Patent Application: US-43355603-A

Abstract:
the invention concerns novel enzymes having an arogenate dehydrogenase activity , in particular arogenate dehydrogenase enzymes of plants , and the genes encoding said enzymes . the inventive arogenate dehydrogenase enzymes catalyze the last stage of the metabolic pathway of tyrosine biosynthesis , and constitute , as such , potential targets of herbicides . hence the invention also concerns a method for identifying herbicide compounds targeting said enzymes , said herbicide compounds preventing tyrosine biosynthesis by being fixed on said enzymes . the invention further concerns transgenic plants tolerant to herbicide compounds targeting an enzyme involved in the tyrosine biosynthesis pathway , in particular an enzyme involved in the transformation of l - tyrosine prephenate , in particular an arogenate dehydrogenase enzyme . said plants become tolerant by expression in their tissues of a prephenate dehydrogenase enzyme , said enzyme being insensitive to said herbicide compounds and enabling the plant to synthetize tyrosine despite being treated with said herbicide compounds .

Description:
the present invention therefore relates to novel isolated polynucleotides encoding an enzyme having arogenate dehydrogenase activity . according to the present invention , the term “ polynucleotide ” is intended to mean a natural or artificial nucleotide sequence which may be of the dna or rna type , preferably of the dna type , in particular double - stranded . the expression “ enzymes having arogenate dehydrogenase activity ” is intended to mean the enzymes capable of converting arogenate to l - tyrosine . the arogenate dehydrogenase activity is measured by any method which makes it possible either to measure a decrease in the amount of the arogenate substrate , or to measure an accumulation of a product derived from the enzyme reaction , namely l - tyrosine or the cofactor nadph . in particular , the arogenate dehydrogenase activity can be measured by the method described in example 4 . according to a particular embodiment of the invention , the polynucleotides encoding an arogenate dehydrogenase enzyme comprise polynucleotides encoding the polypeptide sequence selected from the sequence described in the sequence identifier seq id no : 3 , seq id no : 5 , seq id no : 7 , seq id no : 9 , seq id no : 11 or seq id no : 13 . it is well known to those skilled in the art that this definition includes all the polynucleotides which , although comprising nucleotide sequences which are different as a result of the degeneracy of the genetic code , encode the same amino acid sequence , which sequence is represented by the sequence identifiers seq id no : 3 , seq id no : 5 , seq id no : 7 , seq id no : 9 , seq id no : 11 or seq id no : 13 . the present invention also comprises isolated polynucleotides encoding arogenate dehydrogenase enzymes and capable of hybridizing selectively to one of the polynucleotides described above , or a fragment of these polynucleotides constituting a probe . according to the invention , the expression “ polynucleotide capable of hybridizing selectively ” is intended to mean the polynucleotides which , by one of the usual methods of the state of the art ( sambrook et al ., 1989 , molecular cloning : a laboratory manual , nolan c . ed ., new york : cold spring harbor laboratory press ), hybridize with the polynucleotides above , or with the probes which are derived therefrom , at a level significantly greater than the background noise . the background noise may be associated with the hybridization of other polynucleotides present , for example other cdnas present in a cdna library . the level of the signal generated by the interaction between the polynucleotide capable of hybridizing selectively and the polynucleotides defined by the sequences seq id nos : above according to the invention , or the probes , is generally 10 times , preferably 100 times , more intense than that generated by the interaction with other dna sequences generating the background noise . the level of interaction can be measured , for example , by labeling the polynucleotides described above or the probes with radioactive elements , such as 32 p . selective hybridization is generally obtained using very severe conditions for the medium ( for example 0 . 03 m nacl and 0 . 03 m sodium citrate at approximately 50 ° c .- 60 ° c .). the invention also comprises isolated polynucleotides encoding arogenate dehydrogenase enzymes , and homologs of the polynucleotides described above . according to the invention , the term “ homolog ” is intended to mean polynucleotides exhibiting one or more sequence modifications compared to the nucleotide sequences described above and encoding an enzyme with functional arogenate dehydrogenase activity . these modifications may be natural or obtained artificially according to the usual techniques of mutation leading in particular to the addition , deletion or substitution of one or more nucleotides compared to the sequences of the invention . these modifications determine a degree of homology with respect to the sequences described above . advantageously , the degree of homology will be at least 70 % compared to the sequences described , preferably at least 80 %, more preferentially at least 90 %. the methods for measuring and identifying homologies between nucleic acid sequences are well known to those skilled in the art . use may , for example , be made of the pileup or blast programs ( basic local alignment search tool ; altschul et al ., 1993 , j . mol . evol . 36 : 290 - 300 ; altschul et al ., 1990 , j . mol . biol . 215 : 403 - 10 ; see also http :// www . ncbi . nlm . nih . gov / blast /). the present invention also relates to fragments of the polynucleotides described above . the term “ fragment ” denotes in particular a fragment of at least 20 nucleotides , in particular of at least 50 nucleotides , and preferably of at least 100 nucleotides . according to a particular embodiment of the invention , the polynucleotide according to the invention is represented by the sequence identifier seq id no : 1 , seq id no : 2 , seq id no : 4 , seq id no : 6 , seq id no : 8 , seq id no : 10 or seq id no : 12 . the present invention also relates to polynucleotides comprising at least one of the polynucleotides as described above . all the polynucleotides described above encode arogenate dehydrogenase enzymes . consequently , the invention therefore extends to all the arogenate dehydrogenase enzymes encoded by all of these polynucleotides . according to a particular embodiment of the invention , the arogenate dehydrogenase enzyme is an enzyme the peptide sequence of which is selected from the sequence described by the sequence identifier seq id no : 3 , seq id no : 5 , seq id no : 7 , seq id no : 9 , seq id no : 11 or seq id no : 13 ., or a fragment of these sequences . the term “ fragment ” is intended to mean essentially a biologically active fragment , i . e . a fragment of the sequence of an arogenate dehydrogenase enzyme having the same activity as a complete arogenate dehydrogenase enzyme . according to a particular embodiment of the invention , the polynucleotides and the arogenate dehydrogenase enzymes described above originate from plants . more particularly , they originate from plants of the arabidopsis genus , preferably of the a . thaliana genus , or from plants of the picea genus , preferably picea glauca . according to another particular embodiment of the invention , the polynucleotides and the arogenate dehydrogenase enzymes described above originate from bacteria . more particularly , they originate from bacteria of the synechocystis genus . the present invention also relates to a chimeric gene comprising , functionally linked to one another , at least one promoter which is functional in a host organism , a polynucleotide encoding an arogenate dehydrogenase enzyme as defined in the present inven - tion , and a terminator element which is functional in this same host organism . the various elements that a chimeric gene may contain are , firstly , elements which regulate the transcription , translation and maturation of proteins , such as a promoter , a sequence encoding a signal peptide or a transit peptide , or a terminator element constituting a polyadenylation signal and , secondly , a polynucleotide encoding a protein . the expression “ functionally linked to one another ” means that said elements of the chimeric gene are linked to one another in such a way that the functioning of one of these elements is affected by that of another . by way of example , a promoter is functionally linked to a coding sequence when it is capable of affecting the expression of said coding sequence . the construction of the chimeric gene according to the invention and the assembly of its various elements can be carried out using techniques well known to those skilled in the art , in particular those described by sambrook et al ., ( 1989 , molecular cloning : a laboratory manual , nolan c . ed ., new york : cold spring harbor laboratory press ). the choice of the regulatory elements constituting the chimeric gene depends essentially on the host species in which they must function , and those skilled in the art are capable of selecting regulatory elements which are functional in a given host organism . the term “ functional ” is intended to mean capable of functioning in a given host organism . the promoters which the chimeric gene according to the invention can contain are either constitutive or inducible . a constitutive promoter according to the present invention is a promoter which induces the expression of a coding sequence in all the tissues of a host organism and continuously , i . e . throughout the duration of the life cycle of said organism . some of these promoters may be tissue - specific , i . e . express the coding sequence continuously , but only in a particular tissue of the host organism . constitutive promoters may originate from any type of organism . among the constitutive promoters which can be used in the chimeric gene of the present invention , mention may , for example , be made of bacterial promoters , such as that of the octopine synthase gene or that of the nopaline synthase gene , of viral promoters , such as that of the gene controlling transcription of the 19s or 35s rnas of the cauliflower mosaic virus ( odell et al ., 1985 , nature , 313 , 810 - 812 ), or the promoters of the cassava vein mosaic virus ( as described in patent application wo 97 / 48819 ). among the promoters of plant origin , mention will be made of the promoter of the ribulose - biscarboxylase / oxygenase ( rubisco ) small sub - unit gene , the promoter of a histone gene as described in application ep 0 507 698 , or the promoter of a rice actin gene ( u . s . pat . no . 