Abstract:
Genetically engineered modification of potato for suppressing the formation of amylose-type starch is described. 
     Three fragments for insertion in the antisense direction into the potato genome are also described. Moreover, antisense constructs, genes and vectors comprising said antisense fragments are described. Further a promoter for the gene coding for formation of granule-bound starch synthase and also the gene itself are described. 
     Also cells, plants, tubers, microtubers and seeds of potato comprising said antisense fragments are described. 
     Finally, amylopectin-type starch, both native and derivatised, derived from the potato that is modified in a genetically engineered manner, as well as a method of suppressing amylose formation in potato are described.

Description:
This application is a 371 of PCT/SE91/00892 filed 20 Dec. 1991. 
     The present invention relates to genetically engineered modification of potato, resulting in the formation of practically solely amylopectin-type starch in the potato. The genetically engineered modification implies the insertion of gene fragments into potato, said gene fragments comprising parts of leader sequence, translation start, translation end and trailer sequence as well as coding and noncoding (i.e. exons and introns) parts of the gene for granule-bound starch synthase, inserted in the antisense direction. 
    
    
     BACKGROUND OF THE INVENTION 
     Starch in various forms is of great import in the food and paper industry. In future, starch will also be a great potential for producing polymers which are degradble in nature, e.g. for use as packing material. Many different starch products are known which are produced by derivatisation of native starch originating from, inter alia, maize and potato. Starch from potato and maize, respectively, is competing in most market areas. 
     In the potato tuber, starch is the greatest part of the solid matter. About ¼ to ⅕ of the starch in potato is amylose, while the remainder of the starch is amylopectin. These two components of the starch have different fields of application, and therefore the possibility of producing either pure amylose or pure amylopectin is most interesting. The two starch components can be produced from common starch, which requires a number of process steps and, consequently, is expensive and complicated. 
     It has now proved that by genetic engineering it is possible to modify potato so that the tubers merely produce mainly starch of one or the other type. As a result, a starch quality is obtained which can compete in the areas where potato starch is normally not used today. Starch from such potato which is modified in a genetically engineered manner has great potential as a food additive, since it has not been subjected to any chemical modification process. 
     Starch Synthesis 
     The synthesis of starch and the regulation thereof are presently being studied with great interest, both on the level of basic research and for industrial application. Although much is known about the assistance of certain enzymes in the transformation of saccharose into starch, the biosynthesis of starch has not yet been elucidated. By making researches above all into maize, it has, however, been possible to elucidate part of the ways of synthesis and the enzymes participating in these reactions. The most important starch-synthesising enzymes for producing the starch granules are the starch synthase and the branching enzyme. In maize, three forms of starch synthase have so far been demonstrated and studied, two of which are soluble and one is insolubly associated with the starch granules. Also the branching enzyme consists of three forms which are probably coded by three different genes (Mac Donald &amp; Preiss, 1985; Preiss, 1988). 
     The Waxy Gene in Maize 
     The synthesis of the starch component amylose essentially occurs by the action of the starch synthase alpha-1,4-D-glucane-4-alpha-glucosyl transferase (EC 2.4.1.21) which is associated with the starch granules in the growth cell. The gene coding for this granule-bound enzyme is called “waxy” (= wx   + ), while the enzyme is called “GBSS” (granule-bound starch synthase). 
     Waxy locus in maize has been thoroughly characterised both genetically and biochemically. The waxy gene on chromosome  9  controls the production of amylose in endosperm, pollen and the embryo sac. The starch formed in endosperm in normal maize with the wx +  allele consists to 25% of amylose and to 75% of amylopectin. A mutant form of maize has been found in which the endosperm contains a mutation located to the wx +  gene, and therefore no functioning GBSS is synthesised. Endosperm from this mutant maize therefore contains merely amylopectin as the starch component. This so-called waxy mutant thus contains neither GBSS nor amylose (Echt &amp; Schwartz, 1981). 
     The GBSS protein is coded by the wx +  gene in the cell nucleus but is transported to and active in the amyloplast. The preprotein therefore consists of two components, viz. a 7 kD transit peptide which transfers the protein across the amyloplast membrane, and the actual protein which is 58 kD. The coding region of the wx +  gene in maize is 3.7 kb long and comprises 14 exons and 13 introns. A number of the regulation signals in the promoter region are known, and two different polyadenylating sequences have been described (Klösgen et al, 1986; Schwartz-Sommer et al, 1984; Shure et al, 1983). 
     Amylose Enzyme in Potato 
     In potato, a 60 kD protein has been identified, which constitutes the main granule-bound protein. Since antibodies against this potato enzyme cross-react with GBSS from maize, it is assumed that it is the granule-bound synthase (Vos-Scheperkeuter et al, 1986)., The gene for potato GBSS has, however, so far not been characterised to the same extent as the waxy gene in maize, either in respect of locating or structure. 
     Naturally occurring waxy mutants have been described for barley, rice and sorghum besides maize. In potato no natural mutant has been found, but a mutant has been produced by X-radiation of leaves from a monohaploid (n= 12 ) plant (Visser et al, 1987). Starch isolated from tubers of this mutant contains neither the GBSS protein nor amylose. The mutant is conditioned by a simple recessive gene and is called amf. It may be compared to waxy mutants of other plant species since both the GBSS protein and amylose are lacking. The stability of the chromosome number, however, is weakened since this is quadrupled to the natural number (n= 48 ), which can give negative effects on the potato plants (Jacobsen et al, 1990). 
     Inhibition of Amylose Production 
     The synthesis of amylose can be drastically reduced by inhibition of the granule-bound starch synthase, GBSS, which catalyses the formation of amylose. This inhibition results in the starch mainly being amylopectin. 
     Inhibition of the formation of enzyme can be accomplished in several ways, e.g. by: 
     mutagen treatment which results in a modification of the gene sequence coding for the formation of the enzyme 
     incorporation of a transposon in the gene sequence coding for the enzyme 
     genetically engineered modification so that the gene coding for the enzyme is not expressed, e.g. antisense gene inhibition. 
     FIG. 1 illustrates a specific suppression of normal gene expression in that a complementary antisense nucleotide tide is allowed to hybridise with mRNA for a target gene. 
     The antisense nucleotide thus is antisense RNA which is transcribed in vivo from a “reversed” gene sequence (Izant, 1989). 
     By using the antisense technique, various gene functions in plants have been inhibited. The antisense construct for chalcone synthase, polygalacturonase and phosphinotricin acetyltransferase has been used to inhibit the corresponding enzyme in the plant species petunia, tomato and tobacco. 
     Inhibition of Amylose in Potato 
     In potato, experiments have previously been made to inhibit the synthesis of the granule-bound starch synthase (GBSS protein) with an antisense construct corresponding to the gene coding for GBSS (this gene is hereinafter called the “GBSS gene” ). Hergersberger (1988) describes a method by which a CDNA clone for the GBSS gene in potato has been isolated by means of a cDNA clone for the wx +  gene in maize. An antisense construct based on the entire CDNA clone was transferred to leaf discs of potato by means of Agrobacterium tumefaclens. In microtubers induced in vitro from regenerated potato sprouts, a varying and very weak reduction of the amylose content was observed and shown in a diagram. A complete characterisation of the GBSS gene is not provided. 
     The gene for the GBSS protein in potato has been further characterised in that a genomic wx clone was examined by restriction analysis. However, the DNA sequence of the clone has not been determined (Visser et al, 1989). 
     Further experiments with an antisense construct corresponding to the GBSS gene in potato have been reported. The antisense construct which is based on a cDNA clone together with the CaMV 35S promoter has been transformed by means of Agrobacterium rhizogenes. According to information, the transformation resulted in a lower amylose content in the potato, but no values have been accounted for (Flavell, 1990). 
     None of the methods used so far for genetically engineered modification of potato has resulted in potato with practically no amylose-type starch. 
     The object of the invention therefore is to provide a practically complete suppression of the formation of amylose in potato tubers. 
     SUMMARY OF THE INVENTION 
     According to the invention, the function of the GBSS gene and, thus, the amylose production in potato are inhibited by using completely new antisense constructs. For forming the antisense fragments according to the invention, the genomic GBSS gene is used as a basis in order to achieve an inhibition of GBSS and, consequently, of the amylose production, which is as effective as possible. The antisense constructs according to the invention comprise both coding and noncoding parts of the GBSS gene which correspond to sequences in the region comprising promoter as well as leader sequence, translation start, translation end and trailer sequence in the antisense direction. For a tissue-specific expression, i.e. the amylose production should be inhibited in the potato tubers only, use is made of promoters which are specifically active in the potato tuber. As a result, the starch composition in other parts of the plant is not affected, which otherwise would give negative side-effects. 
     The invention thus comprises a fragment which essentially has one of the nucleotide sequences stated in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. However, the sequences may deviate from those stated by one or more non-adjacent base pairs, without affecting the function of the fragments. 
     The invention also comprises a potato-tuber-specific promoter comprising 987 bp which belongs to the gene according to the invention, which codes for granule-bound starch synthase. Neither the promoter nor the corresponding gene has previously been characterised. The promoter sequence of 987 bp is stated in SEQ ID No. 4, while the gene sequence is stated in SEQ ID No. 5. Also the promoter and gene sequences may deviate from those stated by one or more non-adjacent base pairs, without affecting their function. 
     The invention also comprises vectors including the antisense fragments and the antisense constructs according to the invention. 
     In other aspects the invention comprises cells, plants, tubers, microtubers and seeds whose genome contains the fragments according to the invention inserted in the antisense direction. 
     In still further aspects, the invention comprises amylopectin-type starch, both native and derivatised. 
     Finally, the invention comprises a method of suppressing amylose formation in potato, whereby mainly amylopectin-type starch is formed in the potato. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates the principle of the antisense ene inhibition [SEQ ID No. 21]. 
