centrifuged at 4 ° C for 10 minutes at 24-700 g. A supernatant was used for the starch branching enzyme assays. The assays of the starch branching enzyme were carried out at 25 ° C in a volume of 400 μl composed of 0.1 M of regular pH 7.0, of sodium citrate, 0.75 mg / ml of amylose, 5 mg / ml of albumin bovine serum and potato extract At 0, 1, 30 and 60 minutes the aliquots of 50 μl to 20 μl of 3 N HCl were removed from the reaction, 1 ml in iodine solution was added and the decrease was measured of absorbance at 620 nm with an ELISA spectrophotometer The levels of the starch branching enzyme (SBE) were measured in tuber extracts of 34 transgenic Dianella potato plants transformed with the plasmid pBEA 9. The transformed transgenic lines of pBEA9 produced tubers having SBE levels that are 10% to 15% of the levels of SBE found in transformed Dianella plants.ION
The aforementioned examples refer to the isolation and sequencing of a gene for potato SBE. The examples are further shown that it is possible to prepare antisense constructs of the SBE introns. These constructs can be introduced in the antisense of SBE introns to plants, such as potato plants. After the introduction, a reduction in the level of SBE synthesis and / or the level of SBE activity and / or the composition of the starch in the plants can be achieved. Without wishing to be bound by theory it is believed that the sequence in antisense expressed nucleotides of the present invention is used to sense introns on pre-mRNA and thus avoids the addition of the pre-mRNA and / or the subsequent translation of the mRNA. It is therefore believed that this binding reduces a level of enzyme activity in the plant (in particular the activity of SBE), which at the same time is believed that the activity of SBE influences the amylose: amylopectin ratio and therefore both in the branching pattern of amylopectin. Therefore, the present invention provides a method in which 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 using an antisense RNA technique that uses an antisense intron sequence. In summary, the present invention therefore relates to the surprising use of antisense intron sequences in a method to affect enzymatic 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. For example, it is possible to use antisense promoter sequences to affect enzyme activity, such as SBE promoter in antisense, such as the nucleotide sequence comprising the nucleotide sequence shown as I.D. SEC. No. 28 to a variant, a derivative or a homologue thereof.
The following pages present a number of sequence lists that have been numbered consecutively from I.D. SEC. No. 1 - I.D. SEC. No. 29. In a few words, I.D. SEC. No. 1 - I.D. SEC. No. 13 represent sequences of introns in sense (genomic DNA) I.D. SEC. No. 14 represents the promoter sequence of SBE (genomic sequence); I.D. SEC. No. 15 - I.D. SEC. No. 27 represents the sequences of introns in antisense; and I.D. SEC. No. 28 represents the sequence complementary to the SBE promoter sequence, ie the SBE promoter sequence in the antisense orientation. The complete sequence of genomic nucleotides for SBE including the promoter, exons and introns is shown as I.D. SEC. No. 29 and explained by means of Figures 4 and 12 that highlight the particular features of the genes.
INFORMATION ON SEQUENCES ID. SEC. No. 1 SEQUENCE OF THE INTRON 1 (1167 bp). G7AA77777AC7AA777CA7G77AAt77CAA77A7T777AGCC777GCA777CA77T7CCAA7A7A7C7 GGA7CA7C7CC7TAG_7777_TrA7777A777777A7AA7A7CAAA7A7GGAAGAAAAA7GACAC7TG7AG AsCCATAtGTAAGTATCATsTGACAAAtTTGCAAGGTGGTTGAGTGTATAAAAtTCAAAAATTsAGAGA TGGAGGGGGGGTGGGsGBARAGACAATATTTAGAAAGAGTGTTCTAGGAGGTTATGGAGGACACGGATG AGGGGTAGAAGGTTAGTTAGGTATtTGAGTGTsTCTGsCTTATCCT_TCATACTAGTAGTCGTsGAAT tAtttsGstAGT cttsttttG7TAtttsAtctttGTTAttc ATtttctGttt-ttGTActtcGATt
A77G7A77A7A7A7C77G7CG7AG77A77G77CC7CGG7AAGAA7GCTC7AGCA7GC77C777AG7G7 777A7CA7GCC7TC777A7A77CGCG77GC777GAAA7GC7777AC77TAGCCGAGGG7C7A77AGAAA CAA7C777A7C7CG7AAGG7AGsGs7AAAG7CC7CACCACAC7CCAC77G7GsGA77ACA77G7G77] (] TG77G77s7AAA7CAA77A7s7A7ACA7AA7AAG7GGA777777ACAACACAAA7ACA7GG7CAAGGG Z AAAG77C7GAACACA7AAAGGG77CA77A7A7G7CCAGGGA7A7GATAAAAA77G777C777G7GAAA3 77A7A7AAGA777s77A7GGC7777GC7GGAAACA7AA7AAG7TA7AA7sC7GAGA7AGC7AC7GAAG7 77G777777C7AGCC7777AAA7G7ACCAA7AA7AGA7TCCG7A7CGAACGAG7ATG7777GA77ACC7 GG7CA7GA7G777C7A777777ACA777T777GG7G77GAAC7GCAA7TGAAAATG7TG7A7CC7A7GA sACssA7AG77GAGAA7GTG77C777G7A7sGACC77GAGAAGC7CAAACsC7AC7CCAA7AA7TrC7A 7GAA77CAAA77CAGTT7A7GGC7ACCAG7CAG7CCAGAAACTAGGATA7GC7sCATA7AC77GrrCAA 77A7AC7G7AAAA777C77AAG77C7CAAGA7A7CCA7G7AACC7CGAGAAT77C777GACAG 5. 2 0 INTRON 2 SEQUENCE (321 bp).
G7A7G777GA7AA777A7A7GG77GCA7GGA7AG7A7A7AAA7AG77GGAAAAC77C7GGAC7GG7G ^ CA7GGCA7A? T7GA7C7G7GCACCG7G7GGAGA7G7C. ^ CA7G7GT7ACT7 A.ACC77AAC77GGGAAAGACAGC7C ^^^ ^^ ™ ^^ T7C77 TAC7CC7GTGGGCA77tG77A77TGAA77ACAA 77777GA7A7AAAC7AAC7G7GG7GCA77GC77GCBKKK ID. SEC. No. 3 SEQUENCE OF THE INTRON 3 (504 bp). GTAACAGCCAAAAG77G7sCT77AGsCAG777GACC77AT7TrGGAAGA7GAA77G777A7ACC7AC7T 7GACT77GC7AGAGAA7T_TGCA7ACCGGGGAGTAAG7AG7GGC7CCA77TAGG7GGCACC7GGCCAT7 77777sA7C7TrtAAAAAGCTs777GA7TGGG7C77CAAAAAAG7AGACAAGG77777GGAGAAG7GAC ACACCCCCGGAG7G7CAG7GGCAAAGCAAAGA7777CAC7AAGGAGAT7CAAAA7A7AAAAAAAG7A7A GACA7AAAGAAGC7GAGGGGA77CAACA7G7ACtA7ACAAGCA7CAAA7ATAGTC7TAAAGCAArTTTG 7AGAAA7AAAGAAAG7C77CC77C7G77GC77CACAA7 rCC7TC7ATrA7CA7GAG77AC7C777C7G 77CGAAATAGCT CC77AA7A77AAA77CA7GATACTr77GTrGAGATrrAGCAGTrTTT7 rG7G7A
AAC7GC7C7CTGGGGT7GCAG
0 ID. SEC. Do not . 4 SEQUENCE OF THE INTRON 4 (146 bp).
G7AGGTCC7CG7C7AC7ACAAAA7AG7AG7TTCCATCA7CA7AACAGATptCC7ATTAAAGCATGA7G 77GCAGCA7CAT7GGCT77CT7ACA7G77C7AA7TsCTA7TAAGGT7ATsCT7CTAATTAAC7CATCCA CAA7GCAG
ID. SEC. Do not . 5 SEQUENCE OF THE INTRON 5 (218 bp). GTT7TGTrA77CA7ACC7TGAAGC7GAAtT7TsAACACCATCA7CACAGGCA77TCGA77CA7GtTC77 ACTAGTC77G77A7G7AAGACA7TrtsAAATGCAAAAG77AAAATAAttGTG7C7TTAC7AATTTGsAC TTGA7CCCA7AC7C.Tr7CCC7TAACAAAA7GAG7CAATTCTA7AAG7GC7TGAGAACttAC7ACttCAG
CAAT AAACAG
ID. SEC. No. 6 SEQUENCE OF THE INTRON 6 (198 bp). GTA7TTTAAATTTATrTCTACAACTAAATAATTCTCAGAACAATtsTTAGA7AGAATCCAAATATATAC
? F¡ sTCCTGAAAG7ATAAAAG7ACTTAT7TTCsCCATGGGCCTTCAGAATATTGGTAGCCsCTGAATATCAT GATAAGTrA7T7A7CCAG7GACATpTTATGTTCACTCCTATTATGTCTGCtsGA7ACAG ID. SEC. Do not . 7 SEQUENCE OF THE INTRON 7 (208 bp). GTTTGTCTG777C7A77GCATTT7AAGG7TCATATAGGTTAGCCACGGAAAA7CTCACTCTTTGTGAGG 7AACCAGGG GC7GA7GGA7TAT7CAA7TTTCTCGTTTATCATTTG7T7A7TCTTT7CA7GCATTGTG7
7TC7TTGGCAA7A7CCC7C77ATG7GGAGG7AA77TTTCTCATCTA7TCAC7777AGCTTCTAACCACAG
ID. SEC. Do not . 8 SEQUENCE OF THE INTRON 8 (293 bp).