5 , 641 , 876 ). according to another particular embodiment of the invention , the chimeric gene contains an inducible promoter . an inducible promoter is a promoter which only functions , i . e . which only induces expression of a coding sequence , when it is itself induced by an inducing agent . this inducing agent is generally a substance which can be synthesized in the host organism subsequent to a stimulus external to said organism , this external stimulus possibly being , for example , a pathogenic agent . the inducing agent may also be a substance external to this host organism , capable of penetrating into this host organism . advantageously , the promoter used in the present invention is inducible subsequent to an attack on the host organism by a pathogenic agent . such promoters are known , such as , for example , the promoter of the plant o - methyl - transferase class ii ( comt ii ) gene described in patent application fr 99 03700 , the arabidopsis pr - 1 promoter ( lebel et al ., 1998 , plant j . 16 ( 2 ): 223 - 233 ), the eas4 promoter of the tobacco sesquiterpene synthase gene ( yin et al ., 1997 , plant physiol . 115 ( 2 ): 437 - 451 ), or the promoter of the gene encoding 3 - hydroxy - 3 - methylglutaryl coenzyme a reductase ( nelson et al ., 1994 , plant mol . biol . 25 ( 3 ): 401 - 412 ). among the terminator elements which may be used in the chimeric gene of the present invention , mention may , for example , be made of the nos terminator element of the gene encoding agrobacterium tumefaciens nopaline synthase ( beven et al ., 1983 , nucleic acids res . 11 ( 2 ), 369 - 385 ), or the terminator element of a histone gene as described in application ep 0 633 317 . it also appears to be important for the chimeric gene to additionally comprise a signal peptide or a transit peptide which makes it possible to control and orient the production of the arogenate dehydrogenase enzyme specifically in a part of the host organism , such as , for example , the cytoplasm , a particular compartment of the cytoplasm , or the cell membrane or , in the case of plants , in a particular type of cellular compartment , for example the chloroplasts , or in the extracellular matrix . the transit peptides can be either single or double . the double transit peptides are optionally separated by an intermediate sequence , i . e . they comprise , in the direction of transcription , a sequence encoding a transit peptide of a plant gene encoding an enzyme located in plastids , a portion of sequence of the mature n - terminal portion of a plant gene encoding an enzyme located in plastids , and then a sequence encoding a second transit peptide of a plant gene encoding an enzyme located in plastids . such double transit peptides are , for example , described in patent application ep 0 508 909 . signal peptides of use according to the invention which may be mentioned include in particular the signal peptide of the tobacco pr - 1α gene described by cornelissen et al . ( 1987 , nucleic acid res . 15 , 6799 - 6811 ), in particular when the chimeric gene according to the invention is introduced into plant cells or plants , or the signal peptide of the mat α1 factor precursor ( brake et al ., 1985 , in : gething m .- j . ( eds . ); protein transport and secretion , pp . 103 - 108 , cold spring harbor laboratory press , new york ), when the chimeric gene according to the invention is introduced into yeast . the present invention also relates to a vector containing a chimeric gene according to the invention . the vector according to the invention is of use for transforming a host organism and expressing an arogenate dehydrogenase enzyme in this host organism . this vector may be a plasmid , a cosmid , a bacteriophage or a virus . in general , the main qualities of this vector should be an ability to persist and to self - replicate in the host organism &# 39 ; s cells , in particular by virtue of the presence of an origin of replication , and to express therein an arogenate dehydrogenase enzyme . the choice of such a vector and also the techniques for inserting the chimeric gene according to the invention therein are widely described in sambrook et al . ( 1989 , molecular cloning : a laboratory manual , nolan c . ed ., new york : cold spring harbor laboratory press ) and are part of the general knowledge of those skilled in the art . advantageously , the vector used in the present invention also contains , in addition to the chimeric gene of the invention , a gene encoding a selectable marker . this selectable marker makes it possible to select the host organisms effectively transformed , i . e . those having incorporated the vector . according to a particular embodiment of the invention , the host organism to be transformed is a microorganism , in particular a yeast , a bacterium , a fungus or a virus . according to another embodiment , the host organism is a plant or a plant cell . among the genes encoding selectable markers which can be used , mention may be made of genes for resistance to antibiotics , such as , for example , the hygromycin phosphotransferase ( gritz et al ., 1983 , gene 25 : 179 - 188 ), but also the genes for tolerance to herbicides , such as the bar gene ( white et al ., nar 18 : 1062 , 1990 ) for tolerance to bialaphos , the epsps gene ( u . s . pat . no . 5 , 188 , 642 ) for tolerance to glyphosate or else the hppd gene ( wo 96 / 38567 ) for tolerance to isoxazoles . mention may also be made of genes encoding readily identifiable enzymes such as the gus enzyme , or genes encoding pigments or enzymes which regulate the production of pigments in the transformed cells . such selectable marker genes are in particular described in patent applications wo 91 / 02071 , wo 95 / 06128 , wo 96 / 38567 and wo 97 / 04103 . the present invention also relates to transformed host organisms containing a vector as described above . the term “ host organism ” is intended to mean any lower or higher monocellular or pluricellular organism into which the chimeric gene according to the invention can be introduced , so as to produce arogenate dehydrogenase enzyme . they are in particular bacteria , for example escherichia coli , yeast , in particular of the saccharomyces , kluyveromyces or pichia genera , fungi , in particular aspergillus , a baculovirus , or preferably plant cells and plants . according to the invention , the term “ plant cell ” is intended to mean any cell derived from a plant and able to constitute undifferentiated tissues such as calluses , differentiated tissues such as embryos , parts of plants , plants or seeds . according to the invention , the term “ plant ” is intended to mean any differentiated multicellular organism capable of photosynthesis , in particular monocotyledons or dicotyledons . the term “ transformed host organism ” is intended to mean a host organism which has incorporated into its genome the chimeric gene of the invention and consequently produces an arogenate dehydrogenase enzyme in its tissues , or in a culture medium . those skilled in the art can use one of the many known methods of transformation to obtain the host organisms according to the invention . one of these methods consists in bringing the cells to be transformed into contact with polyethylene glycol ( peg ) and the vectors of the invention ( chang and cohen , 1979 , mol . gen . genet . 168 ( 1 ), 111 - 115 ); mercenier and chassy , 1988 , biochimie 70 ( 4 ), 503 - 517 ). electroporation is another method , which consists in subjecting the cells or tissues to be transformed and the vectors of the invention to an electric field ( andreason and evans , 1988 , biotechniques 6 ( 7 ), 650 - 660 ; shigekawa and dower , 1989 , aust . j . biotechnol . 3 ( 1 ), 56 - 62 ). another method consists in directly injecting the vectors into the host cells or tissues by microinjection ( gordon and ruddle , 1985 , gene 33 ( 2 ), 121 - 136 ). advantageously , the “ biolistic ” method may be used . in consists in bombarding cells or tissues with particles onto which the vectors of the invention are adsorbed ( bruce et al ., 1989 , proc . natl . acad . sci . usa 86 ( 24 ), 9692 - 9697 ; klein et al ., 1992 , biotechnology 10 ( 3 ), 286 - 291 ; u . s . pat . no . 4 , 945 , 050 ). preferentially , the plant transformation will be carried out using bacteria of the agrobacterium genus , preferably by infecting the cells or tissue of said plants by a . tumefaciens ( knopf , 1979 , subcell . biochem . 