     FIG. 2 shows the result of restriction analysis of the potato GBSS gene, 
     FIG. 3 shows two new binary vectors pHo3 and pHo4, 
     FIG. 4 shows the antisense constructs pHoxwA, pHoxwB and pHoxwD, 
     FIG. 5 shows the antisense constructs pHoxwF and pHoxwG, and 
     FIG. 6 shows the antisense constructs pHoxwK and pHoxwL. 
     Moreover, the sequences of the different DNA fragments according to the invention are shown in SEQ ID Nos 1, 2, 3, 4 and 5. There may be deviations from these sequences in one or more non-adjacent base pairs. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Materials 
     In the practical carrying out of the invention the following materials were used: 
     Bacterial Strains 
       E. coli  DH5alfa and DH5alfaF′IQ(BRL).  E. coli  JM105 (Pharmacia). A. tumefaciens LBA4404 (Clontech). 
     Vectors 
     M13mpl8 and mp19 (Pharmacia). pBI101 and pBI121 (Clontech). pBI240.7 (M. W. Bevan). pUC plasmids (Pharmacia). 
     Enzymes 
     Restriction enzymes and EcoRI linker (BRL). UNION™ DNA Ligation Kit (Clontech). Sequenase• DNA Sequencing Kit (USB). T 4 -DNA ligase (Pharmacia). 
     The above-mentioned materials are used according to specifications stated by the manufacturers. 
     Genomic Library 
     A genomic library in EMBL3 has been produced by Clontech on the applicant&#39;s account, while using leaves of the potato Bintje as starting material. 
     Identification and Isolation of the GBSS Gene 
     The genomic library has been screened for the potato GBSS gene by means of cDNA clones for both the 5′ and 3′ end of the gene (said cDNA clones being obtained from M Hergersberger, Max Plank Institute in Cologne) according to a protocol from Clontech. 
     A full-length clone of the potato CBSS gene, wx311, has been identified and isolated from the genomic library. The start of the GBSS gone has been determined at an EcoRI fragment which in called fragment w (3.95 kb). The end of the GBSS gene has also been datrind at an ECORI fragment which in called fragment x (5.0 kb). BgIII-SpeI fragment which is called fragment m (3.9 kb) has also been isolated and shares sequences both from fragment w and from fragment x. The fragments w, m and x have been subcloned in pUC13 (Viera, 1982, Yanisch-Paron at el, 1985) and are called pSw, pSn and pSx, respectively (FIG.  2 ). 
     Characterisation of the GBSS Gene in Potato 
     The GBSS gene in potato has been characterised by restriction analysis and cDNA probes, where the 5′ and 3′ end of the GBSS gene has been determined more accurately (FIG.  2 ). Seuence determination according to Sangar et al, 1977 of the GBSS gene has been made on subclones from pSw and pSx in M13mpl8 and mpl9 as well as pUC19 starting eround the 5′ end (see SEQ ID No. 5). 
     The promoter region has beon determined at a BgIII-NsiI fr nt (see SEQ ID No. 4). Transcription and translation start has been determined at an overlapping BgIII-HindIII fragment. The terminator region has in turn been determined at a SpeI-HindIII fragment. 
     Antisense Constructs for the GBSS Gene in Potato 
     The HBSS gene fragments according to the invention (see SEQ ID Nos 1, 2 and 3. and FIG. 2) have been determined in the following manner. 
     The restriction of pSw with NsiI and HindIII gives fragment I (SEQ ID NO. 1) which subcloned in pUC19 is called 19NH35. Further restriction of 19 NH35 with HpaI-SstI gives a fragment containing 342 bp of the GBSS gene according to the invention. This fragment comprises leader sequence, translation start and the first 125 bp of the coding region. 
     The restriction of pSm with HpaI and NsiI gives fragment II (SEQ ID No. 2) which subcloned in pJRD184 (Heusterspreute et al, 1987) is called pJRDmitt. Further restriction of pJRDmitt with HpaI-SstI gives a fragment containing 2549 bp of the GBSS gene according to the invention. This fragment comprises exons and introns from the middle of the gene. 
     The restriction of pSx with Sstl and SpeI gives fragment III (SEQ ID No. 3) which subcloned in pBluescript (Melton et al, 1984) is called pBlue3′. Further restriction of pBlue3′ with BamHI-SstI gives a fragment containing ing 492 bp of the GBSS gene according to the invention. 
     This fragment comprises the last intron and exon, translation end and 278 bp of trailer sequence. 
     Antisense Constructs with Fragment I (FIG. 4) 
     For the antisense construct pHoxwA, the HpaI-SstI fragment from 19NH35 has been inserted in the antisense direction into the binary vector pBI121 (Jefferson et al, 1987) cleaved with SmaI-SstI. The transcription of the antisense fragment is then initiated by the CaMV 35S promoter and is terminated by the NOS terminator (NOS=nopaline synthase). 
     For the antisense construct pHoxwB, the HpaI-SstI fragment from 19NH35 has been inserted in the antisense direction into the binary vector pHo4 (FIG. 3) cleaved with SmaI-SstI. The patatin I promoter which is tuber specific in potato comes from the vector pBI240.7 obtained from M. Bevan, Institute of Plant Science Research, Norwich. The transcription of the antisense fragment is then initiated by the patatin I promoter and is terminated by the NOS terminator. 
     For the antisense construct pHoxwD, the HpaI-SstI fragment from 19NH35 has been inserted in the antisense direction into the binary vector pHo3 (FIG. 3) cleaved with SmaI-SstI. pHo3 is a new binary vector which is constructed on the basis of pBI101. This vector which contains the promoter according to the invention (see SEQ ID No. 4) (GBSS promoter) of the now characterised potato GBSS gene according to the invention has been restriction-cleaved with SmaI and SstI, the HpaI-SstI fragment from 19NH35 being inserted in the antisense direction. The transcription of the antisense fragment is then initiated by its own GBSS promoter and is terminated by the NOS terminator. This means that the antisense fragment is transcribed only in the potato tuber, since the GBSS promoter like the patatin I promoter is tuber-specific. 
     Antisense Constructs with Fragment II (FIG. 5) 
     For the antisense construct pHoxwF, the HpaI-SstI fragment from pJRDmitt has been inserted in the antisense direction into the binary vector pHo4 cleaved with SmaI-SstI. The transcription of the antisense fragment is then initiated by the patatin I promoter and terminated by the NOS terminator. 
     For the antisense construct pHoxwG, the HpaI-SstI fragment from pJRDmitt has been inserted in the antisense direction into the binary vector pHo3 cleaved with SmaI-SstI. The transcription of the antisense fragment is then initiated by its own GBSS promoter and is terminated by the NOS terminator. 
     Antisense Constructs with Fragment III (FIG. 6) 
     For the antisense construct pHoxwK, the BamHI-SstI fragment from pBlue3′ has been inserted in the antisense direction into the binary vector pHo4 cleaved with BamHI-SstI. The transcription of the antisense fragment is then initiated by the patatin I promoter and is terminated by the NOS terminator. 
     For the antisense construct pHoxwL, the BamHI-SstI fragment from pBlue3′ has been inserted in the antisense direction into the binary vector pHo3 cleaved with BamHI-SstI. The transcription of the antisense fragment is then initiated by its own GBSS promoter and is terminated by the NOS terminator. 
     The formed antisense contructs (FIGS. 4,  5 ,  6 ) have been transformed to Agrobacterium tumefaciens strain LBA4404 by direct transformation with the “freeze-thawing” method (Hoekema et al, 1983, An et al, 1988). 
     Transformation 
     The antisense constructs are transferred to bacteria, suitably by the “freeze-thawing” method (An et al, 1988). The transfer of the recombinant bacterium to potato tissue occurs by incubation of the potato tissue with the recombinant bacterium in a suitable medium after some sort of damage has been inflicted upon the potato tissue. During the incubation, T-DNA from the bacterium enters the DNA of the host plant. After the incubation, the bacteria are killed nd the potato tissue is transferred to a solid medium for callus induction and is incubated for growth of callus. 
     After pausing through further suitable media, sprouts are formed which are cut away from the potato tissue. 
     Checks for testing the expression of the antisense constructs and the transfer thereof to the potato genome are carried out by e.g. southern snd northern hybridisation (Manatis et al (1982)). The number of copies of the antisense construct which has been transferred is determined by southern hybridisation. 
     The testing of the expression on protein level is suitably carried out on microtubers induced in vitro on the transformed sprouts, thus permitting the testing to be performed an quickly as possible. 
     Characterisation of the GBSS Protein 
     The effect of the antisense constructs on the function of the GBSS gene with respect to the activity of the GBSS protein is examined by extracting starch from the microtubers and analysing it regarding the presence of the GBSS protein. In electrophoresis on polyacrylamide gel (Hovenkamp-Hermelink et al, 1987), the GBSS protein forms a distinct band at 60 kD. when the GBSS gene functions. When the GBBS gene is not expressed, i.e. when the antisense GBSS gene is fully expressed, thereby inhibiting the formation of GBSS protein, no 60 kD band is demonstrated on the gel. 
     Characterisation of the Starch 
     The composition of the starch in microtubers is identical with that of ordinary potato tubers, and therefore the effect of the antisense constructs on the amylose production is examined in microtubers. The proportion of amylose to amylopectin can be determined by a spectrophotometric method (e.g. according to Hovenkamp-Hermelink et al, 1988). 
     Extraction of Amylopectin from Amylopectin Potato 
     Amylopectin is extracted from the so-called amylopectin potato (potato in which the formation of amylose has been suppressed by inserting the antisense constructs according to the invention) in a known manner. 
     Derivatisation of Amylopectin 
     Depending on the final use of the amylopectin, its physical and chemical qualities can be modified by derivatisation. By derivatisation is here meant chemical, physical and enzymatic treatment and combinations thereof (modified starches). 
     The chemical derivatisation, i.e. chemical modification of the amylopectin, can be carried out in different ways, for example by oxidation, acid hydrolysis, dextrinisation, different forms of etherification, such as cationisation, hydroxy propylation and hydroxy ethylation, different forms of esterification, for example by vinyl acetate, acetic anhydride, or by monophosphatising, diphosphatising and octenyl succination, and combinations thereof. 
     Physical modification of the amylopectin can be effected by e.g. cylinder-drying or extrusion. 
     In enzymatic derivatisation, degradation (reduction of the viscosity) and chemical modification of the amylopectin are effected by means of existing enzymatic systems. 
     The derivatisation is effected at different temperatures, tures, according to the desired end product. The ordinary range of temperature which is used is 20-45° C., but temperatures up to 180° C. are possible. 
     The invention will be described in more detail in the following Examples. 
     EXAMPLE 1 
     Production of Microtubers with Inserted Antisense Constructs According to the Invention 
     The antisense constructs (see FIGS. 4,  5  and  6 ) are transferred to Agrobacterium tumefaciens LBA 4404 by the “freeze-thawing” method (An et al, 1988). The transfer to potato tissue is carried out according to a modified protocol from Rocha-Sosa et al (1989). 
     Leaf discs from potato plants cultured in vitro are incubated in darkness on a liquid MS-medium (Murashige &amp; Skoog; 1962) with 3% saccharose and 0.5% MES together with 100 μl of a suspension of recombinant Agrobacterium per 10 ml medium for two days. After these two days the bacteria are killed. The leaf discs are transferred to a solid medium for callus induction and incubated for 4-6 weeks, depending on the growth of callus. The solid medium is composed as follows: 
     
