Patent 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.

Full Description:
REFERENCE TO RELATED APPLICATIONS  
       [0001]    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  
         [0002]    1. Field of the Invention  
           [0003]    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.  
           [0004]    2. Description of Related Art  
           [0005]    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.  
           [0006]    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.  
           [0007]    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.  
           [0008]    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.  
           [0009]    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.  
           [0010]    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  
         [0011]    Briefly stated, the invention includes one or more plant cells comprising a polynucleotide that encodes a human acetylcholinesterase.  
           [0012]    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.  
           [0013]    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.  
           [0014]    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  
       [0015]    [0015]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.  
         [0016]    [0016]FIG. 2 shows a bar graph depicting high activity of hAChE in transgenic tomato lines.  
         [0017]    [0017]FIG. 3 shows substrate inhibition of recombinant hAChE obtained from transgenic plants.  
         [0018]    [0018]FIG. 4 shows an inhibition profile of AChE obtained from transgenic plants (diamonds), human erythrocytes (circles) and transgenic mice (squares).  
         [0019]    [0019]FIG. 5A shows a graph of data indicating that a recombinant hAChE derived from transgenic plants is labile at relatively high temperatures.  
         [0020]    [0020]FIG. 5B shows a graph of data indicating that a plant-derived hAChE is relatively stable at room temperature. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0021]    DNA Constructs and Plant Transformation  
         [0022]    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)          
 
         [0023]    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.  
         [0024]    Genomic PCR, DNA and RNA Blot Analysis  
         [0025]    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)          
 
         [0026]    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.  
         [0027]    Acetylcholinesterase Assays and Protein Determination  
         [0028]    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-3one 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 BW284c15from 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.  
         [0029]    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.  
         [0030]    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.  
         [0031]    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.  
         [0032]    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.  
         [0033]    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.  
         [0034]    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.  
         [0035]    Kinetic Properties of the Plant-Produced Recombinant Enzyme  
         [0036]    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).  
         [0037]    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).  
         [0038]    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. 5A). 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. 5B). The activity was very stable at 4 degrees C., with only 20% loss after 24 hours (FIG. 5B). 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.  
         [0039]    Types of Cholinesterases that can be Expressed in Plants  
         [0040]    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.  
         [0041]    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.  
         [0042]    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.  
         [0043]    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.  
         [0044]    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.  
         [0045]    Optimization of the Coding Sequence of hAChE-E4 for Expression in Plants  
         [0046]    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.  
         [0047]    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.  
         [0048]    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.  
         [0049]    Analysis of the cDNA of hAChE-E4 to Assess its Suitability for Expression in plants  
         [0050]    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.  
         [0051]    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                  
 
         [0052]    [0052]                                                           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       +UZ,15/17       Total       21   574   3.6                    
         [0053]    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.  
         [0054]    Purification of Plant-Produced Cholinesterases  
         [0055]    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.  
         [0056]    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.  
         [0057]    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.  
         [0058]    Further Examples of Applications of the Invention  
         [0059]    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.  
         [0060]    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. 5A and 5B). 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.  
         [0061]    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.  
         [0062]    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.  
         [0063]    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.  
         [0064]    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.  
     
       
       
         1 
         
           
             5  
           
           
             1  
             29  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      pAChE-Nco, derived from human AChE gene and 
      modified to introduce an Nco I restriction site.  
             
           
            1 

gatatctgca gccatggcta ggcccccgc                                       29 

 
           
             2  
             31  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      pAChE-Kpn, derived from human AChE gene and 
      modified to introduce a Kpn I restriction site.  
             
           
            2 

cggtacctat caggtagcgc tgagcaattt g                                    31 

 
           
             3  
             5767  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence plasmid 
      vector pTM034.  
             