6 , 143 - 173 ; shaw et al ., 1983 , gene 23 ( 3 ): 315 - 330 ) or a . rhizogenes ( bevan and chilton , 1982 , annu . rev . genet . 16 : 357 - 384 ; tepfer and casse - delbart , 1987 , microbiol . sci . 4 ( 1 ), 24 - 28 ). preferentially , the transformation of plant cells with agrobacterium tumefaciens is carried out according to the protocol described by ishida et al . ( 1996 , nat . biotechnol . 14 ( 6 ), 745 - 750 ). those skilled in the art will choose the appropriate method as a function of the nature of the host organism to be transformed . the present invention therefore also relates to a method for preparing the arogenate dehydrogenase enzyme , comprising the steps of culturing a transformed host organism comprising a gene encoding an arogenate dehydrogenase enzyme as defined above , in a suitable culture medium , recovering the arogenate dehydrogenase enzyme produced from the culture medium by centrifugation or by filtration , and then purifying the recovered enzyme by passing it through at least one chromatography column . these steps bring about the extraction and the purification , which may be total or partial , of the arogenate dehydrogenase enzyme obtained . preferentially , the transformed organism is a microorganism , in particular a bacterium , a yeast , a fungus or a virus . the present invention also comprises a method for identifying a herbicidal compound having as a target an arogenate dehydrogenase enzyme , characterized in that : ( a ) at least two samples , each containing an equivalent amount of arogenate dehydrogenase enzymes in solution , are prepared ; ( c ) the arogenate dehydrogenase activity is measured in each one of said samples ; ( d ) the compound used in step ( b ) is identified as being a herbicidal compound when the activity measured in step ( c ) is significantly less in the treated sample compared to the untreated sample ; ( e ) the herbicidal activity of the compound identified in step ( d ) is validated by treating plants with said compound . according to the present method , the measurement of the arogenate dehydrogenase activity is carried out by any method which makes it possible either to measure a decrease in the amount of arogenate substrate , or to measure an accumulation of a product derived from the enzyme reaction , namely l - tyrosine or the cofactor nadph . in particular , the measurement of the arogenate dehydrogenase activity can be carried out by the method described in example 4 . in addition , the herbicidal activity validated in step ( e ) of the present method may be a lethal activity resulting in the death of the treated plant , or an activity which significantly slows down the growth of the treated plant . according to the invention , the term “ compound ” is intended to mean any chemical compound or mixture of chemical compounds , including peptides and proteins . according to the invention , the term “ mixture of compounds ” is understood to mean at least two different compounds , such as , for example , the ( dia ) stereoisomers of a molecule , mixtures of natural origin derived from the extraction of biological material ( plants , plant tissues , bacterial cultures , yeast cultures or fungal cultures , insects , animal tissues , etc .) or unpurified or totally or partially purified reaction mixtures , or else mixtures of products derived from combinatorial chemistry techniques . according to a particular embodiment of the method according to the invention , the arogenate dehydrogenase enzymes used originate from plants , preferably from arabidopsis thaliana . according to another embodiment of the method according to the invention , the arogenate dehydrogenase enzymes used originate from bacteria , preferably bacteria of the synechocystis genus . preferably , the arogenate dehydrogenase enzymes used in the method according to the invention are the enzymes according to the present invention , in particular those represented by seq id no : 3 , seq id no : 5 , seq id no : 7 , seq id no : 9 , seq id no : 11 or seq id no : 13 . the invention also extends to the herbicidal compounds identified using the method mentioned above , in particular the herbicidal compounds having as a target an arogenate dehydrogenase enzyme , i . e . those which inhibit the activity of this enzyme . preferentially , the herbicidal compounds are not general enzyme inhibitors . also preferentially , the herbicidal compounds according to the invention are not compounds already known to have herbicidal activity . the present invention also relates to herbicidal agrochemical compositions comprising , as active material , at least an effective amount of a herbicidal compound according to the invention . according to the invention , the term “ herbicidal agrochemical composition ” is intended to mean a composition which can be applied preventatively or curatively to the areas on which cultivated plants are being or must be grown , in order to prevent the development of undesirable plants or “ weeds ” on the areas on which said cultivated plants are grown , whatever their state of development . an effective amount of herbicidal compound according to the invention corresponds to an amount of compound which makes it possible to destroy or inhibit the growth of the undesirable plants . the herbicidal agrochemical compositions according to the invention comprise a herbicidal compound according to the invention or one of its agriculturally acceptable salts or a metal or metalloid complex of this compound , in combination with an agriculturally acceptable solid or liquid carrier and / or a surfactant , also agriculturally acceptable . in particular , the usual inert carriers and the usual surfactants can be used . these compositions cover not only the compositions ready to be applied to a plant or a seed to be treated using a suitable device , such as a spraying or dusting device , but also the concentrated commercially available compositions which must be diluted before they are applied to the crop . the herbicidal compositions according to the invention may also contain many other ingredients , such as , for example , protective colloids , adhesives , thickeners , thixotropic agents , penetrating agents , stabilizers or sequestering agents . more generally , the active materials can be combined with any solid or liquid additives which comply with the usual formulating techniques . according to the present invention , the term “ carrier ” denotes a natural or synthetic , organic or inorganic material with which the active material is combined in order to facilitate its application to the parts of the plant . this carrier is therefore generally inert and it must be agriculturally acceptable . the carrier may be solid ( for example clays , natural or synthetic silicates , silica , resins , waxes , solid fertilizers ) or liquid ( for example water , alcohols , in particular butanol ). the surfactant may be an emulsifier , dispersing agent or wetting agent of the ionic or nonionic type , or a mixture of such surfactants . mention may , for example , be made of polyacrylic acid salts , lignosulfonic acid salts , phenolsulfonic or naphthalenesulfonic acid salts , polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines , substituted phenols ( in particular alkylphenols or arylphenols ), salts of sulfosuccinic acid esters , taurine derivatives ( in particular alkyl taurates ), phosphoric esters of alcohols or of phenols which are polyoxyethylated , esters of fatty acids and of polyols , and derivatives of the above compounds containing sulfate , sulfonate and phosphate functions . the presence of at least one surfactant is generally essential when the active material and / or the inert carrier are not water - soluble and when the vector agent for the application is water . the present invention also relates to transgenic plants tolerant to a herbicidal compound having as a target an enzyme involved in one of the metabolic steps of conversion of prephenate to l - tyrosine , characterized in that they contain a gene encoding a prephenate dehydrogenase enzyme and express said enzyme in their tissue . a prephenate dehydrogenase enzyme is an enzyme which catalyzes the reaction of conversion of prephenate to p - hydroxyphenylpyruvate . the identification of an enzyme with prephenate dehydrogenase activity can be carried out by any method which makes it possible either to measure a decrease in the amount of the prephenate substrate , or to measure an accumulation of a product derived from the enzyme reaction , namely p - hydroxyphenylpyruvate or one of the cofactors nadh or nadph . in particular , the measurement of the prephenate dehydrogenase activity can be carried out using the method described in example 4 . according to a particular embodiment of the invention , the transgenic plants according to the invention are tolerant with respect to a herbicidal compound having as a target an arogenate dehydrogenase enzyme , preferably an arogenate dehydrogenase enzyme as described in the present invention . according to another particular embodiment of the invention , the transgenic plants according to the invention are tolerant with respect to a herbicidal compound having as a target a prephenate aminotransferase enzyme . according to a particular embodiment of the invention , the gene encoding the prephenate dehydrogenase enzyme expressed in the tolerant plants according to the invention is a yeast gene . preferably , it is the gene encoding the saccharomyces cerevisiae prephenate dehydrogenase enzyme ( accession no . nc001134 ) as described in mannhaupt et al . ( 1989 , gene 85 , 303 - 311 ) and represented by the sequence identifier seq id no : 14 . according to another particular embodiment of the invention , the gene encoding the prephenate dehydrogenase enzyme expressed in the tolerant plants according to the invention is a bacterial gene . preferably , it is a gene from a bacterium of the bacillus genus , in particular of the species b . subtilis ( accession no . m80245 ) as represented by the sequence identifier seq id no : 16 . preferably , it is a gene from a bacterium of the escherichia genus , in particular of the species e . coli ( accession no . m10431 ) as described in hudson et al . ( 1984 , j . mol . biol . 