       
         
               
               
             
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 MS + 3% saccharose 
               
             
          
           
               
                   
                 2 
                 mg/l 
                 zeatin riboside 
               
               
                   
                 0.02 
                 mg/l 
                 “NAA” 
               
               
                   
                 0.02 
                 mg/l 
                 “GA 3 ” 
               
               
                   
                 500 
                 mg/l 
                 “Claforan” 
               
               
                   
                 50 
                 mg/l 
                 kanamycin 
               
               
                   
                 0.25% 
                   
                 “Gellan” 
               
               
                   
                   
               
             
          
         
       
     
     Subsequently the leaf discs are transferred to a medium having a different composition of hormones, comprising: 
     
       
         
               
               
             
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 MS + 3% saccharose 
               
             
          
           
               
                   
                 5 
                 mg/l 
                 “NAA” 
               
               
                   
                 0.1 
                 mg/l 
                 “BAP” 
               
               
                   
                 500 
                 mg/l 
                 “Claforan” 
               
               
                   
                 50 
                 mg/l 
                 kanamycin 
               
               
                   
                 0.25% 
                   
                 “Gellan” 
               
               
                   
                   
               
             
          
         
       
     
     The leaf discs are stored on this medium for about 4 weeks, whereupon they are transferred to a medium in which the “Claforan” concentration has been reduced to 250 mg/l. If required, the leaf discs are then moved to a fresh medium every 4 or 5 weeks. After the formation of sprouts, these are cut away from the leaf discs and transferred to an identical medium. 
     The condition that the antisense construct has been transferred to the leaf discs is first checked by analysing leaf extracts from the regenerated sprouts in respect of glucuronidase activity by means of the substrates described by Jefferson et al (1987). The activity is demonstrated by visual assessment. 
     Further tests of the expression of the antisense constructs and the transfer thereof to the potato genome are carried out by southern and northern hybridisation according ing to Maniatis et al (1981). The number of copies of the antisense constructs that has been transferred is determined by southern hybridisation. 
     When it has been established that the antisense constructs have been transferred to and expressed in the potato genome, the testing of the expression on protein level begins. The testing is carried out on microtubers which have been induced in vitro on the transformed sprouts, thereby avoiding the necessity of waiting for the development of a complete potato plant with potato tubers. 
     Stem pieces of the potato sprouts are cut off at the nodes and placed on a modified MS medium. There they form 35 microtubers after 2-3 weeks in incubation in darkness at 19° C. (Bourque et al, 1987). The medium is composed as follows: 
     