GTATG7C7TACA7C777AGATA7T77GtGATAA7TACAATTAG_7_T7GGCT7ACTGAACAAGATTCAT7 CCTCAAAATGACC7GAAC7GTTGAACATCAAAGGGGTTGAAACATAGAGGAAAACAACATGATGAATG7 TTCCATtsTCTAGGGAT_ CTATrATGTTGCrGAGAACAAATGTCATCTTAAAAAAAACAT7GTtTACT TTTt7GTAGtATAGAAGATrACTG7ATAGAGtttGCAAGTGTGTCTGTTTTGGAGTAATTG7GAAATG7 TTGA7GAAC7TGTACAG
ID. SEC. Do not . 9 SEQUENCE OF THE INTRON 9 (376 bp). GTrCAAGTATtT7GAA7CGCAGCt7stTAAAtAATCTAGTAATtTtTAsATTsCTTACTtsGAAGTCTA cttsGttctsGGGATGA7AGCtcAtrrcAtcttsttctACttAttttccAAccGAATTt rGATttttG
TTTCGAGATCC_AAGtA77AG? TTCATtTAC? CTtATTACCGCCTCAttTCtACCACtAAGGCC rGAtG
AGCAGCtTAAGTTGATTCtTTGAAGCTATAGTttCAGGCrACCAAtCCACAßCCTsCTATAlTlXil'I ^
ATAC77ACC7TrTCT77ACAA7sAAG7GA7AC7AATrGAAA7GG7CTAAA7C7GA7A7C7A7ATrtCTC
CG7C777CC7CCCCC7CA7GA7GAAA7GCAG
ID. SEC. Do not . 10 INTRON 10 SEQUENCE (172 bp).
G7AAAA7CA7C7AAAG77GAAAG7G77GGG777A7GAAG7GC777AA77C7A7CCAAGGACAAG7AGAA ACC7T7T7ACCT7CCATT7CTTGA7GA7GGATT7CA7AT7ATT7AA7CCAA7AGC7Gs7CAAAT7CGG7
^ 7AGC7G7AC7GAT7AG77AC77CAC777GCAG
ID. SEC. No. 11 SEQUENCE OF THE INTRON 11 (145 bp).
G7A7A7A7G7TTrACT7A7CCA7sAAA77AT7sC7C7GCT7sTTTT7AA7G7AC7sAACAAG7TT7A7G GAGAAG7AAC7GAAACAAA7CA7777CACA77G7C7AA777AAC7C7mT7C7GA7CC7C3CA7GACG AAAACAG
ID. SEC. No. 12 SEQUENCE OF THE INTRON 12 (242 bp).
G7AAGGA777GCTGGAA7AAC7777GA7AA7AAGA7AACAGATG7AGGG7ACAG77C7C7CACCAAAAA GAAC7G7AAT7G7C7CA7CCA7CT77AG77G7A7AAGA7A7CCGAC7G7C7GAGT7CsGAAG7s77TGA GCC7CC7GCCC7CCCCC7GCG77s777AGC7AA77CAAAAAGGAGAAAAC7G777A77GA7GA7C77TG 7C7TCA7GC7GACATACAATC7G77C7CA7GACAG
ID. SEC. No. 13 SEQUENCE OF THE INTRON 13 (797 bp).
GTACAG77C77GCCG7G7GACC7CCC7? TA77GTGG77 GG77CATAG_77_A777GAA7GCGA7AGAA
G77AAC7A7TGATTACCGCCACAA7CGCCAGT7AAG7CCTC7GAAC7AC7AA777GAAAGG7AGGAA7A GCCs7AA7AAGG7C7AC77T7GGCA7C7tAC7sT7ACAAAACAAAAGGA7sCCAAAAAAA77CTrctC7
ATCC7C7Trt7CCC7AAACCAG7sCA7G7AGC77sCACCTsCATAAAC7tAGG7AAA7GA7CAAAAA7G
AAG77sA7GGGAAC7TAAAACCGCCC7GAAGTAAAGC7AGGAA7AG7CA7A7AA7G7CCACC7T7GG7G
7C7GCGC7AACA7CAACAACAACA7ACCtCG7GtAGTCCCACAAAG7GGTTrCAGGGGGAGGG7AGAG7
GTA7GCAAAAC77ACTCC7A7C7CAGAGG7AGAGAGGATTGGGTCAATAGACCC77GGC7CAAGAAAAA
AAG7CCAAAAAGAAGTAACAGAAG7GAAAGCAACATG7G7AGC7AAAGCGACCCAAC77G7TTGGGAC7Ts77s77GAAACAG7GCA7GTAGATGAACACA7G7CAGAAAA7GGACAACACAGTrAT
TTTs7GCAAGTCAAAAAAA7G7AC7AC7A777C77TsTGCAsC7TTATGTATAGAAAAG77AAATAACt (•) AATGAA7TTTGC7AGCAGAAAAA7AGC77GGAGAGAAA7TTGGTA7A77GAAC7AAGC7AAC7A7AT7C
ATC TTCTTT 7GC7TCTTC77C7CC77G7T7GTGAAG
0
ID. SEC. No. 14 DNA SEQUENCE OF THE PROMOTING REGION OF THE SBE GENE
ATCA7GGCCAA77AC7GG77CAAA7GCAT7ACT7CC7T7CAGArrCT7TCGAGT7C7CA7 60
GACCGGTCC7AC7ACAGACGA7AC7AACCCG7GGAAC7G77GCA7C7GCTrC77AGAAC7 120
CTA7GGC7A77Trcs77AGC77GGCG7CGGT77GAACA7AGTr rrG7777CAAAC7CT7 1B0
CATT7ACAG7CAAAA7G77G7A7GGTTTTTGTTTTCC7CAATGA7GTT7ACAG7G77aTG 2 0
7TsTCA7C7s7AC7777GCC7A77AC7tG7t77GAG7TACA7GTrAAAAAAG7G777ATr 300
7TGCCA7A7Trrs77C7C77A77A77A77A7CA7ACA7ACA77ATrACAAGGAAAAGACA 360
AG7ACACAGA7C77AACG7TGA7G77CAA7CAACTGG7GGAGGCA77GACAGG7ACCACA 420
AATTGGGAG77TA7GA77AAG77CAATC77AGAATATGAA7TGAACA7C7ATGA7AGA7G 80
CA7AAAAA7AGCTAA7GA7AGAACA77sACA77TGGCAGAGCTTAGGG7A7GG7A7A7CC 540
AACG77AA777AG7AA77777G77ACG7ACG7ATATGAAA7A7TGAA77AATCACA7GAA 600
CGG7GGA7A77A7A77A7GAG77GsCA7CAGCAAAA7CA77GG7G7AG77sAC7G7AG77 660 .0 GCAGA777AA7AA7AAAA7sG7AArrAACGG7CsA7A77AAAA7AAC7CTCA777AAG7 720
GGGAT7AGAAC7AGT7AT7AAAAAAA7G7A7ACTrrAAG7sATT7GA7GGCA7A7AA7T7 780
AAAG7Tr77CAt77CA7GC7AAAA77GTrAATTAT7G7AA7G7AGAC7GCsAC7GGAA77 840
ATtA7AG7G7AAA777A7GCA77CAG7G7AAAATTAAAGtATTGAACT7G7C7G777tAG 900 AAAATACttrA7AC7TrAA7A7AGGATTrtG7CAtGCGAAT7TAAA77AATCGA7A77GA 960
ACACGGAA7ACCAAAA77AAAAAGGA7ACACA7GGCCT7CA7ATGAACCG7GAACC7TGG 1020
ATAACG7GGAAG77CAAAGAAGG7AAAGTTrAAGAA7AAACTGACAAA7TAA777C77Tr 1080
ATTTGGCCCAC7AC7AAA777GCTG7AC7TGCTAACATG7CAAGTGG7GCCC7C77AG7T 114O 1.5 GAA7GA7A77CA7T777CA7CCClA7AAGTrCAAtt7sA7tG7CA7ACCACCCA7GA7s77 1200
CTGAAAAA7GCT7GGCCATTCACAAAG7TTATCTTAGTtCCTATGAACTTTA7AAGAAGC 1260
7TTAATT7GACA7G77A777ATA77AGATGA7AtAATCCA7GACCCAATAGACAAG7G7A 1320
7tAA7A7TG7AAC7TrG7AA77GAG7G7G7C7ACA7CrrA77CAA7CA77TAAGG7CA77 1 80
AAAA7AAA77A777TrrGACA77CtAAAAC777AAGCAGAA7AAA7AG7T7A7CAA77A7 1440
7AAAAACAAAAAACGAC77AT77A7AAA7CAACAAACAA7TTrAGA77sC7CCAACA7A7 1500
7TtTCCAAAT7AAA7sCAGAAAA7sCA7AATTTTATACTrGA7Ct77A7AGCTrA777Tr 1560
77TAGCCtAACCAACGAA7A777s7AAACTCACAAC77sA7TAAAAGGGAt77ACAACAA 1620 0 GATA7A7A7AAGTAG_7_sACAAA7CTTGATTTrAAATATTTrAATTTsGAGGTCAAAATT7 1680
7ACCA7AA7CA7T G7A777A7AA77AAAT7TTAAATA7CTTATt7A7ACA7A7C7AG7A 1740
AACTT_TAAA7ATACG7A7A7ACAAAA7A7AAAAt7A7TGGCGtTCA7AtTAGG7CAA7A 1B00
AATCCT7AAC7ATA7C7GCCT7ACCACTAGGAGAAAGTAAAAAAC7CTTTACCAAAAA7A 1860
CATG7AtTATG7A7ACAAAAAG7CGATTAGA7TACCTAAATAGAAATTstATAACGAGTA 1920
AGTAAG7AGAAA7A7AAAAAAAC7ACAATACTAAAAAAAATATGT7TtACTTCAA7TrCG 1980