180 ( 4 ), 1023 - 1051 ) and represented by the sequence identifier seq id no : 18 . preferably , it is a gene from a bacterium of the erwinia genus , in particular the species e . herbicola ( accession no . 43343 ) as represented by the sequence identifier seq id no : 20 . according to particular embodiment of the invention , the gene encoding the prephenate dehydrogenase enzyme expressed in the tolerant plants according to the invention is a fungal gene . the transgenic plants according to the invention are obtained by genetic transformation with a gene encoding a prephenate dehydrogenase enzyme . preferably , this gene is a chimeric gene comprising , functionally linked to one another , at least one promoter which is functional in a host organism , a polynucleotide encoding a prephenate dehydrogenase enzyme , and a terminator element which is functional in this same host organism . this gene is generally introduced into a vector , which is used to introduce said gene into said plants by one of the methods of transformation described above . the present invention also relates to a method for producing plants tolerant with respect to herbicidal compounds having as a target an enzyme involved in one of the metabolic steps for conversion of prephenate to l - tyrosine , characterized in that said plants are transformed with a gene encoding a prephenate dehydrogenase enzyme in such a way that they express it in their tissues . according to a particular embodiment of the invention , the present method applies to the production of plants tolerant with respect to a herbicidal compound having as a target an arogenate dehydrogenase enzyme as described in the present invention . according to another particular embodiment of the invention , the present method applies to the production of plants tolerant with respect to a herbicidal compound having as a target a prephenate aminotransferase enzyme . the present method therefore also comprises a method for producing plants tolerant with respect to a herbicidal compound having as a target an arogenate dehydrogenase enzyme , characterized in that said plants are transformed with a gene encoding a prephenate dehydrogenase enzyme in such a way that they express it in their tissues . the transgenic plants according to the invention may also contain , in addition to a gene encoding a prephenate dehydrogenase enzyme , at least one other gene containing a polynucleotide encoding a protein of interest . among these polynucleotides encoding a protein of interest , mention may be made of polynucleotides encoding an enzyme for resistance to a herbicide , for example the polynucleotide encoding the bar enzyme ( white et al ., nar 18 : 1062 , 1990 ) for tolerance to bialaphos , the polynucleotide encoding the epsps enzyme ( u . s . pat . no . 5 , 188 , 642 ; wo 97 / 04103 ) for tolerance to glyphosate , or else the polynucleotide encoding the hppd enzyme ( wo 96 / 38567 ) for tolerance to isoxazoles . mention may also be made of a polynucleotide encoding an insecticidal toxin , for example a polynucleotide encoding a toxin of bacterium bacillus thuringiensis ( for example , see international patent application wo 98 / 40490 ). other polynucleotides for resistance to diseases may also be contained in these plants , for example a polynucleotide encoding the oxaylate oxidase enzyme as described in patent application ep 0 531 498 or u . s . pat . no . 5 , 866 , 778 , or a polynucleotide encoding another antibacterial and / or antifungal peptide , such as those described in patent applications wo 97 / 30082 , wo 99 / 24594 , wo 99 / 02717 , wo 99 / 53053 and wo 99 / 91089 . mention may also be made of polynucleotides encoding agronomic characteristics of the plant , in particular a polynucleotide encoding a delta - 6 desaturase enzyme , as described in u . s . pat . nos . 5 , 552 , 306 and 5 , 614 , 313 , and patent applications wo 98 / 46763 and wo 98 / 46764 , or a polynucleotide encoding a serine acetyltransferase ( sat ) enzyme , as described in patent applications wo 00 / 01833 and pct / fr 99 / 03179 . the following examples make it possible to illustrate the present invention without , however , limiting the scope thereof . identification of the gene encoding the arabidopsis thaliana arogenate dehydrogense enzyme a comparison of the sequences of all the prephenate dehydrogenase and arogenate dehydrogenase enzymes currently available in the public databases ( http :// www / ncbi / nlm / nih / gov ) revealed four short portions of homologous sequences . the enzymes compared are yeast prephenate dehydrogenase ( accession number : z36065 ), bacillus subtilis prephenate dehydrogenase ( accession number : m80245 ) and synechocystis prephenate dehydrogenase ( accession number : d90910 ). these portions of homology made it possible to identify an a . thaliana gene ( accession number : af096371 ) initially noted as encoding an enzyme “ similar to the specific d - isomer 2 - hydroxy acid dehydrogenase ”. this gene consists of two exons separated by a 94 bp intron . the first exon comprises a 1 . 08 kb open reading frame containing a putative chloroplast transit peptide sequence located downstream of the first atg codon . the second exon potentially encodes an 892 bp open reading frame . a very strong homology of approximately 60 % exists between the protein sequences deduced from the two exons . this homology extends to 70 % if the putative chloroplast transit peptide sequence located in the first exon is not taken into account . in addition , each one of the two predicted protein sequences has the size and possesses the four homologous portions characteristic of the prephenate / arogenate dehydrogenase enzymes . this gene was named tyra ( seq id no : 1 ). the size of the transcript of the tyra gene was determined using the northern blotting and pcr techniques . purified mrnas extracted from young leaves of a . thaliana were hybridized with 32 p - radiolabeled probes corresponding to fragments of dna of the two exons of tyra . this analysis made it possible to identify a 1 . 8 - 1 . 9 kb transcript very close to the presumed size of an mrna containing the two exons . in addition , although the complete cdna could not be amplified by pcr , a 1 . 5 kb pcr fragment was obtained . this fragment comprises the 5 ′ oligonucleotide ( p8 = 5 ′- gctaaaactcttctccttcaatacttacctg - 3 ′) beginning at position 513 bp , and the 3 ′ oligonucleotide ( p7 = 5 ′- cagtataattagtagtcaaggatcctgactgagag - 3 ′) complementary to the 3 &# 39 ; utr and beginning at position 2053 bp . this fragment contains a portion of the first coding sequence ( tyra - at1 ) and the complete sequence of the second coding sequence ( tyra - at2 ). analysis of the sequence of this cdna confirmed the splicing of the intron . the results of the analyses by northern blotting and pcr strongly suggests the existence of an mrna transcript containing the two coding regions tyra - at1 ( seq id no : 4 ) and tyra - at2 ( seq id no : 6 ). preparation of constructs containing the various coding sequences of the a . thaliana arogenate dehydrogenase the first exon tyra - at1 was obtained by pcr amplification of the genomic dna of a . thaliana with the oligonucleotide p1 ( 5 ′- tctc catatg atctttcaatctcat - tctcatc - 3 ′) which introduces an nde i restriction site ( underlined ) at the first atg codon , and the oligonucleotide p2 ( 5 ′- ctaactaactaa cta cata - cctcatcatatcc - 3 ′) which is complementary to the 3 ′ end of the first exon and to the 5 ′ end of the intron and introduces a stop codon ( underlined ). three constructs lacking the sequence encoding the transit peptide were also produced with the oligonucleotide p3 ( 5 ′- cctctctttcc atatg ctcccttctc - 3 ′) which introduces an nde i restriction site ( underlined ) at the second atg codon ( m43 ) at position 127 , the oligonucleotide p4 ( 5 ′- ccgccagccacctc catatg accgacaccatcc - 3 ′) which introduces an atg initiating codon and an nde i restriction site ( underlined ) at position 174 from the first atg codon ( v58m ), and the oligonucleotide p5 ( 5 ′- cgccacccct catatg cgtatcgcc - 3 ′) which introduces an atg initiating codon and an nde i restriction site ( underlined ) at position 222 from the first atg codon ( l75m ). all the ocr fragments corresponding to the first exon , which may or may not encode a transit peptide , were cloned into the plasmid ppcr - script ( stratagene ). nde i - bamh i dna fragments containing the coding sequences , with or without the transit peptide sequence , were then cloned into the plasmid pet21 a (+) ( novagen ), leading to the development of the plasmids pet21 - tyra - at1 , with and without transit peptide sequence ( pet21 - tyra - at1 - m1 , pet21 - tyra - at1 - m43 , pet21 - tyra - ati - m58 and pet21 - tyra - ati - m75 ). two other oligonucleotides were used to amplify the second coding sequence ( tyra - at2 ). the oligonucleotide p6 ( 5 ′- gatgcatctttg catatg atgaggtcagaagatg - 3 ′) introduces an nde i restriction site ( underlined ) at the atg codon of the second open reading frame ( at position 1081 from the first atg codon ), and the oligonucleotide p7 ( 5 ′- cagtataattagtagtcaaggatcctgactgagag - 3 ′), complementary to the start of the 3 ′- utr , introduces a bamh i restriction site ( underlined ). the pcr fragment corresponding to the second coding sequence was digested with nde i - bamh i and then cloned into the plasmid pet21 a (+), giving the plasmid pet21 - tyra - at2 . the complete coding sequence was reconstituted by assembly of the missing 5 ′ end of the first exon with a partial tyra - at cdna ( 1 . 