Patent Publication Number: US-2002007053-A1

Title: Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases

Description:
[0001] This is a continuation-in-part of a U.S. patent application Ser. No. 07/749,611, filed Aug. 28, 1991 which is a continuation-in-part of U.S. patent application Ser. No. 07/576,537, filed Aug. 31, 1990, now abandoned. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] This invention relates in general to plant molecular biology and, more particularly, to a new class of glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases.  
       [0003] Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. It is now possible to produce plants which have unique characteristics of agronomic importance. Certainly, one such advantageous trait is more cost effective, environmentally compatible weed control via herbicide tolerance. Herbicide-tolerant plants may reduce the need for tillage to control weeds thereby effectively reducing soil erosion.  
       [0004] One herbicide which is the subject of much investigation in this regard is N-phosphonomethylglycine commonly referred to as glyphosate. Glyphosate inhibits the shikimic acid pathway which leads to the biosynthesis of aromatic compounds including amino acids. plant hormones and vitamins. Specifically, glyphosate curbs the conversion of phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (hereinafter referred to as EPSP synthase or EPSPS). For purposes of the present invention, the term “glyphosate” should be considered to include any herbicidally effective form of N-phosphonomethylglycine (including any salt thereof) and other forms which result in the production of the glyphosate anion in planta.  
       [0005] It has been shown that glyphosate-tolerant plants can be produced by inserting into the genome of the plant the capacity to produce a higher level of EPSP synthase in the chloroplast of the cell (Shah et al., 1986) which enzyme is preferably glyphosate-tolerant (Kishore et al. 1988). Variants of the wild-type EPSPS enzyme have been isolated which are glyphosate-tolerant as a result of alterations in the EPSPS amino acid coding sequence (Kishore and Shah, 1988; Schulz et al., 1984; Sost et al., 1984; Kishore et al., 1986). These variants typically have a higher K i  for glyphosate than the wild-type EPSPS enzyme which confers the glyphosate-tolerant phenotype, but these variants are also characterized by a high K m  for PEP which makes the enzyme kinetically less efficient (Kishore and Shah, 1988; Sost et al., 1984; Schulz et al., 1984; Kishore et al., 1986; Sost and Amrhein, 1990). For example, the apparent K m  for PEP and the apparent Ki for glyphosate for the native EPSPS from  E. coli  are 10 μM and 0.5 μM while for a glyphosate-tolerant isolate having a single amino acid substitution of an alanine for the glycine at position 96 these values are 220 μM and 4.0 mM, respectively. A number of glyphosate-tolerant plant variant EPSPS genes have been constructed by mutagenesis. Again, the glyphosate-tolerant EPSPS was impaired due to an increase in the K m  for PEP and a slight reduction of the V max  of the native plant enzyme (Kishore and Shah, 1988) thereby lowering the catalytic efficiency (V max /K m ) of the enzyme. Since the kinetic constants of the variant enzymes are impaired with respect to PEP, it has been proposed that high levels of overproduction of the variant enzyme, 40-80 fold, would be required to maintain normal catalytic activity in plants in the presence of glyphosate (Kishore et al., 1988).  
       [0006] While such variant EPSP synthases have proved useful in obtaining transgenic plants tolerant to glyphosate, it would be increasingly beneficial to obtain an EPSP synthase that is highly glyphosate-tolerant while still kinetically efficient such that the amount of the glyphosate-tolerant EPSPS needed to be produced to maintain normal catalytic activity in the plant is reduced or that improved tolerance be obtained with the same expression level.  
       [0007] Previous studies have shown that EPSPS enzymes from different sources vary widely with respect to their degree of sensitivity to inhibition by glyphosate. A study of plant and bacterial EPSPS enzyme activity as a function of glyphosate concentration showed that there was a very wide range in the degree of sensitivity to glyphosate. The degree of sensitivity showed no correlation with any genus or species tested (Schulz et al., 1985). Insensitivity to glyphosate inhibition of the activity of the EPSPS from the  Pseudomonas sp.  PG2982 has also been reported but with no details of the studies (Fitzgibbon, 1988). In general, while such natural tolerance has been reported, there is no report suggesting the kinetic superiority of the naturally occurring bacterial glyphosate-tolerant EPSPS enzymes over those of mutated EPSPS enzymes nor have any of the genes been characterized. Similarly, there are no reports on the expression of naturally glyphosate-tolerant EPSPS enzymes in plants to confer glyphosate tolerance.  
       [0008] For purposes of the present invention the term “mature EPSP synthase” relates to the EPSPS polypeptide without the N-terminal chloroplast transit peptide. It is now known that the precursor form of the EPSP synthase in plants (with the transit peptide) is expressed and upon delivery to the chloroplast, the transit peptide is cleaved yielding the mature EPSP synthase. Al numbering of amino acid positions are given with respect to the mature EPSP synthase (without chloroplast transit peptide leader) to facilitate comparison of EPSPS sequences from sources which have chloroplast transit peptides (i.e., plants and fungi) to sources which do not utilize a chloroplast targeting signal (i.e., bacteria).  
       [0009] In the amino acid sequences which follow, the standard single letter or three letter nomenclature are used. All peptide structures represented in the following description are shown in conventional format in which the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus at the right. Likewise, amino acid nomenclature for the naturally occurring amino acids found in protein is as follows: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V). An “X” is used when the amino acid residue is unknown and parentheses designate that an unambiguous assignment is not possible and the amino acid designation within the parentheses is the most probable estimate based on known information.  
       [0010] The term “nonpolar” amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. The term “uncharged polar” amino acids include glycine, serine. threonine, cysteine, tyrosine, asparagine and glutamine. The term “charged polar” amino acids includes the “acidic” and “basic” amino acids. The term “acidic” amino acids includes aspartic acid and glutamic acid. The term “basic” amino acid includes lysine, arginine and histidine. The term “polar” amino acids includes both “charged polar” and “uncharged polar” amino acids.  
       [0011] Deoxyribonucleic acid (DNA) is a polymer comprising four mononucleotide umts. dAMP (2′-Deoxyadenosine-5-monophosphate), dGMP (2′-Deoxyguanosine-5-monophosphate), dCMP (2′-Deoxycytosine-5-monophosphate) and dTMP (2′-Deoxythymosine-5-monophosphate) linked in various sequences by 3′,5′-phosphodiester bridges. The structural DNA consists of multiple nucleotide triplets called “codons” which code for the amino acids. The codons correspond to the various amino acids as follows: Arg (CGA, CGC, CGG, CGT, AGA, AGG); Leu (CTA, CTC, CTG, CTT, TTA, TTG); Ser (TCA, TCC, TCG, TCT, AGC, AGT); Thr (ACA, ACC, ACG, ACT); Pro (CCA, CCC, CCG, CCT); Ala (GCA, GCC, GCG, GCT); Gly (GGA, GGC, GGG, GGT); Ile (ATA, ATC, ATT); Val (GTA, GTC, GTG, GTT); Lys (AAA, AAG); Asn (AAC, AAT); Gln (AA, CAG); His (CAC, CAT); Glu (GAA, GAG); Asp (GAC, GAT); Tyr (TAC, TAT); Cys (TGC, TGT); Phe (rTC, T=i; Met (ATG); and Trp (UGG). Moreover, due to the redundancy of the genetic code (i.e., more than one codon for all but two amino acids), there are many possible DNA sequences which may code for a particular amino acid sequence.  
       SUMMARY OF THE INVENTION  
       [0012] DNA molecules comprising DNA encoding kinetically efficient, glyphosate-tolerant EPSP synthases are disclosed. The EPSP synthases of the present invention reduce the amount of overproduction of the EPSPS enzyme in a transgenic plant necessary for the enzyme to maintain catalytic activity while still conferring glyphosate tolerance. The EPSP synthases described herein represent a new class of EPSPS enzymes, referred to hereinafter as Class II EPSPS enzymes. Class II EPSPS enzymes of the present invention usually share only between about 47% and 55% amino acid similarity or between about 22% and 30% amino acid identity to other known bacterial or plant EPSPS enzymes and exhibit tolerance to glyphosate while maintaining suitable K (PEP) ranges. Suitable ranges of K m  (PEP) for EPSPS for enzymes of the present invention are between 1-150 μM, with a more preferred range of between 1-35 μM. and a most preferred range between 2-25 μM. These kinetic constants are determined under the assay conditions specified hereinafter. An EPSPS of the present invention preferably has a K i  for glyphosate range of between 15-10000 μM. The K i /K m  ratio should be between about 2-500, and more preferably between 25-500. The V max  of the purified enzyme should preferably be in the range of 2-100 units/mg (μmoles/minute.mg at 25° C.) and the K m  for shikimate-3-phosphate should preferably be in the range of 0.1 to 50 μM.  