           
            3 

agcttgcatg cctgcaggtc aacatggtgg agcacgacac tctcgtctac tccaagaata     60 

tcaaagatac agtctcagaa gaccagaggg ctattgagac ttttcaacaa agggtaatat    120 

cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag    180 

aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct atcgttcaag    240 

aatgcctcta ccgacagtgg tcccaaagat ggacccccac ccacgaggaa catcgtggaa    300 

aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgataa cttttcaaca    360 

aagggtaata tcgggaaacc tcctcggatt ccattgccca gctatctgtc acttcatcga    420 

aaggacagta gaaaaggaag atggcttcta caaatgccat cattgcgata aaggaaaggc    480 

tatcgttcaa gaatgcctct accgacagtg gtcccaaaga tggaccccca cccacgagga    540 

acatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata    600 

tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta    660 

tataaggaag ttcatttcat ttggagagga cctcgagaat taattctcaa cacaacatat    720 

acaaaacaaa cgaatctcaa gcaatcaagc attctacttc tattgcagca atttaaatca    780 

tttcttttaa agcaaaagca attttctgaa aattttcacc atttacgaac gatagccatg    840 

gctcccccgc agtgtctgct gcacacgcct tccctggctt ccccactcct tctcctcctc    900 

ctctggctcc tgggtggagg agtgggggct gagggccggg aggatgcaga gctgctggtg    960 

acggtgcgtg ggggccggct gcggggcatt cgcctgaaga cccccggggg ccctgtctct   1020 

gctttcctgg gcatcccctt tgcggagcca cccatgggac cccgtcgctt tctgccaccg   1080 

gagcccaagc agccttggtc aggggtggta gacgctacaa ccttccagag tgtctgctac   1140 

caatatgtgg acaccctata cccaggtttt gagggcaccg agatgtggaa ccccaaccgt   1200 

gagctgagcg aggactgcct gtacctcaac gtgtggacac catacccccg gcctacatcc   1260 

cccacccctg tcctcgtctg gatctatggg ggtggcttct acagtggggc ctcctccttg   1320 

gacgtgtacg atggccgctt cttggtacag gccgagagga ctgtgctggt gtccatgaac   1380 

taccgggtgg gagcctttgg cttcctggcc ctgccgggga gccgagaggc cccgggcaat   1440 

gtgggtctcc tggatcagag gctggccctg cagtgggtgc aggagaacgt ggcagccttc   1500 

gggggtgacc cgacatcagt gacgctgttt ggggagagcg cgggagccgc ctcggtgggc   1560 

atgcacctgc tgtccccgcc cagccggggc ctgttccaca gggccgtgct gcagagcggt   1620 

gcccccaatg gaccctgggc cacggtgggc atgggagagg cccgtcgcag ggccacgcag   1680 

ctggcccacc ttgtgggctg tcctccaggc ggcactggtg ggaatgacac agagctggta   1740 

gcctgccttc ggacacgacc agcgcaggtc ctggtgaacc acgaatggca cgtgctgcct   1800 

caagaaagcg tcttccggtt ctccttcgtg cctgtggtag atggagactt cctcagtgac   1860 

accccagagg ccctcatcaa cgcgggagac ttccacggcc tgcaggtgct ggtgggtgtg   1920 

gtgaaggatg agggctcgta ttttctggtt tacggggccc caggcttcag caaagacaac   1980 

gagtctctca tcagccgggc cgagttcctg gccggggtgc gggtcggggt tccccaggta   2040 

agtgacctgg cagccgaggc tgtggtcctg cattacacag actggctgca tcccgaggac   2100 

ccggcacgcc tgagggaggc cctgagcgat gtggtgggcg accacaatgt cgtgtgcccc   2160 

gtggcccagc tggctgggcg actggctgcc cagggtgccc gggtctacgc ctacgtcttt   2220 

gaacaccgtg cttccacgct ctcctggccc ctgtggatgg gggtgcccca cggctacgag   2280 

atcgagttca tctttgggat ccccctggac ccctctcgaa actacacggc agaggagaaa   2340 

atcttcgccc agcgactgat gcgatactgg gccaactttg cccgcacagg ggatcccaat   2400 

gagccccgag accccaaggc cccacaatgg cccccgtaca cggcgggggc tcagcagtac   2460 

gttagtctgg acctgcggcc gctggaggtg cggcgggggc tgcgcgccca ggcctgcgcc   2520 

ttctggaacc gcttcctccc caaattgctc agcgctacct gataggtacc gagctctctc   2580 

aacaatctag ctagagtttg ctcctatcta tatgtaataa ggtatgctga tatgcactat   2640 

tcaaatagga gcattagcta tgtttgttaa tgtcacttta tgttatgtgg gtaagtcacc   2700 

taagacactc cacgtaccta cgttgttgtc tcttaccggc tttaataaat cttctgccct   2760 

tgttccatat ttactaatta tccctttctt cactaaaaga aaattgttat cattaagtat   2820 

tagtctttag aacatatgag gtctttaatt gggtaggttt tacaaattaa ctaatataaa   2880 

atgtcataaa atccacgtgg ttaaacaaat gcagaaaatc gacgtcgtct attggaccga   2940 

cagttgctat taatataatg ggccaccata gtagactgac aaataaatta cctgacaaca   3000 

tcgtttcact aaataacaaa cacaaaaagg gagtgcattt tccagggcat ttttgtaata   3060 

aaaaacagtt aaaagggagt gcaatagaaa tataggggtg tggaaatagt gatttgagca   3120 

cgtcttgaag cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc   3180 

gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa   3240 

gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg   3300 

atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc   3360 

agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct   3420 

gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc   3480 

tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag   3540 

ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg   3600 

tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata   3660 

cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga   3720 

aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca   3780 

ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat   3840 

cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag   3900 

agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc   3960 

gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct   4020 

cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca   4080 

gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt   4140 

ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat   4200 

gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt   4260 

gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta   4320 

cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga   4380 

ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt   4440 

gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc   4500 

gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct   4560 

gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata   4620 

ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt   4680 

gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc   4740 

gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg   4800 

caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact   4860 

ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg   4920 

tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg   4980 

ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac   5040 

tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca   5100 

cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga   5160 

gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc   5220 

ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct   5280 

gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg   5340 

agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct   5400 

tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc   5460 

tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc   5520 

gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat   5580 

taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt   5640 

aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt   5700 

atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat   5760 

tacgcca                                                             5767 

 
           
             4  
             14446  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               (11862)..(12157)  
               Description of Artificial Sequence plasmid 
      vector pTM036. Identity of sequence residues 11862-12157 unknown.  
             
