Abstract:
Briefly stated, the invention includes a method of making a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase, comprising the steps of introducing into at least one plant cell a polynucleotide that encodes a human acetylcholinesterase, and regenerating from the plant cell a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase in at least one tissue type of the transgenic plant. Another embodiment of the invention includes a method of making a physiologically active human acetylcholinesterase, comprising the steps of introducing into at least one plant cell a polynucleotide that encodes a human acetylcholinesterase, regenerating from the plant cell a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase in at least one tissue type of the transgenic plant, and isolating or purifying from the transgenic plant or a part thereof a physiologically active human acetylcholinesterase.

Description:
REFERENCE TO RELATED APPLICATIONS 
     This application claims an invention that was disclosed in Provisional Application No. 60/190,440, filed Mar. 17, 2000, entitled “EXPRESSION OF RECOMBINANT HUMAN ACETYLCHOLINESTERASE IN TRANSGENIC TOMATOES.” The benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application is hereby claimed, and the aforementioned application is hereby incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention pertains to the field of transgenic plants. More particularly, the invention pertains to the expression of a recombinant form of human acetylcholinesterase in transgenic plants. 
     2. Description of Related Art 
     Acetylcholine (ACh) is one of the major signaling molecules in metazoans, functioning mostly as a neurotransmitter in chemical synapses between neurons and in neuromuscular junctions. To ensure a discrete “all-or-none” response across the synapse, the release of ACh is tightly controlled and the neurotransmitter is efficiently removed by the hydrolyzing enzyme, acetylcholinesterase (AChE). In humans, AChE is encoded by a single gene which yields, through alternative splicing of its pre-mRNA, three polypeptide isoforms having distinct C-termini. See Soreq et al.,  Proc. Nat. Acad. Sci. U.S.A.  87: 9688-9692 (1990); Ben Aziz-Aloya et al.,  Proc. Natl. Acad. Sci. U.S.A.  90: 2471-5 (1993); GenBank Accession No. M55040; and U.S. Pat. No. 5,595,903. The complete disclosure of each of the foregoing references is hereby incorporated herein by reference. 
     Various compounds are well known to inhibit the hydrolyzing activity of AChE. Exposure to such anti-AChE agents leads to over-stimulation of cholinergic pathways, causing muscular tetany, autonomous dysfunction and potentially death. While some naturally occurring AChE inhibitors are very potent, human exposure to them is rare. However, man-made anti-AChE compounds, especially organophosphates (OPs), are widely used as pesticides and pose a substantial occupational and environmental risk. Even more ominous is the fear of deliberate use of OPs as chemical warfare agents against individuals or populations. 
     Current medical interventions, in the case of acute exposure to anticholinesterase agents, include use of the muscarinic receptor antagonist, atropine, and oximes to reactivate the OP-modified AChE. The reversible carbamate, pyridostigmine bromide, is also used as a prophylactic. However, these conventional treatments have limited effectiveness and have serious short and long-term side effects. In fact, the routine treatments, while successfully decreasing anticholinesterase-induced lethality, rarely alleviate post-exposure delayed toxicity, which may result in significant performance deficits, and even permanent brain damage. 
     A different approach in treatment and prevention of anti-AChE toxicity seeks to mimic one of the physiological lines of defense against such agents present in mammals. Butyrylcholinesterase (BuChE) is a serum cholinesterase with a broad hydrolytic spectrum that provides protection against a variety of AChE inhibitors. A similar end may be achieved by a variant of AChE found on the membranes of erythrocytes. Both enzymes are believed to serve as circulating scavengers for anti-AChE agents in protection of the vital synaptic AChE. Therefore, administration of cholinesterases could boost their natural potential to counter-act the toxic effects of anti-cholinergic agents. The efficacy of this treatment to protect against a challenge of OPs was tested in a variety of animal models such as mice, rats, guinea pigs, and primates, and was found to be comparable to or better than the currently-used drug regimens in preventing OP-induced mortality without any detrimental side-effects. 
     Enzyme therapy has the additional benefit of the relatively long half-life time (several days) of the injected enzymes in the blood stream, making it especially useful for prophylaxis. In the foregoing experiments, cholinesterases purified from human or animal blood were used. To be effective, the stoichiometry of cholinesterase to inhibitor must be close to unity. Hence, large amounts of pure, properly folded, stable enzymatic preparations that are free of mammalian pathogens are needed, if enzyme therapy is to be feasible. 
     Genetically engineered plants have recently been recognized as one of the most cost-effective means for the production of useful recombinant proteins and pharmaceuticals. Therefore, we examined the use of transgenic plants as a cost-effective and safe alternative to the production of human acetylcholinesterase (hAChE) from blood or cell cultures, herein providing the first demonstration of the expression in plants of a key protein component of the nervous system of humans. 
     SUMMARY OF THE INVENTION 
     Briefly stated, the invention includes one or more plant cells comprising a polynucleotide that encodes a human acetylcholinesterase. 
     An embodiment of the invention includes a method of making a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase, comprising the steps of introducing into at least one plant cell a polynucleotide that encodes a human acetylcholinesterase, and regenerating from the plant cell a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase in at least one tissue type of the transgenic plant. 
     Another embodiment of the invention includes a method of making a physiologically active human acetylcholinesterase, comprising the steps of introducing into at least one plant cell a polynucleotide that encodes a human acetylcholinesterase, regenerating from the plant cell a transgenic plant that is capable of expressing a physiologically active human acetylcholinesterase in at least one tissue type of the transgenic plant, and isolating or purifying from the transgenic plant or a part thereof a physiologically active human acetylcholinesterase. 
     Another embodiment of the invention includes a method of treating a victim of acetylcholinesterase poisoning, comprising the step of administering a therapeutic amount of a physiologically active human acetylcholinesterase expressed in plant tissue. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 shows a graphic map of pTM036, the pGPTVkan derivative construct used in the generation of transgenic tomato plants that constitutively express hAChE-E4. 
     FIG. 2 shows a bar graph depicting high activity of hAChE in transgenic tomato lines. 
     FIG. 3 shows substrate inhibition of recombinant hAChE obtained from transgenic plants. 
     FIG. 4 shows an inhibition profile of AChE obtained from transgenic plants (diamonds), human erythrocytes (circles) and transgenic mice (squares). 
     FIG. 5A shows a graph of data indicating that a recombinant hAChE derived from transgenic plants is labile at relatively high temperatures. 
     FIG. 5B shows a graph of data indicating that a plant-derived hAChE is relatively stable at room temperature. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     DNA Constructs and Plant Transformation 
     A cDNA encoding human AChE exons II, III and IV was amplified from the plasmid pAChE-E4 (see Sternfeld et al.,  J. Neurosci.  18: 1240-1249 (1990), the complete disclosure of which is hereby incorporated herein by reference) via the polymerase chain reaction (PCR), according to standard methods, which are well known in the art, using the following primers: 
     
       
         
               
               
             
           
               
                 AChE-Nco - (5′-GATATCTGCAGCCATGgctAGGCCCCCGC) (SEQ ID NO: 1) 
                   
               
               
                   
               
               
                 AChE-Kpn - (5′-CggtaccTATCAGGTaGCGCTGAGCAATTTG) (SEQ ID NO: 2) 
               
             
          
         
       
     
     The lower case letters in the foregoing primer sequences represent bases that were introduced to create restriction sites for cloning the gene into plant expression vectors. The PCR product was cloned and sequenced using well known methods. An Nco I-Kpn I fragment from a partial digest of pAChE-E4 was cloned into pIBT210.1 (see Haq et al.,  Science  268: 714-716 (1995), the complete disclosure of which is hereby incorporated herein by reference) behind a CaMV 35S promoter and the 5′ UTR of Tobacco Etch Virus, and in front of the 3′ UTR of the soy bean vspB gene to form pTM034 (FIG. 1, SEQ ID NO:3), according to standard methods, which are well known in the art. A Hind III-Eco RI fragment containing the plant expression cassette was then cloned into the T i  plasmid derivative pGPTV-Kan to form pTM036 (SEQ ID NO:4), using standard methods that are well known in the art. This plasmid was then transferred to  Agrobacterium tumefaciens  strain EHA105, and was used in the subsequent transformation of the  Lycopersicum esculentum  cultivar referred to as “Micro-Tom,” as described by Meissner et al. in  Plant J.  12: 1465-1472 (1997), the complete disclosure of which is hereby incorporated herein by reference. 
     Genomic PCR, DNA and RNA Blot Analysis 
     Screening by genomic PCR was performed on 0.8 μg total DNA isolated from kanamycin resistant plants, using the AChE-Nco and AChE-Kpn primers, according to well known methods. For DNA blot analysis, total DNA was prepared, digested with Nco I, and the digested DNA (˜20 μg) was resolved by electrophoresis, transferred to a nylon hybridization membrane, and hybridized to a digoxigenin-labeled probe, according to standard methods, which are well known in the art. The digoxigenin-labeled probe was synthesized using the following primers: 
     
       
         
               
               
             
           
               
                   
                 AChE585for (5′-CGAGAGGACTGTGCTGGTGTC) 
               
               
                   
                   
               
               
                   
                 AChE1374rev (5′-GTCGCCCACCACATCGCTC) 
               
             
          
         
       