       [0013] Genes coding for Class II EPSPS enzymes have been isolated from five (5) different bacteria:  Agrobacterium tumefaciens  sp. strain CP4, Achromobacter sp. strain LBAA, Pseudomonas sp. strain PG2982,  Bacilus subtilis,  and  Staphylococcus aureus.  The LBAA and PG2982 Class II EPSPS genes have been determined to be identical and the proteins encoded by these two genes are very similar to the CP4 protein and share approximately 84% amino acid identity with it. Class II EPSPS enzymes often may be distinguished from Class I EPSPS&#39;s by their inability to react with polyclonal antibodies prepared from Class I EPSPS enzymes under conditions where other Class I EPSPS enzymes would readily react with the Class I antibodies as well as the presence of certain unique regions of amino acid homology which are conserved in Class II EPSP synthases as discussed hereinafter.  
       [0014] Other Class II EPSPS enzymes can be readily isolated and identified by utilizing a nucleic acid probe from one of the Class II EPSPS genes disclosed herein using standard hybridization techniques. Such a probe from the CP4 strain has been prepared and utilized to isolate the Class II EPSPS genes from strains LBAA and PG2982. These genes may also optionally be adapted for enhanced expression in plants by known methodology. Such a probe has also been used to identify homologous genes in bacteria isolated de novo from soil.  
       [0015] The Class II EPSPS enzymes are preferably fused to a chloroplast transit peptide (CTP) to target the protein to the chloroplasts of the plant into which it may be introduced. Chimeric genes encoding this CTP-Class II EPSPS fusion protein may be prepared with an appropriate promoter and 3′ polyadenylation site for introduction into a desired plant by standard methods.  
       [0016] To obtain the maximal tolerance to glyphosate herbicide it is preferable to transform the desired plant with a plant-expressible Class II EPSPS gene in conjunction with another plant-expressible gene which expresses a protein capable of degrading glyphosate such as a plant-expressible gene encoding a glyphosate oxidoreductase enzyme as described in PCT Application No. WO 92/00377, the disclosure of which is hereby incorporated by reference.  
       [0017] Therefore, in one aspect, the present invention provides a new class of EPSP synthases that exhibit a low K m  for phosphoenolpyruvate (PEP), a high V max /K m  ratio, and a high K i  for glyphosate such that when introduced into a plant, the plant is made glyphosate-tolerant such that the catalytic activity of the enzyme and plant metabolism are maintained in a substantially normal state. For purposes of this discussion, a highly efficient EPSPS refers to its efficiency in the presence of glyphosate.  
       [0018] More particularly, the present invention provides EPSPS enzymes having a K m  for phosphoenolpyruvate (PEP) between 1-150 μM and a K i (glyphosate)/K m (PEP) ratio between 3-500, said enzymes having the sequence domains:  
       [0019] —R—X 1 —H—X 2 —E— (SEQ ID NO:37), in which  
       [0020] X 1  is an uncharged polar or acidic amino acid,  
       [0021] X 2  is serine or threonine; and  
       [0022] —G—D—K—X 3 -(SEQ ID NO:38), in which  
       [0023] X3 is serine or threonine; and  
       [0024] —S—A—Q-X 4 —K— (SEQ ID NO:39), in which  
       [0025] X 4  is any amino acid; and  
       [0026] —N—X 5 T—R— (SEQ ID:40), in which  
       [0027] X 5  is any amino acid.  
       [0028] Exemplary Class II EPSPS enzyme sequences are disclosed from seven sources: Agrobacterium sp. strain designated CP4, Achromobacter sp. strain LBAA, Pseudomonas sp. strain PG2982,  Bacillus subtilis  LA2,  Staphylococcus aureus  (ATCC 35556), Synechocystis sp. PCC6803 and  Dichelobacter nodosus.    
       [0029] In another aspect of the present invention, a double-stranded DNA molecule comprising DNA encoding a Class II EPSPS enzyme is disclosed. Exemplary Class II EPSPS enzyme DNA sequences are disclosed from seven sources: Agrobacterium sp. strain designated CP4, Achromobacter sp. strain LBAA, Pseudomonas sp. strain PG2982,  Bacillus subtilis  1A2 , Staphyloccus aureus  (ATCC 35556), Synechocystis sp. PCC6803 and  Dichelobacter nodosus.    
       [0030] In a further aspect of the present invention, nucleic acid probes from EPSPS Class II genes are presented that are suitable for use in screening for Class II EPSPS genes in other sources by assaying for the ability of a DNA sequence from the other source to hybridize to the probe.  
       [0031] In yet another aspect of the present invention, a recombinant, double-stranded DNA molecule comprising in sequence:  
       [0032] a) a promoter which functions in plant cells to cause the production of an RNA sequence;  
       [0033] b) a structural DNA sequence that causes the production of an RNA sequence which encodes a Class II EPSPS enzyme having the sequence domains:  
       [0034] —R—X 1 —H—X 2 —E— (SEQ ID NO:37), in which  
       [0035] X 1  is an uncharged polar or acidic amino acid.  
       [0036] X 2  is serine or threonine; and  
       [0037] —GSD—K—X 3 -(SEQ ID NO:38), in which  
       [0038] X 3  is serine or threonine; and  
       [0039] —S—A—Q—X 4 -K-(SEQ ID NO:39), in which  
       [0040] X 4  is any amino acid; and  
       [0041] —N—X 5 —T—R—(SEQ ID:40), in which  
       [0042] X 5  is any amino acid: and  
       [0043] a 3′ nontranslated region which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSP synthase polypeptide to enhance the glyphosate tolerance of a plant cell transformed with said DNA molecule.  
       [0044] In still yet another aspect of the present invention, transgenic plants and transformed plant cells are disclosed that are made glyphosatetolerant by the introduction of the above-described plant-expressible Class II EPSPS DNA molecule into the plant&#39;s genome.  
       [0045] In still another aspect of the present invention, a method for selectively controlling weeds in a crop field is presented by planting crop seeds or crop plants transformed with a plant-expressible Class II EPSPS DNA molecule to confer glyphosate tolerance to the plants which allows for glyphosate containing herbicides to be applied to the crop to selectively kill the glyphosate sensitive weeds, but not the crops.  
       [0046] Other and further objects, advantages and aspects of the invention will become apparent from the accompanying drawing figures and the description of the invention. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0047]FIGS. 1A and 1B show the DNA sequence (SEQ ID NO:1) for the full-length promoter of figwort mosaic virus (FDAV35S).  
     [0048]FIG. 2 shows the cosmid cloning vector pMON17020.  
     [0049]FIGS. 3A, 3B,  3 C and  3 D shows the structural DNA sequence (SEQ ID NO:2) for the Class II EPSPS gene from bacterial isolate Agrobacterium sp. strain CP4 and the deduced aminozcd stqunce (SEQ ID NO:3).  
     [0050] FIGS.  4 A- 4 b shows thle structural DNA sequence (SEQ ID NO:4) for the Class II EPSPS gene from the bacterial isolate Achromobacter sp. strain LBAA and the deduced amino acid sequence (SEQ ID NO:5).  
     [0051] FIGS.  5 A- 5 B show the structural DNA sequence (SEQ ID NO:6) for the Class/II EPSPS gene from the bacterial isolate Pseudomonas sp. strain PG2982 and thd ced uno acid sequence (SEQ ID NO:7).  
     [0052] FIGS.  6 A- 6 B show the Bestfit comparison of the CP4 EPSPS amino acid sequen that for the  E. coli  EPSPS (SEQ ID NO:8).  
     [0053] FIGS.  7 A- 7 B show the Bestfit comparison of the CP4 EPSPS amino acid sequence (SEQ ID NO:3) with that for the LBAA EPSPS (SEQ ID NO:5).  
     [0054] FIGS.  8 A- 8 B shows the structural DNA sequence (SEQ ID NO:9) for the synthetic CP4 Class II EPSPS gene.  