5 kb ), obtained by pcr amplification of the arabidopsis cdna with the oligonucleotide p8 ( 5 ′- gctaaaactcttctccttcaatacttacctg - 3 ′) beginning at position 513 bp from the first atg codon , and the 3 ′ oligonucleotide p7 . an ecorv restriction site located at position 812 bp from the first atg codon and present in the 5 ′ end of the partial tyra - at cdna was used for the reconstitution . the partial tyra - at cdna was cloned into the plasmid ppcr - script . an ecorv - ecorv fragment was obtained from the plasmid ppcr - script - tyra - at and then cloned into the plasmid ppcr - script - tyra - at1 digested beforehand with ecorv . this manipulation led to the plasmid ppcr - script - tyra - atc being obtained . an nde1 - bamh1 fragment containing the complete coding sequence was excised from the plasmid ppcr - script - tyra - atc , and then cloned into a plasmid pet21a (+) ( novagen ), digested beforehand with nde1 and bamh1 , producing the plasmid pet2la (+)- tyra - atc . then , in the same way as for the first exon , four plasmids pet21a (+)- tyra - atc were obtained ; a plasmid containing the complete coding sequence with the sequence encoding the putative transit peptide , and three plasmids lacking this transit peptide sequence , which was cleaved at three different sites ( m43 , v58 and l75 , see above ). for all the constructs described above , the cdna inserts were sequenced in order to be sure that no unwanted mutation had been introduced during the pcr amplification . the arogenate dehydrogenase activity is measured at 25 ° c . by spectrophotometric monitoring , at 340 nm , of the formation of nadh or nadph in a solution containing 50 mm of tris - hcl , ph 8 . 6 , 300 μm of arogenate and 1 mm of nad or nadph in a total volume of 200 μl . the prephenate dehydrogenase activity is measured at 25 ° c . by spectrophotometric monitoring , at 340 nm , of the formation of nadh or nadph in a solution containing 50 mm of tris - hcl , ph 8 . 6 , 300 μm of prephenate and 1 mm of nad or nadph in a total volume of 200 μl . [ 0070 ] eshcerichia coli at2471 cells were transformed with each one of the plasmids pet21 - tyra - at obtained in example 3 , and then cultured at 37 ° c . in 2 liters of luria - bertani medium supplemented with 100 μg / ml of carbenicillin . when the culture had reached the equivalent of an absorbance at 600 nm ( a600 ) of 0 . 6 , 1 mm of isopropyl - β - d - thiogalactoside was added to the culture medium in order to induce recombinant protein synthesis . the cells were then cultured for 16 h at 28 ° c ., harvested , and then centrifuged for 20 min at 40 000 g . the pellet was then resuspended in a 50 mm tris - hcl buffer , ph 7 . 5 , containing 1 mm edta , 1 mm dithiothreitol , 1 mm benzamidine hcl and 5 mm aminocaproic acid , and then sonicated ( 100 pulses every 3 seconds at power 5 ) with a vibra - cell disrupter ( sonics and materials , danbury , conn ., usa ). the crude extracts thus obtained were then centrifuged for 20 min at 40 000 g , and the supernatants were used directly for the enzyme assays . the sds - page analyses of total protein extracts of the e . coli strain at 2471 containing the various constructs pet21 - tyra - atc , pet21 - tyra - at1 and pet21 - tyra - at2 revealed the presence of three recombinant proteins having molecular masses of 66 - 68 kda , 35 kda and 33 - 34 kda , respectively . these molecular masses correspond well to the masses deduced from their respective coding sequences ( 68786 da for tyra - atc , 34966 da for tyr - a - at1 , and 34069 da for tyr - a - at2 ). for the transformants containing the complete coding sequence ( tyra - atc ) and the first coding sequence ( tyra - at1 ), recombinant proteins were observed only with the constructs encoding the proteins m58 - tyra - atc and m58 - tyra - at1 . the three recombinant proteins were mainly found in the protein bodies . however , the presence of small amounts of recombinant proteins in the soluble protein extracts of e . coli made it possible to characterize the biochemical properties . identification and biochemical characterization of the arabidopsis thaliana arogenate dehydrogenase enzymes the biochemical characterization of the recombinant arogenate dehydrogenase enzymes was carried out using the soluble protein extracts of the transformed e . coli strains . the arogenate dehydrogenase activity was measured according to the method described in example 4 . a strictly nadp - dependent arogenate dehydrogenase activity was demonstrated for each one of the three recombinant enzymes . no arogenate dehydrogenase activity was detected in the presence of nad , and no prephenate dehydrogenase activity was detected whatever the cofactor used ( nadp or nad ) and whatever the protein tested ( tyra - atc , tyra - at1 or tyra - at2 ). in addition , prephenate at a concentration of 1 mm does not inhibit the arogenate dehydrogenase activity of the three recombinant enzymes . each one of these enzymes has a michaelis - menten - type behavior , and their km value for arogenate and nadp is relatively the same ( fig2 and 3 ). the michaelis constants for nadp are , respectively , 40 μm for tyra - atc , 60 μm for tyra - at1 , and 20 μm for tyra - at2 . the michaelis constants for arogenate are , respectively , 70 μm for tyra - atc , 45 μm for tyra - at1 , and 45 μm for tyra - at2 . in addition , like the other plant arogenate dehydrogenases ( byng et al ., 1981 , phytochemistry 6 , 1289 - 1292 ; connelly and conn , 1986 , z . naturforsch 41c , 69 - 78 ; gaines et al ., 1982 planta 156 , 233 - 240 ), the arabidopsis arogenate dehydrogenases are all very sensitive to tyrosine , the product of the enzyme reaction , and insensitive to 1 mm of phenylalanine and 1 mm of p - hydroxyphenylpyruvate . the inhibition by tyrosine is competitive with respect to arogenate ( ki of 14 μm for tyra - atc , 8 μm for tyra - at1 , and 12 μm for tyra - at2 ), and noncompetitive with respect to nadp . the sequence of the gene encoding the a . thaliana arogenate dehyrogenase identified in example 1 ( tyra ) made it possible to identify an arogenate dehydrogenase gene in the bacterium synechocystis ( accession number : 1652956 ). this gene was originally described as encoding a “ prephenate dehydrogenase ” enzyme . it was isolated from a synechocystis genomic library and the enzyme was produced in the same way as the a . thaliana enzyme , according to the protocol described in example 5 . biochemical characterization of the enzyme produced made it possible to demonstrate that it is an arogenate dehydrogenase enzyme and not a prephenate dehydrogenase enzyme . this biochemical characterization of the synechocystis arogenate dehydrogenase enzyme was carried out using the purified soluble protein extracts of the transformed e . coli strains . the arogenate dehydrogenase activity was measured according to the method described in example 4 . a strictly nadp - dependent arogenate dehydrogenase activity was demonstrated for this enzyme . no arogenate dehydrogenase activity was detected in the presence of nad , and no prephenate dehydrogenase activity was detected whatever the cofactor used ( nadp or nad ). in addition , prephenate at a concentration of 1 mm does not inhibit the arogenate dehydrogenase activity of this enzyme . the synechocystis arogenate dehydrogenase has a michaelis - menten - type behavior ( fig4 ). the michaelis constant is 6 μm for nadp , and 107 μm for arogenate . the sequence of the gene encoding the a . thaliana arogenate dehydrogenase identified in example 1 ( tyra ) made it possible to identify another arogenate dehydrogenase gene in a . thaliana . this new gene ( accession number : ac0342561 ; seq id no : 8 ) was initially noted as “ containing similarity with the embryo abundance protein ( emb20 ) of picea glauca ”. it also has a putative chloroplast transit peptide sequence , but no repeat region . the sequence of the tyra gene also made it possible to identify two other cdnas encoding arogenate dehydrogenase enzymes in the public est ( expressed sequence tags ) databases . one of these cdnas , which is not complete , corresponds to a tomato cdna ( tc41067 ; seq id no : 22 ). the incomplete nature of this cdna does not make it possible to determine whether it is duplicated like tyra , since its 3 ′ end stops just after the codon corresponding to d356 of tyr - at1 . the second cdna corresponds to a complete cdna of picea glauca ( accession number : l47749 ; seq id no : 10 ) and does not possess a repeat region . this picea glauca cdna was noted as being an “ embryo abundance protein ”. atg atc ttt caa tct cat tct cat cat ctt ctt ctc tat caa tcc tca 48 tct tcc tcc tcc ttc ttc ttc ctc cca aag ctc atc acc aaa cct cct 96 ctc tcc ctc tca ttt acc tct ctt tcc tca atg ctc cct tct ctc tct 144 ctc tcc acc gcc aac cgc cac ctc tcc gtc acc gac acc atc