           
            4 

gaattaattc tcaacacaac atatacaaaa caaacgaatc tcaagcaatc aagcattcta     60 

cttctattgc agcaatttaa atcatttctt ttaaagcaaa agcaattttc tgaaaatttt    120 

caccatttac gaacgatagc catggctccc ccgcagtgtc tgctgcacac gccttccctg    180 

gcttccccac tccttctcct cctcctctgg ctcctgggtg gaggagtggg ggctgagggc    240 

cgggaggatg cagagctgct ggtgacggtg cgtgggggcc ggctgcgggg cattcgcctg    300 

aagacccccg ggggccctgt ctctgctttc ctgggcatcc cctttgcgga gccacccatg    360 

ggaccccgtc gctttctgcc accggagccc aagcagcctt ggtcaggggt ggtagacgct    420 

acaaccttcc agagtgtctg ctaccaatat gtggacaccc tatacccagg ttttgagggc    480 

accgagatgt ggaaccccaa ccgtgagctg agcgaggact gcctgtacct caacgtgtgg    540 

acaccatacc cccggcctac atcccccacc cctgtcctcg tctggatcta tgggggtggc    600 

ttctacagtg gggcctcctc cttggacgtg tacgatggcc gcttcttggt acaggccgag    660 

aggactgtgc tggtgtccat gaactaccgg gtgggagcct ttggcttcct ggccctgccg    720 

gggagccgag aggccccggg caatgtgggt ctcctggatc agaggctggc cctgcagtgg    780 

gtgcaggaga acgtggcagc cttcgggggt gacccgacat cagtgacgct gtttggggag    840 

agcgcgggag ccgcctcggt gggcatgcac ctgctgtccc cgcccagccg gggcctgttc    900 

cacagggccg tgctgcagag cggtgccccc aatggaccct gggccacggt gggcatggga    960 

gaggcccgtc gcagggccac gcagctggcc caccttgtgg gctgtcctcc aggcggcact   1020 

ggtgggaatg acacagagct ggtagcctgc cttcggacac gaccagcgca ggtcctggtg   1080 

aaccacgaat ggcacgtgct gcctcaagaa agcgtcttcc ggttctcctt cgtgcctgtg   1140 

gtagatggag acttcctcag tgacacccca gaggccctca tcaacgcggg agacttccac   1200 

ggcctgcagg tgctggtggg tgtggtgaag gatgagggct cgtattttct ggtttacggg   1260 

gccccaggct tcagcaaaga caacgagtct ctcatcagcc gggccgagtt cctggccggg   1320 

gtgcgggtcg gggttcccca ggtaagtgac ctggcagccg aggctgtggt cctgcattac   1380 

acagactggc tgcatcccga ggacccggca cgcctgaggg aggccctgag cgatgtggtg   1440 

ggcgaccaca atgtcgtgtg ccccgtggcc cagctggctg ggcgactggc tgcccagggt   1500 

gcccgggtct acgcctacgt ctttgaacac cgtgcttcca cgctctcctg gcccctgtgg   1560 

atgggggtgc cccacggcta cgagatcgag ttcatctttg ggatccccct ggacccctct   1620 

cgaaactaca cggcagagga gaaaatcttc gcccagcgac tgatgcgata ctgggccaac   1680 

tttgcccgca caggggatcc caatgagccc cgagacccca aggccccaca atggcccccg   1740 

tacacggcgg gggctcagca gtacgttagt ctggacctgc ggccgctgga ggtgcggcgg   1800 

gggctgcgcg cccaggcctg cgccttctgg aaccgcttcc tccccaaatt gctcagcgct   1860 

acctgatagg taccgagctc tctcaacaat ctagctagag tttgctccta tctatatgta   1920 

ataaggtatg ctgatatgca ctattcaaat aggagcatta gctatgtttg ttaatgtcac   1980 

tttatgttat gtgggtaagt cacctaagac actccacgta cctacgttgt tgtctcttac   2040 

cggctttaat aaatcttctg cccttgttcc atatttacta attatccctt tcttcactaa   2100 

aagaaaattg ttatcattaa gtattagtct ttagaacata tgaggtcttt aattgggtag   2160 

gttttacaaa ttaactaata taaaatgtca taaaatccac gtggttaaac aaatgcagaa   2220 

aatcgacgtc gtctattgga ccgacagttg ctattaatat aatgggccac catagtagac   2280 

tgacaaataa attacctgac aacatcgttt cactaaataa caaacacaaa aagggagtgc   2340 

attttccagg gcatttttgt aataaaaaac agttaaaagg gagtgcaata gaaatatagg   2400 

ggtgtggaaa tagtgatttg agcacgtctt gaagcgaatt cgagatcggc cgcggctgag   2460 

tggctccttc aatcgttgcg gttctgtcag ttccaaacgt aaaacggctt gtcccgcgtc   2520 

atcggcgggg gtcataacgt gactccctta attctccgct catgatcaga ttgtcgtttc   2580 

ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga   2640 

aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt tatccgttcg   2700 

tccatttgta tgtgcatgcc aaccacaggg ttccccagat ctggcgccgg ccagcgagac   2760 

gagcaagatt ggccgccgcc cgaaacgatc cgacagcgcg cccagcacag gtgcgcaggc   2820 

aaattgcacc aacgcataca gcgccagcag aatgccatag tgggcggtga cgtcgttcga   2880 

gtgaaccaga tcgcgcagga ggcccggcag caccggcata atcaggccga tgccgacagc   2940 

gtcgagcgcg acagtgctca gaattacgat caggggtatg ttgggtttca cgtctggcct   3000 

ccggaccagc ctccgctggt ccgattgaac gcgcggattc tttatcactg ataagttggt   3060 

ggacatatta tgtttatcag tgataaagtg tcaagcatga caaagttgca gccgaataca   3120 

gtgatccgtg ccgccctgga cctgttgaac gaggtcggcg tagacggtct gacgacacgc   3180 

aaactggcgg aacggttggg ggttcagcag ccggcgcttt actggcactt caggaacaag   3240 

cgggcgctgc tcgacgcact ggccgaagcc atgctggcgg agaatcatac gcattcggtg   3300 

ccgagagccg acgacgactg gcgctcattt ctgatcggga atgcccgcag cttcaggcag   3360 

gcgctgctcg cctaccgcga tggcgcgcgc atccatgccg gcacgcgacc gggcgcaccg   3420 

cagatggaaa cggccgacgc gcagcttcgc ttcctctgcg aggcgggttt ttcggccggg   3480 

gacgccgtca atgcgctgat gacaatcagc tacttcactg ttggggccgt gcttgaggag   3540 

caggccggcg acagcgatgc cggcgagcgc ggcggcaccg ttgaacaggc tccgctctcg   3600 

ccgctgttgc gggccgcgat agacgccttc gacgaagccg gtccggacgc agcgttcgag   3660 

cagggactcg cggtgattgt cgatggattg gcgaaaagga ggctcgttgt caggaacgtt   3720 

gaaggaccga gaaagggtga cgattgatca ggaccgctgc cggagcgcaa cccactcact   3780 

acagcagagc catgtagaca acatcccctc cccctttcca ccgcgtcaga cgcccgtagc   3840 

agcccgctac gggctttttc atgccctgcc ctagcgtcca agcctcacgg ccgcgctcgg   3900 

cctctctggc ggccttctgg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg   3960 

tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag   4020 

aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc   4080 

gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca   4140 

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt   4200 

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc   4260 

tgtccgcctt tctcccttcg ggaagcgtgg cgcttttccg ctgcataacc ctgcttcggg   4320 

gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac aggattttgc   4380 

caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg caggataggt   4440 

gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc gcacctggcg   4500 

gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt aacagatgag   4560 

ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc tatcaaggtg   4620 

tactgccttc cagacgaacg aagagcgatt gaggaaaagg cggcggcggc cggcatgagc   4680 

ctgtcggcct acctgctggc cgtcggccag ggctacaaaa tcacgggcgt cgtggactat   4740 

gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg gccgcctggg cggcctgctg   4800 

aaactctggc tcaccgacga cccgcgcacg gcgcggttcg gtgatgccac gatcctcgcc   4860 

ctgctggcga agatcgaaga gaagcaggac gagcttggca aggtcatgat gggcgtggtc   4920 

cgcccgaggg cagagccatg acttttttag ccgctaaaac ggccgggggg tgcgcgtgat   4980 

tgccaagcac gtccccatgc gctccatcaa gaagagcgac ttcgcggagc tggtgaagta   5040 

catcaccgac gagcaaggca agaccgagcg cctttgcgac gctcaccggg ctggttgccc   5100 

tcgccgctgg gctggcggcc gtctatggcc ctgcaaacgc gccagaaacg ccgtcgaagc   5160 

cgtgtgcgag acaccgcggc cgccggcgtt gtggatacct cgcggaaaac ttggccctca   5220 

ctgacagatg aggggcggac gttgacactt gaggggccga ctcacccggc gcggcgttga   5280 

cagatgaggg gcaggctcga tttcggccgg cgacgtggag ctggccagcc tcgcaaatcg   5340 

gcgaaaacgc ctgattttac gcgagtttcc cacagatgat gtggacaagc ctggggataa   5400 

gtgccctgcg gtattgacac ttgaggggcg cgactactga cagatgaggg gcgcgatcct   5460 

tgacacttga ggggcagagt gctgacagat gaggggcgca cctattgaca tttgaggggc   5520 

tgtccacagg cagaaaatcc agcatttgca agggtttccg cccgtttttc ggccaccgct   5580 

aacctgtctt ttaacctgct tttaaaccaa tatttataaa ccttgttttt aaccagggct   5640 

gcgccctgtg cgcgtgaccg cgcacgccga aggggggtgc ccccccttct cgaaccctcc   5700 

cggcccgcta acgcgggcct cccatccccc caggggctgc gcccctcggc cgcgaacggc   5760 

ctcaccccaa aaatggcagc gctggcagtc cttgccattg ccgggatcgg ggcagtaacg   5820 

ggatgggcga tcagcccgag cgcgacgccc ggaagcattg acgtgccgca ggtgctggca   5880 

tcgacattca gcgaccaggt gccgggcagt gagggcggcg gcctgggtgg cggcctgccc   5940 

ttcacttcgg ccgtcggggc attcacggac ttcatggcgg ggccggcaat ttttaccttg   6000 

ggcattcttg gcatagtggt cgcgggtgcc gtgctcgtgt tcgggggtgc gataaaccca   6060 

gcgaaccatt tgaggtgata ggtaagatta taccgaggta tgaaaacgag aattggacct   6120 

ttacagaatt actctatgaa gcgccatatt taaaaagcta ccaagacgaa gaggatgaag   6180 

aggatgagga ggcagattgc cttgaatata ttgacaatac tgataagata atatatcttt   6240 

tatatagaag atatcgccgt atgtaaggat ttcagggggc aaggcatagg cagcgcgctt   6300 

atcaatatat ctatagaatg ggcaaagcat aaaaacttgc atggactaat gcttgaaacc   6360 

caggacaata accttatagc ttgtaaattc tatcataatt gggtaatgac tccaacttat   6420 

tgatagtgtt ttatgttcag ataatgcccg atgactttgt catgcagctc caccgatttt   6480 

gagaacgaca gcgacttccg tcccagccgt gccaggtgct gcctcagatt caggttatgc   6540 

cgctcaattc gctgcgtata tcgcttgctg attacgtgca gctttccctt caggcgggat   6600 

tcatacagcg gccagccatc cgtcatccat atcaccacgt caaagggtga cagcaggctc   6660 

ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata cgtgcgcaac aaccgtcttc   6720 