     
     Hybridization and detection were performed according to well known methods. Total RNA was isolated and 5 μg samples were resolved by denaturing formaldehyde gel electrophoresis and transferred to nylon hybridization membranes, according to well known methods. 
     Acetylcholinesterase Assays and Protein Determination 
     Plant samples were homogenized in the presence ice-cold extraction buffer (100 mM NaCl, 25 mM Tris, 0.1 mM EDTA, 10 μg/ml leupeptin, pH 7.4, 3 ml per 1 g tissue) using ceramic beads in a bead-beater, and cleared supernatants were collected followed by centrifugation (14,000 rpm). Scaled-down microtiter plate Ellman assays were performed, according to standard methods, which are well known in the art. Cleared extracts (˜20 μl) were incubated for 30 minutes at room temperature with 80 μl assay buffer (0.1 M phosphate buffer, pH 7.4) with or without 2×10 −5  M 1,5-bis(allyldimethylammiumphenyl)pentan-3 one dibromide (BW284c51), which is a specific inhibitor of mammalian AChE. At the end of the 30 minute incubation period, 100 μl of 1 mM 5-5′-dithio-bis(2-nitrobenzoate) (Ellman&#39;s reagent) and 2 mM acetylthiocholine in assay buffer was added. Hydrolysis was monitored by measuring optical density at 405 nm at 5 minute intervals for 30 minutes, using a microtiter plate spectrophotometer, plotted against time, and initial rates were calculated from the slope of the linear portion of the graph. Net hydrolysis rates were calculated by subtracting the rates measured in the presence of BW284c15 from those obtained in its absence. To determine the K m , the concentration of the acetylthiocholine substrate in the Ellman&#39;s reagent was varied in the range of 0.05-50 mM. 
     Inhibition curves were obtained by performing the Ellman assay with 1 mM acetylthiocholine in the presence of the indicated concentrations of diethyl p-nitrophenyl phosphate (paraoxon), neostigmine, phehylmethylsulfonyl fluoride (PMSF) or tetraisopropyl pyrophosphoramide (Iso-OMPA). To determine K I  of BW284c51, assays were performed in the presence of 1, 0.33 and 0.25 mM acetylthiocholine, and the inhibitor at 10 −4  to 10 −10  M. Results were then analyzed according to the method of Ordentlich et al. (see Ordentlich et al.,  J. Biol. Chem.  268: 17083-17095 (1993), the complete disclosure of which is hereby incorporated herein by reference). In these experiments, acetylcholinesterase from human erythrocytes was used. 
     To evaluate the heat stability of the enzyme, plant extracts were incubated for 30 minutes at the indicated temperatures and then assayed as described above. Stability of the enzymatic activity was determined at 4 degrees C. and at 25 degrees C. by incubating plant extracts at the respective temperatures and assaying samples at the indicated time points. 
     A cDNA encoding exons 2-4 of the human AChE29 gene was inserted into a plant expression cassette driven by the constitutive cauliflower mosaic virus 35S promoter. Referring now to FIG. 1, a graphic map is shown of pTM036, the pGPTVkan derivative construct used in the generation of transgenic tomato plants that constitutively express hAChE-E4. Empty arrowheads denote positions of the PCR primers AChE-Nco and AChE-Kpn used for amplification of the full length coding region of hAChE-E4. Filled arrowheads denote the positions of the PCR primers AChE585 for and AChE1374rev used for the generation of DIG-labeled probe. 
     We used  Agrobacterium tumefaciens  to construct the tomato explants, and regenerated 27 kanamycin resistant tomato lines. We screened the transformants for the insertion of the recombinant human gene AChE-E4 by PCR. Twelve out of 17 plants tested were positive for the appropriate gene insertion event. The product of the AChE-E4 construct was previously demonstrated to be a monomeric soluble protein, which is fully active in acetylcholine hydrolysis. Therefore, we screened the putative transgenic plants for the expression of specific acetylcholinesterase activity in the soluble protein fraction of leaf extracts of kanamycin-resistant lines. 
     Referring now to FIG. 2, kanamycin-resistant lines were assayed for specific esterase activity (i.e., total minus activity in the presence of the inhibitor BW284c51) in leaves by the method of Ellman, using acetylthiocholine as a substrate. Protein samples from the indicated transgenic plant lines (AChE-53, 54, 62, 68 and 83), untransformed plant (UT) and a commercially available preparation of AChE from human erythrocytes (E5) were resolved on a non-denaturing gel which was then stained for AChE activity. Plant-derived AChE migrates as a discrete band in non-denaturing gel electrophoresis. On a per soluble protein basis, high activity, comparable to a third of the activity present in mammalian brain and five times more than that present in muscles, was registered in several of the lines, including AChE-53, AChE-54, AChE-62 and AChE-68. In these lines, activity was on the order of 100 mU/g leaf tissue (fresh weight). Acetylcholinesterase present in the transgenic lines appeared as a discrete band in non-denaturing polyacrylamide gels stained for cholinesterase activity. This result demonstrates the apparent uniformity of the protein produced by the plants. No activity was detected in the untransformed line, or in line AChE-83. Unexpectedly, in contrast to the sharp bands of the plant derived recombinant enzyme, the activity of the commercially available preparation of AChE from human erythrocytes appeared as a diffuse smear. 
     DNA blot analysis revealed that three of the lines that express high levels of activity, AChE-54, AChE-62 and AChE-68, each have one copy of the hAChE-E4 gene inserted in their genomes. Total DNA was isolated from the indicated lines, digested with Nco I, resolved by agarose gel electrophoresis, blotted to nylon membrane and probed with digoxigenin-labeled probe, according to well known methods. Referring to FIG. 2, AChE-83, a transgenic line that does not exhibit AChE activity, has at least two copies of the gene inserted in its genome. However, in this line, the mRNA encoding hAChE-E4 failed to accumulate to detectable levels, as demonstrated by RNA blot analysis, suggesting that transgene silencing in this line might have occurred. RNA blot analysis of several kanamycin-resistant tomato lines indicated that mRNA accumulated to similar levels in all the other lines that were tested. Total RNA was isolated from the indicated lines, resolved by agarose gel electrophoresis, blotted to nylon membrane and stained with methylene blue. The membrane was then probed with AChE specific DIG-labeled probe. 
     Kinetic Properties of the Plant-Produced Recombinant Enzyme 
     We calculated the K m  of the plant-derived enzyme for four of our expressing lines to be 0.44±0.10 mM (FIG. 3, inset). This value is similar to that reported for the same molecular form of the enzyme expressed in injected oocytes of  Xenopus laevis  and also to those reported for other forms of the human enzyme. Hydrolysis was inhibited by the presence of substrate at high concentration (FIG.  3 ), as previously reported for native and recombinant AChE. Enzyme activity was assayed in the presence of acetylthiocholine at 0.05-50 mM, and hydrolysis in the presence of the inhibitor BW284c51 was subtracted at each concentration. A representative high expression line (AChE-54) is shown in FIG.  3 . The inset of FIG. 3 shows Lineweaver-Burk analysis for the determination of the K m  for four lines: AChE-53 (squares), AChE-54 (diamonds), AChE-62 (triangles) and AChE-68 (crosses). 
     AChE inhibitors of various classes, including the reversible inhibitors neostigmine (a carbamate), BW284C51 (an AChE-specific bisquaternary inhibitor), as well as the irreversible inhibitors paraoxon (an organophosphate, the activated form of the pesticide parathion) and PMSF (a general serine hydrolase inhibitor) can inhibit the plant derived recombinant AChE (rAChE), and the inhibition profile is very similar to that of a commercially available preparation of human AChE derived from erythrocytes (FIG.  4 ). The K I  calculated for BW284c51 is 16 nM, which is in close agreement with the values for the recombinant human synaptic enzyme transiently expressed in mammalian cell cultures (10 nM) and for the erythrocyte form (5 nM). As expected, the butyrylcholinesterase-specific organophosphate, Iso-OMPA, had no effect on either the plant-derived or the erythrocyte-derived enzyme preparations (up to 100 μM), and only partial inhibition was registered at 10 mM (FIG.  4 ). The plant-derived E4 enzyme was somewhat less susceptible to paraoxon than an equivalent recombinant enzyme obtained from transgenic mice (FIG.  4 ). 
     The plant-derived hAChE in total soluble protein extracts retained 50% of its initial activity after incubation at 42 degrees for at least 30 minutes (FIG.  5 A). Crude leaf extracts were incubated at the indicated temperatures for 30 minutes and then subjected to Ellman&#39;s AChE assay. Incubation of plant extracts at room temperature (˜25 degrees C.) resulted in gradual loss of AChE activity, with 20% residual activity remaining after 25 hours (FIG.  5 B). The activity was very stable at 4 degrees C., with only 20% loss after 24 hours (FIG.  5 B). Crude leaf extracts were incubated at 4 degrees C. or at 25 degrees C. for the indicated time periods and then assayed for AChE activity. 
     Types of Cholinesterases that can be Expressed in Plants 
     Traditionally, cholinesterases are classified as either acetylcholinesterase (EC 3.1.1.7, AChE) or as butyrylcholine hydrolases (EC 3.1.1.8, BChE, formerly referred to as pseudo-acetylcholinesterase) on the basis of their substrate specificity. While BChE can efficiently hydrolyze substrates with a longer acyl group, the catalytic efficiency of AChE is limited to acetylcholine and, to a lesser degree, propionylcholine. More recently inhibitors have been identified that can selectively inhibit the two types of cholinesterases. 
     The genes encoding AChE and BChE from several mammals, including humans, have been cloned. Cholinesterases from non-vertebrates and lower vertebrates, even when possessing several different genes, have mixed characteristics. A further complication of the molecular picture is presented by the alternative splicing that the transcript of the AChE gene can undergo leading, in mammals, to three distinct isoforms. These isoforms share a common N-terminal catalytic domain, but diverge in their C-termini, which impact their quaternary structure and membrane association. 
     The catalytic distinction between the enzymes is not restricted to acyl-choline substrates but to other types of esters. Thus, BChE can catalyze the hydrolysis of cocaine whereas AChE cannot. On the other hand, it was recently demonstrated that the erythrocyte form of AChE can hydrolyze heroin (3,6-diacylmorphine) to morphine, while BChE can hydrolyze heroin only to the intermediate 6-NAM (6-monoacetylmorphine). Interestingly, the synaptic isoform of AChE cannot hydrolyze heroin, making heroin hydrolysis the first reported catalytic distinction between the different isoforms of AChE. 
     The literature on the non-cholinergic functions of cholinesterases, and especially of AChE, is becoming richer all the time. These proteins apparently play important roles in the developing nervous system and its maintenance, especially in directing the growth of neurons and establishing synaptic connections. The different isoforms have distinct roles through their different C-termini. For example, addition of the synaptic isoform of AChE to cultured neurons has a marked activation effect on neurite outgrowth, and a similar effect has been noted in transgenic frog embryos. In contrast, frog embryos expressing soluble forms of the enzyme do not exhibit such effects. 
     These small nuances make all of these different isoforms valuable, and we anticipate that plant production of them will be useful for many different ends, including, but not limited to, the following: 1) scavengers of anticholinesterase agents including organophosphates; 2) the hydrolysis of cocaine and heroin in treatment of cases of overdose intoxication by drug abusers; and 3) regeneration of damaged neuronal tissue. 
     Optimization of the Coding Sequence of hAChE-E4 for Expression in Plants 
     In most cases, the accumulation of foreign proteins in transgenic proteins is a desirable objective, as it tends to maximize yield and reduce costs of production. Accumulation of proteins is a complex function of many factors that effect synthesis and degradation. By “synthesis” we mean all the biochemical steps which lead to the formation of a mature protein, from transcription of a gene, accumulation of mRNA, translation of messages, localization of products, and many co- and post-translational modifications. Not all of these steps can easily be controlled directly (some, for example, are inherent to the polypeptide in question) and, as yet, not all can be manipulated to enhance accumulation. However, experience has shown that certain optimization measures can have a profound effect on the overall levels of foreign protein accumulation in plants. 
     Up to the translation stage, the expression of a gene is dependent on the nucleotide sequence not only of the control elements, such as promoter, enhancer elements and 3′ sequences, but also on the coding region as well. Molecular cues are encoded by the nucleic acid sequence to allow molecular events, such as termination of transcription, splicing of intervening sequences, rapid turnover of mRNA, and its translatability. Many of these features are common to many different types of organisms, while others are specific for plants, including, for example, intron splice sites, plant-specific RNA stabilizing sequences, and even plant specific biases in codon usage. Thus, optimization of gene sequences entails conforming the coding sequence to those of plant genes. 
     Numerous methods for the optimization of DNA sequences for increased expression in plants are well known in the art. For example, see U.S. Pat. Nos. 6,180,774; 6,166,302; 6,121,014; 6,110,668; 6,075,185; 6,051,760; 6,043,415; 6,015,891; 6,013,523; 5,994,526; 5,952,547; 5,880,275; 5,877,306; 5,866,421; 5,859,347; 5,859,336; 5,689,052; 5,633,446; 5,625,136; 5,567,862; 5,567,600; 5,545,817; 5,500,365; and 5,380,831, the disclosures of each of which are hereby incorporated herein by reference. 
     Analysis of the cDNA of hAChE-E4 to Assess its Suitability for Expression in Plants 
     Although we present herein an example wherein the hAChE gene is expressed in tomato plants, the tomato serves here only as a model organism. The expression of hAChE in all major crop plants is intended to be within the scope of the present invention, including (but not restricted to): dicotyledonous plants, such as, for example, tomato, potato, tobacco, legumes (i.e., soybean, peanut, alfalfa), and sweet potato. These plants are typically engineered by Agrobacterium transformation, various suitable methods for which are well known in the art. Monocotyledonous plants are also intended to be within the scope of the present invention, including (but not restricted to): maize, rice, wheat, and barley. These plants are typically engineered by biollistic transformation, various suitable methods for which are well known in the art. 
     The hAChE-E4 nucleotide sequence includes a total of 574 codons and has an A+T content of 34.8%. Codon use in hAChE-E4 generally is unfavorable for expression in dicots, but acceptable for expression in monocots. In summary, 3.6% of the codons are monocot-unfavorable (including Arg—17.5%, Lys—42.9% and Ser—15.6%), while 12.7% are dicot-unfavorable (including Arg—72.5%, Gly—32.8, Pro—17.6% and Thr—32%), when favorability is defined as making up less than 10% of codon choice for a particular amino acid. Monocot and Dicot preferences were analyzed separately, so as to reveal any potential monocot vs. dicot problems. Tables I and II below summarize total codon use: 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Dicot 
               