AAACtAA7GGGGTC7GAG7sAAATA7TCAGAAAGsGsAGGACTAACAAAAGGs7CA7AAT 20 0
GTTTrrtTA7AAAAAGCCACTAAAA7GAGGAAATCAAGAATCAGAACATACAAGAAGGCA 2100
TK GCAGCTGAAGCAAAGTACCATAATT7AAtCAATGGAAATTAATTrCAAAGTT7TA7CAAA 2160 ACCCATTCG ID. SEC. No. 15 INTRON 1 ANTICIPATION SEQUENCE (1167 bp). CtG7CAAAGAAA77C7CGAGG77ACA7GGA7A7C7tGAGAAC7TAAGAAA77T7ACAG7A7AATTGAAC
AAG7A7A7GCAGCA7A7CC7AA777C7GGACTGACtGG7AGCCA7AAAC7GAA777GAA77CATAGAAA
77A7TGGAG7AGCG777sAGC77CrrCAAGG7CCAtACAAAGAACACA77C7CAAC7A7CCsTCTCATAG
GATACAACA7TI CAA77GCAG77CAAC? CCAAAAAAA7G7AAAAAA7AGAAACA7CA7GACCAGGTAA
7CAAAACA7AC7CG77CGA7ACGGAATC7A7TA77ss7ACA7T7AAAAGGC7AGAAAAAACAAACTTCA
G7AGC7A7C7CAGCA7tA7AAC77ATTATGTT7CCAGCAAAAGCCATAACAAA7C77A7A7AAC7TrCA
CAAAGAAACAAT7777A7CA7A7CCCTGGACA7ATAATsAACCC77TA7G7G77CAGAAC77TGCCCTr
GACCA7G7A7TrG7G77G7AAAAAA7CCACtTATTATG7ATACA7AA77GA7T7ACAACAACAAACACA
ATG7AA7CCCACAAG7GsAG7G7GG7GAGGAC7TrACCCC7ACC77ACGAGA7AGAGAGA77GTrTC7A
ATAGACCC7CGGC7AAAG7AAAAGCA77TCAAAGCAACGCGAA7A7AAAGAAGsCA7GA7AAAACACTA
AAGGAAGCA7GC7AGAGCA77CTrACCGAGGAACAAtAAC7ACGACAAGA7A7A7AA7ACAA7AA7CGA
AG7ACAAGAAACAGAAAA7AGAA7AACAAAGA7CAAA7AACAAAACAAGAAAC7ACCCAAA7AAGGCCA
CGAC7AC7AGtA7GAAAGGA7AAGCCAGACAACACTCAAA7ACCTAAC7AACC77C7ACCCC7CATCCG
7GTCC7CCA7AACCrCC7AGAACAC7 rT7CTAAATATTG7CTYTVCCCCCACCCCCCC7CCATCTC7C
AATT777GAA7777A7ACAC7CAACCACCT7GCAAATTTG7CACA7GATAC77ACA7A7GGC7CTACAA
GTGTCA7mTC77CCA7AT7TsAtA77ATAAAAAATAAAATAAAAAACTAAGGAGA7GA7CCAGATATAAA7GCAAAGGC7AAAAATAA7tsAAATTAACATGAAAT7AG7AAAAA77AC
ID. SEC. No. 16 SEQUENCE IN RNTISENTIDES OF THE INTRON 2 (321 bp).
MMTlVGCAAGCAA7GCACCACAGrrAGTtTATA7CAAAAAGAAGAAAGGTATrAACGGAGCTAAAAACTG 7tATA7ACCACA7GAAAGAAGT7GATAATGTGAAAACACCATGCTCAtAAAGA7Ts7AA7TCAAATAAC AAA7GCCCACAGGAG7AAAGAGC7GtctTTCCCAAGTtAAGGTATTATAAA7TGGCGGAACGAAGTAAC 20 ACA7s777GACA7C7CCACACGG7GCACAGA7CAAATATGCCATGAGCACCAG7CCAGAAGTrTTCCAA
CTATTrATA7ACTA7CCA7GCAACCA7AtAAA7tATCAAACATAC
t r¡ ID. SEC. No. 17 INTRON 3 ANTICIPATION SEQUENCE (504 bp).
CTGCAAAAAAAGAGAGCAG7TTACAC_AAGAAAAAACTGCTAAAtCTCAACAAAAG7A7CA7GAATtTAA 7ATTAAGGAAGC7ATT7CGAACAGAAAGAG7AACTCATGATAATAGAAGGAAAT7G7GAAGCAACAGAA GGAAGACr7TCTTTATT7CTACAAAAT GCTTTAAGACTATAT TGATGCTTGTATAGTACATGTTGAA TCCCCTCAGCTTCTTTA7GTCTATAC rTTTTTATATTTTGAATCTCCTTAGTGAAAATC7TTGCTTTG CCAC7GACACTCCGGGGG7GTG7C? C rCTCCAAAAACC rGTC-7AC-TTTriTGAAGACCCAATCAAAC AGCTtttTAAAAGATCAAAAAAA7GsCCAsstGCCACCTAAATGGAGCCACTACT7AC7CCCCGsTATG CAAAAtTCTCTAGC_AAAGTCAAAG7AGGTATAAACAATtCAtCTTCCAAAATAAGG7CAAACrrsCCTAA AGCACAAC7TTTGGCTG77AC
ID. SEC. Do not . 18 INTRON 4 ANTICIPAL SEQUENCE (146 bp).
C7GCA77G7GGA7GAG77AATTAGAAGCA7AACCTTAA7AGCAATTAGAACA7G7AAGAAAGCCAATGA 7GC7GCAACA7CA7GC777AAtAGGAAAA7C7s7tATGA7GA7GsAAACTACtATrrts7AGtAGACGA GGACCTAC
ID. SEC. No. 19 INTRON 5 ANTI-SEQUENCE SEQUENCE (218 bp). Cts777AA77GC7GAAG7AGTAAG77CTCAAGCACTTATAGAATTGACTCATTT GTTAAGGGAAAGAG 7ATGGGA7CAAGtCCAAATTAG_7_AAAGACACAA7TATTrTAACTtTTGCATT7CAAAA7s7C77ACATA ACAAGAC7AGTAAGAACATGAA7CGAAA7GCC7G7GA7GATGsTGtTCAAAAT7CAGCt7CAAGGtA7G AA7AACAAAAC
ID. SEC. Do not . 20 ANTENNA SEQUENCE OF THE INTRON 6 (198 bp).
C7G7A7CCAGCAGACA7AATAGGAG7GAACA7AAAAAtsTCACTGGATAAATAAC7TA7CA7GATATrC AGCGGCTACCAATATTCtsAAGGCCCATsGCGAAAATAAGTACTTTrAtACTTTCAGGACG7AtAtATT 7GGA77C7A7C7AACAA7TGTTC7GAGAA77ATrTAGTtGTAGAAATAAATTTAAAA7AC
ID. SEC. Do not . 21 INTRON 7 ANTI-SEQUENCE SEQUENCE (208 bp).
CTG7GGTtAGAAGCTAAAAGTGAATAGA7GAGAAAATTACCTCCAAATAAGAGGGATATTGAAAAAGA AACACAA7GCATGAAAAGAATAAACAAA7GAtAAACGAGAAAATTGAATAATCCA7CAGAACCCTGGTT
ACC7CACAAAGAG7sAGA7TtTCCG7GsCTAACC7ATATGAACC7TAAAA7GCAA7AGAAACAGACAAAC
ID. SEC. Do not . 22 INTRON 8 ANTI-SEQUENCE SEQUENCE (293 bp).
CTstACAAGTtCAtCAAACAtTTCACAATTACTCC_AAAACAGACACACTtGCAAACTC7A7ACAGTAAT CTTCTATAC7ACAAAAAAGTAAAC_ tsTTr7TTttAAGATGACATTTsTTCtCAGCAACAtAATAGAA ATCCCTAGACAATGGAAAC TTC TC TGTTG'rti CCCC CCTCTATGTTTC_ ^ T GATGTTCAACAG TtCAGGTCATTTTGAGGAATGAATCTTGT7C -????? "- 3TAAGCCAAACTAATTstAATTATCACAAAATATCT AAAGA7G7AAGACATAC
ID. SEC. Do not . 23 ANTI-SENSE SEQUENCE OF THE INTRON 9 (376 bp). CTGCATTTCAtC? TsAsGsGGAGGAAAsACsGAsAAATATAGATATCAGATTTAsACCATrrCAAtTAG_7_ATCACTT _ATTGTAAAGAAAAGGtAAGTATCCAACAAATAtAGCAGGCTsTGGAtTGstAGCCTsAAA ^ ATAGCTTa_ftAGAATCAACttAAGC sCtCATCAAGsCCTTAGTGGtAsAAATGAGsCGsTAATAAs
? rAAATGAATC A7ACTTGGATC7CsAAACAAAAAT U3AAA7TCGs7TGGAAAA7AAGTAGAACAA
GA7GAAA7GAGC7A7CA7CCCCAGAACCAAG7AGACTrCCAAG7AAGCAA7C7AAAAA7TAC7AGA77A
7t7AACAAGC7GCGA77CAAAA7AC77GAAC
ID. SEC. Do not . 24 INTRON 10 ANTI-SEQUENCE SEQUENCE (172 bp).
C7GCAAAG7GAAG7AAC7AA7CAG7ACAGC7A77ACCGAATT7GACCAGC7AT7GGAT7AAA7AATA7G AAA7CCA7CA7CAAGAAA7GGAAGG7AAAAAGs7TTC7AC77G7CC77sGA7AGAA77AAAGCAC77CA 7AAACCCAACAC777CAAC777AGA7GA7T77AC
ID. SEC. No. 25 INTRON 11 ANTIQUESTION SEQUENCE (145 bp).