cct ctt 192 leu ser thr ala asn arg his leu ser val thr asp thr ile pro leu ccc aac tcc aac tcc aac gcc acc cct cct ctc cgt atc gcc atc atc 240 gga ttc gga aac tac ggc caa ttc ctt gcc gaa acc cta att tct caa 288 gly phe gly asn tyr gly gln phe leu ala glu thr leu ile ser gln ggc cac att ctc ttc gct cac tcc cga tcc gat cac tcc tcc gcc gct 336 cgc cgt ctc ggt gtc tca tac ttc acc gat ctt cac gat ctc tgc gaa 384 arg arg leu gly val ser tyr phe thr asp leu his asp leu cys glu cgt cat cct gac gta gtc ctt ctc tgt act tca atc ctc tcc ata gag 432 arg his pro asp val val leu leu cys thr ser ile leu ser ile glu aat att ctc aaa acg ttg ccg ttt cag aga ctc cgt cgc aac act ctc 480 ttc gtt gat gtt ctc tcc gtt aaa gag ttt gct aaa act ctt ctc ctt 528 caa tac tta cct gaa gat ttc gat att ctt tgt aca cat cca atg ttt 576 gln tyr leu pro glu asp phe asp ile leu cys thr his pro met phe ggt cct cag agt gtg agt tca aat cat ggc tgg aga gga tta aga ttt 624 gly pro gln ser val ser ser asn his gly trp arg gly leu arg phe gtg tat gat aaa gtt agg att ggg gaa gag aga ttg aga gtc tca agg 672 tgt gag agt ttt ctt gag att ttt gtt aga gaa gga tgt gag atg gtg 720 gag atg agt gtt act gat cat gat aag ttt gct gct gaa tca cag ttt 768 glu met ser val thr asp his asp lys phe ala ala glu ser gln phe ata act cat act ctt ggt agg ctt ttg ggg atg ttg aag ttg ata tcg 816 acg ccg att aat acg aaa ggg tac gag gcg ttg ctt gat tta gct gag 864 thr pro ile asn thr lys gly tyr glu ala leu leu asp leu ala glu aat att tgt ggg gat agt ttt gat ttg tat tat ggg ttg ttt gtg tat 912 aat aac aac tct ttg gag gtg tta gag agg att gat ttg gct ttc gag 960 gct ttg cgt aag gag ctt ttt agt cgg ctt cac ggt gtt gtg agg aag 1008 cag tct ttt gaa ggt gaa gca aag aaa gtt cat gtt ttt cca aat tgt 1056 gln ser phe glu gly glu ala lys lys val his val phe pro asn cys ggt gaa aat gat gct tct ttg gat atg atg agg tca gaa gat gtt gtt 1104 gtg aag tat gaa tat aac tcc cag gtg tct ggt agt gtt aat gac ggt 1152 tcg agg ctc aag att ggt atc gtc ggg ttt gga aat ttt gga cag ttt 1200 cta ggt aaa acc atg gtc aag cag ggt cac act gtg tta gct tat tcc 1248 leu gly lys thr met val lys gln gly his thr val leu ala tyr ser aga agt gac tac act gat gaa gca gca aag ctc ggt gtt tcg tat ttt 1296 arg ser asp tyr thr asp glu ala ala lys leu gly val ser tyr phe tca gat ctt gat gat cta ttt gaa gag cat cct gaa gtt att att ctc 1344 tgt acg tca atc ctt tcg act gaa aaa gtt ctc gag tca cta ccg ttt 1392 cag aga ctg aag aga agc aca ctt ttt gtg gat gta ctc tca gta aaa 1440 gag ttc ccg agg aat tta ttt ctt caa act ctc cca caa gat ttt gat 1488 att ttg tgc acg cat cct atg ttt ggg cca gag agt ggt aaa aat gga 1536 ile leu cys thr his pro met phe gly pro glu ser gly lys asn gly tgg aac aat ctt gcc ttt gtg ttt gat aag gtt agg att gga atg gat 1584 trp asn asn leu ala phe val phe asp lys val arg ile gly met asp gat aga aga aaa tcg agg tgt aac agt ttt ctt gat att ttt gcc cgt 1632 gaa gga tgt cgt atg gtg gag atg tcg tgt gct gaa cat gat tgg cat 1680 glu gly cys arg met val glu met ser cys ala glu his asp trp his gct gct gga tca cag ttt atc aca cac aca gtg gga agg ctt ctg gag 1728 ala ala gly ser gln phe ile thr his thr val gly arg leu leu glu aag ctg agc ttg gaa tct act cct ata gat acc aaa ggt tat gag aca 1776 ttg cta aaa ctg gtg gag aat act gct ggt gac agc ttt gat ctg tac 1824 leu leu lys leu val glu asn thr ala gly asp ser phe asp leu tyr tat gga cta ttt tta tac aat cct aat gca atg gaa cag ctt gag agg 1872 tyr gly leu phe leu tyr asn pro asn ala met glu gln leu glu arg ttt cat gtg gct ttt gaa tca ttg aag aca cag ctc ttt gga cga cta 1920 phe his val ala phe glu ser leu lys thr gln leu phe gly arg leu cat tct caa cat tct cat gag cta gct aaa tca tct tcc cca aag aca 1968 leu ser thr ala asn arg his leu ser val thr asp thr ile pro leu gly phe gly asn tyr gly gln phe leu ala glu thr leu ile ser gln arg arg leu gly val ser tyr phe thr asp leu his asp leu cys glu arg his pro asp val val leu leu cys thr ser ile leu ser ile glu gln tyr leu pro glu asp phe asp ile leu cys thr his pro met phe gly pro gln ser val ser ser asn his gly trp arg gly leu arg phe glu met ser val thr asp his asp lys phe ala ala glu ser gln phe thr pro ile asn thr lys gly tyr glu ala leu leu asp leu ala glu gln ser phe glu gly glu ala lys lys val his val phe pro asn cys leu gly lys thr met val lys gln gly his thr val leu ala tyr ser arg ser asp tyr thr asp glu ala ala lys leu gly val ser tyr phe ile leu cys thr his pro met phe gly pro glu ser gly lys asn gly trp asn asn leu ala phe val phe asp lys val arg ile gly met asp glu gly cys arg met val glu met ser cys ala glu his asp trp his ala ala gly ser gln phe ile thr his thr val gly arg leu leu glu leu leu lys leu val glu asn thr ala gly asp ser phe asp leu tyr tyr gly leu phe leu tyr asn pro asn ala met glu gln leu glu arg phe his val ala phe glu ser leu lys thr gln leu phe gly arg leu atg acc gac acc atc cct ctt ccc aac tcc aac tcc aac gcc acc cct 48 cct ctc cgt atc gcc atc atc gga ttc gga aac tac ggc caa ttc ctt 96 gcc gaa acc cta att tct caa ggc cac att ctc ttc gct cac tcc cga 144 ala glu thr leu ile ser gln gly his ile leu phe ala his ser arg tcc gat cac tcc tcc gcc gct cgc cgt ctc ggt gtc tca tac ttc acc 192 ser asp his ser ser ala ala arg arg leu gly val ser tyr phe thr gat ctt cac gat ctc tgc gaa cgt cat cct gac gta gtc ctt ctc tgt 240 act tca atc ctc tcc ata gag aat att ctc aaa acg ttg ccg ttt cag 288 aga ctc cgt cgc aac act ctc ttc gtt gat gtt ctc tcc gtt aaa gag 336 ttt gct aaa act ctt ctc ctt caa tac tta cct gaa gat ttc gat att 384 phe ala lys thr leu leu leu gln tyr leu pro glu asp phe asp ile ctt tgt aca cat cca atg ttt ggt cct cag agt gtg agt tca aat cat 432 leu cys thr his pro met phe gly pro gln ser val ser ser asn his ggc tgg aga gga tta aga ttt gtg tat gat aaa gtt agg att ggg gaa 480 gly trp arg gly leu arg phe val tyr asp lys val arg ile gly glu gag aga ttg aga gtc tca agg tgt gag agt ttt ctt gag att ttt gtt 528 aga gaa gga tgt gag atg gtg gag atg agt gtt act gat cat gat aag 576 arg glu gly cys glu met val glu met ser val thr asp his asp lys ttt gct gct gaa tca cag ttt ata act cat act ctt ggt agg ctt ttg 624 phe ala ala glu ser gln phe ile thr his thr leu gly arg leu leu ggg atg ttg aag ttg ata tcg acg ccg att aat acg aaa ggg tac gag 672 gly met leu lys leu ile ser thr pro ile asn thr lys gly tyr glu gcg ttg ctt gat tta gct gag aat att tgt ggg gat agt ttt gat ttg 720 tat tat ggg ttg ttt gtg tat aat aac aac tct ttg gag gtg tta gag 768 agg att gat ttg gct ttc gag gct ttg cgt aag gag ctt ttt agt cgg 816 ctt cac ggt gtt gtg agg aag cag tct ttt gaa ggt gaa gca aag aaa 864 leu his gly val val arg lys gln ser phe glu gly glu ala lys lys gtt cat gtt ttt cca aat tgt ggt gaa aat gat gct tct ttg gat atg 912 val his val phe pro asn cys gly glu asn asp ala ser leu asp met ala glu thr leu ile ser gln gly his ile leu phe ala his ser arg ser asp his ser ser ala ala arg arg leu gly val ser tyr phe thr phe ala lys thr leu leu leu gln tyr leu pro glu asp phe asp ile leu cys thr his pro met phe gly pro gln ser val ser ser asn his gly trp arg gly leu arg phe val tyr asp lys val arg ile gly glu arg glu gly cys glu met val glu met ser val thr asp his asp lys phe ala ala glu ser gln phe ile thr his thr leu gly arg leu leu gly met leu lys leu ile ser thr pro ile asn thr lys gly tyr glu leu his gly val val arg lys gln ser phe glu gly glu ala lys lys val his val phe pro asn cys gly glu asn asp ala ser leu asp met atg atg agg tca gaa gat gtt gtt gtg aag tat gaa tat aac tcc cag 48 gtg tct ggt agt gtt aat gac ggt tcg agg ctc aag att ggt atc gtc 96 ggg ttt gga aat ttt gga cag ttt cta ggt aaa acc atg gtc aag cag 144 ggt cac act gtg tta gct tat tcc aga agt gac tac act gat gaa gca 192 gly his thr val leu ala tyr ser arg ser asp tyr thr asp glu ala gca aag ctc ggt gtt tcg tat ttt tca gat ctt gat gat cta ttt gaa 240 gag cat cct gaa gtt att att ctc tgt acg tca atc ctt tcg act gaa 288 aaa gtt ctc gag tca cta ccg ttt cag aga ctg aag aga agc aca ctt 336 ttt gtg gat gta ctc tca gta aaa gag ttc ccg agg aat tta ttt ctt 384 caa act ctc cca caa gat ttt gat att ttg tgc acg cat cct atg ttt 432 ggg cca gag agt ggt aaa aat gga tgg aac aat ctt gcc ttt gtg ttt 480 gly pro glu ser gly lys asn gly trp asn asn leu ala phe val phe gat aag gtt agg att gga atg gat gat aga aga aaa tcg agg tgt aac 528 agt ttt ctt gat att ttt gcc cgt gaa gga tgt cgt atg gtg gag atg 576 ser phe leu asp ile phe ala arg glu gly cys arg met val glu met tcg tgt gct gaa cat gat tgg cat gct gct gga tca cag ttt atc aca 624 ser cys ala glu his asp trp his ala ala gly ser gln phe ile thr cac aca gtg gga agg ctt ctg gag aag ctg agc ttg gaa tct act cct 672 ata gat acc aaa ggt tat gag aca ttg cta aaa ctg gtg gag aat act 720 gct ggt gac agc ttt gat ctg tac tat gga cta ttt tta tac aat cct 768 aat gca atg gaa cag ctt gag agg ttt cat gtg gct ttt gaa tca ttg 816 asn ala met glu gln leu glu arg phe his val ala phe glu ser leu aag aca cag ctc ttt gga cga cta cat tct caa cat tct cat gag cta 864 gct aaa tca tct tcc cca aag aca act aag cta tta act agc taa 909 gly his thr val leu ala tyr ser arg ser asp tyr thr asp glu ala gly pro glu ser gly lys asn gly trp asn asn leu ala phe val phe ser phe leu asp ile phe ala arg glu gly cys arg met val glu met ser cys ala glu his asp trp his ala ala gly ser gln phe ile thr asn ala met glu gln leu glu arg phe his val ala phe glu ser leu atg cta ctc cat ttc tct ccg gcg aaa ccc ctc att tct cca ccc aat 48 ctc cgc cgc aat tca ccc aca ttc ctc att tcc ccg ccg cga tct ctt 96 cga att cga gca atc gac gcc gcc caa atc ttc gat tac gaa acc caa 144 ctc aaa tcc gag tac cgc aaa tcc tct gct ctc aaa atc gcc gtc ttg 192 ggt ttc ggc aat ttc ggc caa ttc ctc tcc aaa