cggagactgt catacgcgta aaacagccag cgctggcgcg atttagcccc gacatagccc   6780 

cactgttcgt ccatttccgc gcagacgatg acgtcactgc ccggctgtat gcgcgaggtt   6840 

accgactgcg gcctgagttt tttaagtgac gtaaaatcgt gttgaggcca acgcccataa   6900 

tgcgggctgt tgcccggcat ccaacgccat tcatggccat atcaatgatt ttctggtgcg   6960 

taccgggttg agaagcggtg taagtgaact gcagttgcca tgttttacgg cagtgagagc   7020 

agagatagcg ctgatgtccg gcggtgcttt tgccgttacg caccaccccg tcagtagctg   7080 

aacaggaggg acagctgata gacacagaag ccactggagc acctcaaaaa caccatcata   7140 

cactaaatca gtaagttggc agcatcaccc ataattgtgg tttcaaaatc ggctccgtcg   7200 

atactatgtt atacgccaac tttgaaaaca actttgaaaa agctgttttc tggtatttaa   7260 

ggttttagaa tgcaaggaac agtgaattgg agttcgtctt gttataatta gcttcttggg   7320 

gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa tgagaatatc   7380 

accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg aaggaatgtc   7440 

tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa aaatgacgga   7500 

cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga tgctatggct   7560 

ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa cggcatgatg gctggagcaa   7620 

tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag atgaacaaag   7680 

ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact ccatcgacat   7740 

atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg attacttact   7800 

gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacactc catttaaaga   7860 

tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac ttgtcttttc   7920 

ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa gtggctttat   7980 

tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct gcgtccggtc   8040 

gatcagggag gatatcgggg aagaacagta tgtcgagcta ttttttgact tactggggat   8100 

caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt tttagtacct   8160 

agatgtggcg caacgatgcc ggcgacaagc aggagcgcac cgacttcttc cgcatcaagt   8220 

gttttggctc tcaggccgag gcccacggca agtatttggg caaggggtcg ctggtattcg   8280 

tgcagggcaa gattcggaat accaagtacg agaaggacgg ccagacggtc tacgggaccg   8340 

acttcattgc cgataaggtg gattatctgg acaccaaggc accaggcggg tcaaatcagg   8400 

aataagggca cattgccccg gcgtgagtcg gggcaatccc gcaaggaggg tgaatgaatc   8460 

ggacgtttga ccggaaggca tacaggcaag aactgatcga cgcggggttt tccgccgagg   8520 

atgccgaaac catcgcaagc cgcaccgtca tgcgtgcgcc ccgcgaaacc ttccagtccg   8580 

tcggctcgat ggtccagcaa gctacggcca agatcgagcg cgacagcgtg caactggctc   8640 

cccctgccct gcccgcgcca tcggccgccg tggagcgttc gcgtcgtctc gaacaggagg   8700 

cggcaggttt ggcgaagtcg atgaccatcg acacgcgagg aactatgacg accaagaagc   8760 

gaaaaaccgc cggcgaggac ctggcaaaac aggtcagcga ggccaagcag gccgcgttgc   8820 

tgaaacacac gaagcagcag atcaaggaaa tgcagctttc cttgttcgat attgcgccgt   8880 

ggccggacac gatgcgagcg atgccaaacg acacggcccg ctctgccctg ttcaccacgc   8940 

gcaacaagaa aatcccgcgc gaggcgctgc aaaacaaggt cattttccac gtcaacaagg   9000 

acgtgaagat cacctacacc ggcgtcgagc tgcgggccga cgatgacgaa ctggtgtggc   9060 

agcaggtgtt ggagtacgcg aagcgcaccc ctatcggcga gccgatcacc ttcacgttct   9120 

acgagctttg ccaggacctg ggctggtcga tcaatggccg gtattacacg aaggccgagg   9180 

aatgcctgtc gcgcctacag gcgacggcga tgggcttcac gtccgaccgc gttgggcacc   9240 

tggaatcggt gtcgctgctg caccgcttcc gcgtcctgga ccgtggcaag aaaacgtccc   9300 

gttgccaggt cctgatcgac gaggaaatcg tcgtgctgtt tgctggcgac cactacacga   9360 

aattcatatg ggagaagtac cgcaagctgt cgccgacggc ccgacggatg ttcgactatt   9420 

tcagctcgca ccgggagccg tacccgctca agctggaaac cttccgcctc atgtgcggat   9480 

cggattccac ccgcgtgaag aagtggcgcg agcaggtcgg cgaagcctgc gaagagttgc   9540 

gaggcagcgg cctggtggaa cacgcctggg tcaatgatga cctggtgcat tgcaaacgct   9600 

agggccttgt ggggtcagtt ccggctgggg gttcagcagc cagcgcttta ctggcatttc   9660 

aggaacaagc gggcactgct cgacgcactt gcttcgctca gtatcgctcg ggacgcacgg   9720 

cgcgctctac gaactgccga taaacagagg attaaaattg acaattgtga ttaaggctca   9780 

gattcgacgg cttggagcgg ccgacgtgca ggatttccgc gagatccgat tgtcggccct   9840 

gaagaaagct ccagagatgt tcgggtccgt ttacgagcac gaggagaaaa agcccatgga   9900 

ggcgttcgct gaacggttgc gagatgccgt ggcattcggc gcctacatcg acggcgagat   9960 

cattgggctg tcggtcttca aacaggagga cggccccaag gacgctcaca aggcgcatct  10020 

gtccggcgtt ttcgtggagc ccgaacagcg aggccgaggg gtcgccggta tgctgctgcg  10080 

ggcgttgccg gcgggtttat tgctcgtgat gatcgtccga cagattccaa cgggaatctg  10140 

gtggatgcgc atcttcatcc tcggcgcact taatatttcg ctattctgga gcttgttgtt  10200 

tatttcggtc taccgcctgc cgggcggggt cgcggcgacg gtaggcgctg tgcagccgct  10260 

gatggtcgtg ttcatctctg ccgctctgct aggtagcccg atacgattga tggcggtcct  10320 

gggggctatt tgcggaactg cgggcgtggc gctgttggtg ttgacaccaa acgcagcgct  10380 

agatcctgtc ggcgtcgcag cgggcctggc gggggcggtt tccatggcgt tcggaaccgt  10440 

gctgacccgc aagtggcaac ctcccgtgcc tctgctcacc tttaccgcct ggcaactggc  10500 

ggccggagga cttctgctcg ttccagtagc tttagtgttt gatccgccaa tcccgatgcc  10560 