     [0055]FIG. 9 shows the DNA sequence (SEQ ID NO:10) of the chloroplast transit peptide (CTP) and encoded amino acid sequence (SEQ ID NO:11) derived from the Arabidopsis thaliana EPSPS CTP and containing a SphI restriction site at the chloroplast processing site, hereinafter referred to as CTP2. , o  
     [0056]FIGS. 10A and 10B shows the DNA sequence (SEQ ID NO:12) of the chloroplast transit peptide and encoded amino acid sequence (SEQ ID NO:13) derived from the Arabidopsis thaliana EPSPS gene and containing an EcoRI restriction site within the mature region of the EPSPS, hereinafter referred to as CTP3.  
     [0057]FIG. 11 shows the DNA sequence (SEQ ID NO: 14) of the chloroplast transit peptide and encoded amino acid sequence (SEQ ID NO:15) derived from the Petunia hybrida EPSPS CTP and containing a SphI restriction site at the chloroplast processing site and in which the amino acids at the processing site are change to -Cys-Met hereinafter referred to as CTP4.  
     [0058]FIGS. 12A and 12B show the DNA sequence (SEQ ID NO:16) of the chloroplast transit peptide and encoded amino acid sequence (SEQ ID NO:17) derived from the Petunia hybrida EPSPS gene with the naturally occurring EcoRI site in the mature region of the EPSPS gene. hereinafter referred to as CTP5.  
     [0059]FIG. 13 shows a plasmid map of CP4 plant transformation/expression vector pMON17110.  
     [0060]FIG. 14 shows a plasmid map of CP4 synthetic EPSPS gene plant transformation/expression vector pMON17131.  
     [0061]FIG. 15 shows a plasmid map of CP4 EPSPS free DNA plant transformation expression vector pMON13640.  
     [0062]FIG. 16 shows a plasmid map of CP4 plant transformation/direct selection vector pMON17227.  
     [0063]FIG. 17 shows a plasmid map of CP4 plant transformation/expression vector MON19653.  
     [0064] FIGS.  18 A- 18 D show the structural DNA sequence (SEQ ID NO:41) for the Class II EPSPS gene from the bacterial isolate  Bacillus subtilis  and the deduced amino acid sequence (SEQ ID NO:42).  
     [0065] FIGS.  19 A- 19 D show the structural DNA sequence (SEQ ID NO:43) for the Class II EPSPS gene from the bacterial isolate  Staphylococcus aureus  and the deduced amino acid sequence (SEQ ID NO:44).  
     [0066] FIGS.  20 A- 20  B show the Bestfit comparison of the representative Class II EPSPS amino acid sequences Pseucomonas sp. strain PG2982 (SEQ ID NO:7), Achromobacter sp. strain LBAA (SEQ ID NO:5), Agrobacterium sp. strain designated CP4 (SEQ ID NO:3),  Bacillus subtilis  (SEQ ID NO:42), and  Staphylococcus aureus  (SEQ ID NO:44) with that for representative Class I EPSPS amino acid sequences [ Sacchromyces cerevisiae  (SEQ ID NO:49),  Aspergillus nidulans  (SEQ ID NO:50),  Brassica napus  (SEQ ID NO:51),  Arabidopsis thaliana  (SEQ ID NO:52),  Nicotina tobacum  (SEQ ID NO:53),  L. esculentum  (SEQ ID NO:54),  Petunia hybrida  (SEQ ID NO:55),  Zea mays  (SEQ ID NO:56),  Salmenella gallinarum  (SEQ ID NO:57),  Salmenella typhimurium  (SEQ ID NO:58),  Salmenella typhi  (SEQ ID NO:65),  E. coli  (SEQ ID NO:8),  K. pneumoniae  (SEQ ID NO:59),  Y. enterocolitica  (SEQ ID NO:60),  H. influenzae  (SEQ ID NO:61).  P. multocida  (SEQ ID NO:62),  Aeromonas salmonicida  (SEQ ID NO:63),  Bacillus pertussis  (SEQ ID NO:64)] and illustrates the conserved regions among Class II EPSPS sequences which are unique to Class II EPSPS sequences. To aid in a comparison of the EPSPS sequences, only mature EPSPS sequences were compared. That is, the sequence corresponding to the chloroplast transit peptide, if present in a subject EPSPS, was removed prior to m atpnent.  
     [0067] FIGS.  21 A- 21 E show the structural DNA sequence (SEQ ID NO:66) for the Class II EPSPS gene from the bacterial isolate Synechocystis sp. PCC6803 and the deduced amino acsquenf (SEQ ID NO:67).  
     [0068] FIGS.  22 A- 22 E show the structural DNA sequence (SEQ ID NO:68) for the Class II EPSPS gene from the bacterial isolate  Dichelobacter nodosus  and the deduced acidsequence)(SEQ ID NO:69).  
     [0069] FIGS.  23 A- 23 D show the Bestfit comparison of the representative Class II EPSPS amino acid sequences Pseudomonas sp. strain PG2982 (SEQ ID NO:7), Achromobacter sp. strain LBAA (SEQ ID NO:5), Agrobacterium sp. strain designated CP4 (SEQ ID NO:3), Synechocystis sp. PCC6803 (SEQ ID NO:67),  Bacillus subtilis  (SEQ ID NO:42),  Dichelobacter nodosus  (SEQ ID NO:69) and  Staphylococcus aureus  (SEQ ID NO:44).  
     [0070]FIG. 24 a plasmid map of canola plant transformation/expression vector pMON17209.  
     [0071]FIG. 25 a plasmid map of canola plant transformation/expression vector pMON17237.  
     STATEMENT OF THE INVENTION  
     [0072] The expression of a plant gene which exists in double-stranded DNA form involves synthesis of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the MRNA primary transcript inside the nucleus. This processing involves a 3′ non-translated region which adds polyadenylate nucleotides to the 3′ end of the RNA.  
     [0073] Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the “promoter.” The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription into mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA. A number of promoters which are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters. (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide) and the full-length transcript promoter from the figwort mosaic virus (FMV35S), promoters from the maize ubiquitin and rice actin genes. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants; see, e.g., PCT publication WO 84/02913 (Rogers et al., Monsanto).  
     [0074] Promoters which are known or found to cause transcription of DNA in plant cells can be used in the present invention. Such promoters may be obtained from a variety of sources such as plants and plant DNA viruses and include, but are not limited to, the CaMV35S and FMV35S promoters and promoters isolated from plant genes such as ssRLTBISCO genes and the maize ubiquitin and rice actin genes. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of a Class II EPSPS to render the plant substantially tolerant to glyphosate herbicides. The amount of Class II EPSPS needed to induce the desired tolerance may vary with the plant species. It is preferred that the promoters utilized have relatively high expression in all meristematic tissues in addition to other tissues inasmuch as it is now known that glyphosate is translocated and accumulated in this type of plant tissue. Alternatively, a combination of chimeric genes can be used to cumulatively result in the necessary overall expression level of the selected Class II EPSPS enzyme to result in the glyphosate-tolerant phenotype.  
     [0075] The mRNA produced by a DNA construct of the present invention also contains a 5′ non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the MRNA. The 5′ non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs, as presented in the following examples, wherein the non-translated region is derived from both the 5′ non-translated sequence that accompanies the promoter sequence and part of the 5′ non-translated region of the virus coat protein gene. Rather, the non-translated leader sequence can be derived from an unrelated promoter or coding sequence as discussed above.  
     [0076] Preferred promoters for use in the present invention the the fill-length transcript (SEQ ID NO:1) promoter from the figwort mosaic virus (FMV35S) and the full-length transcript (35S) promoter from cauliflower mosaic virus (CaMV), including the enhanced CaMV35S promoter (Kay et al. 1987). The FMV35S promoter functions as strong and uniform promoter with particularly good expression in meristematic tissue for chimeric genes inserted into plants, particularly dicotyledons. The resulting transgenic plant in general expresses the protein encoded by the inserted gene at a higher and more uniform level throughout the tissues and cells of the transformed plant than the same gene driven by an enhanced CaMV35S promoter. Referring to FIG. 1, the DNA sequence (SEQ ID NO:1) of the FMV35S promoter is located between nucleotides 6368 and 6930 of the FMV genome. A 5′ non-translated leader sequence is preferably coupled with the promoter. The leader sequence can be from the FMV35S genome itself or can be from a source other than FMV35S.  