C7s7777CG7CA7sCGAGGA7CAGAAAAAAGAGT7AAAT7AGACAA7G7GAAAA7GATT7G7T7CAs77 AC77C7CCA7AAAAC77G77CAG7ACAT7AAAAACAAGCAsAGCAA7AA777CA7GGA7AAG7AAAACA 7A7A7AC
ID. SEC. No. 26 INTRON 12 ANTICIPATION SEQUENCE (242 bp). C7G7CA7GAGAACAGA77s7ATG7CAGCAtsAAsACAAAGA7CATCAA7AAACAG77TrC7CCTTTtts AATTAGC7AAACAACGCAGGGGGAGGGCAGGAGGCTCAAACAC77CCGAAC7CAGACAGTCGGATATC7 TA7ACAAC7AAAGA7GGA7GAGACAATtACAG7TCT77T7GG7sAGAGAAC7G7ACCC7ACA7CTGT7A 7CT7AT7A7CAAAAGT7ATTCAAGCAAATCCTTAC ID. SEC. No. 27 SEQUENCE IN THE ANTIQUESTION OF THE INTRON 13 (797 bp).
C77CACAAACAAGGAGAAGAAGAAGCAAAAAGAAAGA7GAA7A7AG77AGC77AG77CAA7A7AAAAAA
77tC7C7CCAAGC7A77777C7GC7AGCAAAA7TCATTAG_77_A777AAC7TrtC7A7ACATAAAGC7sC
ACAAAGAAA7AG7AG7ACAll'T7T ': "n, GACTTGCACAAAA7AAC7G7G77G7CCA7777C7GACA7G7G7CAC7G777CAACAACAACAACTACT7CAGtCCCAAACAAG77sGG7CGC777AGC7AC
ACA7G77GC777CAC77C7GT7AC TCrtrrTGGACTTT7T_TC7TGAGCCAAGGG7C7A77sAAAAAA
7CCTC7C7ACC7C7sAGA7AGGAG7AAGT7ttsCATACAC7C7ACCC7CCCCCrrsAAACCA rtTGTGG
GAC7ACACGAGG7A7GTrGTTG77GATGTTAGCGCAGACACCAAAGGTGGACA7tA7ATGAC7ATtCC7
AGC777AC77CAGGGCGG7T7TAAG7TCCCATCAACrrrCATTptsATCA7TrACC7AAG777ATsCAG
G7GCAAGC7ACA7GCAC7GG7TrAGGGAAAAAGAGGAtAGAGAAGAA77T77TTGGCA7CC7TTTGT77
7G7AACAG7AAGA7GCCAAAAGTAGACC7TATTACGGC7A77CC7ACC777CAAA77AG7AG7TCAGAG
GAC77AAC7GGCGA77G7GGCGG7AATCAA7AGT7AAC7TC7A7CGCA77CAAATAACtATGAACAAAA
CCACAATAAAAAGGGAGG7CACACGGCAAGAACTGTAC
ID. SEC. No. 28 SEQUENCE OF ANCIENT DNA IN THE REGION PROMOTING THE GENE
SBE. CGAA7GGG7777GA7AAAACT77GAAAT7AATT7CCATTGATTAAAT7A7GG7ACT77GC 60 7tCAGC7GC7GCCTrC7TG7A7G77CTGA77C7TGATrTCCTCA77TTAG_7_GGCTpTTA 120 7AAAAAAACA7TATGACCCTITrs77AG7CC7CCCCTITCTGAA7ATTrCAC7CAGACCC 180 CATTAG_777_CGAAAtTGAAGtAAAACA7Aa "P'ti? RrAGtAT7s7AG77T777TATA7tT 2 0 CTAC77AC77ACTCG77A7ACAA777CTATtTAGGTAA7CTAA7CGACTTtTTG7A7ACA 7tAAGGAT77A7TsACC7AA7A7sAACsCCAA7AAinTATAT7Trs7A7ATACsTA7A7 420 360 300 7AA7ACA7G7A7TTrTGG7AAAGAG77TTTTAC777C7CC7AG7GG7AAGGCAGA7A7AG T7AAAAGTT7ACTAGATA7G7A7AAA7AAGA7ATTrAAAATT7AATTATAAA7ACAAA7s AT7A7GG7AAAAT7TTGACC7CCAAATTAAAATATGAAAATCAAGA7TTG7CACTAC77 480 540 600 A7ATA7A7C77sT7GTAAAtCCCT7TTAATCAAGTrG7GAGT77ACAAATAT7CG77GG7? N 7AGGC7AAAAAAAA7AAGC7ATAAAGATCAAG7A7AAAATTA7GCATTT7CTGCArTTAA 7TrGGAAAAA7ATG rsGAGCAA7C7AAAATTsnts77GATTrA7AAATAAG7CG7Tr7 660 720 780 77G777TrAA7AA77GA7AAAC7A77TAT7CTGC7TAAAG7rrrAGAA7GTCAAAAAA7A ATT7A77T7AA7GACC77AAATGA77sAAtAAGA7GTAGACACAC7CAATTACAAAGrrA CAATAT7AA7ACACTrG7CTAT7sGGTCATGGATrATAtCA7C7AATATAAA7AACAts7 840 900 CAAA7TAAAGC 7TC7TA7AAAG77CATAGGAAC7AAGA7AAAC7TTGTGAA7GGCCAAGC 960 ATTT77_AGAACA7CA7GGGTGG7A7GAC? ATCAAATtsAAC77ATGGGATGAAAAAtGA ATA7CATTCAACTAAGAGGGCACAACTTGACA7GTTAGAAAG7AAAGCAAArrrAG7AG7 1020 1140 1080 5 GGGCCAAA7AAAAGAAA77AATTrG7CAG7TTA77CT7AAAC_TTACC7TCtTTGAACTr CCACG77A7CAAAGG77CACGGTrCATATGAAGGCCAtGTsTA7CC77TTTAA7TTrGG7 1200 ATrCCG7G77CAAtA7CGATTAA7TrAAATTCGCA7GACAAAA7CCTATATTAAAGTATA AAG7A77T7C7AAAACAGACAAG77CAATAC7TtAATTrTACAC7sAATGCAtAAAT7TA 1260 1320 1380 CAC7A7AA7AA7tCCAG7CGCAG7CtACA7TACAATAA7TAACAATrTTAGCA7GAAA7G AAAAACTr7AAATTATA7sCCA7CAAAtCACT7AAAG7A7ACA7TTmTAA7AAC7AG7 7CTAA7CCCAC77GAAA7GAGAG77ATT77AA7A7CGACCG77AATTACCA7777A77AT 1440 1500 1560 0 7ATCCACCGT7CATGTGATTAATTCAATATT7CATA7ACGtACG7AACAAAAATTAC7AA 1620 7AAA7C7GCAACTACAG7CAAC7ACACCAA7sA7TrtsC7sA7GCCAAC7CA7AA7A7AA ATTAACG77GGA7ATACCATACCC7AAGCTCTsCCAAA7GTCAA7GtTCTATCATTAGC7 ATT777A7GCA7CTA7AATAGATG7TAAATTCATAttCTAAGATrGAACTTAATCAtAAA 1680 1740 1800 CTCAAAA777s7GGTACCTGtCAATGCCTCCAAAAGtTGATTGAACATAAACGTTAAGAT CTG7G7A ? CTTGTC7TTTCC7TG7AATAATGTATGTATGAtAATAATAATAAGAGAACAAA ATA7GGCAAAAtAAACACtTTTTTAACATGTAACTC AAACAAGTAAtAGGCAAAAGTAC 1860 1920 2040 AGA7sACAACACAACAC7G7AAACA7CA7TGAGGAAAACAAAAACCA7ACAACA77TrGA 19T0 CTGTAAATGAAGAGTTTGAAAACAAAAACTATGTTCAAACCGACGCCAAGCTAACGAAAA 5 7AGCCATAGAGTTCTAAGAAGCAGATs < _AACAGTTCCACsGGTTAG_7_ATCGTC7sTAGTA 2100 GGACCGG7CATGAGAACTCGAAAGAATCTGAAAGGAAGTAATsCATTTGAACCAGTAATT ZL60 GGCCA7GAT ID. SEC. Do not . 29 GEN SBE GENOMICO
A7CA7GGCCA A77AC7GG77 CAAATGCA77 ACTTCCTTTC AGATTCTTTC GAG77CTCA7 60
GACCGGTCC7 AC7ACAGACG ATACTAACCC G7GGAACTG7 7GCA7C7GCT 7CTTAGAAC7 120
CTA7GGC7A7 777CGTTAGC 7TGGCG7CGG T7TGAACA7A GT? TTGTGG TCAAACTCT7 180 CATTTACAG7 CAAAA7G7TG 7ATGGTTTTT G7TTTCC7CA A7GATGTTTA CAGTGTTGTG 240
7TG7CA7C7G 7AC7TTTGCC 7ATTAC77G7 TTTGAGTTAC ATGTTAAAAA AGTGTTTAT7 300
7TGCCA7A77 T7GT7C7CT7 ATTA7TA7TA TCATACATAC A7TATTACAA GGAAAAGACA 360
AGTACACAGA 7C7TAACGTT 7ATGT7CAA7 CAACtTTTGG AGGCAT7GAC AGGTACCACA 420
AATTTTGAG7 77A7GA7TAA GTTCAATCT7 AGAATATGAA TTTAACATCT ATTATAGATG 480
CATAAAAA7A GCTAA7GA7A GAACAT7GAC ATTTGGCAGA GC7TAGGG7A 7GGTATATCC 540
AACG77AA77 7AG7AA7777 7GTTACG7AC GTATATGAAA TATTGAATTA ATCACATGAA 600
CGG7GGA7A7 7ATA77A7GA GT7GGCA7CA GCAAAATCA7 7GGTGTAGT7 GACTGTAG_77_660 GCAGAT77AA 7AA7AAAA7G GTAA7TAACG G7CGATA77A AAATAAC7CT CATTTCAAG7 720C7AG7TA77A AAAAAA7G7A 7ACTTTAAG7 GATTTGATGG CATATAATT7 780
AAAGTTT77C ATT7CA7GC7 AAAATTGTtA ATTATTGTAA 7GTAGACTGC GACTGGAA7T 840
ATTATAG_7_G7 AAATTTATGC ATTCAGTG7A AAATTAAAG7 ATTGAACT7G 7CTGTTtTAG_900_AAAA7ACTT7 ATACTT7AA7 ATAGGA7TT7 GTCATGCGAA 7TTAAATTAA 7CGATATTGA 960