       
         
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 MS + 6% saccharose 
               
               
                   
                 2.5 mg/l kinetin 
               
               
                   
                 2.5 mg/l “Gellan” 
               
               
                   
                   
               
             
          
         
       
     
     The effect of the antisense constructs on the functions of the GBSS gene in respect of the activity of the GBSS protein is analysed by means of electrophoresis on polyacrylamide gel (Hovenkamp-Hermelink et al, 1987). Starch is extracted from the microtubers and analysed regarding the presence of the GBSS protein. In a polyacrylamide gel, the GBSS protein forms a distinct band at 60 kD, when the GBSS gene functions. If the GBSS gene is not expressed, i.e. when the antisense GBSS gene is fully expressed so that the formation of GBSS protein is inhibited, no 60 kD band can be seen on the gel. 
     The composition of the starch, i.e. the proportion of amylose to amylopectin, is determined by a spectrophotometric method according to Hovenkamp-Hermelink et al (1988), the content of each starch component being determined on the basis of a standard graph. 
     EXAMPLE 2 
     Extraction of Amylopectin from Amylopectin Potato 
     Potato whose main starch component is amylopectin, below called amylopectin potato, modified in a genetically engineered manner according to the invention, is grated, thereby releasing the starch from the cell walls. 
     The cell walls (fibres) are separated from fruit juice and starch in centrifugal screens (centrisiler). The fruit juice is separated from the starch in two steps, viz. first in hydrocyclones and subsequently in specially designed band-type vacuum filters. 
     Then a finishing refining is carried out in hydrocyclones in which the remainder of the fruit juice and fibres are separated. 
     The product is dried in two steps, first by predrying on a vacuum filter and subsequently by final drying in a hot-air current. 
     EXAMPLE 3 
     Chemical Derivatisation of Amylopectin 
     Amylopectin is sludged in water to a concentration of 20-50%. The pH is adjusted to 10.0-12.0 and a quatenary ammonium compound is added in such a quantity that the end product obtains a degree of substitution of 0.004-0.2. The reaction temperature is set at 20-45° C. When the reaction is completed, the pH is adjusted to 4-8, whereupon the product is washed and dried. In this manner the cationic starch derivative 2-hydroxy-3-trimethyl ammonium propyl ether is obtained. 
     EXAMPLE 4 
     Chemical Derivatisation of Amylopectin 
     Amylopectin is sludged in water to a water content of 10-25% by weight. The pH is adjusted to 10.0-12.0, and a quatenary ammonium compound is added in such a quantity that the end product obtains a degree of substitution of 0.004-0.2. The reaction temperature is set at 20-45° C. When the reaction is completed, the pH is adjusted to 4-8. The end product is 2-hydroxy-3-trimethyl ammonium propyl ether. 
     EXAMPLE 5 
     Chemical Derivatisation of Amylopectin 
     Amylopectin is sludged in water to a concentration 25 of 20-50% by weight. The pH is adjusted to 5.0-12.0, and sodium hypochlorite is added so that the end product obtains the desired viscosity. The reaction temperature is set at 20-45° C. When the reaction is completed, the pH is adjusted to 4-8, whereupon the end product is washed and dried. In this manner, oxidised starch is obtained. 
     EXAMPLE 6 
     Physical Derivatisation of Amylopectin 
     Amylopectin is sludged in water to a concentration of 20-50% by weight, whereupon the sludge is applied to a heated cylinder where it is dried to a film. 
     EXAMPLE 7 
     Chemical and Physical Derivatisation of Amylopectin 
     Amylopectin is treated according to the process described in one of Examples 3-5 for chemical modification and is then further treated according to Example 6 for physical derivatisation. 
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     Flavell, R. B., 1990, AgBiotech. News and Information 2(5):629-630 
     Hergersberger, M., 1988, Molekulare Analyse des waxy Gens aus Solanum tuberosum und Expression von waxy antisense RNA in transgenen Kartoffeln. Thesis for a doctorate from the University in Cologne 
     Visser, R. G. F., Hergersberger, M., van der Leij, F. R., Jacobsen, E., Witholt, B. and Feenstra, W. J., 1989, Plant. Sci. 64:185-192 
     An, G., Ebert, P. R., Mitra, A. and Ha, S. B., 1987, Plant Mol. Biol. Manual A3:1-19 
     Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J. and Schilperoort, R. A., 1983, Nature 303:179-180 
     Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., 1987, EMBO J. 6:3201-3207 
     Sanger, F., Nicklen, S. and Coulson, A. R., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467 
     Viera, J. and Messing, J., 1982, Gene 19:259-268 
     Yanisch-Perron, C., Viera, J. and Messing, J., 1985, Gene 33:103-119 
     Heusterspreute et al (1987) Gene 53:294-300 
     Melton, D. A. et al (1984), Nucleic Acids Res. 12:7035-7056 (the plasmide is sold by Stratagene) 
     Murashige, T. and Skoog, F., 1962, Physiol. Plant 15:473-497. 
     Rocha-Sosa, M., Sonnewald, U., Frommer, W., Stratmann, M., Shell, J. and Willmitzer, L., 1989, EMBO J., 8(1):23-29 
     Jefferson, R. A., Kavanagh, R. A. and Bevan, M. W., 1987, EMBO J. 6:3901-3907 
     Maniatis, T., Fritsch, E. F. and Sambrook, J., 1982, Molecular Cloning, A Laboratory Handbook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 
     Bourque, J. E., Miller, J. C. and Park, W. D., 1987, In Vitro Cellular &amp; Development Biology 23(5):381-386 
     Hovenkamp-Hermelink, J. H. M., Jacobsen, E., Ponstein, A. S., Visser, R. G. F., Vos-Scheperkeuter, G. H., Bijmolt, E. W., de Vries, J. N., Witholt, B. J. &amp; Feenstra, W. J., 1987, Theor. Appl. Genet. 75:217-221 
     Hovenkamp-Hermelink, J. H. M., de Vries, J. N., Adamse, P., Jacobsen, E., Witholt, B. and Feenstra, W. J., 1988, Potato Research 31:241-246 
     Modified starches: Properties and use D. B. Wurzburg 
     Bevan, M. W., 1984. Nucleic Acids Res. 12:8711-8721. 
     