     [0077] For expression of heterologous genes in moncotyledonous plants the use of an intron has been found to enhance expression of the heterologous gene. While one may use any of a number of introns which have been isloated from plant genes. the use of the first intron from the maize heat shock 70 gene is preferred.  
     [0078] The 3′ non-translated region of the chimeric plant gene contains a polyadenylation signal which functions in plants to cause the addition of polyadenylate nucleotides to the 3′ end of the viral RNA. Examples of suitable 3′ regions are (1) the 3′ transcribed, non-translated regions containing the polyadenylated signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean storage protein genes and the small subunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. An example of a preferred 3′ region is that from the ssRUBISCO gene from pea (E9), described in greater detail below.  
     [0079] The DNA constructs of the present invention also contain a structural coding sequence in double-stranded DNA form which encodes a glyphosate-tolerant, highly efficient Class II EPSPS enzyme.  
     [0080] Identification of glyphosatr-tolerant, highly efficient EPSPS enzymes  
     [0081] In an attempt to identify and isolate glyphosate-tolerant, highly efficient EPSPS enzymes, kinetic analysis of the EPSPS enzymes from a number of bacteria exhibiting tolerance to glyphosate or that had been isolated from suitable sources was undertaken. It was discovered that in some cases the EPSPS enzymes showed no tolerance to inhibition by glyphosate and it was concluded that the tolerance phenotype of the bacterium was due to an impermeability to glyphosate or other factors. In a number of cases, however, microorganisms were identified whose EPSPS enzyme showed a greater degree of tolerance to inhibition by glyphosate and that displayed a low K m  for PEP when compared to that previously reported for other microbial and plant sources. The EPSPS enzymes from these microorganisms were then subjected to further study and analysis.  
     [0082] Table I displays the data obtained for the EPSPS enzymes identified and isolated as a result of the above described analysis. Table I includes data for three identified Class II EPSPS enzymes that were observed to have a high tolerance to inhibition to glyphosate and a low K m  for PEP as well as data for the native Petunia EPSPS and a glyphosate-tolerant variant of the Petunia EPSPS referred to as GA101. The GA101 variant is so named because it exhibits the substitution of an alanine residue for a glycine residue at position 101 (with respect to Petunia). When the change introduced into the Petunia EPSPS (GA101) was introduced into a number of other EPSPS enzymes, similar changes in kinetics were observed, an elevation of the K i  for glyphosate of the K m  for PEP.  
               TABLE I                          Kinetic characterization of EPSPS enzymes                                     ENZYME   K m  PEP   K i  Glyphosate               SOURCE   (μM)   (μM)   K i K m                         Petunia   5   0.4   0.08           Petunia GA101   200   2000   10           PG2982   2.1-3.1 1     25-82   ˜8-40           LBAA   ˜7.3-8 2     60 (est) 7     ˜7.9           CP4   12 3     2720   227             B. subtilis  1A2   13 4     440   33.8             S. aureus     5 5     200   40                                                                                              
 
     [0083] The Agrobacterium sp. strain CP4 was initially identified by its ability to grow on glyphosate as a carbon source (10 mM) in the presence of 1 mM phosphate. The strain CP4 was identified from a collection obtained from a fixed-bed immobilized cell column that employed Mannville R-635 diatomaceous earth beads. The column had been run for three months on a waste-water feed from a glyphosate production plant. The column contained 50 mg/ml glyphosate and NH3 as NH 4 Cl. Total organic carbon was 300 mg/ml and BOD&#39;s (Biological Oxygen Demand—a measure of “soft” carbon availability) were less than 30 mg/ml. This treatment column has been described (Heitkamp et al., 1990). Dworkin-Foster minimal salts medium containing glyphosate at 10 nM and with phosphate at 1 mM was used to select for microbes from a wash of this column that were capable of growing on glyphosate as sole carbon source. Dworkin-Foster minimal medium was made up by combining in 1 liter (with autoclaved H 2 O), 1 ml each of A, B and C and 10 ml of D (as per below) and thiamine HCI (5 mg).  
                                      A.   D-F Salts (1000× stock; per 100 ml; autoclaved):                                 H 3 BO 3     1   mg           MnSO 4 .7H 2 O   1   mg           ZnSO 4 .7H 2 O   12.5   mg           CuSO 4 .5H 2 O   8   mg           NaMoO 3 .3H 2 O   1.7   mg                             B.   FeSO 4 .7H 2 O (1000× stock: per 100 ml; autoclaved)   0.1   g       C.   MgSO 4 .7H 2 O (1000× stock; per 100 ml; autoclaved)   20   g       D.   (NH 4 ) 2 SO 4  (100× stock; per 100 ml; autoclaved)   20   g                  
 
     [0084] Yeast Extract (YE; Difco) was added to a final concentration of 0.01 or 0.001%. The strain CP4 was also grown on media composed of D-F salts, amended as described above, containing glucose, gluconate and citrate (each at 0.1 %) as carbon sources and with inorganic phosphate (0.2-1.0 mM) as the phosphorous source.  
     [0085] Other Class II EPSPS containing microorganisms were identified as Achromobacter sp. strain LBAA (Hallas et aL., 1988), Pseudomonas sp. strain PG2982 (Moore et al., 1983; Fitzgibbon 1988),  Bacillus subtilis  IA2 (Henner et al., 1984) and  Staphylococcus aureus  (O&#39;Connell et al., 1993). It had been reported previously, from measurements in crude lysates, that the EPSPS enzyme from strain PG2982 was less sensitive to inhibition to glyphosate than that of  E. coli , but there has been no report of the details of this lack of sensitivity and there has been no report on the K m  for PEP for this enzyme or of the DNA sequence for the gene for this enzyme (Fitzgibbon, 1988; Fitzgibbon and Braymer, 1990).  
     [0086] Relationship of the Class II EPSPS to those previously studied  
     [0087] All EPSPS proteins studied to date have shown a remarkable degree of homology. For example, bacterial and plant EPSPS&#39;s are about 54% identical and with similarity as high as 80%. Within bacterial EPSPS&#39;s and plant EPSPS&#39;s themselyes the degree of identity and similarity is much greater (see Table II).  
               TABLE II                          Comparison between exemplary Class I EPSPS       protein sequences 1                               similarity   identity                                               E. coli  vs.  S. typhimurium     93   88             P. hybrida  vs.  E. coli      72   55             P. hybrida  vs.  L. esculentum     93   88                                  
 
     [0088] When crude extracts of CP4 and LBAA bacteria (50 μg protein) were probed using rabbit anti-EPSPS antibody (Padgette et al., 1987) to the Petunia EPSPS protein in a Western analysis, no positive signal could be detected, even with extended exposure times (Protein A-1251 development system) and under conditions where the control EPSPS (Petunia EPSPS, 20 ng; a Class I EPSPS) was readily detected. The presence of EPSPS activity in these extracts was confirmed by enzyme assay. This surprising result, indicating a lack of similarity between the EPSPS&#39;s from these bacterial isolates and those previously studied, coupled with the combination of a low K m  for PEP and a high K i  for glyphosate, illustrates that these new EPSPS enzymes are different from known EPSPS enzymes (now referred to as Class I EPSPS).  
     [0089] Glyphosate-tolerant Enzymes in Microbial Isolates  
     [0090] For clarity and brevity of disclosure, the following description of the isolation of genes encoding Class II EPSPS enzymes is directed to the isolation of such a gene from a bacterial isolate. Those skilled in the art will recognize that the same or similar strategy can be utilized to isolate such genes from other microbial isolates, plant or fungal sources.  
     [0091] Cloning of the Agrobacterium sp. strain CP4 EPSPS (Gene(s) in  E. coli    
     [0092] Having established the existence of a suitable EPSPS in Agrobacterium sp. strain CP4, two parallel approaches were undertaken to clone the gene: cloning based on the expected phenotype for a glyphosate-tolerant EPSPS; and purification of the enzyme to provide material to raise antibodies and to obtain amino acid sequences from the protein to facilitate the verification of clones. Cloning and genetic techniques, unless otherwise indicated, are generally those described in Maniatis et al., 1982 or Sambrook et al., 1987. The cloning strategy was as follows: introduction of a cosmid bank of strain Agrobacterilum sp. strain CP4 into  E. coli  and selection for the EPSPS gene by selection for growth on inhibitory concentrations of glyphosate.  