             
          
           
               
                 AA 
                 DNA 
                 Unfavorable 
                 Total 
                 % 
               
               
                   
               
             
          
           
               
                 Ala 
                 GCG 
                 5 
                 53 
                 9.5 
               
               
                 Arg 
                 CGA 
                 7 
                 40 
                 72.5 
               
               
                   
                 CGC 
                 8 
               
               
                   
                 CGG 
                 14 
               
               
                 Gly 
                 GGG 
                 19 
                 58 
                 32.8 
               
               
                 Leu 
                 CTA 
                 1 
                 67 
                 1.5 
               
               
                 Pro 
                 CGG 
                 9 
                 51 
                 17.6 
               
               
                 Ser 
                 TCG 
                 2 
                 32 
                 6.3 
               
               
                 Thr 
                 ACG 
                 8 
                 25 
                 32 
               
               
                 Other 
                   
                   
               
               
                 Total 
                   
                 73 
                 574 
                 12.7 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 Monocot 
               
             
          
           
               
                 AA 
                 DNA 
                 Unfavorable 
                 Total 
                 % 
               
               
                   
               
             
          
           
               
                 Arg 
                 CGA 
                 7 
                 40 
                 17.5 
               
               
                 Ile 
                 ATA 
                 0 
                 9 
                 0 
               
               
                 Leu 
                 CTA 
                 1 
                 67 
                 1.5 
               
               
                   
                 TTA 
                 0 
               
               
                 Lys 
                 AAA 
                 3 
                 7 
                 42.9 
               
               
                 Ser 
                 AGT 
                 5 
                 32 
                 15.6 
               
               
                 Val 
                 GTA 
                 5 
                 53 
                 9.4 
               
               
                 STOP 
                 TAA 
                 0 
                 0 
                 0 
               
               
                 Other 
                   
                   
               
               
                 Total 
                   
                 21 
                 574 
                 3.6 
               
               
                   
               
             
          
         
       
     
     Based on the data in the foregoing tables, it is evident that some optimization of the native hAChE-E4 nucleotide sequence is desirable, particularly if the gene is to be expressed in dicots. Thus, we present herein an example of a synthetic DNA sequence encoding human acetylcholinesterase that is optimized for expression in plants, referred to herein as SEQ ID NO:5. 
     Purification of Plant-Produced Cholinesterases 
     While for some of the potential applications of cholinesterases, no purification would be necessary (e.g., in-vivo bioremediation), and for other applications only partially purified preparations of the enzymes would be necessary (e.g., certain industrial uses, oral administration, topical applications in creams, etc.), for other applications relatively pure enzymes are preferable, and may be required. This is especially true for treating individuals by intra-venous or intra-muscular injections of cholinesterases. 
     Several published procedures for the purification of acetylcholinesterase are known in the art. See, for example, Fischer et al.,  Biotechnol. Appl. Biochem.  21: 295-311 (1995) and Heim et al.,  Biochim. Biophys. Acta  1396: 306-319 (1998), the complete disclosures of which are hereby incorporated herein by reference. A large scale purification protocol for butyrylcholinesterase based on ammonium sulfate fractionation followed by an batch affinity chromatography should be applicable also for acetylcholinesterase with minor modifications. See, for example, Grunwald et al.,  J. Biochem. Biophys. Methods  34: 123-135 (1997), the complete disclosure of which is hereby incorporated herein by reference. 
     Additional purification schemes, which are well known in the art, involve engineering a tag to the recombinant enzyme by creating translational fusions. Commercially available plasmids directing such fusions exist mainly for bacterial expression, but can easily be adapted to expression in plants. For example, well known tags that can be used include histidine tags (whereby purification is typically conducted by a nickel-based affinity chromatography); intein-chitin binding tags (whereby purification is conducted by chitin based affinity chromatography and cleavage by a reducing agent, such as dithiothreitol or beta-mercaptoethanol); cellulose binding domains (whereby purification involves affinity chromatography with cellulose). The latter is likely the most useful approach, as it can be done without the addition of any exogenous affinity matrix, since the cholinesterase-CBD fusion binds to cell walls of the plant extract. Release is then mediated by either addition of cellobiose or brief acidification. Cleavage is also possible. For some applications, the cellulose immobilized enzyme-CBD will be extremely useful as a catalytic platform for filters, cellulose based cleaning aids etc. 
     Further Examples of Applications of the Invention 
     Administration of exogenous cholinesterases is an efficacious and safe treatment for the prevention of anti-AChE toxicity. In fact, a single pre-treatment injection of either AChE or BuChE may be sufficient for full protection without any post-exposure treatment. However, to ensure maximum protection against a high dosage (equal to several LD50) of OPs, large amounts of the enzymes are required to satisfy the 1:1 stoichiometry required between the enzyme and the inhibitors in the blood. The enzymes can be purified from human or animal blood, or alternatively, they can be expressed in a variety of cell cultures. However, these systems inherently suffer from high costs and risks of contamination with human pathogens. Recombinant cholinesterases of various sources have been expressed in  Escherichia coli , however, the enzymes thus produced must be denatured and refolded to obtain even partial activity. In addition, they are very labile as compared to the native enzymes. Production by fermentation of yeast cell cultures is also possible, but costs are high and scaling up is expensive. 
     Therefore, we introduced transgenic plants as a novel production system for human acetylcholinesterase, a key component of cholinergic synapses. Some of the transgenic tomato lines obtained express high levels of AChE activity, with accumulation levels (on a fresh weight basis) as reported for the yeast-derived enzyme (FIGS.  1  and  2 ). This activity represents authentic human acetylcholinesterase activity, as judged by its enzymatic properties (FIGS.  3  and  4 ). The plant-derived enzyme is also very stable in the crude plant extract (FIGS.  5 A and  5 B). Expression levels of the gene product can be increased further by optimizing the coding sequence of the human gene for expression in plants, according to methods that are well known in the art, and by regulating and restricting expression of the gene product to certain tissues. 
     In humans, AChE is encoded by a single gene which yields, through alternative splicing of its pre-mRNA, three polypeptide isoforms with distinct C-termini. We expressed the engineered AChE-E4 form, encoded by exons 2-4 of the human gene. However, one of ordinary skill in the art will appreciate that the other isoforms can be used as well. AChE-E4 consists of the globular N-terminal domain shared among the three physiological variants of AChE. Expressed by itself, the soluble AChE-E4 polypeptide is a fully competent acetylcholine hydrolase with kinetic properties which are similar to those of the natural forms. This recombinant AChE-E4 variant is especially suited for application as a protective decoy for the neutralization of AChE inhibitors, because its kinetic properties are practically identical to those of synaptic AChE (unlike BuChE). Because it is soluble, it may be cleared more slowly from circulation (unlike the membrane bound AChE forms, which are cleared 50 times faster than soluble BuChE). Because it has the same amino acid sequence as the human enzyme, the plant-derived recombinant hAChE-E4 isoform is expected to be less immunogenic than the heterologous cholinesterases used in previous studies. There are three potential glycosylation sites in human AChE, and glycosylation, which does not affect the enzymatic properties of the enzyme, is important for both the stability of AChE and its pharmacokinetics and its immunogenic properties. As eukaryotes, plants offer the advantage of all forms of post-translational modification, including glycosylation, which, however, differs in details from that in mammals. 
     Use of the inexpensively produced enzyme is not limited to application by injection, as efficacy of other routes of entry into the body (e.g., orally, inhalation) is expected as well. Lastly, cholinesterases can be incorporated into cleansing preparations, protective skin-creams, filtration devices, and biosensors. For these purposes, the plant-derived enzyme is especially useful, due to lower costs of partial purification and its higher stability. 
     The extensive use of anticholinesterase pesticides and the concurrent accidental poisoning, the unfortunate threat of OP chemical warfare agents by terrorists and rogue governments, as well as environmental concerns, are the driving force for the development of effective, inexpensive and safe countermeasures and bioremediation solutions. Plant-derived recombinant human AChE is an important step in this direction. 
     Accordingly, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention. 
     