       
         
           
             21 
           
           
             
               342 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                217..342
 
             
             1
TGCATGTTTC CCTACATTCT ATTTAGAATC GTGTTGTGGT GTATAAACGT TGTTTCATAT     60
CTCATCTCAT CTATTCTGAT TTTGATTCTC TTGCCTACTG TAATCGGTGA TAAATGTGAA    120
TGCTTCCTTT CTTCTCAGAA ATCAATTTCT GTTTTGTTTT TGTTCATCTG TAGCTTATTC    180
TCTGGTAGAT TCCCCTTTTT GTAGACCACA CATCAC ATG GCA AGC ATC ACA GCT      234
                                        Met Ala Ser Ile Thr Ala
                                          1               5
TCA CAC CAC TTT GTG TCA AGA AGC CAA ACT TCA CTA GAC ACC AAA TCA      282
Ser His His Phe Val Ser Arg Ser Gln Thr Ser Leu Asp Thr Lys Ser
             10                  15                  20
ACC TTG TCA CAG ATA GGA CTC AGG AAC CAT ACT CTG ACT CAC AAT GGT      330
Thr Leu Ser Gln Ile Gly Leu Arg Asn His Thr Leu Thr His Asn Gly
         25                  30                  35
TTA AGG GCT GTT                                                      342
Leu Arg Ala Val
     40
 
           
           
             
               2549 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             2
AACAAGCTTG ATGGGCTCCA ATCAACAACT AATACTAAGG TAACACCCAA GATGGCATCC     60
AGAACTGAGA CCAAGAGACC TGGATGCTCA GCTACCATTG TTTGTGGAAA GGGAATGAAC    120
TTGATCTTTG TGGGTACTGA GGTTGGTCCT TGGAGCAAAA CTGGTGGACT AGGTGATGTT    180
CTTGGTGGAC TACCACCAGC CCTTGCAGTA AGTCTTTCTT TCATTTGGTT ACCTACTCAT    240
TCATTACTTA TTTTGTTTAG TTAGTTTCTA CTGCATCAGT CTTTTTATCA TTTAGGCCCG    300
CGGACATCGG GTAATGACAA TATCCCCCCG TTATGACCAA TACAAAGATG CTTGGGATAC    360
TGGCGTTGCG GTTGAGGTAC ATCTTCCTAT ATTGATACGG TACAATATTG TTCTCTTACA    420
TTTCCTGATT CAAGAATGTG ATCATCTGCA GGTCAAAGTT GGAGACAGCA TTGAAATTGT    480
TCGTTTCTTT CACTGCTATA AACGTGGGGT TGATCGTGTT TTTGTTGACC ACCCAATGTT    540
CTTGGAGAAA GTAAGCATAT TATGATTATG AATCCGTCCT GAGGGATACG CAGAACAGGT    600
CATTTTGAGT ATCTTTTAAC TCTACTGGTG CTTTTACTCT TTTAAGGTTT GGGGCAAAAC    660
TGGTTCAAAA ATCTATGGCC CCAAAGCTGG ACTAGATTAT CTGGACAATG AACTTAGGTT    720
CAGCTTGTTG TGTCAAGTAA GTTAGTTACT CTTGATTTTT ATGTGGCATT TTACTCTTTT    780
GTCTTTAATC GTTTTTTTAA CCTTGTTTTC TCAGGCAGCC CTAGAGGCAC CTAAAGTTTT    840
GAATTTGAAC AGTAGCAACT ACTTCTCAGG ACCATATGGT AATTAACACA TCCTAGTTTC    900
AGAAAACTCC TTACTATATC ATTGTAGGTA ATCATCTTTA TTTTGCCTAT TCCTGCAGGA    960
GAGGATGTTC TCTTCATTGC CAATGATTGG CACACAGCTC TCATTCCTTG CTACTTGAAG   1020
TCAATGTACC AGTCCAGAGG AATCTACTTG AATGCCAAGG TAAAATTTCT TTGTATTCAC   1080
TCGATTGCAC GTTACCCTGC AAATCAGTAA GGTTGTATTA ATATATGATA AATTTCACAT   1140
TGCCTCCAGG TTGCTTTCTG CATCCATAAC ATTGCCTACC AAGGTCGATT TTCTTTCTCT   1200
GACTTCCCTC TTCTCAATCT TCCTGATGAA TTCAGGGGTT CTTTTGATTT CATTGATGGG   1260
TATGTATTTA TGCTTGAAAT CAGACCTCCA ACTTTTGAAG CTCTTTTGAT GCTAGTAAAT   1320
TGAGTTTTTA AAATTTTGCA GATATGAGAA GCCTGTTAAG GGTAGGAAAA TCAACTGGAT   1380
GAAGGCTGGG ATATTAGAAT CACATAGGGT GGTTACAGTG AGCCCATACT ATGCCCAAGA   1440
ACTTGTCTCT GCTGTTGACA AGGGAGTTGA ATTGGACAGT GTCCTTCGTA AGACTTGCAT   1500
AACTGGGATT GTGAATGGCA TGGATACACA AGAGTGGAAC CCAGCGACTG ACAAATACAC   1560
AGATGTCAAA TACGATATAA CCACTGTAAG ATAAGATTTT TCCGACTCCA GTATATACTA   1620
AATTATTTTG TATGTTTATG AAATTAAAGA GTTCTTGCTA ATCAAAATCT CTATACAGGT   1680
CATGGACGCA AAACCTTTAC TAAAGGAGGC TCTTCAAGCA GCAGTTGGCT TGCCTGTTGA   1740
CAAGAAGATC CCTTTGATTG GCTTCATCGG CAGACTTGAG GAGCAGAAAG GTTCAGATAT   1800
TCTTGTTGCT GCAATTCACA AGTTCATCGG ATTGGATGTT CAAATTGTAG TCCTTGTAAG   1860
TACCAAATGG ACTCATGGTA TCTCTCTTGT TGAGTTTACT TGTGCCGAAA CTGAAATTGA   1920
CCTGCTACTC ATCCTATGCA TCAGGGAACT GGCAAAAAGG AGTTTGAGCA GGAGATTGAA   1980
CAGCTCGAAG TGTTGTACCC TAACAAAGCT AAAGGAGTGG CAAAATTCAA TGTCCCTTTG   2040
GCTCACATGA TCACTGCTGG TGCTGATTTT ATGTTGGTTC CAAGCAGATT TGAACCTTGT   2100
GGTCTCATTC AGTTACATGC TATGCGATAT GGAACAGTAA GAACCAGAAG AGCTTGTACC   2160
TTTTTACTGA GTTTTTAAAA AAAGAATCAT AAGACCTTGT TTTCCATCTA AAGTTTAATA   2220
ACCAACTAAA TGTTACTGCA GCAAGCTTTT CATTTCTGAA AATTGGTTAT CTGATTTTAA   2280
CGTAATCACA TGTGAGTCAG GTACCAATCT GTGCATCGAC TGGTGGACTT GTTGACACTG   2340
TGAAAGAAGG CTATACTGGA TTCCATATGG GAGCCTTCAA TGTTGAAGTA TGTGATTTTA   2400
CATCAATTGT GTACTTGTAC ATGGTCCATT CTCGTCTTGA TATACCCCTT GTTGCATAAA   2460
CATTAACTTA TTGCTTCTTG AATTTGGTTA GTGCGATGTT GTTGACCCAG CTGATGTGCT   2520
TAAGATAGTA ACAACAGTTG CTAGAGCTC                                     2549
 
           
           
             
               492 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               CDS 
                1..15
 
             
             
               CDS 
                101..218
 
             
             3
GAG CTC TCC TGG AAG GTAAGTGTGA ATTTGATAAT TTGCGTAGGT ACTTCAGTTT       55
Glu Leu Ser Trp Lys
  1               5
GTTGTTCTCG TCAGCACTGA TGGATTCCAA CTGGTGTTCT TGCAG GAA CCT GCC        109
                                                  Glu Pro Ala
                                                    1
AAG AAA TGG GAG ACA TTG CTA TTG GGC TTA GGA GCT TCT GGC AGT GAA      157
Lys Lys Trp Glu Thr Leu Leu Leu Gly Leu Gly Ala Ser Gly Ser Glu
      5                  10                  15
CCC GGT GTT GAA GGG GAA GAA ATC GCT CCA CTT GCC AAG GAA AAT GTA      205
Pro Gly Val Glu Gly Glu Glu Ile Ala Pro Leu Ala Lys Glu Asn Val
 20                  25                  30                  35
GCC ACT CCT TAAATGAGCT TTGGTTATCC TTGTTTCAAC AATAAGATCA              254
Ala Thr Pro  *
TTAAGCAAAC GTATTTACTA GCGAACTATG TAGAACCCTA TTATGGGGTC TCAATCATCT    314
ACAAAATGAT TGGTTTTTGC TGGGGAGCAG CAGCATATAA GGCTGTAAAA TCCTGGTTAA    374
TGTTTTTGTA GGTAAGGGCT ATTTAAGGTG GTGTGGATCA AAGTCAATAG AAAATAGTTA    434
TTACTAACGT TTGCAACTAA ATACTTAGTA ATGTAGCATA AATAATACTA GAACTAGT      492
 