acc cta att cga cac 240 gly phe gly asn phe gly gln phe leu ser lys thr leu ile arg his ggc cac gat cta atc act cac tcc cgc tcc gat tac tcc gac gcc gca 288 aac tca atc gga gct cgt ttc ttc gat aac cct cac gat ctc tgt gaa 336 asn ser ile gly ala arg phe phe asp asn pro his asp leu cys glu caa cat ccc gac gtt gtc ctc ctc tgt acc tca atc ctc tcc aca gaa 384 gln his pro asp val val leu leu cys thr ser ile leu ser thr glu tca gtc ctc aga tca ttc cct ttc caa cgt ctc cgt cgt agc aca ctc 432 ttc gtc gat gtt ctc tcc gtt aag gaa ttc cca aaa gcc ctc ttc att 480 aaa tac ctt cct aag gag ttt gac att ctc tgt act cat cca atg ttt 528 lys tyr leu pro lys glu phe asp ile leu cys thr his pro met phe gga cct gag agt ggt aag cat tct tgg tct ggc ttg ccc ttt gtc tac 576 gly pro glu ser gly lys his ser trp ser gly leu pro phe val tyr gat aag gtg aga atc gga gac gca gct tca aga caa gag agg tgt gag 624 asp lys val arg ile gly asp ala ala ser arg gln glu arg cys glu aag ttt cta aga att ttt gag aat gaa ggt tgc aag atg gtt gaa atg 672 lys phe leu arg ile phe glu asn glu gly cys lys met val glu met agc tgt gag aag cat gat tat tac gca gct gga tcg caa ttc gtg acg 720 ser cys glu lys his asp tyr tyr ala ala gly ser gln phe val thr cat act atg gga agg gtt ttg gag aaa tat gga gtt gag tct tcg ccg 768 his thr met gly arg val leu glu lys tyr gly val glu ser ser pro att aac acc aaa ggt tat gag acg ttg ttg gat ttg gtg gag aac aca 816 tcg agt gat agc ttt gag ctt ttc tac ggt ttg ttt atg tat aat ccg 864 aat gct ctt gaa cag ttg gag aga ttg gat atg gct ttt gag tct gtt 912 asn ala leu glu gln leu glu arg leu asp met ala phe glu ser val aag aag gag ctg ttt ggg aga tta cat cag caa tac agg aag caa atg 960 ttt ggt ggg gag gtt caa tcg ccc aag aaa act gag cag aaa ttg ctc 1008 gly phe gly asn phe gly gln phe leu ser lys thr leu ile arg his asn ser ile gly ala arg phe phe asp asn pro his asp leu cys glu gln his pro asp val val leu leu cys thr ser ile leu ser thr glu lys tyr leu pro lys glu phe asp ile leu cys thr his pro met phe gly pro glu ser gly lys his ser trp ser gly leu pro phe val tyr asp lys val arg ile gly asp ala ala ser arg gln glu arg cys glu lys phe leu arg ile phe glu asn glu gly cys lys met val glu met ser cys glu lys his asp tyr tyr ala ala gly ser gln phe val thr his thr met gly arg val leu glu lys tyr gly val glu ser ser pro asn ala leu glu gln leu glu arg leu asp met ala phe glu ser val ccaagccctc ttctcagaaa actccctccg cggca atg cct ctt cat ttc tca 113 tgg aat cca aca gaa gac cct cac aca gta cgc cct act gag gct ctc 161 trp asn pro thr glu asp pro his thr val arg pro thr glu ala leu agg aat cag agc aat gga cgt cgc ggg gcc cct cga tta aga aga ata 209 aaa tcc att aaa tat tgg cat cgt agg gtt tgg aaa cta cca cca att 257 tct ggt gaa aac cat ggt gaa gcc ggg cca ccc ggt gct cgc cca ttc 305 cag gac gga cta tac gga ggc cac tgc gag atc ggg gtt caa ttc ttc 353 gln asp gly leu tyr gly gly his cys glu ile gly val gln phe phe aga gac gcg gac gat ttc tgc gaa gag cat cca gag atc ata ctg atg 401 arg asp ala asp asp phe cys glu glu his pro glu ile ile leu met tgc gca tcc atc act ttg gtg gga gga cgt gct gaa gtc tct gcc aac 449 cys ala ser ile thr leu val gly gly arg ala glu val ser ala asn aca gcg cct gaa gag gag tac gct ttt cgc aga cgt cct gtc tgt gaa 497 aga gtt tcc gca ccg gtt gtt cct gca ggt ttt gtc gcc cga gtc gat 545 gtg ctg tgc act cat ccc atg ttt ggt cca gag agc agc aag gac gat 593 val leu cys thr his pro met phe gly pro glu ser ser lys asp asp ttg ggc gac ctc cct ttc gtt tac gat aag gtt agg gtt tct aac gaa 641 leu gly asp leu pro phe val tyr asp lys val arg val ser asn glu ggt ttg aga gcc aag cac tgc gag cgt ttt ctc aac ata ttt tcg tgc 689 gly leu arg ala lys his cys glu arg phe leu asn ile phe ser cys gag ggc tgc cgg atg gtc gag atg tcg tgt gca gaa cat gat cgc tat 737 glu gly cys arg met val glu met ser cys ala glu his asp arg tyr gtc gcg gag agc caa ttc att acc cac acc gtt ggg agg atg ttg ggg 785 val ala glu ser gln phe ile thr his thr val gly arg met leu gly agg ctg ggc ttg gag tcc act ccg att gct acc aag ggt tat gag aaa 833 arg leu gly leu glu ser thr pro ile ala thr lys gly tyr glu lys tta ctg gaa gtg gcc tgg aat att gcc ggg gat agt ttt gat att tat 881 leu leu glu val ala trp asn ile ala gly asp ser phe asp ile tyr tat gga ctc ttc atg tat aat gtc aat tcg att gaa caa atc gag agg 929 tyr gly leu phe met tyr asn val asn ser ile glu gln ile glu arg tta gat atg gcg ttc aat tca ctc aag aac gag gtt tcg ggt tca aat 977 leu asp met ala phe asn ser leu lys asn glu val ser gly ser asn met pro leu his phe ser trp asn pro thr glu asp pro his thr val trp lys leu pro pro ile ser gly glu asn his gly glu ala gly pro pro gly ala arg pro phe gln asp gly leu tyr gly gly his cys glu ile gly val gln phe phe arg asp ala asp asp phe cys glu glu his pro glu ile ile leu met cys ala ser ile thr leu val gly gly arg phe val ala arg val asp val leu cys thr his pro met phe gly pro val arg val ser asn glu gly leu arg ala lys his cys glu arg phe leu asn ile phe ser cys glu gly cys arg met val glu met ser cys ala glu his asp arg tyr val ala glu ser gln phe ile thr his thr val gly arg met leu gly arg leu gly leu glu ser thr pro ile ala thr lys gly tyr glu lys leu leu glu val ala trp asn ile ala gly ile glu gln ile glu arg leu asp met ala phe asn ser leu lys asn atg aaa att ggt gtt gtt ggt ttg ggt tta att ggg gct tcc ttg gcg 48 gga gac ttg cgt cgt cgg ggc cat tat ttg att ggg gtt tct cgg caa 96 caa agc acc tgt gaa aaa gca gtg gaa aga caa ttg gtg gat gaa gcg 144 gln ser thr cys glu lys ala val glu arg gln leu val asp glu ala ggt caa gat tta tct ctt ctc caa aca gca aaa ata att ttt ctt tgt 192 gly gln asp leu ser leu leu gln thr ala lys ile ile phe leu cys act cct ata caa tta att ttg cct acc cta gag aag ctt att ccc cat 240 cta tcg ccc aca gcc att gtc act gat gtg gcc tct gta aaa acg gcg 288 atc gcc gag ccg gcc agt caa ctt tgg tct ggg ttc att ggt ggt cac 336 ile ala glu pro ala ser gln leu trp ser gly phe ile gly gly his ccc atg gcc ggc aca gcg gcc cag ggc atc gac ggg gcg gaa gaa aat 384 tta ttt gtc aac gct ccc tat gtg ctc act ccc acc gaa tat act gac 432 cca gag caa ttg gct tgt tta cgt tca gtg ttg gaa ccc ctg ggg gta 480 aaa att tac ctc tgc act ccc gca gac cat gac caa gca gta gcc tgg 528 lys ile tyr leu cys thr pro ala asp his asp gln ala val ala trp att tcc cat tta cct gta atg gtg agt gct gct tta atc caa gcc tgt 576 gcc ggt gaa aaa gat ggg gat att ctc aaa cta gcc caa aat ttg gcc 624 agt tcg ggt ttt cgg gat acc agt cgg gtg gga ggc ggc aac ccg gag 672 ttg ggc acc atg atg gcc acc tat aac caa cgg gct ttg cta aaa agt 720 leu gly thr met met ala thr tyr asn gln arg ala leu leu lys ser ttg caa gac tat cgt cag cac ctg gat cag cta att acc cta att agt 768 aac caa caa tgg cct gaa ctc cat cgt ctt tta caa caa acc aac ggc 816 gln ser thr cys glu lys ala val glu arg gln leu val asp glu ala gly gln asp leu ser leu leu gln thr ala lys ile ile phe leu cys ile ala glu pro ala ser gln leu trp ser gly phe ile gly gly his lys ile tyr leu cys thr pro ala asp his asp gln ala val ala trp leu gly thr met met ala thr tyr asn gln arg ala leu leu lys ser atg gta tca gag gat aag att gag caa tgg aaa gcc aca aaa gtc att 48 met val ser glu asp lys ile glu gln trp lys ala thr lys val ile ggt ata att ggt ctg ggt gat atg ggc cta tta tac gct aat aaa ttt 96 aca gat gct gga tgg ggt gtt ata tgt tgt gat agg gaa gaa tat tat 144 thr asp ala gly trp gly val ile cys cys asp arg glu glu tyr tyr gat gaa ctg aaa gaa aaa tat gcc tca gct aaa ttc gaa ctg gtg aaa 192 aat ggt cat ttg gta tcc agg caa agc gac tat att atc tat agt gtt 240 asn gly his leu val ser arg gln ser asp tyr ile ile tyr ser val gaa gca tcc aat att agt aag atc gtc gca acg tat gga cca tct tct 288 glu ala ser asn ile ser lys ile val ala thr tyr gly pro ser ser aag gtt gga aca att gtt ggg ggt caa acg agt tgt aag ctg ccg gaa 336 lys val gly thr ile val gly gly gln thr ser cys lys leu pro glu atc gag gct ttc gaa aag tat tta ccc aag gac tgc gac atc att acc 384 ile glu ala phe glu lys tyr leu pro lys asp cys asp ile ile thr gtg cat tcc ctt cat ggg cct aaa gtt aat act gaa ggc caa cca cta 432 val his ser leu his gly pro lys val asn thr glu gly gln pro leu gtt att atc aat cac aga tca cag tac cca gaa tct ttt gag ttc gtt 480 val ile ile asn his arg ser gln tyr pro glu ser phe glu phe val aat tct gtt atg gca tgt ttg aaa agt aag caa gtt tat ttg aca tat 528 asn ser val met ala cys leu lys ser lys gln val tyr leu thr tyr gaa gag cat gac aag att acc gct gat aca caa gct gtg aca cat gct 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ctt ggt att gat cca tat gat cat atg att tgt tcg acg cca tta ttc 1056 leu gly ile asp pro tyr asp his met ile cys ser thr pro leu phe aga ata ttc ctg ggt gtg tcc gaa tat ctt ttt tta aaa cct ggc tta 1104 arg ile phe leu gly val ser glu tyr leu phe leu lys pro gly leu tta gaa cag aca att gat gca gct atc cat gat aaa tca ttc ata aaa 1152 leu glu gln thr ile asp ala ala ile his asp lys ser phe ile lys gat gat tta gaa ttt gtt att tcg gct aga gaa tgg agc tcg gtt gtt 1200 tct ttt gcc aat ttt gat ata tac aaa aag caa ttt cag agt gtt caa 1248 aag ttc ttt gag cca atg ctt cca gag gct aat ctc att ggc aac gag 1296 atg ata aaa acc att ctg agt cat tct agt gac cgt tcg gcc gct gaa 1344 met ile lys thr ile leu ser his ser ser asp arg ser ala ala glu met val ser glu asp lys ile glu gln trp lys ala thr lys val ile thr asp ala gly trp gly val ile cys cys asp arg glu glu tyr tyr asn gly his leu val ser arg gln ser asp tyr ile ile tyr ser val glu ala ser asn ile ser lys ile val ala thr tyr gly pro ser ser lys val gly thr ile val gly gly gln thr ser cys lys leu pro glu ile glu ala phe glu lys tyr leu pro lys asp cys asp ile ile thr val his ser leu his