       
         
               
             
           
               
                   
               
             
             
               
                                    
               
               
                 #             SEQUENCE LISTING  
               
               
                   
               
               
                   
               
               
                 &lt;160&gt; NUMBER OF SEQ ID NOS: 5  
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 1  
               
               
                 &lt;211&gt; LENGTH: 29  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial Sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Description of Artificial  
               
               
                 #Sequence: primer  
               
               
                       pAChE-Nco, derived from human AChE  
               
               
                 #gene and  
               
               
                       modified to introduce an Nco I  
               
               
                 #restriction site.  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 1  
               
               
                   
               
               
                 gatatctgca gccatggcta ggcccccgc          
               
               
                 #                   
               
               
                 #            29  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 2  
               
               
                 &lt;211&gt; LENGTH: 31  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial Sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Description of Artificial  
               
               
                 #Sequence: primer  
               
               
                       pAChE-Kpn, derived from human AChE  
               
               
                 #gene and  
               
               
                       modified to introduce a Kpn I  
               
               
                 #restriction site.  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 2  
               
               
                   
               
               
                 cggtacctat caggtagcgc tgagcaattt g         
               
               
                 #                   
               
               
                 #          31  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 3  
               
               
                 &lt;211&gt; LENGTH: 5767  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial Sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Description of Artificial  
               
               
                 #Sequence: plasmid  
               
               
                       vector pTM034.  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 3  
               
               
                   
               
               
                 agcttgcatg cctgcaggtc aacatggtgg agcacgacac tctcgtctac tc  
               
               
                 #caagaata     60  
               
               
                   
               
               
                 tcaaagatac agtctcagaa gaccagaggg ctattgagac ttttcaacaa ag  
               
               
                 #ggtaatat    120  
               
               
                   
               
               
                 cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa ag  
               
               
                 #gacagtag    180  
               
               
                   
               
               
                 aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct at  
               
               
                 #cgttcaag    240  
               
               
                   
               
               
                 aatgcctcta ccgacagtgg tcccaaagat ggacccccac ccacgaggaa ca  
               
               
                 #tcgtggaa    300  
               
               
                   
               
               
                 aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgataa ct  
               
               
                 #tttcaaca    360  
               
               
                   
               
               
                 aagggtaata tcgggaaacc tcctcggatt ccattgccca gctatctgtc ac  
               
               
                 #ttcatcga    420  
               
               
                   
               
               
                 aaggacagta gaaaaggaag atggcttcta caaatgccat cattgcgata aa  
               
               
                 #ggaaaggc    480  
               
               
                   
               
               
                 tatcgttcaa gaatgcctct accgacagtg gtcccaaaga tggaccccca cc  
               
               
                 #cacgagga    540  
               
               
                   
               
               
                 acatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tg  
               
               
                 #atgtgata    600  
               
               
                   
               
               
                 tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac cc  
               
               
                 #ttcctcta    660  
               
               
                   
               
               
                 tataaggaag ttcatttcat ttggagagga cctcgagaat taattctcaa ca  
               
               
                 #caacatat    720  
               
               
                   
               
               
                 acaaaacaaa cgaatctcaa gcaatcaagc attctacttc tattgcagca at  
               
               
                 #ttaaatca    780  
               
               
                   
               
               
                 tttcttttaa agcaaaagca attttctgaa aattttcacc atttacgaac ga  
               
               
                 #tagccatg    840  
               
               
                   
               
               
                 gctcccccgc agtgtctgct gcacacgcct tccctggctt ccccactcct tc  
               
               
                 #tcctcctc    900  
               
               
                   
               
               
                 ctctggctcc tgggtggagg agtgggggct gagggccggg aggatgcaga gc  
               
               
                 #tgctggtg    960  
               
               
                   
               
               
                 acggtgcgtg ggggccggct gcggggcatt cgcctgaaga cccccggggg cc  
               
               
                 #ctgtctct   1020  
               
               
                   
               
               
                 gctttcctgg gcatcccctt tgcggagcca cccatgggac cccgtcgctt tc  
               
               
                 #tgccaccg   1080  
               
               
                   
               
               
                 gagcccaagc agccttggtc aggggtggta gacgctacaa ccttccagag tg  
               
               
                 #tctgctac   1140  
               
               
                   
               
               
                 caatatgtgg acaccctata cccaggtttt gagggcaccg agatgtggaa cc  
               
               
                 #ccaaccgt   1200  
               
               
                   
               
               
                 gagctgagcg aggactgcct gtacctcaac gtgtggacac catacccccg gc  
               
               
                 #ctacatcc   1260  
               
               
                   
               
               
                 cccacccctg tcctcgtctg gatctatggg ggtggcttct acagtggggc ct  
               
               
                 #cctccttg   1320  
               
               
                   
               
               
                 gacgtgtacg atggccgctt cttggtacag gccgagagga ctgtgctggt gt  
               
               
                 #ccatgaac   1380  
               
               
                   
               
               
                 taccgggtgg gagcctttgg cttcctggcc ctgccgggga gccgagaggc cc  
               
               
                 #cgggcaat   1440  
               
               
                   
               
               
                 gtgggtctcc tggatcagag gctggccctg cagtgggtgc aggagaacgt gg  
               
               
                 #cagccttc   1500  
               
               
                   
               
               
                 gggggtgacc cgacatcagt gacgctgttt ggggagagcg cgggagccgc ct  
               
               
                 #cggtgggc   1560  
               
               
                   
               
               
                 atgcacctgc tgtccccgcc cagccggggc ctgttccaca gggccgtgct gc  
               
               
                 #agagcggt   1620  
               
               
                   
               
               
                 gcccccaatg gaccctgggc cacggtgggc atgggagagg cccgtcgcag gg  
               
               
                 #ccacgcag   1680  
               
               
                   
               
               
                 ctggcccacc ttgtgggctg tcctccaggc ggcactggtg ggaatgacac ag  
               
               
                 #agctggta   1740  
               
               
                   
               
               
                 gcctgccttc ggacacgacc agcgcaggtc ctggtgaacc acgaatggca cg  
               
               
                 #tgctgcct   1800  
               
               
                   
               
               
                 caagaaagcg tcttccggtt ctccttcgtg cctgtggtag atggagactt cc  
               
               
                 #tcagtgac   1860  
               
               
                   
               
               
                 accccagagg ccctcatcaa cgcgggagac ttccacggcc tgcaggtgct gg  
               
               
                 #tgggtgtg   1920  
               
               
                   
               
               
                 gtgaaggatg agggctcgta ttttctggtt tacggggccc caggcttcag ca  
               
               
                 #aagacaac   1980  
               
               
                   
               
               
                 gagtctctca tcagccgggc cgagttcctg gccggggtgc gggtcggggt tc  
               
               
                 #cccaggta   2040  
               
               
                   
               
               
                 agtgacctgg cagccgaggc tgtggtcctg cattacacag actggctgca tc  
               
               
                 #ccgaggac   2100  
               
               
                   
               
               
                 ccggcacgcc tgagggaggc cctgagcgat gtggtgggcg accacaatgt cg  
               
               
                 #tgtgcccc   2160  
               
               
                   
               
               
                 gtggcccagc tggctgggcg actggctgcc cagggtgccc gggtctacgc ct  
               
               
                 #acgtcttt   2220  
               
               
                   
               
               
                 gaacaccgtg cttccacgct ctcctggccc ctgtggatgg gggtgcccca cg  
               
               
                 #gctacgag   2280  
               
               
                   
               
               
                 atcgagttca tctttgggat ccccctggac ccctctcgaa actacacggc ag  
               
               
                 #aggagaaa   2340  
               
               
                   
               
               
                 atcttcgccc agcgactgat gcgatactgg gccaactttg cccgcacagg gg  
               
               
                 #atcccaat   2400  
               
               
                   
               
               
                 gagccccgag accccaaggc cccacaatgg cccccgtaca cggcgggggc tc  
               
               
                 #agcagtac   2460  
               
               
                   
               
               
                 gttagtctgg acctgcggcc gctggaggtg cggcgggggc tgcgcgccca gg  
               
               
                 #cctgcgcc   2520  
               
               
                   
               
               
                 ttctggaacc gcttcctccc caaattgctc agcgctacct gataggtacc ga  
               
               
                 #gctctctc   2580  
               
               
                   
               
               
                 aacaatctag ctagagtttg ctcctatcta tatgtaataa ggtatgctga ta  
               
               
                 #tgcactat   2640  
               
               
                   
               
               
                 tcaaatagga gcattagcta tgtttgttaa tgtcacttta tgttatgtgg gt  
               
               
                 #aagtcacc   2700  
               
               
                   
               
               
                 taagacactc cacgtaccta cgttgttgtc tcttaccggc tttaataaat ct  
               
               
                 #tctgccct   2760  
               
               
                   
               
               
                 tgttccatat ttactaatta tccctttctt cactaaaaga aaattgttat ca  
               
               
                 #ttaagtat   2820  
               
               
                   
               
               
                 tagtctttag aacatatgag gtctttaatt gggtaggttt tacaaattaa ct  
               
               
                 #aatataaa   2880  
               
               
                   
               
               
                 atgtcataaa atccacgtgg ttaaacaaat gcagaaaatc gacgtcgtct at  
               
               
                 #tggaccga   2940  
               
               
                   
               
               
                 cagttgctat taatataatg ggccaccata gtagactgac aaataaatta cc  
               
               
                 #tgacaaca   3000  
               
               
                   
               
               
                 tcgtttcact aaataacaaa cacaaaaagg gagtgcattt tccagggcat tt  
               
               
                 #ttgtaata   3060  
               
               
                   
               
               
                 aaaaacagtt aaaagggagt gcaatagaaa tataggggtg tggaaatagt ga  
               
               
                 #tttgagca   3120  
               
               
                   
               
               
                 cgtcttgaag cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aa  
               
               
                 #accctggc   3180  
               
               
                   
               
               
                 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg ta  
               
               
                 #atagcgaa   3240  
               
               
                   
               
               
                 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga at  
               
               
                 #ggcgcctg   3300  
               
               
                   
               
               
                 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gt  
               
               
                 #gcactctc   3360  
               
               
                   
               
               
                 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aa  
               
               
                 #cacccgct   3420  
               
               
                   
               
               
                 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tg  
               
               
                 #tgaccgtc   3480  
               
               
                   
               
               
                 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc ga  
               
               
                 #gacgaaag   3540  
               
               
                   
               
               
                 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt tt  
               
               
                 #cttagacg   3600  
               
               
                   
               
               
                 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tt  
               
               
                 #tctaaata   3660  
               
               
                   
               
               
                 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca at  
               
               
                 #aatattga   3720  
               
               
                   
               
               
                 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt tt  
               
               
                 #ttgcggca   3780  
               
               
                   
               
               
                 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tg  
               
               
                 #ctgaagat   3840  
               
               
                   
               
               
                 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa ga  
               
               
                 #tccttgag   3900  
               
               
                   
               
               
                 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gc  
               
               
                 #tatgtggc   3960  
               
               
                   
               
               
                 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat ac  
               
               
                 #actattct   4020  
               
               
                   
               
               
                 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tg  
               
               
                 #gcatgaca   4080  
               
               
                   
               
               
                 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc ca  
               
               
                 #acttactt   4140  
               
               
                   
               
               
                 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gg  
               
               
                 #gggatcat   4200  
               
               
                   
               
               
                 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cg  
               
               
                 #acgagcgt   4260  
               
               
                   
               
               
                 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tg  
               
               
                 #gcgaacta   4320  
               
               
                   
               
               
                 cttactctag cttcccggca acaattaata gactggatgg aggcggataa ag  
               
               
                 #ttgcagga   4380  
               
               
                   