           
           
             
               987 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             4
AAGCTTTAAC GAGATAGAAA ATTATGTTAC TCCGTTTTGT TCATTACTTA ACAAATGCAA     60
CAGTATCTTG TACCAAATCC TTTCTCTCTT TTCAAACTTT TCTATTTGGC TGTTGACGGA    120
GTAATCAGGA TACAAACCAC AAGTATTTAA TTGACTCCTC CGCCAGATAT TATGATTTAT    180
GAATCCTCGA AAAGCCTATC CATTAAGTCC TCATCTATGG ATATACTTGA CAGTATCTTC    240
CTGTTTGGGT ATTTTTTTTT CCTGCCAAGT GGAACGGAGA CATGTTATGA TGTATACGGG    300
AAGCTCGTTA AAAAAAAATA CAATAGGAAG AAATGTAACA AACATTGAAT GTTGTTTTTA    360
ACCATCCTTC CTTTAGCAGT GTATCAATTT TGTAATAGAA CCATGCATCT CAATCTTAAT    420
ACTAAAATGC AACTTAATAT AGGCTAAACC AAGATAAAGT AATGTATTCA ACCTTTAGAA    480
TTGTGCATTC ATAATTAGAT CTTGTTTGTC GTAAAAAATT AGAAAATATA TTTACAGTAA    540
TTTGGAATAC AAAGCTAAGG GGGAAGTAAC TAATATTCTA GTGGAGGGAG GGACCAGTAC    600
CAGTACCTAG ATATTATTTT TAATTACTAT AATAATAATT TAATTAACAC GAGACATAGG    660
AATGTCAAGT GGTAGCGTAG GAGGGAGTTG GTTTAGTTTT TTAGATACTA GGAGACAGAA    720
CCGGACGGCC CATTGCAAGG CCAAGTTGAA GTCCAGCCGT GAATCAACAA AGAGAGGGCC    780
CATAATACTG TCGATGAGCA TTTCCCTATA ATACAGTGTC CACAGTTGCC TTCTGCTAAG    840
GGATAGCCAC CCGCTATTCT CTTGACACGT GTCACTGAAA CCTGCTACAA ATAAGGCAGG    900
CACCTCCTCA TTCTCACTCA CTCACTCACA CAGCTCAACA AGTGGTAACT TTTACTCATC    960
TCCTCCAATT ATTTCTGATT TCATGCA                                        987
 
           
           