tacaggaacc aatgttctcg gcctggcgtg gctcggcctg atcggagcgg gtttaaccta  10620 

cttcctttgg ttccggggga tctcgcgact cgaacctaca gttgtttcct tactgggctt  10680 

tctcagcccc agatctgggg tcgatcagcc ggggatgcat caggccgaca gtcggaactt  10740 

cgggtccccg acctgtacca ttcggtgagc aatggatagg ggagttgata tcgtcaacgt  10800 

tcacttctaa agaaatagcg ccactcagct tcctcagcgg ctttatccag cgatttccta  10860 

ttatgtcggc atagttctca agatcgacag cctgtcacgg ttaagcgaga aatgaataag  10920 

aaggctgata attcggatct ctgcgaggga gatgatattt gatcacaggc agcaacgctc  10980 

tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca  11040 

gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa  11100 

cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg  11160 

tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa  11220 

acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg tactggggtg  11280 

gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga  11340 

gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg  11400 

gtggttccga aatcggcaaa atcccttata aatcaaaaga atagcccgag atagggttga  11460 

gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag  11520 

ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt  11580 

ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta  11640 

gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag  11700 

cgggcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta  11760 

ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg  11820 

ttttcccagt cacgacgttg taaaacgacg gccagtgaat tnnnnnnnnn nnnnnnnnnn  11880 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn  11940 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn  12000 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn  12060 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn  12120 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnngga tccagatccc gtgggcgaag  12180 

aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt  12240 

ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg  12300 

ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa  12360 

ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc  12420 

ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct  12480 

gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt  12540 

ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatca tcgccgtcgg  12600 

gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt  12660 

ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat  12720 

gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg  12780 

catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc  12840 

ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag  12900 

ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt  12960 

cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca  13020 

gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata  13080 

gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa  13140 

aagatctgga ttgagagtga atatgagact ctaattggat accgagggga atttatggaa  13200 

cgtcagtgga gcatttttga caagaaatat ttgctagctg atagtgacct taggcgactt  13260 

ttgaacgcgc aataatggtt tctgacgtat gtgcttagct cattaaactc cagaaacccg  13320 

cggctgagtg gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc  13380 

ccgcgtcatc ggcgggggtc ataacgtgac tcccttaatt ctccgctcat gatcttgatc  13440 

ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc  13500 

caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg  13560 

cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg  13620 

cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct  13680 

gcggactggc tttctacgtg ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc  13740 