ACACGGAA7A CCAAAATTAA AAAGGATACA CATGGCCTTC ATATGAACCG tGAACCtTTG 1020
ATAACG7GGA AGT7CAAAGA AGGTAAAGTt 7AAGAATAAA CTGACAAATT AATTTCTri 1080
ATTTGGCCCA CTACTAAATT TGCT7TACT7 7CTAACATGT CAAGTTGTGC CCTCTTAGT7 1140 GAATGATA77 CAT7TT7CAT CCCATAAGTT CAATTTGATT GTCATACCAC CCATGATGTT 1200
CTGAAAAA7G CTTGGCCATT CACAAAGTTT ATCTTAGTTC CTATGAACTT TATAAGAAGC 1260
TTTAATTTGA CATG77A7TT ATATTAGATG ATATAATCCA TGACCCAATA GACAAGTGTA 1320
7TAA7A77G7 AAC7TTG7AA TTGAGTGTGT CTACA7CT7A 7TCAATCA7T 7AAGGTCAT7 1380
AAAA7AAA77 AT77TT7GAC A7TCTAAAAC TTTAAGCAGA ATAAATAG_7_T 7ATCAATTA7 1440
7AAAAACAAA AAACGACTTA 7tTATAAA7C AACAAACAAT 7TTAGATTGC 7CCAACATA7 1500
TTTTCCAAA7 7AAA7GCAGA AAATGCATAA 7TTTATAC77 GATCTTTATA GCTTATTT77 1560
TTTAGCCTAA CCAACGAATA TTTGTAAACT CACAACTTGA TTAAAAGGGA TTTACAACAA 1620 GATATATATA AGTAGTGACA AATCTTGATT TTAAATATTT TAATTTGGAG GTCAAAATTT 1680
TACCATAATC ATTTGTATTT ATAATTAAAT TTTAAATATC TTATTTATAC ATATCTAGTA 1740
AACTTTTAAA TATACGTATA TACAAAATAT AAAATTATTG GCGTTCATAT TAGGTCAATA 1800
AATCCTTAAC TATATCTGCC TTACCACTAG GAGAAAGTAA AAAACTCTTT ACCAAAAATA 1860
CATGTATTAT GTATACAAAA AGTCGATTAG ATTACCTAAA TAGAAATTGT ATAACGAGTA 1920 AGTAAGTAGA AATATAAAAA AACTACAATA CTAAAAAAAA TATGTTTTAC TTCAATTTCG 1980
AAACTAATGG GGTCTGAGTG AAATATTCAG AAAGGGGAGG ACTAACAAAA GGGTCATAAT 2040
GTTTTTTTTAT AAAAAGCCAC TAAAATGAGG AAATCAAGAA TCAGAACATA CAAGAAGGCA 2100
GCAGCTGAAG CAAAGTACCA TAATTTAATC AATGGAAATT AATTTCAAAG TTTTATCAAA 2160
ACCCATTCGA GGATCTTTTC CATCTTTCTC ACCTAAAGTT TCTTCAGGGG TAATTTTTAC 2220
TAATTTCATG TTAATTTCAA TTATTTTTAG CCTTTGCATT TCATTTTCCA ATATATCTGG 2280
ATCATCTCCT TAGTTTTTTA TTTTATTTTT TATAATATCA AATATGGAAG AAAAATGACA 2340
CTTGTAGAGC CATATGTAAG TATCATGTGA CAAATTTGCA AGGTGGTTGA GTGTATAAAA 2400
TTCAAAAATT GAGAGATGGA GGGGGGGTGG GGGBARAGAC AATATTTAGA AAGAGTGTTC 2460
TAGGAGGTTA TGGAGGACAC GGATGAGGGG TAGAAGGT7A GTTAGG7ATT TGAGTGTTGT 2520
CTGGCTTATC CTTTCATACT AGTAGTCGTG GAATTAT7TG GGTAGTTTC7 TGTTTTGTTA 2580
TTTGATCTTT GTTATTC7AT 777CTGTTTC TTGTACTTCG ATTATTGTA7 TATATATCTT 2640
GTCGTAGTTA TTGTTCCTCG GTAAGAATGC TCTAGCATGC TTCCTTTAG_7_GTTTTATCAT 2700
GCCTTCTTTA TATTCGCG7T GC7T7GAAAT GCTTTTACTT TAGCCGAGGG TCTATTAGAA 2760
ACAATCTCTC TATCTCGTAA GG7AGGGG7A AAGTCC7CAC CACACTCCAC TTGTGGGATT 2820
ACATTGTGTT 7GTTG7TG7A AATCAATTAT GTATACATAA TAAGTGGA777TTTACAACA 2BB0
CAAATACATG GTCAAGGGCA AAGTTCTGAA CACATAAAGG GTTCATTATA TGTCCAGGGA 2940
TATGATAAAA ATTGTTTCTT TGTGAAAGTT ATATAAGATT TGTTATGGCT TTTGCTGGAA 3000
ACATAATAAG TTATAATGCT GAGATAGCTA CTGAAGTTTG TTTTTTCTCG CCTTTTAAAT 3060
GTACCAATAA TAGAT7CCG7 ATCGAACGAG TATGTTTTGA TTACCTGGTC ATGATGTTTC 3120
TATTTTTTAC ATTTTTTTTGG TGTTGAACTG CAATTGAAAA TGTTGTATCC TATGAGACGG 3180
ATAGTTGAGA AtGTGTTCTT TGTATGGACC TTGAGAAGCT CAAACGCTAC TCCAATAATT 3240
TCTATGAATT CAAATTCAGT TTATGGCTAC CAGTCAGTCC AGAAATTAGG ATATGCTGCA 3300
TATACTTGTT CAATTATACT GTAAAATTTC TTAAGTTCTC AAGATATCCA TGTAACCTCG 3360
AGAATTTCTT TGACAGGCT7 CTAGAAATAA GATATGTTTT CCTTCTCAAC ATAGTACTGG 3420
ACTGAAGTTT GGATCTCAGG AACGGTCTTG GGATATTTCT TCCACCCCAA AATCAAGAGT 3480
TAGAAAAGAT GAAAGGGTA7 G7TTGATAAT TTATATGGTT GCATGGATAG TATATAAATA 3540
GTTGGAAAAC TTCTGGACTG GTGCTCATGG CATATTTGAT CTGTGCACCG 7GTGGAGATG 3600
TCAAACATGT GTTACTTCGT 7CCGCCAATT TATAATACCT TAACTTGGGA AAGACAGCTC 3660
TTTACTCCTG TGGGCATTTG TTATTTGAAT TACAATCTTT ATGAGCATGG TGTTTTCACA 3720
TTATCAACTT CTTTCATGTG GTATATAACA GTTTTTAGCT CCGTTAATAC CTTTCTTCTT 3780
TTTGATATAA ACTAACTGTG GTGCATTGCT TGCBKKKATG AAGCACAGTT CAGCTATTTC 3840
CGCTGTTTTG ACCGATGACG ACAATTCGAC AATGGCACCC CTAGAGGAAG ATGTCAAGAC 3900
TGAAAATATT GGCCTCCTAA ATTTGGATCC AACTTTGGAA CCTTATCTAG ATCACTTCAG 3960
ACACAGAATG AAGAGATATG TGGATCAGAA AATGCTCATT GAAAAATATG AGGGACCCCT 4020
TGAGGAATTT GCTCAAGGTA ACAGCCAAAA GTTGTGCTTT AGGCAGTTTG ACCTTATTTT 4080
GGAAGATGAA TTGTTTATAC CTACTTTGAC TTTGCTAGAG AATTTTGCAT ACCGGGGAGT 4140
AAGTAGTGGC TCCATTTAGG TGGCACCTGG CCATTT7TTT GATCTTTTAA AAAGCTGTTT 4200
GATTGGGTCT TCAAAAAAGT AGACAAGGTT TTTGGAGAAG TGACACACCC CCGGAGTGTC 4260
AGTGGCAAAG CAAAGATTTT CACTAAGGAG ATTCAAAATA TAAAAAAAGT ATAGACATAA 4320
AGAAGCTGAG GGGATTCAAC ATGTACTATA CAAGCATCAA ATATAGTCTT AAAGCAATTT 4380 TGTAGAAATA AAGAAAGTCT TCCTTCTGTT GCTTCACAAT TTCCTTCTAT TATCATGAGT 4440 TACTCTTTCT GTTCGAAATA GCTTCCTTAA TATTAAATTC ATGATACTTT TGTTGAGATT 4500 TAGCAGTTTT TTCTTGTGTA AACTGCTCTC TTTTTTTGCA GGTTATTTAA AATTTGGATT 560 CAACAGGGAA GATGGTTGCA TAGTCTATCG TGAATGGGCT CCTGCTGCTC AGTAGGTCCT 4620 CGTCTACTAC AAAATAGTAG TTTCCATCAT CATAACAGAT TTTCCTATTA AAGCATGATG 4680 TTGCAGCATC ATTGGCTTTC TTACATGTTC TAATTGCTAT TAAGGTTATG CTTCTAATTA 4740 ACTCATCCAC AATGCAGGGA AGCAGAAGTT ATTGGCGATT TCAATGGATG GAACGGTTCT 4800
AACCACATGA TGGAGAAGGA CCAGTTTGGT GTTTGGAGTA TTAGAATTCC TGATGTTGAC 860
AGTAAGCCAG TCATTCCACA CAACTCCAGA GTTAAGTTTC GTTTCAAACA TGGTAATGGA 4920
GTGTGGGTAG ATCGTATCCC TGCTTGGATA AAGTATGCCA CTGCAGACGC CACAAAGTTT '4980
GCAGCACCAT ATGATGGTGT CTACTGGGAC CCACCACCTT CAGAAAGGTT TTGTTATTCA 5040
TACCTTGAAG CTGAATTTTG AACACCATCA TCACAGGCAT TTCGATTCAT GTTCTTACTA 5100
GTCTTGTTAT GTAAGACATT TTGAAATGCA AAAGTTAAAA TAATTGTGTC TTTACTAATT 5160