             
               4964 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             5
AAGCTTTAAC GAGATAGAAA ATTATGTTAC TCCGTTTTGT TCATTACTTA ACAAATGCAA     60
CAGTATCTTG TACCAAATCC TTTCTCTCTT TTCAAACTTT TCTATTTGGC TGTTGACGGA    120
GTAATCAGGA TACAAACCAC AAGTATTTAA TTGACTCCTC CGCCAGATAT TATGATTTAT    180
GAATCCTCGA AAAGCCTATC CATTAAGTCC TCATCTATGG ATATACTTGA CAGTATCTTC    240
CTGTTTGGGT ATTTTTTTTT CCTGCCAAGT GGAACGGAGA CATGTTATGA TGTATACGGG    300
AAGCTCGTTA AAAAAAAATA CAATAGGAAG AAATGTAACA AACATTGAAT GTTGTTTTTA    360
ACCATCCTTC CTTTAGCAGT GTATCAATTT TGTAATAGAA CCATGCATCT CAATCTTAAT    420
ACTAAAATGC AACTTAATAT AGGCTAAACC AAGATAAAGT AATGTATTCA ACCTTTAGAA    480
TTGTGCATTC ATAATTAGAT CTTGTTTGTC GTAAAAAATT AGAAAATATA TTTACAGTAA    540
TTTGGAATAC AAAGCTAAGG GGGAAGTAAC TAATATTCTA GTGGAGGGAG GGACCAGTAC    600
CAGTACCTAG ATATTATTTT TAATTACTAT AATAATAATT TAATTAACAC GAGACATAGG    660
AATGTCAAGT GGTAGCGTAG GAGGGAGTTG GTTTAGTTTT TTAGATACTA GGAGACAGAA    720
CCGGACGGCC CATTGCAAGG CCAAGTTGAA GTCCAGCCGT GAATCAACAA AGAGAGGGCC    780
CATAATACTG TCGATGAGCA TTTCCCTATA ATACAGTGTC CACAGTTGCC TTCTGCTAAG    840
GGATAGCCAC CCGCTATTCT CTTGACACGT GTCACTGAAA CCTGCTACAA ATAAGGCAGG    900
CACCTCCTCA TTCTCACTCA CTCACTCACA CAGCTCAACA AGTGGTAACT TTTACTCATC    960
TCCTCCAATT ATTTCTGATT TCATGCATGT TTCCCTACAT TCTATTATGA ATCGTGTTGT   1020
GGTGTATAAA CGTTGTTTCA TATCTCATCT CATCTATTCT GATTTTGATT CTCTTGCCTA   1080
CTGTAATCGG TGATAAATGT GAATGCTTCC TTTCTTCTCA GAAATCAATT TCTGTTTTGT   1140
TTTTGTTCAT CTGTAGCTTA TTCTCTGGTA GATTCCCCTT TTTGTAGACC ACACATCACA   1200
TGGCAAGCAT CACAGCTTCA CACCACTTTG TGTCAAGAAG CCAAACTTCA CTAGACACCA   1260
AATCAACCTT GTCACAGATA GGACTCAGGA ACCATACTCT GACTCACAAT GGTTTAAGGG   1320
CTGTTAACAA GCTTGATGGG CTCCAATCAA CAACTAATAC TAAGGTAACA CCCAAGATGG   1380
CATCCAGAAC TGAGACCAAG AGACCTGGAT GCTCAGCTAC CATTGTTTGT GGAAAGGGAA   1440
TGAACTTGAT CTTTGTGGGT ACTGAGGTTG GTCCTTGGAG CAAAACTGGT GGACTAGGTG   1500
ATGTTCTTGG TGGACTACCA CCAGCCCTTG CAGTAAGTCT TTCTTTCATT TGGTTACCTA   1560
CTCATTCATT ACTTATTTTG TTTAGTTAGT TTCTACTGCA TCAGTCTTTT TATCATTTAG   1620
GCCCGCGGAC AGCGGGTAAT GACAATATCC CCCCGTTATG ACCAATACAA AGATGCTTGG   1680
GATACTGGCG TTGCGGTTGA GGTACATCTT CCTATATTGA TACGGTACAA TATTGTTCTC   1740
TTACATTTCC TGATTCAAGA ATGTGATCAT CTGCAGGTCA AAGTTGGAGA CAGCATTGAA   1800
ATTGTTCGTT TCTTTCACTG CTATAAACGT GGGGTTGATC GTGTTTTTGT TGACCACCCA   1860
ATGTTCTTGG AGAAAGTAAG CATATTATGA TTATGAATCC GTCCTGAGGG ATACGCAGAA   1920
CAGGTCATTT TGAGTATCTT TTAACTCTAC TGGTGCTTTT ACTCTTTTAA GGTTTGGGGC   1980
AAAACTGGTT CAAAAATCTA TGGCCCCAAA GCTGGACTAG ATTATCTGGA CAATGAACTT   2040
AGGTTCAGCT TGTTGTGTCA AGTAAGTTAG TTACTCTTGA TTTTTATGTG GCATTTTACT   2100
CTTTTGTCTT TAATCGTTTT TTTAACCTTG TTTTCTCAGG CAGCCCTAGA GGCACCTAAA   2160
GTTTTGAATT TGAACAGTAG CAACTACTTC TCAGGACCAT ATGGTAATTA ACACATCCTA   2220
GTTTCAGAAA ACTCCTTACT ATATCATTGT AGGTAATCAT CTTTATTTTG CCTATTCCTG   2280
CAGGAGAGGA TGTTCTCTTC ATTGCCAATG ATTGGCACAC AGCTCTCATT CCTTGCTACT   2340
TGAAGTCAAT GTACCAGTCC AGAGGAATCT ACTTGAATGC CAAGGTAAAA TTTCTTTGTA   2400
TTCACTCGAT TGCACGTTAC CCTGCAAATC AGTAAGGTTG TATTAATATA TGATAAATTT   2460
CACATTGCCT CCAGGTTGCT TTCTGCATCC ATAACATTGC CTACCAAGGT CGATTTTCTT   2520
TCTCTGACTT CCCTCTTCTC AATCTTCCTG ATGAATTCAG GGGTTCTTTT GATTTCATTG   2580
ATGGGTATGT ATTTATGCTT GAAATCAGAC CTCCAACTTT TGAAGCTCTT TTGATGCTAG   2640
TAAATTGAGT TTTTAAAATT TTGCAGATAT GAGAAGCCTG TTAAGGGTAG GAAAATCAAC   2700
TGGATGAAGG CTGGGATATT AGAATCACAT AGGGTGGTTA CAGTGAGCCC ATACTATGCC   2760
CAAGAACTTG TCTCTGCTGT TGACAAGGGA GTTGAATTGG ACAGTGTCCT TCGTAAGACT   2820
TGCATAACTG GGATTGTGAA TGGCATGGAT ACACAAGAGT GGAACCCAGC GACTGACAAA   2880
TACACAGATG TCAAATACGA TATAACCACT GTAAGATAAG ATTTTTCCGA CTCCAGTATA   2940
TACTAAATTA TTTTGTATGT TTATGAAATT AAAGAGTTCT TGCTAATCAA AATCTCTATA   3000
CAGGTCATGG ACGCAAAACC TTTACTAAAG GAGGCTCTTC AAGCAGCAGT TGGCTTGCCT   3060
GTTGACAAGA AGATCCCTTT GATTGGCTTC ATCGGCAGAC TTGAGGAGCA GAAAGGTTCA   3120
GATATTCTTG TTGCTGCAAT TCACAAGTTC ATCGGATTGG ATGTTCAAAT TGTAGTCCTT   3180
GTAAGTACCA AATGGACTCA TGGTATCTCT CTTGTTGAGT TTACTTGTGC CGAAACTGAA   3240
ATTGACCTGC TACTCATCCT ATGCATCAGG GAACTGGCAA AAAGGATTTT GAGCAGGAGA   3300
TTGAACAGCT CGAAGTGTTG TACCCTAACA AAGCTAAAGG AGTGGCAAAA TTCAATGTCC   3360
CTTTGGCTCA CATGATCACT GCTGGTGCTG ATTTTATGTT GGTTCCAAGC AGATTTGAAC   3420
CTTGTGGTCT CATTCAGTTA CATGCTATGC GATATGGAAC AGTAAGAACC AGAAGAGCTT   3480
GTACCTTTTT ACTGAGTTTT TAAAAAAAGA ATCATAAGAC CTTGTTTTCC ATCTAAAGTT   3540
TAATAACCAA CTAAATGTTA CTGCAGCAAG CTTTTCATTT CTGAAAATTG GTTATCTGAT   3600
TTTAACGTAA TCACATGTGA GTCAGGTACC AATCTGTGCA TCGACTGGTG GACTTGTTGA   3660
CACTGTGAAA GAAGGCTATA CTGGATTCCA TATGGGAGCC TTCAATGTTG AAGTATGTGA   3720
TTTTACATCA ATTGTGTACT TGTACATGGT CCATTCTCGT CTTGATATAC CCCTTGTTGC   3780
ATAAACATTA ACTTATTGCT TCTTGAATTT GGTTAGTGCG ATGTTGTTGA CCCAGCTGAT   3840
GTGCTTAAGA TAGTAACAAC AGTTGCTAGA GCTCTTGCAG TCTATGGCAC CCTCGCATTT   3900
GCTGAGATGA TAAAAAATTG CATGTCAGAG GAGCTCTCCT GGAAGGTAAG TGTGAATTTG   3960
ATAATTTGCG TAGGTACTTC AGTTTGTTGT TCTCGTCAGC ACTGATGGAT TCCAACTGGT   4020
GTTCTTGCAG GAACCTGCCA AGAAATGGGA GACATTGCTA TTGGGCTTAG GAGCTTCTGG   4080
CAGTGAACCC GGTGTTGAAG GGGAAGAAAT CGCTCCACTT GCCAAGGAAA ATGTAGCCAC   4140
TCCTTAAATG AGCTTTGGTT ATCCTTGTTT CAACAATAAG ATCATTAAGC AAACGTATTT   4200
ACTAGCGAAC TATGTAGAAC CCTATTATGG GGTCTCAATC ATCTACAAAA TGATTGGTTT   4260
TTGCTGGGGA GCAGCAGCAT ATAAGGCTGT AAAATCCTGG TTAATGTTTT TGTAGGTAAG   4320
GGCTATTTAA GGTGGTGTGG ATCAAAGTCA ATAGAAAATA GTTATTACTA ACGTTTGCAA   4380
CTAAATACTT AGTAATGTAG CATAAATAAT ACTAGAACTA GTAGCTAATA TATATGCGTG   4440
AATTTGTTGT ACCTTTTCTT GCATAATTAT TTGCAGTACA TATATAATGA AAATTACCCA   4500
AGGAATCAAT GTTTCTTGCT CCGTCCTCCT TTGATGATTT TTTACGCAAT ACAGAGCTAG   4560
TGTGTTATGT TATAAATTTT GTTTAAAAGA AGTAATCAAA TTCAAATTAG TTGTTTGGTC   4620
ATATGAAAGA AGCTGCCAGG CTAACTTTGA GGAGATGGCT ATTGAATTTC AAAATGATTA   4680
TGTGAAAACA ATGCAACATC TATGTCAATC AACACTTAAA TTATTGCATT TAGAAAGATA   4740
TTTTTGAGCC CATGACACAT TCATTCATAA AGTAAGGTAG TATGTATGAT TGAATGGACT   4800
ACAGCTCAAT CAAAGCATCT CCTTTACATA ACGGCACTGT CTCTTGTCTA CTACTCTATT   4860
GGTAGTAGTA GTAGTAATTT TACAATCCAA ATTGAATAGT AATAAGATGC TCTCTATTTA   4920
CTAAAGTAGT AGTATTATTC TTTCGTTACT CTAAAGCAAC AAAA                    4964
 
           
           
             
               69 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..69 
                /note= “Amino acid sequence encoded
               by nucleotides 1-207 of SEQ ID NO. 2.”
 
             
             6
Asn Lys Leu Asp Gly Leu Gln Ser Thr Thr Asn Thr Lys Val Thr Pro
1               5                   10                  15
Lys Met Ala Ser Arg Thr Glu Thr Lys Arg Pro Gly Cys Ser Ala Thr
            20                  25                  30
Ile Val Cys Gly Lys Gly Met Asn Leu Ile Phe Val Gly Thr Glu Val
        35                  40                  45
Gly Pro Trp Ser Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu
    50                  55                  60
Pro Pro Ala Leu Ala
65
 
           
           
             
               27 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..27 
                /note= “Amino acid sequence encoded
               by nucleotides 296-377 of SEQ ID NO. 2.”
 