ggcagcgtga agcttgcatg cctgcaggtc aacatggtgg agcacgacac tctcgtctac  13800 

tccaagaata tcaaagatac agtctcagaa gaccagaggg ctattgagac ttttcaacaa  13860 

agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa  13920 

aggacagtag aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct  13980 

atcgttcaag aatgcctcta ccgacagtgg tcccaaagat ggacccccac ccacgaggaa  14040 

catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgataa  14100 

cttttcaaca aagggtaata tcgggaaacc tcctcggatt ccattgccca gctatctgtc  14160 

acttcatcga aaggacagta gaaaaggaag atggcttcta caaatgccat cattgcgata  14220 

aaggaaaggc tatcgttcaa gaatgcctct accgacagtg gtcccaaaga tggaccccca  14280 

cccacgagga acatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat  14340 

tgatgtgata tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac  14400 

ccttcctcta tataaggaag ttcatttcat ttggagagga cctcga                 14446 

 
           
             5  
             1725  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence synthetic 
      human acetylcholinesterase gene optimized for 
      expression in plants  
             
           
            5 

atgaggcccc cgcagtgtct gctgcacacg ccttccctgg cttccccact ccttctcctc     60 

ctcctctggc tcctgggtgg aggagtgggg gctgagggcc gggaggatgc agagctgctg    120 

gtgacggtgc gtgggggccg gctgcggggc attcgcctga agacccccgg gggccctgtc    180 

tctgctttcc tgggcatccc ctttgcggag ccacccatgg gaccccgtcg ctttctgcca    240 

ccggagccca agcagccttg gtcaggggtg gtagacgcta caaccttcca gagtgtctgc    300 

taccaatatg tggacaccct atacccaggt tttgagggca ccgagatgtg gaaccccaac    360 

cgtgagctga gcgaggactg cctgtacctc aacgtgtgga caccataccc ccggcctaca    420 

tcccccaccc ctgtcctcgt ctggatctat gggggtggct tctacagtgg ggcctcctcc    480 

ttggacgtgt acgatggccg cttcttggta caggccgaga ggactgtgct ggtgtccatg    540 

aactaccggg tgggagcctt tggcttcctg gccctgccgg ggagccgaga ggccccgggc    600 

aatgtgggtc tcctggatca gaggctggcc ctgcagtggg tgcaggagaa cgtggcagcc    660 

ttcgggggtg acccgacatc agtgacgctg tttggggaga gcgcgggagc cgcctcggtg    720 

ggcatgcacc tgctgtcccc gcccagccgg ggcctgttcc acagggccgt gctgcagagc    780 

ggtgccccca atggaccctg ggccacggtg ggcatgggag aggcccgtcg cagggccacg    840 

cagctggccc accttgtggg ctgtcctcca ggcggcactg gtgggaatga cacagagctg    900 

gtagcctgcc ttcggacacg accagcgcag gtcctggtga accacgaatg gcacgtgctg    960 

cctcaagaaa gcgtcttccg gttctccttc gtgcctgtgg tagatggaga cttcctcagt   1020 

gacaccccag aggccctcat caacgcggga gacttccacg gcctgcaggt gctggtgggt   1080 

gtggtgaagg atgagggctc gtattttctg gtttacgggg ccccaggctt cagcaaagac   1140 

aacgagtctc tcatcagccg ggccgagttc ctggccgggg tgcgggtcgg ggttccccag   1200 

gtaagtgacc tggcagccga ggctgtggtc ctgcattaca cagactggct gcatcccgag   1260 

gacccggcac gcctgaggga ggccctgagc gatgtggtgg gcgaccacaa tgtcgtgtgc   1320 

cccgtggccc agctggctgg gcgactggct gcccagggtg cccgggtcta cgcctacgtc   1380 

tttgaacacc gtgcttccac gctctcctgg cccctgtgga tgggggtgcc ccacggctac   1440 

gagatcgagt tcatctttgg gatccccctg gacccctctc gaaactacac ggcagaggag   1500 

aaaatcttcg cccagcgact gatgcgatac tgggccaact ttgcccgcac aggggatccc   1560 

aatgagcccc gagaccccaa ggccccacaa tggcccccgt acacggcggg ggctcagcag   1620 

tacgttagtc tggacctgcg gccgctggag gtgcggcggg ggctgcgcgc ccaggcctgc   1680 

gccttctgga accgcttcct ccccaaattg ctcagcgcca cctga                   1725

Technology Classification (CPC): 2