     [0093] Chromosomal DNA was prepared from strain Agrobacterium sp. strain CP4 as follows: The cell pellet from a 200 ml L-Broth (Miller, 1972), late log phase culture of Agrobacterium sp. strain CP4 was resuspended in 10 ml of Solution I; 50 mnM Glucose, 10 mini EDTA, 25 mM Tris -CL pH 8.0 (Birnboim and Doly, 1979). SDS was added to a final concentration of 1% and the suspension was subjected to three freeze-thaw cycles, each consisting of immersion in dry ice for 15 minutes and in water at 70° C. for 10 mninutes. The lysate was then extracted four times with equal volumes of phenol:chloroform (1:1; phenol saturated with TE; TE =10 mM Tris pH8.0; 1.0 mM EDTA) and the phases separated by centrifugation (15000g; 10 minutes). The ethanol-precipitable material was pelleted from the supernatant by brief centrifugation (8000 g, 5 minutes) following addition of two volumes of ethanol. The pellet was resuspended in 5 ml TE and dialyzed for 16 hours at 4° C. against 2 liters TE. This preparation yielded a 5 ml DNA solution of 552 μg/ml.  
     [0094] Partially-restricted DNA was prepared as follows. Three 100 μg aliquot samples of CP4 DNA were treated for 1 hour at 37° C. with restriction endonuclease HindIII at rates of 4, 2 and 1 enzyme unit/μg DNA, respectively. The DNA samples were pooled, made 0.25 mM with EDTA and extracted with an equal volume of phenol:chloroform. Following the addition of sodium acetate and ethanol, the DNA was precipitated with two volumes of ethanol and pelleted by centrifugation (12000 g; 10 minutes). The dried DNA pellet was resuspended in 500 μl TE and layered on a 10-40% Sucrose gradient (in 5% increments of 5.5 ml each) in 0.5 M NaCl, 50 mM Tris pH8.0, 5 mM EDTA. Following centrifugation for 20 hours at 26,000 rpm in a SW28 rotor, the tubes were punctured and 1.5 ml fractions collected. Samples (20 μl) of each second fraction were run on 0.7% agarose gel and the size of the DNA determined by comparison with linearized lambda DNA and HindIII-digested lambda DNA standards. Fractions containing DNA of 25-35 kb fragments were pooled, desalted on AMICONIO columns (7000 rpm; 20° C.; 45 minutes) and concentrated by precipitation. This procedure yielded 15 jg of CP4 DNA of the required size. A cosmid bank was constructed using the vector pMON17020. This vector. a map of which is presented in FIG. 2, is based on the pBR327 replicon and contains the spectinomycin/streptomycin (Spr;spc) resistance gene from Tn7 (Fling et al., 1985). the chloramphenicol resistance gene (Cmr;cat) from Tn9 (Alton et al., 1979), the gene10 promoter region from phage T7 (Dunn et al., 1983), and the 1.6 kb BglII phage lambda cos fragment from pHC79 (Hohn and Collins, 1980). A number of cloning sites are located downstream of the cat gene. Since the predominant block to the expression of genes from other microbial sources in  E. coli  appears to be at the level of transcription, the use of the T7 promoter and supplying the T7 polymerase in trans from the pdP1-2 plasmid (Tabor and Richardson, 1985), enables the expression of large DNA segments of foreign DNA, even those containing RNA polymerase transcription termination sequences. The expression of the spc gene is impaired by transcription from the T7 promoter such that only Cm 4  can be selected in strains containing pGP1-2. The use of antibiotic resistances such as Cm resistance which do not employ a membrane component is preferred due to the observation that high level expression of resistance genes that involye a membrane component. i.e. J3-lactamase and Amp resistance, give rise to a glyphosate-tolerant phenotype. Presumably, this is due to the exclusion of glyphosate from the cell by the membrane localized resistance protein. It is also preferred that the selectable marker be oriented in the same direction as the T7 promoter.  
     [0095] The vector was then cut with HindIII and treated with calf alkaline phosphatase (CAP) in preparation for cloning. Vector and target sequences were ligated by combining the following:  
                                                          Vector DNA (HindIII/CAP)   3   μg           Size fractionated CP4 HindIII fragments   1.5   μg           10× ligation buffer   2.2   μl           T4 DNA ligase (New England Biolabs) (400 U/μl)   1.0   μl                      
 
     [0096] and adding H 2 O. This mixture was incubated for 18 hours at 16° C. 10× ligation buffer is 250 mM Tris-HCI, pH 8.0; 100 mM MgCI 2 ; 100 mM Dithiothreitol: 2 mM Spermidine. The ligated DNA (5 μl) was packaged into lambda phage particles (Stratagene; Gigapack Gold) using the manufacturer&#39;s procedure.  
     [0097] A sample (200μl) of  E. coli  HB11 (Boyer and Rolland-Dussoix, 1973) containing the T7 polymerase expression plasmid pGP1-2 (Tabor and Richardson, 1985) and grown overnight in L-Broth (with maltose at 0.2% and kanamycin at 50 μg/ml) was infected with 50 μl of the packaged DNA. Transformants were selected at 30° C. on M9 (Miller, 1972) agar containing kanamycin (50 μg/ml), chloramphenicol (25 μg/ml), L-proline (50 μg/ml), L-leucine (50 μg(ml) and B1 (5 μg/ml), and with glyphosate at 3.0 mM. Aliquot samples were also plated on the same media lacking glyphosate to titer the packaged cosmids. Cosmid transformants were isolated on this latter medium at a rate of ˜-5×10 5  per fig CP4 HindIII DNA after 3 days at 30° C. Colonies arose on the glyphosate agar from day 3 until day 15 with a final rate of ˜1 per 200 cosmids. DNA was prepared from 14 glyphosate-tolerant clones and, following verification of this phenotype, was transformed into  E. coli  GB1OOfpGP1-2 ( E. coli  GB100 is an aroA derivative of MM294 (Talmadge and Gilbert, 19801) and tested for complementation for growth in the absence of added aromatic amino acids and aminobenzoic acids. Other aroA strains such as SR481 (Bachman et al., 1980; Padgette et al., 1987), could be used and would be suitable for this experiment. The use of GB100 is merely exemplary and should not be viewed in a limiting sense. This aroA strain usually requires that growth media be supplemented with L-phenylalanine, L-tyrosine and L-tryptophan each at 100 μg/ml and with para-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid and para-aminobenzoic acid each at 5 μg/ml for growth in minimal media. Of the fourteen cosmids tested only one showed complementation of the aroA-phenotype. Transformants of this cosmid, pMON17076, showed weak but uniform growth on the unsupplemented minimal media after 10 days.  
     [0098] The proteins encoded by the cosmids were determined in uivo using a T7 expression system (Tabor and Richardson, 1985). Cultures of  E. coli  containing pGP1-2 (Tabor and Richardson, 1985) and test and control cosmids were grown at 30° C. in L-broth (2 ml) with chloramphenicol and kanamycin (25 and 50 μl, respectively) to a Klett reading of 50. An aliquot was removed and the cells collected by centrifugation, washed with M9 salts (Miller, 1972) and resuspended in 1 ml M9 medium containing glucose at 0.2%, thiamine at 20 μg(ml and containing the 18 amino acids at 0.01% (minus cysteine and methionine). Following incubation at 30° C. for 90 minutes, the cultures were transferred to a 42° C. water bath and held there for 15 minutes. Rifampicin (Sigma) was added to 200 μg(ml and the cultures held at 42° C. for 10 additional mminutes and then transferred to 30° C. for 20 minutes. Samples were pulsed with 10 μCi of  35 S-methionine for 5 minutes at 30° C. The cells were collected by centrifugation and suspended in 60-120 pi cracking buffer (60 mM Tris-HCI 6.8, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue). Aliquot samples were electrophoresed on 12.5% SDIS-PAGE and following soaking for 60 minutes in 10 volumes of Acetic Acid-Methanol-water (10:30:60), the gel was soaked in ENLIGHTNING T™ (DUPONT) following manufacturer&#39;s directions, dried, and exposed at −70° C. to X-Ray film. Proteins of about 45 kd in size, labeled with 35S-methionine, were detected in number of the cosmids. including pMON17076.  