TGGACTTGAT CCCATACTCT TTCCCTTAAC AAAATGAGTC AATTCTATAA GTGCTTGAGA 5220
ACTTACTACT TCAGCAATTA AACAGGTACC ACTTCAAATA CCCTCGCCCT CCCAAACCCC 5280
GAGCCCCACG AATC7ATGAA GCACATGTCG GCATGAGCAG CTCTGAGCCA CGTGTAAATT 5340 0 CGTATCGTGA G777GCAGAT GATGTTTTAC CTCGGATTAA GGCAAATAAC TATAATACTG 5400
TCCAG77GA7 GGCCATAATG GAACATTCTT ACTATGGATC ATTTGGATAT CATGTTACAA 5460
ACTTTT7TGC TG7GAGCAG7 AGATATGGAA ACCCGGAGGA CCTAAAGTAT CTGATAGATA 5520
AAGCACATAG C77GGG7TTA CAGGTTCTGG TGGATGTAGT TCACAGTCAT GCAAGCAATA 55B0
ATGTCACTGA TGGCC7CAAT GGCTTTGATA TTGGCCAAGG TTCTCAAGAA TCCTACT7TC 5640
ATGCTGGAGA GCGAGGGTAC CATAAGTTGT GGGATAGCAG GCTGTTCAAC TATGCCAATT 5700
GGGAGGTTC7 7CGTTTCCTT CTTTCCAACT TGAGGTGG7G GCTAGAAGAG TATAACTTTG 5760
ACGGATTTCG ATTTGATGGA ATAACTTCTA TGCTGTATGT TCATCATGGA ATCAATATGG 5820 5 GATTTACAGG AAACTATAAT GAGTATTTCA GCGAGGCTAC AGATGTTGAT GCTGTGGTCT 5880
ATTTAATGTT GGCCAATAAT CTGATTCACA AGATTTTCCC AGATGCAACT GTTATTGCCG 5940
AAGATGTTTC TGGTATGCCG GGCC7TGGCC GGCCTGTTTC TGAGGGAGGA ATTGGTTTTG S000
TTTACCGCCT GGCAATGGCA ATCCCAGATA AGTGGATAGA TTATTTAAAG AATAAGAATG 6060
ATGAAGATTG GTCCATGAAG GAAGTAACAT CGAGTTTGAC AAATAGGAGA TATACAGAGA S120
AGTGTATAGC ATA7GCGGAG ACCCATGATC AGGTATTT7A AATTTATTTC TACAACTAAA 6180
TAATTCTCAG AACAATTGTT AGATAGAATC CAAATATATA CGTCCTGAAA GTATAAAAGT 6240
ACTTATTTTC GCCATGGGCC TTCAGAATAT TGGTAGCCGC TGAATATCAT GATAAGTTAT 6300
TTATCCAGTG ACATTTTTAT GTTCACTCCT ATTATGTCTG CTGGATACAG TCTATTGT7G 6360
GTGACAAGAC CATTGCATTT CTCCTAATGG ACAAAGAGAT GTATTCTGGC ATGTCTTGCT 6420
TGACAGATGC TTCTCCTGTT GTTGATCGAG GAATTGCGCT TCACAAGGTT TGTCTGTTTC 64B0
TATTGCATTT TAAGGTTCAT ATAGGTTAGC CACGGAAAAT CTCACTCTTT GTGAGGTAAC 6540
CAGGGTTCTG ATGGATTATT CAATTTTCTC GTTTATCATT TGTTTATTCT TTTCATGCAT 6600
TGTGTTTCTT TTTCAATATC CCTCTTATTT GGAGGTAATT TTTCTCATCT ATTCACTTTT 6660
AGCTTCTAAC CACAGATGAT CCATTTTTTC ACAATGGCCT TGGGAGGAGA GGGGTACCTC 6720
AATTTCATGG GTAACGAGGT ATGTCTTACA TCTTTAGATA TTTTGTGATA ATTACAATTA 6780
? r \ GTTTGGCTTA CTTGAACAAG ATTCATTCCT CAAAATGACC TGAACTGTTG AACATCAAAG S840
GGGTTGAAAC ATAGAGGAAA ACAACATGAT GAATGTTTCC ATTGTCTAGG GATTTCTATT S900 ATGTTGCTGA GAACAAATGT CATCTTAAAA AAAACATTGT TTACTTTTTT GTAGTATAGA 6960
AGATTACTGT ATAGAGTTTG CAAGTGTGTC TGTTTTGGAG TAATTGTGAA ATGTTTGATG 7020
AACTTGTACA GTTTGGCCAT CCTGAGTGGA TTGACTTCCC TAGAGAGGGC AATAATTGGA 7080
GTTATGACAA ATGTAGACGC CAGTGGAACC TCGCGGATAG CGAACACTTG AGATACAAGG 7140
TTCAAGTATT TTGAATCGCA GCTTGTTAAA TAATCTAGTA ATTTTTAGAT TGCTTACTTG 7200
GAAGTCTACT TGGTTCTGGG GATGATAGCT CATTTCATCT TGTTCTACTT ATTTTCCAAC 7260
CGAATTTCTG ATTTT-'GTTT CGAGATCCAA GTATTAGATT CATTTACACT TATTACCGCC 7320CACTAAGGCC TTGATGAGCA GCTTAAGTTG ATTCTTTGAA GCTATAGTTT 7380
CAGGCTACCA ATCCACAGCC TGCTATATTT GTTGGATACT TACCTTTTCT TTACAATGAA 7440
GTGATACTAA TTGAAATGGT CTAAATCTGA TATCTATATT TCTCCGTCTT TCCTCCCCCT 7500
CATGATGAAA TGCAGTTTAT GAATGCATTT GATAGAGC7A TGAATTCGCT CGATGAAAAG 7560
TTCTCATTCC TCGCATCAGG AAAACAGATA GTAAGCAGCA TGGATGATGA TAATAAGGTA 7620
AAATCATCTA AAGTTGAAAG TGTTGGGTTT ATGAAGTGCT TTAATTCTAT CCAAGGACAA 7680
GTAGAAACCT TTTTACCTTC CATTTCTTGA TGATGGATTT CATATTATTT AATCCAATAG_7740_CTGGTCAAAT TCGGTAATAG CTGTACTGAT TAGTTACTTC ACTTTGCAGG TTGTTGTGTT 7800
TGAACGTGGT GACCTGGTAT TTGTATTCAA CTTCCACCCA AAGAACACAT ACGAAGGGTA 7860
TATATGTTTT ACTTATCCAT GAAATTATTG CTCTGCTTGT TTTTAATGTA CTGAACAAGT 7920
TTTATGGAGA AGTAACTGAA ACAAATCATT TTCACATTGT CTAATTTAAC TCTTTTTTTCT 7980
GATCCTCGCA TGACGAAAAC AGGTATAAAG TTGGATGTGA CTTGCCAGGG AAGTACAGAG 8040
TTGCACTGGA CAGTGATGCT TGGGAAT7TG G7GGCCATGG AAGAGTAAGG ATTTGCT7GA B100
ATAACTTTTG ATAATAAGAT AACAGATGTA GGGTACAGTT CTCTCACCAA AAAGAAC7G7 8160
AATTGTCTCA TCCATCTTTA G7TGTATAAG ATATCCGACT GTCTGAGTTC GGAAGTGTTT 8220
GAGCCTCCTG CCCTCCCCCT GCGTTGTTTA GCTAATTCAA AAAGGAGAAA ACTGTTTA77 8280
GATGATCTTT GTCTTCATGC 7GACATACAA TCTGTTCTCA TGACAGACTG GTCATGATGT 8340
TGACCATTTC ACATCACCAG AAGGAATACC TGGAGTTCCA GAAACAAATT TCAATGGTCG 8400
TCCAAATTCC TTCAAAGTGC TG7C7CCTGC GCGAACATGT GTGGTACAGT TCTTGCCGTG 8460
TGACCTCCCT TTTTATTGTG GTTT7GTTCA TAGTTATTTG AATGCGATAG AAGTTAACTA 8520
TTGATTACCG CCACAATCGC CAGTTAAG7C CTCTGAACTA CTAATTTGAA AGGTAGGAAT 8580
AGCCGTAATA AGGTCTACTT T7GGCA7CTT ACTGTTACAA AACAAAAGGA TGCCAAAAAA 8640
ATTCTTCTCT ATCC7CTTTT 7CCC7AAACC AGTGCATGTA GCTTGCACCT GCATAAAC77 8700
AGGTAAATGA TCAAAAATGA AGTTGATGGG AACTTAAAAC CGCCCTGAAG TAAAGCTAGG 8760
AATAGTCATA TAATGTCCAC CTTTGGTGTC TGCGCTAACA TCAACAACAA CATACCTCGT 8820
GTAGTCCCAC AAAG7GG77T CAGGGGGAGG GTAGAGTGTA TGCAAAACTT ACTCCTATCT SB80
CAGAGGTAGA GAGGAT7TTT TCAATAGACC CTTGGCTCAA GAAAAAAAGT CCAAAAAGAA 8940
GTAACAGAAG TGAAAGCAAC ATGTGTAGCT AAAGCGACCC AACTTGTTTG GGACTGAAGT 9000
AGTTGTTGTt GTTGAAACAG TGCATGTAGA TGAACACATG TCAGAAAATG GACAACACAG 9060
TTATTTTGTG CAAGTCAAAA AAATGTACTA CTATTTCTTT GTGCAGCTTT ATGTATAGAA 9120
AAGTTAAATA ACTAATGAAT TTTGCTAGCA GAAAAATAGC TTGGAGAGAA ATTTTTTATA 9180
TTGAACTAAG CTAACTATAT TCATCTTTCT TTTTGCTTCT TCTTCTCCTT GTTTGTGAAG 9240
GCTTATTACA GAGTTGATGA ACGCATGTCA GAAACTGAAG ATTACCAGAC AGACATTTGT 9300
AGTGAGCTAC TACCAACAGC CAATATCGAG GAGAGTGACG AGAAACTTAA AGATTCGTTA 9360