               
               
                 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tg  
               
               
                 #gagccggt   4440  
               
               
                   
               
               
                 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ct  
               
               
                 #cccgtatc   4500  
               
               
                   
               
               
                 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag ac  
               
               
                 #agatcgct   4560  
               
               
                   
               
               
                 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ct  
               
               
                 #catatata   4620  
               
               
                   
               
               
                 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa ga  
               
               
                 #tccttttt   4680  
               
               
                   
               
               
                 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gt  
               
               
                 #cagacccc   4740  
               
               
                   
               
               
                 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ct  
               
               
                 #gctgcttg   4800  
               
               
                   
               
               
                 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gc  
               
               
                 #taccaact   4860  
               
               
                   
               
               
                 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt cc  
               
               
                 #ttctagtg   4920  
               
               
                   
               
               
                 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cc  
               
               
                 #tcgctctg   4980  
               
               
                   
               
               
                 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cg  
               
               
                 #ggttggac   5040  
               
               
                   
               
               
                 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg tt  
               
               
                 #cgtgcaca   5100  
               
               
                   
               
               
                 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tg  
               
               
                 #agctatga   5160  
               
               
                   
               
               
                 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cg  
               
               
                 #gcagggtc   5220  
               
               
                   
               
               
                 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct tt  
               
               
                 #atagtcct   5280  
               
               
                   
               
               
                 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc ag  
               
               
                 #gggggcgg   5340  
               
               
                   
               
               
                 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt tt  
               
               
                 #gctggcct   5400  
               
               
                   
               
               
                 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg ta  
               
               
                 #ttaccgcc   5460  
               
               
                   
               
               
                 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gt  
               
               
                 #cagtgagc   5520  
               
               
                   
               
               
                 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gc  
               
               
                 #cgattcat   5580  
               
               
                   
               
               
                 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg ca  
               
               
                 #acgcaatt   5640  
               
               
                   
               
               
                 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tc  
               
               
                 #cggctcgt   5700  
               
               
                   
               
               
                 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tg  
               
               
                 #accatgat   5760  
               
               
                   
               
               
                 tacgcca                  
               
               
                 #                   
               
               
                 #                   
               
               
                 #        5767  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 4  
               
               
                 &lt;211&gt; LENGTH: 14446  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial Sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;221&gt; NAME/KEY: misc_feature  
               
               
                 &lt;222&gt; LOCATION: (11862)..(12157)  
               
               
                 &lt;223&gt; OTHER INFORMATION: Description of Artificial  
               
               
                 #Sequence: plasmid  
               
               
                       vector pTM036. Identity of sequence  
               
               
                 #residues 11862-12157 unknown.  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 4  
               
               
                   
               
               
                 gaattaattc tcaacacaac atatacaaaa caaacgaatc tcaagcaatc aa  
               
               
                 #gcattcta     60  
               
               
                   
               
               
                 cttctattgc agcaatttaa atcatttctt ttaaagcaaa agcaattttc tg  
               
               
                 #aaaatttt    120  
               
               
                   
               
               
                 caccatttac gaacgatagc catggctccc ccgcagtgtc tgctgcacac gc  
               
               
                 #cttccctg    180  
               
               
                   
               
               
                 gcttccccac tccttctcct cctcctctgg ctcctgggtg gaggagtggg gg  
               
               
                 #ctgagggc    240  
               
               
                   
               
               
                 cgggaggatg cagagctgct ggtgacggtg cgtgggggcc ggctgcgggg ca  
               
               
                 #ttcgcctg    300  
               
               
                   
               
               
                 aagacccccg ggggccctgt ctctgctttc ctgggcatcc cctttgcgga gc  
               
               
                 #cacccatg    360  
               
               
                   
               
               
                 ggaccccgtc gctttctgcc accggagccc aagcagcctt ggtcaggggt gg  
               
               
                 #tagacgct    420  
               
               
                   
               
               
                 acaaccttcc agagtgtctg ctaccaatat gtggacaccc tatacccagg tt  
               
               
                 #ttgagggc    480  
               
               
                   
               
               
                 accgagatgt ggaaccccaa ccgtgagctg agcgaggact gcctgtacct ca  
               
               
                 #acgtgtgg    540  
               
               
                   
               
               
                 acaccatacc cccggcctac atcccccacc cctgtcctcg tctggatcta tg  
               
               
                 #ggggtggc    600  
               
               
                   
               
               
                 ttctacagtg gggcctcctc cttggacgtg tacgatggcc gcttcttggt ac  
               
               
                 #aggccgag    660  
               
               
                   
               
               
                 aggactgtgc tggtgtccat gaactaccgg gtgggagcct ttggcttcct gg  
               
               
                 #ccctgccg    720  
               
               
                   
               
               
                 gggagccgag aggccccggg caatgtgggt ctcctggatc agaggctggc cc  
               
               
                 #tgcagtgg    780  
               
               
                   
               
               
                 gtgcaggaga acgtggcagc cttcgggggt gacccgacat cagtgacgct gt  
               
               
                 #ttggggag    840  
               
               
                   
               
               
                 agcgcgggag ccgcctcggt gggcatgcac ctgctgtccc cgcccagccg gg  
               
               
                 #gcctgttc    900  
               
               
                   
               
               
                 cacagggccg tgctgcagag cggtgccccc aatggaccct gggccacggt gg  
               
               
                 #gcatggga    960  
               
               
                   
               
               
                 gaggcccgtc gcagggccac gcagctggcc caccttgtgg gctgtcctcc ag  
               
               
                 #gcggcact   1020  
               
               
                   
               
               
                 ggtgggaatg acacagagct ggtagcctgc cttcggacac gaccagcgca gg  
               
               
                 #tcctggtg   1080  
               
               
                   
               
               
                 aaccacgaat ggcacgtgct gcctcaagaa agcgtcttcc ggttctcctt cg  
               
               
                 #tgcctgtg   1140  
               
               
                   
               
               
                 gtagatggag acttcctcag tgacacccca gaggccctca tcaacgcggg ag  
               
               
                 #acttccac   1200  
               
               
                   
               
               
                 ggcctgcagg tgctggtggg tgtggtgaag gatgagggct cgtattttct gg  
               
               
                 #tttacggg   1260  
               
               
                   
               
               
                 gccccaggct tcagcaaaga caacgagtct ctcatcagcc gggccgagtt cc  
               
               
                 #tggccggg   1320  
               
               
                   
               
               
                 gtgcgggtcg gggttcccca ggtaagtgac ctggcagccg aggctgtggt cc  
               
               
                 #tgcattac   1380  
               
               
                   
               
               
                 acagactggc tgcatcccga ggacccggca cgcctgaggg aggccctgag cg  
               
               
                 #atgtggtg   1440  
               
               
                   
               
               
                 ggcgaccaca atgtcgtgtg ccccgtggcc cagctggctg ggcgactggc tg  
               
               
                 #cccagggt   1500  
               
               
                   
               
               
                 gcccgggtct acgcctacgt ctttgaacac cgtgcttcca cgctctcctg gc  
               
               
                 #ccctgtgg   1560  
               
               
                   
               
               
                 atgggggtgc cccacggcta cgagatcgag ttcatctttg ggatccccct gg  
               
               
                 #acccctct   1620  
               
               
                   
               
               
                 cgaaactaca cggcagagga gaaaatcttc gcccagcgac tgatgcgata ct  
               
               
                 #gggccaac   1680  
               
               
                   
               
               
                 tttgcccgca caggggatcc caatgagccc cgagacccca aggccccaca at  
               
               
                 #ggcccccg   1740  
               
               
                   
               
               
                 tacacggcgg gggctcagca gtacgttagt ctggacctgc ggccgctgga gg  
               
               
                 #tgcggcgg   1800  
               
               
                   
               
               
                 gggctgcgcg cccaggcctg cgccttctgg aaccgcttcc tccccaaatt gc  
               
               
                 #tcagcgct   1860  
               
               
                   
               
               
                 acctgatagg taccgagctc tctcaacaat ctagctagag tttgctccta tc  
               
               
                 #tatatgta   1920  
               
               
                   
               
               
                 ataaggtatg ctgatatgca ctattcaaat aggagcatta gctatgtttg tt  
               
               
                 #aatgtcac   1980  
               
               
                   
               
               
                 tttatgttat gtgggtaagt cacctaagac actccacgta cctacgttgt tg  
               
               
                 #tctcttac   2040  
               
               
                   
               
               
                 cggctttaat aaatcttctg cccttgttcc atatttacta attatccctt tc  
               
               
                 #ttcactaa   2100  
               
               
                   
               
               
                 aagaaaattg ttatcattaa gtattagtct ttagaacata tgaggtcttt aa  
               
               
                 #ttgggtag   2160  
               
               
                   
               
               
                 gttttacaaa ttaactaata taaaatgtca taaaatccac gtggttaaac aa  
               
               
                 #atgcagaa   2220  
               
               
                   
               
               
                 aatcgacgtc gtctattgga ccgacagttg ctattaatat aatgggccac ca  
               
               
                 #tagtagac   2280  
               
               
                   
               
               
                 tgacaaataa attacctgac aacatcgttt cactaaataa caaacacaaa aa  
               
               
                 #gggagtgc   2340  
               
               
                   
               
               
                 attttccagg gcatttttgt aataaaaaac agttaaaagg gagtgcaata ga  
               
               
                 #aatatagg   2400  
               
               
                   
               
               
                 ggtgtggaaa tagtgatttg agcacgtctt gaagcgaatt cgagatcggc cg  
               
               
                 #cggctgag   2460  
               
               
                   
               
               
                 tggctccttc aatcgttgcg gttctgtcag ttccaaacgt aaaacggctt gt  
               
               
                 #cccgcgtc   2520  
               
               
                   
               
               
                 atcggcgggg gtcataacgt gactccctta attctccgct catgatcaga tt  
               
               
                 #gtcgtttc   2580  
               
               
                   
               
               
                 ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa ac  
               
               
                 #ctaagaga   2640  
               
               
                   
               
               
                 aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt ta  
               
               
                 #tccgttcg   2700  
               
               
                   
               
               
                 tccatttgta tgtgcatgcc aaccacaggg ttccccagat ctggcgccgg cc  
               
               
                 #agcgagac   2760  
               
               
                   
               
               
                 gagcaagatt ggccgccgcc cgaaacgatc cgacagcgcg cccagcacag gt  
               
               
                 #gcgcaggc   2820  
               
               
                   
               
               
                 aaattgcacc aacgcataca gcgccagcag aatgccatag tgggcggtga cg  
               
               
                 #tcgttcga   2880  
               
               
                   
               
               
                 gtgaaccaga tcgcgcagga ggcccggcag caccggcata atcaggccga tg  
               
               
                 #ccgacagc   2940  
               
               
                   
               
               
                 gtcgagcgcg acagtgctca gaattacgat caggggtatg ttgggtttca cg  
               
               
                 #tctggcct   3000  
               
               
                   