     [0099] Purification of EPSPS from Arobacterium spa strain CP4  
     [0100] All protein purification procedures were carried out at 3-5° C. EPSPS enzyme assays were performed using either the phosphate release or radioactive HPLC method, as previously described in Padgette et al., 1987, using 1 mM phosphoenol pyruvate (PEP, Boehringer) and 2 mM shikimate-3-phosphate 1S3P) substrate concentrations. For radioactive HPLC assays, 14C-PEP (Amersham) was utilized. S3P was synthesized as previously described in Wibbenmeyer et al. 1988. N-terminal amino acid sequencing was performed by loading samples onto a Polybrene precycled filter in aliquots while drying. Automated Edman degradation chemistry was used to determine the N-terminal protein sequence, using an Applied Biosystems Model 470A gas phase sequencer (Hunkapiller et at., 1983) with an Applied Biosystems 120A PTH analyzer.  
     [0101] Five 100litre fermentations were carried out on a spontaneous “smooth” isolate of strain CP4 that displayed less clumping when grown in liquid culture. This reduced clumping and smooth colony morphology may be due to reduced polysaccharide production by this isolate. In the following section dealing with the purification of the EPSPS enzyme, CP4 refers to the “smooth” isolate—CP4-S1. The cells from the three batches showing the highest specific activities were pooled. Cell paste of Agrobacterium sp. CP4 (300 g) was washed twice with 0.5 L of 0.9% salne and collected by centrifuation (30 minutes, 8000 rpm in a GS3 Sorvall rotor). The cell pellet was suspended in 0.9 L extraction buffer (100 maM TrisCl, 1 mM EDTA, 1 mM BAM (Benzamidine), 5 mM DTT, 10% glycerol, pH 7.5) and lysed by 2 passes through a Manton Gaulin cell. The resulting solution was centrifuiged (30 minutes, 8000 rpm) and the supernatant was treated with 0.21 L of 1.5% protamine sulfate (in 100 rmM TrisCl, pH 7.5, 0.2% w/v final protamine sulfate concentration). After stirring for 1 hour, the m e was centrifuged (50 minutes, 8000 rpm) and the resulting supernatant treated with solid ammonium sulfate to 40% saturation and stirred for I hour. After centrifugation (50 minutes, 8000 rpm), the resulting supernatant was treated with solid ammonium sulfate to 70% saturation, stirred for 50 minutes, and the insoluble protein was collected by centrifugation (1 hour, 8000 rpm). This 40-70% ammonium sulfate fraction was then dissolyed in extraction buffer to give a final volume of 0.2 L, and dialyzed twice (Spectrum 10,000 MW cutoff dialysis tubing) against 2 L of extraction buffer for a total of 12 hours.  
     [0102] To the resulting dialyzed 40-70% ammonium sulfate fraction tO.29 L) was added solid ammonium sulfate to give a final concentration of 1 M. This material was loaded (2 ml/min) onto a coluimn (5 cm×15 cm, 295 ml) packed with phenyl Sepharose CL-4B (Pharmacia) resin equilibrated with extraction buffer containing 1 M ammonium sulfate, and washed with the same buffer (1.5 L, 2 ml/min). EPSPS was eluted with a linear gradient of extraction buffer going from 1 M to 0.00 M ammonium sulfate (total volume of 1.5 L, 2 ml/min). Fractions were collected (20 ml) and assayed for EPSPS activity by the phosphate release assay. The fractions with the highest EPSPS activity (fractions 3650) were pooled and dialyzed against 3×2 L (18 hours) of 10 mM TrisCl, 25 mM KCr, 1 mM EDTA, 5 mM DTT, 10% glycerol, pH 7.8.  
     [0103] The dialyzed EPSPS extract (350 ml) was loaded (5 ml/min) onto a column (2.4 cm×30 cm, 136 ml) packed with Q-Sepharose Fast Flow (Pharmacia) resin equilibrated with 10 mM TrisCl, 25 mM KCR, 5 mM DTT, 10% glycerol, pH 7.8 (Q Sepharose buffer), and washed with 1 L of the same buffer. EPSPS was eluted with a linear gradient of Q Sepharose buffer going from 0.025 M to 0.40 M KCI (total volume of 1.4 L, 5 ml/min). Fractions were collected (15 ml) and assayed for EPSPS activity by the phosphate release assay. The fractions with the highest EPSPS activity (fractions 47-60) were pooled and the protein was precipitated by adding solid ammonium sulfate to 80% saturation and stirring for 1 hour. The precipitated protein was collected by centrifugation (20 minutes, 12000 rpm in a GSA Sorvall rotor), dissolyed in Q Sepharose buffer (total volume of 14 ml), and dialyzed against the same buffer (2×1 L, 18 hours).  
     [0104] The resulting dialyzed partially purified EPSPS extract (19 ml) was loaded (1.7 ml/min) onto a Mono Q 10/10 column (Pharmacia) equilibrated with Q Sepharose buffer, and washed with the same buffer t35 ml). EPSPS was eluted with a linear gradient of 0.025 M to 0.35 M KCI (total volume of 119 ml, 1.7 ml/min). Fractions were collected (1.7 ml) and assayed for EPSPS activity by the phosphate release assay. The fractions with the highest EPSPS activity (fractions 30-37) were pooled (6 ml).  
     [0105] The Mono Q pool was made 1 M in ammonium sulfate by the addition of solid ammonium sulfate and 2 ml aliquots were chromatographed on a Phenyl Superose 5/5 column (Pharmacia) equilibrated with 100 mM TrisCl, 5 mM DTT, 1 M ammonium sulfate, 10% glycerol, pH 7.5 (Phenyl Superose buffer). Samples were loaded (1 ml/min), washed with Phenyl Superose buffer (10 ml), and eluted with a linear gradient of Phenyl Superose buffer going from 1 M to 0.00 M ammonium sulfate (total volume of 60 ml, 1 ml/min). Fractions were collected (1 ml) and assayed for EPSPS activity by the phosphate release assay. The fractions from each run with the highest EPSPS activity (fractions ˜36-40) were pooled together (10 ml, 2.5 mg protein). For N-terminal amino acid sequence determination, a portion of one fraction (#39 from run 1) was dialyzed against 50 mM NaHCO 3  (2×1 L). The resulting pure EPSPS sample (0.9 ml, 77 μg protein) was found to exhibit a single N-terminal amino acid sequence of:  
     [0106] XH(G)ASSRPATARKSS(G)LX(G)(T)VR)IPG(D)(K)(M) (SEQ ID NO:18).  
     [0107] The remaining Phenyl Superose EPSPS pool was dialyzed against 50 mM TrisCl, 2 mM DIT, 10 mM KCl, 10% glycerol, pH 7.5 (2×1 L). An aliquot (0.55 ml, 0.61 mg protein) was loaded (1 ml/min) onto a Mono Q 5/5 column (Pharmacia) equilibrated with Q Sepharose buffer, washed with the same buffer (5 ml), and eluted with a linear gradient of Q Sepharose buffer going from 0-0.14 M KCI in 10 minutes, then holding at 0.14 M KCl (1 ml/min). Fractions were collected (1 ml) and assayed for EPSPS activity by the phosphate release assay and were subjected to SDS-PAGE (10-15%, Phast System, Pharmacia, with silyer staining) to determine protein purity. Fractions exhibiting a single band of protein by SDS-PAGE (22-25, 222 Ag) were pooled and dialyzed against 100 mM ammonium bicarbonate, pH 8.1 (2×1 L, 9 hours).  
     [0108] Trysinolysis and neotide seguencinz of Aarobacterium ST strain CP 4 EPSPS    
     [0109] To the resulting pure Agrobacterium sp. strain CP4 EPSPS (111 μg) was added 3 μg of trypsin (Calbiochem), and the trypsinolysis reaction was allowed to proceed for 16 hours at 37° C. The tryptic digest was then chromatographed (lml/min) on a C18 reverse phase HPLC column (Vydac) as previously described in Padgette et al., 1988 for  E. coli  EPSPS. For all peptide purifications, 0.1% trifluoroacetic acid (TFA, Pierce) was designated buffer “RP--A” and 0.1% TFA in acetonitrile was buffer “RP-B”. The gradient used for elution of the trypsinized Agrobacterium sp. CP4 EPSPS was: 0-8 minutes, 0% RP-B; 8-28 minutes, 0-15% RP-B; 28-40 minutes, 15-21% RP-B; 40-68 minutes, 21-49% RP-B; 68-72 minutes, 49-75% RP-B; 72-74 minutes, 75-100% RP-B. Fractions were collected (1 ml) and, based on the elution profile at 210 nm, at least 70 distinct peptides were produced from the trypsinized EPSPS. Fractions 40-70 were evaporated to dryness and redissolyed in 150 μl each of 10% acetonitrile, 0.1% trifluoroacetic acid.  