gly pro lys val asn thr glu gly gln pro leu val ile ile asn his arg ser gln tyr pro glu ser phe glu phe val asn ser val met ala cys leu lys ser lys gln val tyr leu thr tyr ala phe leu ser met gly ser ala trp ala lys ile lys ile tyr pro val asn ile ser leu arg ile tyr ser asn lys trp his val tyr ala gly leu ala ile thr asn pro ser ala his gln gln ile leu gln tyr ala thr ser ala thr glu leu phe ser leu met ile asp asn lys glu gln glu leu thr asp arg leu leu lys ala lys gln phe val phe gly pro asn ser his leu ser leu leu ala ile val asp ser trp phe gln leu gly ile asp pro tyr asp his met ile cys ser thr pro leu phe arg ile phe leu gly val ser glu tyr leu phe leu lys pro gly leu leu glu gln thr ile asp ala ala ile his asp lys ser phe ile lys met ile lys thr ile leu ser his ser ser asp arg ser ala ala glu atg aat caa atg aaa gat aca ata ttg ctc gcc ggt ctc gga ttg ata 48 ggc ggt tcg att gcc cta gcc atc aaa aaa aat cat ccc ggc aaa cgg 96 att atc gga atc gac atc tct gat gaa cag gcg gta gcg gca tta aaa 144 tta ggc gtg ata gac gat cgt gct gat tcg ttt att agc ggt gtg aaa 192 gag gca gct aca gta atc att gcg aca cct gtt gaa caa aca ctg gtt 240 atg ctt gaa gag ctg gct cat tca gga att gaa cat gag ctt ttg att 288 acg gat gta gga agc aca aag caa aaa gtg gtt gat tac gct gat caa 336 gtg ctg cct agc cgc tat caa ttt gtc gga ggg cat ccg atg gcg ggt 384 val leu pro ser arg tyr gln phe val gly gly his pro met ala gly tca cat aaa tca gga gtg gcc gct gcg aag gag ttc ctg ttt gaa aat 432 gca ttt tat att tta acg cca ggc cag aaa acg gac aaa caa gct gtg 480 ala phe tyr ile leu thr pro gly gln lys thr asp lys gln ala val gaa cag tta aaa aac ctg ctg aag ggg acg aat gcc cat ttt gtg gaa 528 glu gln leu lys asn leu leu lys gly thr asn ala his phe val glu atg tcg cca gag gag cat gat ggc gtt aca agc gta atc agt cat ttt 576 met ser pro glu glu his asp gly val thr ser val ile ser his phe ccg cat att gta gca gct agc ctt gtt cac caa acc cat cat tcg gaa 624 aac ctg tat ccg ctt gtt aag cgt ttt gct gcc ggc ggg ttc aga gat 672 asn leu tyr pro leu val lys arg phe ala ala gly gly phe arg asp att aca agg att gca tca agc agc ccg gca atg tgg cgg gat att tta 720 tta cat aat aaa gat aaa atc tta gac cgt ttt gat gag tgg att cgt 768 gaa att gac aag atc cgt aca tat gta gaa caa gaa gat gcg gaa aat 816 glu ile asp lys ile arg thr tyr val glu gln glu asp ala glu asn cta ttt cgt tat ttt aaa aca gcc aag gat tat cgc gac ggg ctg ccg 864 ctt cgg cag aag gga gcg ata cct gca ttt tat gat tta tat gtg gat 912 leu arg gln lys gly ala ile pro ala phe tyr asp leu tyr val asp gta ccc gat cat ccg ggt gta ata tcc gag ata aca gcg atc tta gct 960 val pro asp his pro gly val ile ser glu ile thr ala ile leu ala gcg gag cgc atc agt atc acg aat atc cgc att atc gaa aca cga gag 1008 gat att aac ggg att tta agg atc agt ttt cag tct gat gac gac cgc 1056 aaa agg gca gaa caa tgc att gaa gcc cgg gcg gaa tat gaa act ttt 1104 val leu pro ser arg tyr gln phe val gly gly his pro met ala gly ala phe tyr ile leu thr pro gly gln lys thr asp lys gln ala val glu gln leu lys asn leu leu lys gly thr asn ala his phe val glu met ser pro glu glu his asp gly val thr ser val ile ser his phe asn leu tyr pro leu val lys arg phe ala ala gly gly phe arg asp glu ile asp lys ile arg thr tyr val glu gln glu asp ala glu asn leu arg gln lys gly ala ile pro ala phe tyr asp leu tyr val asp val pro asp his pro gly val ile ser glu ile thr ala ile leu ala atg gtt gct gaa ttg acc gca tta cgc gat caa att gat gaa gtc gat 48 aaa gcg ctg ctg aat tta tta gcg aag cgt ctg gaa ctg gtt gct gaa 96 gtg ggc gag gtg aaa agc cgc ttt gga ctg cct att tat gtt ccg gag 144 val gly glu val lys ser arg phe gly leu pro ile tyr val pro glu cgc gag gca tct atg ttg gcc tcg cgt cgt gca gag gcg gaa gct ctg 192 ggt gta ccg cca gat ctg att gag gat gtt ttg cgt cgg gtg atg cgt 240 gaa tct tac tcc agt gaa aac gac aaa gga ttt aaa aca ctt tgt ccg 288 glu ser tyr ser ser glu asn asp lys gly phe lys thr leu cys pro tca ctg cgt ccg gtg gtt atc gtc ggc ggt ggc ggt cag atg gga cgc 336 ctg ttc gag aag atg ctg acc ctc tcg ggt tat cag gtg cgg att ctg 384 leu phe glu lys met leu thr leu ser gly tyr gln val arg ile leu gag caa cat gac tgg gat cga gcg gct gat att gtt gcc gat gcc gga 432 atg gtg att gtt agt gtg cca atc cac gtt act gag caa gtt att ggc 480 aaa tta ccg cct tta ccg aaa gat tgt att ctg gtc gat ctg gca tca 528 gtg aaa aat ggg cca tta cag gcc atg ctg gtg gcg cat gat ggt ccg 576 val lys asn gly pro leu gln ala met leu val ala his asp gly pro gtg ctg ggg cta cac ccg atg ttc ggt ccg gac agc ggt agc ctg gca 624 aag caa gtt gtg gtc tgg tgt gat gga cgt aaa ccg gaa gca tac caa 672 lys gln val val val trp cys asp gly arg lys pro glu ala tyr gln tgg ttt ctg gag caa att cag gtc tgg ggc gct cgg ctg cat cgt att 720 trp phe leu glu gln ile gln val trp gly ala arg leu his arg ile agc gcc gtc gag cac gat cag aat atg gcg ttt att cag gca ctg cgc 768 ser ala val glu his asp gln asn met ala phe ile gln ala leu arg cac ttt gct act ttt gct tac ggg ctg cac ctg gca gaa gaa aat gtt 816 cag ctt gag caa ctt ctg gcg ctc tct tcg ccg att tac cgc ctt gag 864 ctg gcg atg gtc ggg cga ctg ttt gct cag gat ccg cag ctt tat gcc 912 leu ala met val gly arg leu phe ala gln asp pro gln leu tyr ala gac atc att atg tcg tca gag cgt aat ctg gcg tta atc aaa cgt tac 960 asp ile ile met ser ser glu arg asn leu ala leu ile lys arg tyr tat aag cgt ttc ggc gag gcg att gag ttg ctg gag cag ggc gat aag 1008 tyr lys arg phe gly glu ala ile glu leu leu glu gln gly asp lys cag gcg ttt att gac agt ttc cgc aag gtg gag cac tgg ttc ggc gat 1056 gln ala phe ile asp ser phe arg lys val glu his trp phe gly asp tac gca cag cgt ttt cag agt gaa agc cgc gtg tta ttg cgt cag gcg 1104 val gly glu val lys ser arg phe gly leu pro ile tyr val pro glu glu ser tyr ser ser glu asn asp lys gly phe lys thr leu cys pro leu phe glu lys met leu thr leu ser gly tyr gln val arg ile leu val lys asn gly pro leu gln ala met leu val ala his asp gly pro lys gln val val val trp cys asp gly arg lys pro glu ala tyr gln trp phe leu glu gln ile gln val trp gly ala arg leu his arg ile ser ala val glu his asp gln asn met ala phe ile gln ala leu arg leu ala met val gly arg leu phe ala gln asp pro gln leu tyr ala asp ile ile met ser ser glu arg asn leu ala leu ile lys arg tyr tyr lys arg phe gly glu ala ile glu leu leu glu gln gly asp lys gln ala phe ile asp ser phe arg lys val glu his trp phe gly asp atg gtg gct gaa ctg acc gcg tta cgc gat caa att gac agt gta gat 48 met val ala glu leu thr ala leu arg asp gln ile asp ser val asp aaa gcg ctg ctg gat ctg ctg gct aag cga ctg gaa ctg gtg gcc gag 96 gta ggt gag gtg aag agc cgt tac ggc ctg cct atc tat gtg cct gag 144 cgt gag gcg tcg atg ctg gct tcg cgt cgc aaa gag gcc gaa gcg ctc 192 ggc gta cca ccg gat ctg att gag gat gtg ctg cgt cgc gtg atg cgg 240 gaa tcc tat acc agc gag aat gat aaa ggc ttt aaa acc ctc tgt cct 288 glu ser tyr thr ser glu asn asp lys gly phe lys thr leu cys pro gaa ctg cgc ccg gtg gtg att gtc ggt ggt aag ggc cag atg ggc cgg 336 ctg ttt gaa aaa atg ctc ggg cta tca ggc tac acg gtt aaa acg ctg 384 gat aaa gag gac tgg cct cag gct gag act ctg ctc agc gat gcc gga 432 asp lys glu asp trp pro gln ala glu thr leu leu ser asp ala gly atg gtg atc att agc gtg ccg att cac ctg acc gag cag gtg att gcc 480 met val ile ile ser val pro ile his leu thr glu gln val ile ala caa ctg cca cca ctg ccg gaa gat tgt att ctg gtc gat ctg gcg tca 528 gtc aaa aac cgg cct ctg cag gca atg ctg gct gcc cat aac ggg cct 576 val lys asn arg pro leu gln ala met leu ala ala his asn gly pro gta ctg ggt ctg cat ccg atg ttt ggc ccg gac agc ggc agc ctg gca 624 aaa cag gtg gtg gtc tgg tgt gat gga aga caa ccg gaa gcg tat cag 672 lys gln val val val trp cys asp gly arg gln pro glu ala tyr gln tgg ttc ctg gag cag att cag gtc tgg ggt gcg cgt ctg cat cgt atc 720 trp phe leu glu gln ile gln val trp gly ala arg leu his arg ile agc gct gtt gag cat gac cag aac atg gca ttc att cag gcg ctg cgt 768 ser ala val glu his asp gln asn met ala phe ile gln ala leu arg cac ttt gct acc ttc gct tat ggt ctg cat tta gcc gaa gag aac gtc 816 aat ctg gat cag ctg ctg gcg ctc tcg tcg ccc att tac cgg ctt gaa 864 asn leu asp gln leu leu ala leu ser ser pro ile tyr arg leu glu ctg gcg atg gtg ggg cgg ttg ttc gct cag gat ccg caa ctc tat gcg 912 leu ala met val gly arg leu phe ala gln asp pro gln leu tyr ala gat atc atc atg tct tca gag agt aat ctg gcg ctg ata aaa cgc tat 960 asp ile ile met ser ser glu ser asn leu ala leu ile lys arg tyr tac cag cgg ttt ggt gaa gcg att gcg ctg ctg gag cag ggc gac aag 1008 tyr gln arg phe gly glu ala ile ala leu leu glu gln gly asp lys cag gcg ttt atc gcc agc ttt aac cgg gtt gaa cag tgg ttt ggc gat 1056 gln ala phe ile ala ser phe asn arg val glu gln trp phe gly asp cac gca aaa cgc ttc ctg gtc gaa agc cga agc ctg ttg cga tcg gcc 1104 aat gac agc cgc cca taaaaaaaag gcatccagtt ggatgccttt ttt 1152 met val ala glu leu thr ala leu arg asp gln ile asp ser val asp glu ser tyr thr ser glu asn asp lys gly phe lys thr leu cys pro asp lys glu asp trp pro gln ala glu thr leu leu ser asp ala gly met val ile ile ser val pro ile his leu thr glu gln val ile ala val lys asn arg pro leu gln ala met leu ala ala his asn gly pro lys gln val val val trp cys asp gly arg gln pro glu ala tyr gln trp phe leu glu gln ile gln val trp gly ala arg leu his arg ile ser ala val glu his asp gln asn met ala phe ile gln ala leu arg asn leu asp gln leu leu ala leu ser ser pro ile tyr arg leu glu leu ala met val gly arg leu phe ala gln asp pro gln leu tyr ala asp ile ile met ser ser glu ser asn leu ala leu ile lys arg tyr tyr gln arg phe gly glu ala ile ala leu leu glu gln gly asp lys gln ala phe ile ala ser phe asn arg val glu gln trp phe gly asp