               
               
                 ccggaccagc ctccgctggt ccgattgaac gcgcggattc tttatcactg at  
               
               
                 #aagttggt   3060  
               
               
                   
               
               
                 ggacatatta tgtttatcag tgataaagtg tcaagcatga caaagttgca gc  
               
               
                 #cgaataca   3120  
               
               
                   
               
               
                 gtgatccgtg ccgccctgga cctgttgaac gaggtcggcg tagacggtct ga  
               
               
                 #cgacacgc   3180  
               
               
                   
               
               
                 aaactggcgg aacggttggg ggttcagcag ccggcgcttt actggcactt ca  
               
               
                 #ggaacaag   3240  
               
               
                   
               
               
                 cgggcgctgc tcgacgcact ggccgaagcc atgctggcgg agaatcatac gc  
               
               
                 #attcggtg   3300  
               
               
                   
               
               
                 ccgagagccg acgacgactg gcgctcattt ctgatcggga atgcccgcag ct  
               
               
                 #tcaggcag   3360  
               
               
                   
               
               
                 gcgctgctcg cctaccgcga tggcgcgcgc atccatgccg gcacgcgacc gg  
               
               
                 #gcgcaccg   3420  
               
               
                   
               
               
                 cagatggaaa cggccgacgc gcagcttcgc ttcctctgcg aggcgggttt tt  
               
               
                 #cggccggg   3480  
               
               
                   
               
               
                 gacgccgtca atgcgctgat gacaatcagc tacttcactg ttggggccgt gc  
               
               
                 #ttgaggag   3540  
               
               
                   
               
               
                 caggccggcg acagcgatgc cggcgagcgc ggcggcaccg ttgaacaggc tc  
               
               
                 #cgctctcg   3600  
               
               
                   
               
               
                 ccgctgttgc gggccgcgat agacgccttc gacgaagccg gtccggacgc ag  
               
               
                 #cgttcgag   3660  
               
               
                   
               
               
                 cagggactcg cggtgattgt cgatggattg gcgaaaagga ggctcgttgt ca  
               
               
                 #ggaacgtt   3720  
               
               
                   
               
               
                 gaaggaccga gaaagggtga cgattgatca ggaccgctgc cggagcgcaa cc  
               
               
                 #cactcact   3780  
               
               
                   
               
               
                 acagcagagc catgtagaca acatcccctc cccctttcca ccgcgtcaga cg  
               
               
                 #cccgtagc   3840  
               
               
                   
               
               
                 agcccgctac gggctttttc atgccctgcc ctagcgtcca agcctcacgg cc  
               
               
                 #gcgctcgg   3900  
               
               
                   
               
               
                 cctctctggc ggccttctgg cgctcttccg cttcctcgct cactgactcg ct  
               
               
                 #gcgctcgg   3960  
               
               
                   
               
               
                 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg tt  
               
               
                 #atccacag   4020  
               
               
                   
               
               
                 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gc  
               
               
                 #caggaacc   4080  
               
               
                   
               
               
                 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac ga  
               
               
                 #gcatcaca   4140  
               
               
                   
               
               
                 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga ta  
               
               
                 #ccaggcgt   4200  
               
               
                   
               
               
                 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt ac  
               
               
                 #cggatacc   4260  
               
               
                   
               
               
                 tgtccgcctt tctcccttcg ggaagcgtgg cgcttttccg ctgcataacc ct  
               
               
                 #gcttcggg   4320  
               
               
                   
               
               
                 gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac ag  
               
               
                 #gattttgc   4380  
               
               
                   
               
               
                 caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg ca  
               
               
                 #ggataggt   4440  
               
               
                   
               
               
                 gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc gc  
               
               
                 #acctggcg   4500  
               
               
                   
               
               
                 gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt aa  
               
               
                 #cagatgag   4560  
               
               
                   
               
               
                 ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc ta  
               
               
                 #tcaaggtg   4620  
               
               
                   
               
               
                 tactgccttc cagacgaacg aagagcgatt gaggaaaagg cggcggcggc cg  
               
               
                 #gcatgagc   4680  
               
               
                   
               
               
                 ctgtcggcct acctgctggc cgtcggccag ggctacaaaa tcacgggcgt cg  
               
               
                 #tggactat   4740  
               
               
                   
               
               
                 gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg gccgcctggg cg  
               
               
                 #gcctgctg   4800  
               
               
                   
               
               
                 aaactctggc tcaccgacga cccgcgcacg gcgcggttcg gtgatgccac ga  
               
               
                 #tcctcgcc   4860  
               
               
                   
               
               
                 ctgctggcga agatcgaaga gaagcaggac gagcttggca aggtcatgat gg  
               
               
                 #gcgtggtc   4920  
               
               
                   
               
               
                 cgcccgaggg cagagccatg acttttttag ccgctaaaac ggccgggggg tg  
               
               
                 #cgcgtgat   4980  
               
               
                   
               
               
                 tgccaagcac gtccccatgc gctccatcaa gaagagcgac ttcgcggagc tg  
               
               
                 #gtgaagta   5040  
               
               
                   
               
               
                 catcaccgac gagcaaggca agaccgagcg cctttgcgac gctcaccggg ct  
               
               
                 #ggttgccc   5100  
               
               
                   
               
               
                 tcgccgctgg gctggcggcc gtctatggcc ctgcaaacgc gccagaaacg cc  
               
               
                 #gtcgaagc   5160  
               
               
                   
               
               
                 cgtgtgcgag acaccgcggc cgccggcgtt gtggatacct cgcggaaaac tt  
               
               
                 #ggccctca   5220  
               
               
                   
               
               
                 ctgacagatg aggggcggac gttgacactt gaggggccga ctcacccggc gc  
               
               
                 #ggcgttga   5280  
               
               
                   
               
               
                 cagatgaggg gcaggctcga tttcggccgg cgacgtggag ctggccagcc tc  
               
               
                 #gcaaatcg   5340  
               
               
                   
               
               
                 gcgaaaacgc ctgattttac gcgagtttcc cacagatgat gtggacaagc ct  
               
               
                 #ggggataa   5400  
               
               
                   
               
               
                 gtgccctgcg gtattgacac ttgaggggcg cgactactga cagatgaggg gc  
               
               
                 #gcgatcct   5460  
               
               
                   
               
               
                 tgacacttga ggggcagagt gctgacagat gaggggcgca cctattgaca tt  
               
               
                 #tgaggggc   5520  
               
               
                   
               
               
                 tgtccacagg cagaaaatcc agcatttgca agggtttccg cccgtttttc gg  
               
               
                 #ccaccgct   5580  
               
               
                   
               
               
                 aacctgtctt ttaacctgct tttaaaccaa tatttataaa ccttgttttt aa  
               
               
                 #ccagggct   5640  
               
               
                   
               
               
                 gcgccctgtg cgcgtgaccg cgcacgccga aggggggtgc ccccccttct cg  
               
               
                 #aaccctcc   5700  
               
               
                   
               
               
                 cggcccgcta acgcgggcct cccatccccc caggggctgc gcccctcggc cg  
               
               
                 #cgaacggc   5760  
               
               
                   
               
               
                 ctcaccccaa aaatggcagc gctggcagtc cttgccattg ccgggatcgg gg  
               
               
                 #cagtaacg   5820  
               
               
                   
               
               
                 ggatgggcga tcagcccgag cgcgacgccc ggaagcattg acgtgccgca gg  
               
               
                 #tgctggca   5880  
               
               
                   
               
               
                 tcgacattca gcgaccaggt gccgggcagt gagggcggcg gcctgggtgg cg  
               
               
                 #gcctgccc   5940  
               
               
                   
               
               
                 ttcacttcgg ccgtcggggc attcacggac ttcatggcgg ggccggcaat tt  
               
               
                 #ttaccttg   6000  
               
               
                   
               
               
                 ggcattcttg gcatagtggt cgcgggtgcc gtgctcgtgt tcgggggtgc ga  
               
               
                 #taaaccca   6060  
               
               
                   
               
               
                 gcgaaccatt tgaggtgata ggtaagatta taccgaggta tgaaaacgag aa  
               
               
                 #ttggacct   6120  
               
               
                   
               
               
                 ttacagaatt actctatgaa gcgccatatt taaaaagcta ccaagacgaa ga  
               
               
                 #ggatgaag   6180  
               
               
                   
               
               
                 aggatgagga ggcagattgc cttgaatata ttgacaatac tgataagata at  
               
               
                 #atatcttt   6240  
               
               
                   
               
               
                 tatatagaag atatcgccgt atgtaaggat ttcagggggc aaggcatagg ca  
               
               
                 #gcgcgctt   6300  
               
               
                   
               
               
                 atcaatatat ctatagaatg ggcaaagcat aaaaacttgc atggactaat gc  
               
               
                 #ttgaaacc   6360  
               
               
                   
               
               
                 caggacaata accttatagc ttgtaaattc tatcataatt gggtaatgac tc  
               
               
                 #caacttat   6420  
               
               
                   
               
               
                 tgatagtgtt ttatgttcag ataatgcccg atgactttgt catgcagctc ca  
               
               
                 #ccgatttt   6480  
               
               
                   
               
               
                 gagaacgaca gcgacttccg tcccagccgt gccaggtgct gcctcagatt ca  
               
               
                 #ggttatgc   6540  
               
               
                   
               
               
                 cgctcaattc gctgcgtata tcgcttgctg attacgtgca gctttccctt ca  
               
               
                 #ggcgggat   6600  
               
               
                   
               
               
                 tcatacagcg gccagccatc cgtcatccat atcaccacgt caaagggtga ca  
               
               
                 #gcaggctc   6660  
               
               
                   
               
               
                 ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata cgtgcgcaac aa  
               
               
                 #ccgtcttc   6720  
               
               
                   
               
               
                 cggagactgt catacgcgta aaacagccag cgctggcgcg atttagcccc ga  
               
               
                 #catagccc   6780  
               
               
                   
               
               
                 cactgttcgt ccatttccgc gcagacgatg acgtcactgc ccggctgtat gc  
               
               
                 #gcgaggtt   6840  
               
               
                   
               
               
                 accgactgcg gcctgagttt tttaagtgac gtaaaatcgt gttgaggcca ac  
               
               
                 #gcccataa   6900  
               
               
                   
               
               
                 tgcgggctgt tgcccggcat ccaacgccat tcatggccat atcaatgatt tt  
               
               
                 #ctggtgcg   6960  
               
               
                   
               
               
                 taccgggttg agaagcggtg taagtgaact gcagttgcca tgttttacgg ca  
               
               
                 #gtgagagc   7020  
               
               
                   
               
               
                 agagatagcg ctgatgtccg gcggtgcttt tgccgttacg caccaccccg tc  
               
               
                 #agtagctg   7080  
               
               
                   
               
               
                 aacaggaggg acagctgata gacacagaag ccactggagc acctcaaaaa ca  
               
               
                 #ccatcata   7140  
               
               
                   