     [0110] The fraction 61 peptide was further purified on the C18 column by the gradient: 0-5 minutes, 0% RP-B; 5-10 minutes, 0-38% RP-B; 10-30 minutes, 38-45% B. Fractions were collected based on the UV signal at 210 nm. A large peptide peak in fraction 24 eluted at 42% RP-B and was dried down, resuspended as described above, and rechromatographed on the C18 column with the gradient: 0-5 minutes, 0% RP-B; 5-12 min, 0-38% RP-B; 12-15 min, 38-39% RP-B; 15-18 minutes, 39% RP-B; 18-20 minutes. 39-41% RP-B; 20-24 minutes, 41% RP-B; 24-28 minutes, 42% RP-B. The peptide in fraction 25, eluting at 41% RP-B and designated peptide 61-24-25, was subjected to N-terminal amino acid sequencing, and the following sequence was determined:  
     [0111] APSM(I)(D)EYPILAV (SEQ ID NO:19)  
     [0112] The CP4 EPSPS fraction 53 tryptic peptide was further purified by C18 HPLC by the gradient 0% B (5 minutes). 0-30% B (5-17 minutes), 30-40% B (17-37 minutes). The peptide in fraction 28. eluting at 34% B and designated peptide 53-28, was subjected to N-terminal amino acid sequencing, and the following sequence was determined:  
     [0113] ITGLLEGEDVINTGK (SEQ ID NO:20).  
     [0114] In order to verify the CP4 EPSPS cosmid clone, a number of oligonucleotide probes were designed on the basis of the sequence of two of the tryptic sequences from the CP4 enzyme (Table III). The probe identified as MID was very low degeneracy and was used for initial screening. The probes identified as EDV-C and EDV-T were based on the same amino acid sequences and differ in one position (underlined in Table III below) and were used as confirmatory probes, with a positive to be expected only from one of these two probes. In the oligonucleotides below, alternate acceptable nucleotides at a particular position are designated by a “/” such as A(C/T.  
               TABLE III                       Selected CP4 EPSPS peptide seaquences and DNA probes                                        PEPTIDE 61-24-25 APSM(I)(D)EYPILAV   (SEQ ID NO:19)               Probe MID; 17-mer; mixed probe; 24-fold degenerate               ATGATA/C/TGAC/TGAG/ATAC/TCC   (SEQ ID NO:21)               PEPTIDE 53-28 ITGLLEGEDVINTGK   (SEQ ID NO:20)               Probe EDV-C; 17-mer; mixed probe; 48-fold degenerate               GAA/GGAC/TGTA/C/G/TATA/C/TAA C AC   (SEQ ID NO:22)               Probe EDV-T; 17-mer; mixed probe; 48-fold degenerate               GAA/GGAC/TGTA/C/G/TATA/C/TAA T AC   (SEQ ID NO:23)                  
 
     [0115] The probes were labeled using gamma-32P-ATP and polynucleotide kinase. DNA from fourteen of the cosmids described above was restricted with EcoRI, transferred to membrane and probed with the oligonucleotide probes. The conditions used were as follows: prehybridization was carried out in 6X SSC, 10X Denhardt&#39;s for 2-18 hour periods at 60° C., and hybridization was for 48-72 hours in 6X SSC. 10X Denhardt&#39;s, 100 utgml tRNA at 100C below the T d  for the probe. The T d  of the probe was approximated by the formula 20° C.×(A+T) +4° C.×(G+C). The filters were then washed three times with 6X SSC for ten minutes each at room temperature, dried and autoradiographed. Using the MID probe, an ˜9.9 kb fragment in the pMON17076 cosmid gave the only positive signal. This cosmid DNA was then probed with the EDVC (SEQ ID NO:22) and EDV-T (SEQ ID NO:23) probes separately and again this ˜9.9 kb band gave a signal and only with the EDV-T probe.  
     [0116] The combihed data on the glyphosate-tolerant phenotype, the complementation of the  E. coli  aroA-phenotype, the expression of a -45 Kd protein, and the hybridization to two probes derived from the CP4 EPSPS amino acid sequence strongly suggested that the pMON17076 cosmid contained the EPSPS gene.  
     [0117] Localization and subcloning of the CP4 EPSPS gene  
     [0118] The CP4 EPSPS gene was further localized as follows: a number of additional Southern analyses were carried out on different restriction digests of pMON17076 using the MID (SEQ ID NO:21) and EDV-T (SEQ ID NO:23) probes separately. Based on these analyses and on subsequent detailed restriction mapping of the pBlueScript (Stratagene) subclones of the ˜9.9 kb fragment from pMON17076, a 3.8 kb EcoRI-Sall fragment was identified to which both probes hybridized. This analysis also showed that MID (SEQ ID NO:21) and EDV-T (SEQ ID NO:23) probes hybridized to different sides of BamHI, ClaI, and SacII sites. This 3.8 kb fragment was cloned in both orientations in pBlueScript to form pMON17081 and pMON17082. The phenotypes imparted to  E. coli  by these clones were then determined. Glyphosate tolerance was determined following transformation into  E. coli  MM294 containing pGP1-2 (pBlueScript also contains a T7 promoter) on M9 agar media containing glyphosate at 3 mM. Both pMON17081 and pMON17082 showed glyphosate-tolerant colonies at three days at 30° C. at about half the size of the controls on the same media lacking glyphosate. This result suggested that the 3.8 kb fragment contained an intact EPSPS gene. The apparent lack of orientation-dependence of this phenotype could be explained by the presence of the T7 promoter at one side of the cloning sites and the lac promoter at the other. The aroA phenotype was determined in transformants of  E. coli  GB100 on M9 agar media lacking aromatic supplements. In this experiment, carried out with and without the Plac inducer IPTG, pMON17082 showed much greater growth than pMON17081, suggesting that the EPSPS gene was expressed from the Sall site towards the EcoRi site.  
     [0119] Nucleotide sequencing was begun from a number of restriction site ends, including the BamHI site discussed above. Sequences encoding protein sequences that closely matched the N-terminus protein sequence and that for the tryptic fragment 53-28 (SEQ ID NO:20) (the basis of the EDV-T probe) (SEQ ID NO:23) were localized to the SalI side of this BamHI site. These data provided conclusive evidence for the cloning of the CP4 EPSPS gene and for the direction of transcription of this gene. These data coupled with the restriction mapping data also indicated that the complete gene was located on an ˜2.3 kb XhoI fragment and this fragment was subeloned into pBlueScript. The nucleotide sequence of almost 2 kb of this fragment was determined by a combination of sequencing from cloned restriction fragments and by the use of specific primers to extend the sequence. The nucleotide sequence of the CP4 EPSPS gene and flanking regions is shown in FIG. 3 (SEQ ID NO:2). The sequence corresponding to peptide 61-24-25 (SEQ ID NO:19) was also located. The sequence wvas determined using both the SEQUENASE™ kit from IBI (International Biotechnologies Inc.) and the T7 sequencing/Deaza K i  t from Pharmacia.  
     [0120] That the cloned gene encoded the EPSPS activity purified from the Agrobacterium sp. strain CP4 was verified in the following manner: By a series of site directed mutageneses, BglII and NcoI sites were placed at the N-terminus with the fMet contained within the NcoI recognition sequence, the first internal VcoI site was removed (the-second internal NcoI site was removed later), and a Sacl site was placed after the stop codons. At a later stage the internal NotI site was also removed by site-directed mutagenesis. The following list includes the primers for the site-directed mutagenesis (addition or removal of restriction sites) of the CP4 EPSPS gene. Mutagenesis was carried out by the procedures of Kunkel et al. (1987), essentially as described in Sambrook et al. (1989).  