TCTACAAATA TCAGTAACAT TGACGAACGC ATGTCAGAAA CTGAAGTTTA CCAGACAGAC 9420 ATTTCTAGTG AGCTACTACC AACAGCCAAT ATTGAGGAGA GTGACGAGAA ACTTAAAGAT 9480
TCGTTATCTA CAAATATCAG TAACATTGAT CAGACTGTTG TAGTTTCTGT TGAGGAGAGA 9540
GACAAGGAAC TTAAAGATTC ACCGTCTGTA AGCATCATTA GTGATGTTGT TCCAGCTGAA 9600
TGGGATGATT CAGATGCAAA CGTCTGGGGT GAGGACTAGT CAGATGATTG ATCGACCCTT 9660
CTACCGATTG GTGATCGCTA TCCTTGCTCT CTGAGAAATA GGTGAGGCGA AACAAAAAAT 9720
AATTTGCATG ATAAAAAGTC TGATTTTATG ATCGCTATCC TCGCTCTCTG AGAAAGAAGC 9780
GAAACAAAGG CGACTCCTGG ACTCGAATCT ATAAGATAAC AAAGGCGACT CCTGGGACTC 9840 GAATCTATAA GATAACAAAG GCAATTCCAA GACTTGAATC TATAAAAAAT TTAGTTAAGA 9900
ATGATTAACG TCCGATCCTA ATTCGAATCG AGGCATCTTA CCACTCCATT GATAATTATA 9960
TAAGTCAATA AGTCATATAA WAGTATTAAA AACTAAATTG ACTTGATCGG TCTATCAAAA 10020
ATMAGATMAA ATTGTGTTCA TATGTAACAT TTTTGTTGTC ACAATTAGCT TAATTACATC 10080
TTTCATGTGC AATAACAAAG AAATGATAGG AATTTAGAGA TTCCAATTTT TTTGTTGCCA 10140
CAATTAACTT AATTACATCT 7TCATTTGCA ATAACAAAGA AATGATAGGA ATTTAGAGAT 10200
CCAGTGTCAA TACACAACCT AGGCCAACAT CGAAAGCATA ACTGTAAACT CATGCATGAA 10260
GAAATCAGTC GTAAAAATGA ATAAATGCGA CATAAAAACA AATTGCATGT ATCATTAATG 10320 tsACTTAACT ACAAGTAAAA ATAAATTTAA CAAATGTAAC TTSACTACAA GTAAAAATAA 10380
ATTGCTTCTA TCATTAACAA ACAAACAGAA TTAAAAAGAA AAAAACATAC TAAATCT7AC 10440
CGTCATTCGA TAAAAAAAAA TACCAAATTC ATAATGCAAG GAAAACGAAA CGCGTCCTGA 10500
TCGGGTATCA ACGATGAAAT GGACCAGTTG GATCGACTGC CTGCACAACG TTAGGTATGC 10560
CAAAAAAAAG AACACGATCC TTTGCACCCG TTCGATGATT ATCAGTATGT TCACAAAAAA 10620
AACTTAAGTT CATCCCAGTG TACAACAGCC CCAACATCTG CCCCAAGTAA CAAAAAACAA 106B0
CCAATTTATC TTATTCTTAT CTGCCACAAA ATAATCGGTT TCACACTATT CTCTTGTTAT 10740
ACAAAATTGA CAAGTAGGAA GGAGAGGAGT CATCCAAATA AACGGTGCAC GTTCTTTGAG 10800 AAAAGTCTTA T7TTTCGTAA GATCCAATTT CAACAAACTT TTCTTCAAGT CAAAATTCCT 10860
GATAGTGTAT CTCCTCTCGA CGACCTCTTG CATTGAACGA TCTCCGCTTA TCATGAAAAG 10920
TTGCTTGGAT AACAAGTATT GCAAGGGGGG GACAGTAGCT ATTAAGTTAG TCGGCCCAAG 10980
GAAATGGAGG AGTGATAGTC TCGAATATTA TTCACCTCTT TAGCATTACC CGGTCTGGC7 11040
TTAAGGAGTT ACGTCTTTTA CGCTCGCCAA TTTCTri l TAGAATGGTT GGTGTCAAAA 11100
TCGCGAGTTG TGGAAGGTTC AAGTTACTCG ATTCGTGATT TTCAAGTATG AGTGGTGAGA 11160
GAGATTCGAT ATTTTCACGA GGTGTATTCG AGGTCTAGTA GAACGAAGGG TGTCACTAAT 11220
GAAAGTTTCA AGAGTTCATC ATCATCTTCT TCTAGTAGAT TTTCGCTTTC AAATGAGTA7 11280 GAAAATTCTT CCTCTTTTCT ATTGATTTTC TTCATTGTTT TCTTCATTGT TGTGGTTstT 11340
ATTGAAAAGA AAGAAAATTT ATAACAGAAA AAGATGTCAA AAAAAAGGTA AAATGAAAGA 11400
GTATCATATA CTTAAAGAGT TGCGTAGAGA TAAGTCAAAA GAAACAGAAT TATAGTAATT 11460
TCAGCTAAGT TAGAATTC 11478
Claims (22)
1. - A method for carrying out the enzymatic activity in a plant (or a cell, a tissue or an organ thereof) comprising expressing in the plant (a cell a tissue or an organ thereof) a nucleotide sequence further characterized in that the nucleotide sequence is encoded, partially or completely, for an intron in an antisense orientation; and as a result of nucleotides it does not contain a sequence that is antisense to a sequence of exons normally associated with the intron.
2. A method according to claim 1, further characterized by affecting the activity of the starch branching enzyme and / or by affecting the levels of amylopectin and / or changing the composition of the starch.
3. A method to affect the enzymatic activity in an organism that produces starch (a cell, a tissue or an organ thereof) that comprises expressing in the organism (or a cell, a tissue or an organ thereof) that produces starch a nucleotide sequence, further characterized in that the nucleotide sequence is encoded, partially or completely, for an intron in an antisense orientation; and because the activity of the starch branching enzyme is affected and / or the levels of amylopectin are affected and / or the composition of the starch is changed.
4. A method according to claim 3, further characterized in that the nucleotide sequence does not contain a sequence that is antisense to a sequence of exons normally associated with the intron.
5. A method according to any of the preceding claims, further characterized in that the enzymatic activity is reduced or eliminated.
6. A method according to any of the preceding claims, further characterized in that the nucleotide sequence is encoded by at least substantially at least an entire intron in an antisense orientation.
7. A method according to any of the preceding claims, further characterized in that the nucleotide sequence is encoded at least for an entire intron in an antisense orientation.
8. A method according to any of the preceding claims, further characterized in that the nucleotide sequence comprises the sequence shown as any of ID. SEC. No. 15 to ID. SEC. No. 27 or a variant, a derivative or a homologue thereof, including combinations thereof.
9. A method according to any of the preceding claims, further characterized in that the nucleotide sequence is expressed by a promoter having a sequence shown as ID. SEC. No. 14 or a variant, a derivative or a homologue thereof.