               
               
                 cactaaatca gtaagttggc agcatcaccc ataattgtgg tttcaaaatc gg  
               
               
                 #ctccgtcg   7200  
               
               
                   
               
               
                 atactatgtt atacgccaac tttgaaaaca actttgaaaa agctgttttc tg  
               
               
                 #gtatttaa   7260  
               
               
                   
               
               
                 ggttttagaa tgcaaggaac agtgaattgg agttcgtctt gttataatta gc  
               
               
                 #ttcttggg   7320  
               
               
                   
               
               
                 gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa tg  
               
               
                 #agaatatc   7380  
               
               
                   
               
               
                 accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg aa  
               
               
                 #ggaatgtc   7440  
               
               
                   
               
               
                 tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa aa  
               
               
                 #atgacgga   7500  
               
               
                   
               
               
                 cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga tg  
               
               
                 #ctatggct   7560  
               
               
                   
               
               
                 ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa cggcatgatg gc  
               
               
                 #tggagcaa   7620  
               
               
                   
               
               
                 tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag at  
               
               
                 #gaacaaag   7680  
               
               
                   
               
               
                 ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact cc  
               
               
                 #atcgacat   7740  
               
               
                   
               
               
                 atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg at  
               
               
                 #tacttact   7800  
               
               
                   
               
               
                 gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacactc ca  
               
               
                 #tttaaaga   7860  
               
               
                   
               
               
                 tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac tt  
               
               
                 #gtcttttc   7920  
               
               
                   
               
               
                 ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa gt  
               
               
                 #ggctttat   7980  
               
               
                   
               
               
                 tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct gc  
               
               
                 #gtccggtc   8040  
               
               
                   
               
               
                 gatcagggag gatatcgggg aagaacagta tgtcgagcta ttttttgact ta  
               
               
                 #ctggggat   8100  
               
               
                   
               
               
                 caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt tt  
               
               
                 #tagtacct   8160  
               
               
                   
               
               
                 agatgtggcg caacgatgcc ggcgacaagc aggagcgcac cgacttcttc cg  
               
               
                 #catcaagt   8220  
               
               
                   
               
               
                 gttttggctc tcaggccgag gcccacggca agtatttggg caaggggtcg ct  
               
               
                 #ggtattcg   8280  
               
               
                   
               
               
                 tgcagggcaa gattcggaat accaagtacg agaaggacgg ccagacggtc ta  
               
               
                 #cgggaccg   8340  
               
               
                   
               
               
                 acttcattgc cgataaggtg gattatctgg acaccaaggc accaggcggg tc  
               
               
                 #aaatcagg   8400  
               
               
                   
               
               
                 aataagggca cattgccccg gcgtgagtcg gggcaatccc gcaaggaggg tg  
               
               
                 #aatgaatc   8460  
               
               
                   
               
               
                 ggacgtttga ccggaaggca tacaggcaag aactgatcga cgcggggttt tc  
               
               
                 #cgccgagg   8520  
               
               
                   
               
               
                 atgccgaaac catcgcaagc cgcaccgtca tgcgtgcgcc ccgcgaaacc tt  
               
               
                 #ccagtccg   8580  
               
               
                   
               
               
                 tcggctcgat ggtccagcaa gctacggcca agatcgagcg cgacagcgtg ca  
               
               
                 #actggctc   8640  
               
               
                   
               
               
                 cccctgccct gcccgcgcca tcggccgccg tggagcgttc gcgtcgtctc ga  
               
               
                 #acaggagg   8700  
               
               
                   
               
               
                 cggcaggttt ggcgaagtcg atgaccatcg acacgcgagg aactatgacg ac  
               
               
                 #caagaagc   8760  
               
               
                   
               
               
                 gaaaaaccgc cggcgaggac ctggcaaaac aggtcagcga ggccaagcag gc  
               
               
                 #cgcgttgc   8820  
               
               
                   
               
               
                 tgaaacacac gaagcagcag atcaaggaaa tgcagctttc cttgttcgat at  
               
               
                 #tgcgccgt   8880  
               
               
                   
               
               
                 ggccggacac gatgcgagcg atgccaaacg acacggcccg ctctgccctg tt  
               
               
                 #caccacgc   8940  
               
               
                   
               
               
                 gcaacaagaa aatcccgcgc gaggcgctgc aaaacaaggt cattttccac gt  
               
               
                 #caacaagg   9000  
               
               
                   
               
               
                 acgtgaagat cacctacacc ggcgtcgagc tgcgggccga cgatgacgaa ct  
               
               
                 #ggtgtggc   9060  
               
               
                   
               
               
                 agcaggtgtt ggagtacgcg aagcgcaccc ctatcggcga gccgatcacc tt  
               
               
                 #cacgttct   9120  
               
               
                   
               
               
                 acgagctttg ccaggacctg ggctggtcga tcaatggccg gtattacacg aa  
               
               
                 #ggccgagg   9180  
               
               
                   
               
               
                 aatgcctgtc gcgcctacag gcgacggcga tgggcttcac gtccgaccgc gt  
               
               
                 #tgggcacc   9240  
               
               
                   
               
               
                 tggaatcggt gtcgctgctg caccgcttcc gcgtcctgga ccgtggcaag aa  
               
               
                 #aacgtccc   9300  
               
               
                   
               
               
                 gttgccaggt cctgatcgac gaggaaatcg tcgtgctgtt tgctggcgac ca  
               
               
                 #ctacacga   9360  
               
               
                   
               
               
                 aattcatatg ggagaagtac cgcaagctgt cgccgacggc ccgacggatg tt  
               
               
                 #cgactatt   9420  
               
               
                   
               
               
                 tcagctcgca ccgggagccg tacccgctca agctggaaac cttccgcctc at  
               
               
                 #gtgcggat   9480  
               
               
                   
               
               
                 cggattccac ccgcgtgaag aagtggcgcg agcaggtcgg cgaagcctgc ga  
               
               
                 #agagttgc   9540  
               
               
                   
               
               
                 gaggcagcgg cctggtggaa cacgcctggg tcaatgatga cctggtgcat tg  
               
               
                 #caaacgct   9600  
               
               
                   
               
               
                 agggccttgt ggggtcagtt ccggctgggg gttcagcagc cagcgcttta ct  
               
               
                 #ggcatttc   9660  
               
               
                   
               
               
                 aggaacaagc gggcactgct cgacgcactt gcttcgctca gtatcgctcg gg  
               
               
                 #acgcacgg   9720  
               
               
                   
               
               
                 cgcgctctac gaactgccga taaacagagg attaaaattg acaattgtga tt  
               
               
                 #aaggctca   9780  
               
               
                   
               
               
                 gattcgacgg cttggagcgg ccgacgtgca ggatttccgc gagatccgat tg  
               
               
                 #tcggccct   9840  
               
               
                   
               
               
                 gaagaaagct ccagagatgt tcgggtccgt ttacgagcac gaggagaaaa ag  
               
               
                 #cccatgga   9900  
               
               
                   
               
               
                 ggcgttcgct gaacggttgc gagatgccgt ggcattcggc gcctacatcg ac  
               
               
                 #ggcgagat   9960  
               
               
                   
               
               
                 cattgggctg tcggtcttca aacaggagga cggccccaag gacgctcaca ag  
               
               
                 #gcgcatct  10020  
               
               
                   
               
               
                 gtccggcgtt ttcgtggagc ccgaacagcg aggccgaggg gtcgccggta tg  
               
               
                 #ctgctgcg  10080  
               
               
                   
               
               
                 ggcgttgccg gcgggtttat tgctcgtgat gatcgtccga cagattccaa cg  
               
               
                 #ggaatctg  10140  
               
               
                   
               
               
                 gtggatgcgc atcttcatcc tcggcgcact taatatttcg ctattctgga gc  
               
               
                 #ttgttgtt  10200  
               
               
                   
               
               
                 tatttcggtc taccgcctgc cgggcggggt cgcggcgacg gtaggcgctg tg  
               
               
                 #cagccgct  10260  
               
               
                   
               
               
                 gatggtcgtg ttcatctctg ccgctctgct aggtagcccg atacgattga tg  
               
               
                 #gcggtcct  10320  
               
               
                   
               
               
                 gggggctatt tgcggaactg cgggcgtggc gctgttggtg ttgacaccaa ac  
               
               
                 #gcagcgct  10380  
               
               
                   
               
               
                 agatcctgtc ggcgtcgcag cgggcctggc gggggcggtt tccatggcgt tc  
               
               
                 #ggaaccgt  10440  
               
               
                   
               
               
                 gctgacccgc aagtggcaac ctcccgtgcc tctgctcacc tttaccgcct gg  
               
               
                 #caactggc  10500  
               
               
                   
               
               
                 ggccggagga cttctgctcg ttccagtagc tttagtgttt gatccgccaa tc  
               
               
                 #ccgatgcc  10560  
               
               
                   
               
               
                 tacaggaacc aatgttctcg gcctggcgtg gctcggcctg atcggagcgg gt  
               
               
                 #ttaaccta  10620  
               
               
                   
               
               
                 cttcctttgg ttccggggga tctcgcgact cgaacctaca gttgtttcct ta  
               
               
                 #ctgggctt  10680  
               
               
                   
               
               
                 tctcagcccc agatctgggg tcgatcagcc ggggatgcat caggccgaca gt  
               
               
                 #cggaactt  10740  
               
               
                   
               
               
                 cgggtccccg acctgtacca ttcggtgagc aatggatagg ggagttgata tc  
               
               
                 #gtcaacgt  10800  
               
               
                   
               
               
                 tcacttctaa agaaatagcg ccactcagct tcctcagcgg ctttatccag cg  
               
               
                 #atttccta  10860  
               
               
                   
               
               
                 ttatgtcggc atagttctca agatcgacag cctgtcacgg ttaagcgaga aa  
               
               
                 #tgaataag  10920  
               
               
                   
               
               
                 aaggctgata attcggatct ctgcgaggga gatgatattt gatcacaggc ag  
               
               
                 #caacgctc  10980  
               
               
                   
               
               
                 tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aa  
               
               
                 #acccggca  11040  
               
               
                   
               
               
                 gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cg  
               
               
                 #ccttacaa  11100  
               
               
                   
               
               
                 cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt gg  
               
               
                 #tgattttg  11160  
               
               
                   
               
               
                 tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tg  
               
               
                 #tggtgtaa  11220  
               
               
                   
               
               
                 acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg ta  
               
               
                 #ctggggtg  11280  
               
               
                   
               
               
                 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ct  
               
               
                 #ggccctga  11340  
               
               
                   
               
               
                 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ct  
               
               
                 #gtttgatg  11400  
               
               
                   
               
               
                 gtggttccga aatcggcaaa atcccttata aatcaaaaga atagcccgag at  
               
               
                 #agggttga  11460  
               
               
                   