             
             7
Ala Arg Gly His Arg Val Met Thr Ile Ser Pro Arg Tyr Asp Gln Tyr
1               5                   10                  15
Lys Asp Ala Trp Asp Thr Gly Val Ala Val Glu
            20                  25
 
           
           
             
               33 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..33 
                /note= “Amino acid sequence encoded
               by nucleotides 452-550 of SEQ ID NO. 2.”
 
             
             8
Val Lys Val Gly Asp Ser Ile Glu Ile Val Arg Phe Phe His Cys Tyr
1               5                   10                  15
Lys Arg Gly Val Asp Arg Val Phe Val Asp His Pro Met Phe Leu Glu
            20                  25                  30
Lys
 
           
           
             
               30 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..30 
                /note= “Amino acid sequence encoded
               by nucleotides 647-736 of SEQ ID NO. 2.”
 
             
             9
Val Trp Gly Lys Thr Gly Ser Lys Ile Tyr Gly Pro Lys Ala Gly Leu
1               5                   10                  15
Asp Tyr Leu Asp Asn Glu Leu Arg Phe Ser Leu Leu Cys Gln
            20                  25                  30
 
           
           
             
               21 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..21 
                /note= “Amino acid sequence encoded
               by nucleotides 815-878 of SEQ ID NO. 2.”
 
             
             10
Ala Ala Leu Glu Ala Pro Lys Val Leu Asn Leu Asn Ser Ser Asn Tyr
1               5                   10                  15
Phe Ser Gly Pro Tyr
            20
 
           
           
             
               34 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..34 
                /note= “Amino acid sequence encoded
               by nucleotides 878 and 959-1059 of SEQ ID NO. 2.”
 
             
             11
Gly Glu Asp Val Leu Phe Ile Ala Asn Asp Trp His Thr Ala Leu Ile
1               5                   10                  15
Pro Cys Tyr Leu Lys Ser Met Tyr Gln Ser Arg Gly Ile Tyr Leu Asn
            20                  25                  30
Ala Lys
 
           
           
             
               38 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..38 
                /note= “Amino acid sequence encoded
               by nucleotides 1150-1263 of SEQ ID NO 2.”
 
             
             12
Val Ala Phe Cys Ile His Asn Ile Ala Tyr Gln Gly Arg Phe Ser Phe
1               5                   10                  15
Ser Asp Phe Pro Leu Leu Asn Leu Pro Asp Glu Phe Arg Gly Ser Phe
            20                  25                  30
Asp Phe Ile Asp Gly Tyr
        35
 
           
           
             
               79 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..79 
                /note= “Amino acid sequence encoded
               by nucleotides 1349-1585 of SEQ ID NO 2.”
 
             
             13
Lys Pro Val Lys Gly Arg Lys Ile Asn Trp Met Lys Ala Gly Ile Leu
1               5                   10                  15
Glu Ser His Arg Val Val Thr Val Ser Pro Tyr Tyr Ala Gln Glu Leu
            20                  25                  30
Val Ser Ala Val Asp Lys Gly Val Glu Leu Asp Ser Val Leu Arg Lys
        35                  40                  45
Thr Cys Ile Thr Gly Ile Val Asn Gly Met Asp Thr Gln Glu Trp Asn
    50                  55                  60
Pro Ala Thr Asp Lys Tyr Thr Asp Val Lys Tyr Asp Ile Thr Thr
65                  70                  75
 
           
           
             
               59 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..59 
                /note= “Amino acid sequence encoded
               by nucleotides 1676-1855 of SEQ ID NO 2.”
 
             
             14
Val Met Asp Ala Lys Pro Leu Leu Lys Glu Ala Leu Gln Ala Ala Val
1               5                   10                  15
Gly Leu Pro Val Asp Lys Lys Ile Pro Leu Ile Gly Phe Ile Gly Arg
            20                  25                  30
Leu Glu Glu Gln Lys Gly Ser Asp Ile Leu Ala Val Ala Ile His Lys
        35                  40                  45
Phe Ile Gly Leu Asp Val Gln Ile Val Val Leu
    50                  55
 
           
           
             
               64 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..64 
                /note= “Amino acid sequence encoded
               by nucleotides 1945-2136 of SEQ ID NO 2.”
 
             
             15
Gly Thr Gly Lys Lys Glu Phe Glu Gln Glu Ile Glu Gln Leu Glu Val
1               5                   10                  15
Leu Tyr Pro Asn Lys Ala Lys Gly Val Ala Lys Phe Asn Val Pro Leu
            20                  25                  30
Ala His Met Ile Thr Ala Gly Ala Asp Phe Met Leu Val Pro Ser Arg
        35                  40                  45
Phe Glu Pro Cys Gly Leu Ile Gln Leu His Ala Met Arg Tyr Gly Thr
    50                  55                  60
 
           
           
             
               29 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..29 
                /note= “Amino acid sequence encoded
               by nucleotides 2301-2386 of SEQ ID NO 2.”
 
             
             16
Val Pro Ile Cys Ala Ser Thr Gly Gly Leu Val Asp Thr Val Lys Glu
1               5                   10                  15
Gly Tyr Thr Gly Phe His Met Gly Ala Phe Asn Val Glu
            20                  25
 
           
           
             
               19 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..19 
                /note= “Amino acid sequence encoded
               by nucleotides 2492-2459 of SEQ ID NO 2.”
 
             
             17
Cys Asp Val Val Asp Pro Ala Asp Val Leu Lys Ile Val Thr Thr Val
1               5                   10                  15
Ala Arg Ala
 
           
           
             
               111 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..111 
                /note= “Amino acid sequence encoded
               by nucleotides 1200-1532 of SEQ ID NO 5.”
 
             
             18
Met Ala Ser Ile Thr Ala Ser His His Phe Val Ser Arg Ser Gln Thr
1               5                   10                  15
Ser Leu Asp Thr Lys Ser Thr Leu Ser Gln Ile Gly Leu Arg Asn His
            20                  25                  30
Thr Leu Thr His Asn Gly Leu Arg Ala Val Asn Lys Leu Asp Gly Leu
        35                  40                  45
Gln Ser Thr Thr Asn Thr Lys Val Thr Pro Lys Met Ala Ser Arg Thr
    50                  55                  60
Glu Thr Lys Arg Pro Gly Cys Ser Ala Thr Ile Val Cys Gly Lys Gly
65                  70                  75                  80
Met Asn Leu Ile Phe Val Gly Thr Glu Val Gly Pro Trp Ser Lys Thr
                85                  90                  95
Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Leu Ala
            100                 105                 110
 
           
           
             
               43 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..43 
                /note= “Amino acid sequence encoded
               by nucleotides 3817-3945 of SEQ ID NO. 5.”
 
             
             19
Cys Asp Val Val Asp Pro Ala Asp Val Leu Lys Ile Val Thr Thr Val
1               5                   10                  15
Ala Arg Ala Leu Ala Val Tyr Gly Thr Leu Ala Phe Ala Glu Met Ile
            20                  25                  30
Lys Asn Cys Met Ser Glu Glu Leu Ser Trp Lys
        35                  40
 
           
           
             
               38 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               Modified-site 
                1..38 
                /note= “Amino acid sequence encoded
               by nucleotides 4031-4144 of SEQ ID NO. 5.”
 
             
             20
Glu Pro Ala Lys Lys Trp Glu Thr Leu Leu Leu Gly Leu Gly Ala Ser
1               5                   10                  15
Gly Ser Glu Pro Gly Val Glu Gly Glu Glu Ile Ala Pro Leu Ala Lys
            20                  25                  30
Glu Asn Val Ala Thr Pro
        35
 
           
           
             
               17 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               RNA 
             
             
               misc_RNA 
                /note= “Nucleotide 1 is a 7-methyl
               guanine added by 5′-5′ linkage as an RNA cap.”
 
             
             21
GAUGGCAAGA AAAAAAA                                                    17