10. An antisense sequence comprising the nucleotide sequence defined in claim 8 or a variant, a derivative or a homologue thereof. 11.- A promoter that has a sequence shown as ID. SEC. No. 14, or a variant, a derivative, a homologue thereof. 12. A promoter according to claim 11 in combination with a gene of interest ("GOI"). 13. A construction capable of understanding or expressing the invention according to any of claims 10 to 12. 14. A vector comprising or expressing the invention according to any of claims 10 to 13. 15.- A combination of nucleotide sequences comprising a first nucleotide sequence that is encoded for a recombinant enzyme; and a second nucleotide sequence corresponding to an intron in antisense orientation; because the intron is an intron that is associated with a genomic gene that encodes an enzyme corresponding to a recombinant enzyme; and because the second nucleotide sequence does not contain a sequence that is antisense to a sequence of exons normally associated with the intron. 16.- One cell, a tissue or an organ comprising or expressing the invention according to any of claims 10 to 15. 17. A transgenic starch-producing organism comprising and expressing the invention according to any of claims 10 to 16. .- A transgenic organism producing starch according to the application 17, further characterized because the organism is a plant. 19. A starch obtained from the invention according to any of the preceding claims. 20.- pBEA8 (NCIMB 40753) or pBEA9 (NCIMB 40815). 21. A nucleotide sequence that is antisense to any or any of the sequences of introns obtainable from \ -SBE 3.2 (NCIMB 40751) or \ -SBE 3.4 (NCIMB 40752) or a variant, a derivative or a homologue thereof. 22. A method for expressing a recombinant protein or enzyme in a host organism comprising expressing a nucleotide sequence that is encoded for a protein or a recombinant enzyme; and expressing an additional nucleotide sequence, further characterized in that the additional nucleotide sequence is encoded, partially or completely, for an intron in an antisense orientation; because the intron is an intron normally associated with the genomic gene that encodes a protein or an enzyme corresponding to the protein or recombinant enzyme; and because the additional nucleotide sequence does not contain a sequence that is antisense to a sequence of exons normally associated with the intron.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9514435.8 | 1995-07-14 | ||
| GBGB9514435.8A GB9514435D0 (en) | 1995-07-14 | 1995-07-14 | Inhibition of gene expression |
| PCT/EP1996/003052 WO1997004112A2 (en) | 1995-07-14 | 1996-07-12 | Inhibition of gene expression |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| MXPA98000500A true MXPA98000500A (en) | 1998-04-01 |
| MX9800500A MX9800500A (en) | 1998-04-30 |
Family
ID=10777672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| MX9800500A MX9800500A (en) | 1995-07-14 | 1996-07-12 | Inhibition of gene expression. |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US6147279A (en) |
| EP (1) | EP0839202A2 (en) |
| JP (1) | JPH11509103A (en) |
| CN (1) | CN1196093A (en) |
| AU (1) | AU709798B2 (en) |
| BR (1) | BR9609726A (en) |
| CA (1) | CA2226832A1 (en) |
| GB (1) | GB9514435D0 (en) |
| MX (1) | MX9800500A (en) |
| NZ (1) | NZ313626A (en) |
| WO (1) | WO1997004112A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1248293A (en) * | 1997-02-21 | 2000-03-22 | 丹尼斯科有限公司 | Sense intron inhibition of starch branching enzyme expression |
| US6483009B1 (en) | 1997-02-21 | 2002-11-19 | Danisco A/S | Antisense intron inhibition of starch branching enzyme expression |
| US6303848B1 (en) | 1998-01-16 | 2001-10-16 | Large Scale Biology Corporation | Method for conferring herbicide, pest, or disease resistance in plant hosts |
| US6426185B1 (en) | 1998-01-16 | 2002-07-30 | Large Scale Biology Corporation | Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation |
| US20020159625A1 (en) * | 2001-04-02 | 2002-10-31 | Cytoprint, Inc. | Method and apparatus for discovering, identifying and comparing biological activity mechanisms |
| EP1925672A1 (en) | 2001-06-22 | 2008-05-28 | Syngeta Participations AG | Abiotic stress responsive polynucleotides and polypeptides |
| EP2216405A1 (en) | 2002-05-03 | 2010-08-11 | Monsanto Technology LLC | Speed specific USP promoters for expressing genes in plants |
| US7078234B2 (en) | 2002-12-18 | 2006-07-18 | Monsanto Technology Llc | Maize embryo-specific promoter compositions and methods for use thereof |
| DK1578973T3 (en) | 2002-12-19 | 2008-11-24 | Bayer Cropscience Ag | Plant cells and plants that synthesize a starch with increased final viscosity |
| WO2004087878A2 (en) | 2003-03-28 | 2004-10-14 | Monsanto Technology, Llc | Novel plant promoters for use in early seed development |
| ATE517994T1 (en) | 2003-06-30 | 2011-08-15 | Commw Scient Ind Res Org | WHEAT WITH ALTERED BRANCHING ENZYM ACTIVITY AND STARCH AND PRODUCTS CONTAINING STARCH OBTAINED FROM IT |
| US20090031440A1 (en) | 2005-02-26 | 2009-01-29 | Basf Plant Science Gmbh | Expression Cassettes for Seed-Preferential Expression in Plants |
| EP1707632A1 (en) | 2005-04-01 | 2006-10-04 | Bayer CropScience GmbH | Phosphorylated waxy potato starch |
| ATE552343T1 (en) * | 2005-04-19 | 2012-04-15 | Basf Plant Science Gmbh | ENDOSPERM-SPECIFIC EXPRESSION AND/OR EXPRESSION IN GERMINATING EMBRYOS OF MONOCOTYLEDONE PLANTS |
| US7902423B2 (en) | 2005-04-20 | 2011-03-08 | Basf Plant Science Gmbh | Expression cassettes for seed-preferential expression that utilize the promoter from a flax tonoplast intrinsic protein gene |
| WO2006120197A2 (en) | 2005-05-10 | 2006-11-16 | Basf Plant Science Gmbh | Expression cassettes for seed-preferential expression in plants |
| BRPI0808008B1 (en) | 2007-02-16 | 2016-08-09 | Basf Plant Science Gmbh | isolated nucleic acid molecule, expression cassette, expression vector, methods for excising target sequences from a plant to produce a plant with increased yield, and / or increased stress tolerance, and / or increased nutritional quality, and / or increased or modified oil content of a seed or sprout for the plant, and use of the nucleic acid molecule |
| WO2010118477A1 (en) | 2009-04-17 | 2010-10-21 | Molecular Plant Breeding Nominees Ltd | Plant promoter operable in endosperm and uses thereof |
| US8921657B2 (en) | 2009-07-10 | 2014-12-30 | Basf Plant Science Company Gmbh | Expression cassettes for endosperm-specific expression in plants |
| BR112012013156A2 (en) | 2009-12-03 | 2017-06-13 | Basf Plant Science Co Gmbh | expression cassette, vector, host cell, transgenic plant tissue and method for producing a transgenic plant tissue |
| EP2766486A1 (en) | 2011-10-12 | 2014-08-20 | Bayer CropScience AG | Plants with decreased activity of a starch dephosphorylating enzyme |
| AU2012323042B2 (en) | 2011-10-12 | 2015-01-15 | Bayer Cropscience Ag | Plants with decreased activity of a starch dephosphorylating enzyme |
| CN109476715B (en) | 2016-05-26 | 2023-06-27 | 纽海姆有限公司 | Plants producing seedless fruits |
| WO2019238832A1 (en) | 2018-06-15 | 2019-12-19 | Nunhems B.V. | Seedless watermelon plants comprising modifications in an abc transporter gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4740463A (en) * | 1984-04-13 | 1988-04-26 | Massachusetts Institute Of Technology | Methods and artificial genes for antagonizing the function of an oncogene |
| IL81737A (en) * | 1986-03-28 | 1992-11-15 | Calgene Inc | Regulation of gene expression in plant cells |
| JPH04504355A (en) * | 1989-02-02 | 1992-08-06 | パラディン ハイブリッズ インコーポレーテッド | Molecular methods of hybrid seed production |
| SE9004095L (en) * | 1990-12-21 | 1992-06-01 | Amylogene Hb | GENETIC MODIFICATION OF POTATOIS BEFORE EDUCATION OF AMYLOST TYPE |
| DE4104782B4 (en) * | 1991-02-13 | 2006-05-11 | Bayer Cropscience Gmbh | Novel plasmids containing DNA sequences that cause changes in carbohydrate concentration and carbohydrate composition in plants, as well as plants and plant cells containing these plasmids |
| EP0575518A1 (en) * | 1991-03-06 | 1993-12-29 | Board Of Regents, The University Of Texas System | Methods and compositions for the selective inhibition of gene expression |
| JPH0698656A (en) * | 1992-03-30 | 1994-04-12 | Mitsui Giyousai Shokubutsu Bio Kenkyusho:Kk | Rice plant treated with gene |
| DK0664835T3 (en) * | 1992-10-14 | 2004-09-27 | Syngenta Ltd | New plants and methods for obtaining them |
| GB9223454D0 (en) * | 1992-11-09 | 1992-12-23 | Ici Plc | Novel plants and processes for obtaining them |
| EP1897942A1 (en) * | 1993-05-11 | 2008-03-12 | The University Of North Carolina At Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
| WO1995026407A1 (en) * | 1994-03-25 | 1995-10-05 | National Starch And Chemical Investment Holding Corporation | Method for producing altered starch from potato plants |
-
1995
- 1995-07-14 GB GBGB9514435.8A patent/GB9514435D0/en active Pending
-
1996
- 1996-07-12 US US08/981,803 patent/US6147279A/en not_active Expired - Fee Related
- 1996-07-12 AU AU66145/96A patent/AU709798B2/en not_active Ceased
- 1996-07-12 JP JP9506260A patent/JPH11509103A/en active Pending
- 1996-07-12 NZ NZ313626A patent/NZ313626A/en unknown
- 1996-07-12 CA CA002226832A patent/CA2226832A1/en not_active Abandoned
- 1996-07-12 MX MX9800500A patent/MX9800500A/en unknown
- 1996-07-12 BR BR9609726A patent/BR9609726A/en unknown
- 1996-07-12 WO PCT/EP1996/003052 patent/WO1997004112A2/en not_active Application Discontinuation
- 1996-07-12 EP EP96925731A patent/EP0839202A2/en not_active Withdrawn
- 1996-07-12 CN CN96196986A patent/CN1196093A/en active Pending
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