               
               
                 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aa  
               
               
                 #cgtcaaag  11520  
               
               
                   
               
               
                 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aa  
               
               
                 #atcaagtt  11580  
               
               
                   
               
               
                 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc cc  
               
               
                 #ccgattta  11640  
               
               
                   
               
               
                 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gc  
               
               
                 #gaaaggag  11700  
               
               
                   
               
               
                 cgggcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ct  
               
               
                 #cttcgcta  11760  
               
               
                   
               
               
                 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aa  
               
               
                 #cgccaggg  11820  
               
               
                   
               
               
                 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tnnnnnnnnn nn  
               
               
                 #nnnnnnnn  11880  
               
               
                   
               
               
                 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn  
               
               
                 #nnnnnnnn  11940  
               
               
                   
               
               
                 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn  
               
               
                 #nnnnnnnn  12000  
               
               
                   
               
               
                 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn  
               
               
                 #nnnnnnnn  12060  
               
               
                   
               
               
                 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn  
               
               
                 #nnnnnnnn  12120  
               
               
                   
               
               
                 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngga tccagatccc gt  
               
               
                 #gggcgaag  12180  
               
               
                   
               
               
                 aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg ga  
               
               
                 #aaacgatt  12240  
               
               
                   
               
               
                 ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga tg  
               
               
                 #gcaggttg  12300  
               
               
                   
               
               
                 ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tc  
               
               
                 #gtcaagaa  12360  
               
               
                   
               
               
                 ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc ac  
               
               
                 #gaggaagc  12420  
               
               
                   
               
               
                 ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gc  
               
               
                 #tatgtcct  12480  
               
               
                   
               
               
                 gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cg  
               
               
                 #gccatttt  12540  
               
               
                   
               
               
                 ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatca tc  
               
               
                 #gccgtcgg  12600  
               
               
                   
               
               
                 gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tg  
               
               
                 #ctcttcgt  12660  
               
               
                   
               
               
                 ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tc  
               
               
                 #gatgcgat  12720  
               
               
                   
               
               
                 gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cg  
               
               
                 #ccgcattg  12780  
               
               
                   
               
               
                 catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg ag  
               
               
                 #atcctgcc  12840  
               
               
                   
               
               
                 ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tc  
               
               
                 #gagcacag  12900  
               
               
                   
               
               
                 ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tc  
               
               
                 #ctgcagtt  12960  
               
               
                   
               
               
                 cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tg  
               
               
                 #cgctgaca  13020  
               
               
                   
               
               
                 gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca ta  
               
               
                 #gccgaata  13080  
               
               
                   
               
               
                 gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca at  
               
               
                 #catgcgaa  13140  
               
               
                   
               
               
                 aagatctgga ttgagagtga atatgagact ctaattggat accgagggga at  
               
               
                 #ttatggaa  13200  
               
               
                   
               
               
                 cgtcagtgga gcatttttga caagaaatat ttgctagctg atagtgacct ta  
               
               
                 #ggcgactt  13260  
               
               
                   
               
               
                 ttgaacgcgc aataatggtt tctgacgtat gtgcttagct cattaaactc ca  
               
               
                 #gaaacccg  13320  
               
               
                   
               
               
                 cggctgagtg gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa ac  
               
               
                 #ggcttgtc  13380  
               
               
                   
               
               
                 ccgcgtcatc ggcgggggtc ataacgtgac tcccttaatt ctccgctcat ga  
               
               
                 #tcttgatc  13440  
               
               
                   
               
               
                 ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg ca  
               
               
                 #gggcttcc  13500  
               
               
                   
               
               
                 caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc ca  
               
               
                 #taaaaccg  13560  
               
               
                   
               
               
                 cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tt  
               
               
                 #tgcgcttg  13620  
               
               
                   
               
               
                 cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag ca  
               
               
                 #ccgtttct  13680  
               
               
                   
               
               
                 gcggactggc tttctacgtg ttccgcttcc tttagcagcc cttgcgccct ga  
               
               
                 #gtgcttgc  13740  
               
               
                   
               
               
                 ggcagcgtga agcttgcatg cctgcaggtc aacatggtgg agcacgacac tc  
               
               
                 #tcgtctac  13800  
               
               
                   
               
               
                 tccaagaata tcaaagatac agtctcagaa gaccagaggg ctattgagac tt  
               
               
                 #ttcaacaa  13860  
               
               
                   
               
               
                 agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca ct  
               
               
                 #tcatcgaa  13920  
               
               
                   
               
               
                 aggacagtag aaaaggaaga tggcttctac aaatgccatc attgcgataa ag  
               
               
                 #gaaaggct  13980  
               
               
                   
               
               
                 atcgttcaag aatgcctcta ccgacagtgg tcccaaagat ggacccccac cc  
               
               
                 #acgaggaa  14040  
               
               
                   
               
               
                 catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt ga  
               
               
                 #tgtgataa  14100  
               
               
                   
               
               
                 cttttcaaca aagggtaata tcgggaaacc tcctcggatt ccattgccca gc  
               
               
                 #tatctgtc  14160  
               
               
                   
               
               
                 acttcatcga aaggacagta gaaaaggaag atggcttcta caaatgccat ca  
               
               
                 #ttgcgata  14220  
               
               
                   
               
               
                 aaggaaaggc tatcgttcaa gaatgcctct accgacagtg gtcccaaaga tg  
               
               
                 #gaccccca  14280  
               
               
                   
               
               
                 cccacgagga acatcgtgga aaaagaagac gttccaacca cgtcttcaaa gc  
               
               
                 #aagtggat  14340  
               
               
                   
               
               
                 tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc tt  
               
               
                 #cgcaagac  14400  
               
               
                   
               
               
                 ccttcctcta tataaggaag ttcatttcat ttggagagga cctcga    
               
               
                 #              14446  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 5  
               
               
                 &lt;211&gt; LENGTH: 1725  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial Sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Description of Artificial  
               
               
                 #Sequence: synthetic  
               
               
                       human acetylcholinesterase gene optimiz  
               
               
                 #ed for  
               
               
                       expression in plants  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 5  
               
               
                   
               
               
                 atgaggcccc cgcagtgtct gctgcacacg ccttccctgg cttccccact cc  
               
               
                 #ttctcctc     60  
               
               
                   
               
               
                 ctcctctggc tcctgggtgg aggagtgggg gctgagggcc gggaggatgc ag  
               
               
                 #agctgctg    120  
               
               
                   
               
               
                 gtgacggtgc gtgggggccg gctgcggggc attcgcctga agacccccgg gg  
               
               
                 #gccctgtc    180  
               
               
                   
               
               
                 tctgctttcc tgggcatccc ctttgcggag ccacccatgg gaccccgtcg ct  
               
               
                 #ttctgcca    240  
               
               
                   
               
               
                 ccggagccca agcagccttg gtcaggggtg gtagacgcta caaccttcca ga  
               
               
                 #gtgtctgc    300  
               
               
                   
               
               
                 taccaatatg tggacaccct atacccaggt tttgagggca ccgagatgtg ga  
               
               
                 #accccaac    360  
               
               
                   
               
               
                 cgtgagctga gcgaggactg cctgtacctc aacgtgtgga caccataccc cc  
               
               
                 #ggcctaca    420  
               
               
                   
               
               
                 tcccccaccc ctgtcctcgt ctggatctat gggggtggct tctacagtgg gg  
               
               
                 #cctcctcc    480  
               
               
                   
               
               
                 ttggacgtgt acgatggccg cttcttggta caggccgaga ggactgtgct gg  
               
               
                 #tgtccatg    540  
               
               
                   
               
               
                 aactaccggg tgggagcctt tggcttcctg gccctgccgg ggagccgaga gg  
               
               
                 #ccccgggc    600  
               
               
                   
               
               
                 aatgtgggtc tcctggatca gaggctggcc ctgcagtggg tgcaggagaa cg  
               
               
                 #tggcagcc    660  
               
               
                   
               
               
                 ttcgggggtg acccgacatc agtgacgctg tttggggaga gcgcgggagc cg  
               
               
                 #cctcggtg    720  
               
               
                   
               
               
                 ggcatgcacc tgctgtcccc gcccagccgg ggcctgttcc acagggccgt gc  
               
               
                 #tgcagagc    780  
               
               
                   
               
               
                 ggtgccccca atggaccctg ggccacggtg ggcatgggag aggcccgtcg ca  
               
               
                 #gggccacg    840  
               
               
                   
               
               
                 cagctggccc accttgtggg ctgtcctcca ggcggcactg gtgggaatga ca  
               
               
                 #cagagctg    900  
               
               
                   
               
               
                 gtagcctgcc ttcggacacg accagcgcag gtcctggtga accacgaatg gc  
               
               
                 #acgtgctg    960  
               
               
                   
               
               
                 cctcaagaaa gcgtcttccg gttctccttc gtgcctgtgg tagatggaga ct  
               
               
                 #tcctcagt   1020  
               
               
                   
               
               
                 gacaccccag aggccctcat caacgcggga gacttccacg gcctgcaggt gc  
               
               
                 #tggtgggt   1080  
               
               
                   
               
               
                 gtggtgaagg atgagggctc gtattttctg gtttacgggg ccccaggctt ca  
               
               
                 #gcaaagac   1140  
               
               
                   
               
               
                 aacgagtctc tcatcagccg ggccgagttc ctggccgggg tgcgggtcgg gg  
               
               
                 #ttccccag   1200  
               
               
                   
               
               
                 gtaagtgacc tggcagccga ggctgtggtc ctgcattaca cagactggct gc  
               
               
                 #atcccgag   1260  
               
               
                   
               
               
                 gacccggcac gcctgaggga ggccctgagc gatgtggtgg gcgaccacaa tg  
               
               
                 #tcgtgtgc   1320  
               
               
                   
               
               
                 cccgtggccc agctggctgg gcgactggct gcccagggtg cccgggtcta cg  
               
               
                 #cctacgtc   1380  
               
               
                   
               
               
                 tttgaacacc gtgcttccac gctctcctgg cccctgtgga tgggggtgcc cc  
               
               
                 #acggctac   1440  
               
               
                   
               
               
                 gagatcgagt tcatctttgg gatccccctg gacccctctc gaaactacac gg  
               
               
                 #cagaggag   1500  
               
               
                   
               
               
                 aaaatcttcg cccagcgact gatgcgatac tgggccaact ttgcccgcac ag  
               
               
                 #gggatccc   1560  
               
               
                   
               
               
                 aatgagcccc gagaccccaa ggccccacaa tggcccccgt acacggcggg gg  
               
               
                 #ctcagcag   1620  
               
               
                   
               
               
                 tacgttagtc tggacctgcg gccgctggag gtgcggcggg ggctgcgcgc cc  
               
               
                 #aggcctgc   1680  
               
               
                   
               
               
                 gccttctgga accgcttcct ccccaaattg ctcagcgcca cctga    
               
               
                 #                1725