                      PRIMER BgNc (addition of BglII and NcoI sites to N-terminus)       CGTGGATAGATCTAGGAAGACAACCATGGCTCACGGTC       (SEQ ID NO:24)               PRIMER Sph2 (addition of SphI site to N-terminus)       GGATAGATTAAGGAAGACGCGCATGCTTCACGGTGCAAGCAGCC       (SEQ ID NO:25)               PRIMER S1 (addition of SacI site immediately after stop codons)       GGCTGCCTGATGAGCTCCACAATCGCCATCGATGG       (SEQ ID NO:26)               PRIMER N1 (removal of internal NotI recognition site)       CGTCGCTCGTCGTGCGTGGCCGCCCTGACGGC       (SEQ ID NO:27)               PRIMER Nco1 (removal of first internal NcoI recognition site)       CGGGCAAGGCCATGCAGGCTATGGGCGCC       (SEQ ID NO:28)               PRIMER Nco2 (removal of second internal NcoI recognition site)       CGGGCTGCCGCCTGACTATGGGCCTCGTCGG       (SEQ ID NO:29)          
 
     [0121] This CP4 EPSPS gene was then cloned as a NcoI-BamER N-terminal fragment plus a BamHI-SacI C-terminal fragment into a PrecA-gene10L expression vector similar to those described (Wong et al., 1988; Olins et al., 1988) to form pMON17101. The K m  for PEP and the K, for glyphosate were determined for the EPSPS activity in crude lysates of pMON17101/GB100 transformants following induction with nalidixic acid (Wong et al., 1988) and found to be the same as that determined for the purified and crude enzyme preparations from Agrobacterium sp. strain CP4.  
     [0122] Characterization of the EPSPS vene from Achromobacter sp. strain LBAA and from Pseudomonas sp. strain PG2982  
     [0123] A cosmid bank of partially HindIII-restricted LBAA DNA was constructed in  E. coli  MM294 in the vector pHC79 (Hohn and Collins, 1980). This bank was probed with a full length CP4 EPSPS gene probe by colony hybridization and positive clones were identified at a rate of ˜1 per 400 cosmids. The LBAA EPSPS gene was further localized in these cosmids by Southern analysis. The gene was located on an ˜2.8 kb Xhol fragment and by a series of sequencing steps, both from restriction fragment ends and by using the oligonucleotide primers from the sequencing of the CP4 EPSPS gene, the nucleotide sequence of the LBAA EPSPS gene was completed and is presented in FIG. 4 (SEQ ID NO:4).  
     [0124] The EPSPS gene from PG2982 was also cloned. The EPSPS protein was purified, essentially as described for the CP4 enzyme, with the following differences: Following the Sepharose CL-4B column, the fractions with the highest EPSPS activity were pooled and the protein precipitated by adding solid ammonium sulfate to 85% saturation and stirring for 1 hour. The precipitated protein was collected by centrifugation, resuspended in Q Sepharose buffer and following dialysis against the same buffer was loaded onto the column (as for the CP4 enzyme). After purification on the Q Sepharose column, ˜40 mg of protein in 100 mnM Tris pH 7.8, 10% glycerol, 1 mM EDTA, 1 mM DTT, and 1 M ammonium sulfate, was loaded onto a Phenyl Superose (Pharmacia) column. The column was eluted at 1.0 ml/minutes with a 40 ml gradient from 1.0 M to 0.00 M ammonium sulfate in the above buffer.  
     [0125] Approximately 1.0 mg of protein from the active fractions of the Phenyl Superose 10/10 column was loaded onto a Pharmacia Mono P 5/10 Chromatofocusing column with a flow rate of 0.75 ml/minutes. The starting buffer was 25 mM bis-Tris at pH 6.3, and the column was eluted with 39 ml of Polybuffer 74, pH 4.0. Approximately 50 g of the peak fraction from the Chromatofocusing column was dialyzed into 25 mM ammonium bicarbonate. This sample was then used to determine the N-terminal amino acid sequence.  
     [0126] The N-terminal sequence obtained was:  
     [0127] XHSASPKPATARRSE (where X an unidentified residue) (SEQ ID NO:30)  
     [0128] A number of degenerate oligonucleotide probes were designed based on this sequence and used to probe a library of PG2982 partial-HindIII DNA in the cosmid pHC79 (Hohn and Collins, 1980) by colony hybridization under nonstringent conditions. Final washing conditions were 15 minutes with 1X SSC, 0.1% SDS at 55° C. One probe with the sequence GCGGTBGCSGGYTrSGG (where B═C, G, or T; S═C or G, and Y═C or T) (SEQ ID NO:31) identified a set of cosmid clones.  
     [0129] The cosmid set identified in this way was made up of cosmids of diverse HindIII fragments. However, when this set was probed with the CP4 EPSPS gene probe, a cosmid containing the PG2982 EPSPS gene was identified (designated as cosmid 9C1 originally and later as pMON20107). By a series of restriction mappings and Southern analysis this gene was localized to a ˜2.8 kb XhoI fragment and the nucleotide sequence of this gene was determined. This DNA sequence (SEQ ID NO:6) is shown in FIG. 5. There are no nucleotide differences between the EPSPS gene sequences from LBAA (SEQ ID NO:4) and PG2982 (SEQ ID NO:6). The kinetic parameters of the two enzymes are within the range of experimental error.  
     [0130] A gene from PG2982 that imparts glyphosate tolerance in  E. coli  has been sequenced rFitzgibbon, 1988; Fitzgibbon and Braymer, 1990). The sequence of the PG2982 EPSPS Class II gene shows no homology to the previously reported sequence suggesting that the glyphosate-tolerant phenotype of the previous work is not related to EPSPS.  
     [0131] Charcterization of he EPSPS from  Bacillus subtilis    
     [0132] Bacillus subtilis 1A2 (prototroph) was obtained from the Bacillus Genetic Stock Center at Ohio State University. Standard EPSPS assay reactions contained crude bacterial extract with, 1 mM phosphoenolpyruvate (PEP), 2 mM shikimate-3-phosphate (S3P), 0.1 mM ammonium molybdate, 5 mM potassium fluoride, and 50 mM HEPES, pH 7.0 at 25° C. One unit (U) of EPSPS activity is defined as one 1 mol EPSP formed per minute under these conditions. For kinetic determinations, reactions contained crude bacterial, 2 mM S3P, varying concentrations of PEP, and 50 pmM HEPES, pH 7.0 at 25° C. The EPSPS specific activity was found to be 0.003 U/mg. When the assays were performed in the presence of 1 mM glyphosate, 100% of the EPSPS activity was retained. The appKm(PEP) of the B. subtilis EPSPS was determined by measuring the reaction velocity at varying concentrations of PEP. The results were analyzed graphically by the hyperbolic, Lineweaver-Burk and Eadie-Hofstee plots. which yielded appKr(PEP) values of 15.3 μM, 10.8 LM and 12.2 μM, respectively. These three data treatments are in good agreement, and yield an average value for appK m (PEP) of 13 μM. The appK i (glyphosate) was estimated by determining the reaction rates of  B. subtilis  1A2 EPSPS in the presence of several concentrations of glyphosate, at a PEP concentration of 2 μM. These results were compared to the calculated V max  of the EPSPS, and making the assumption that glyphosate is a competitive inhibitor versus PEP for  B. subtilis  EPSPS, as it is for all other characterized EPSPSs, an app(glyphosate) was determined graphically. The appK;(glyphosate) was found to be 0.44 mM.  
     [0133] The EPSPS expressed from the  B. subtilis  aroE gene described by Henner et al. (1986) was also studied. The source of the  B. subtilis  aroE (EPSPS) gene was the  E. coli  plasmid-bearing strain ECE13 (original code=MM294[trp100]; Henner, et al., 1984; obtained from the Bacillus Genetic Stock Center at Ohio State University; the culture genotype is [pBR322 trp100] Ap [in MM294] [pBR322::6 kb insert with trpFBA-hisH]). Two strategies were taken to express the enzyme in  E. coli  GB100 (aroA-): 1) the gene was isolated by PCR and cloned into an overexpression vector, and 2) the gene was subcloned into an overexpression vector. For the PCR cloning of the  B. subtilis  aroE from ECE13, two oligonucleotides were synthesized which incorporated two restriction enzyme recognition sites (NdeI and E,coRI) to the sequences of the following oligonucleotides:  
                          GGAACATATGAAACGAGATAAGGTGCAG   (SEQ ID NO:45)               GGAATTCAAACTTCAGGATCTTGAGATAGAAAATG   (SEQ ID NO:46)          
 
     [0134] The other approach to the isolation of the  B. subtilis  aroE gene, subcloning from ECE13 into pUC118, was performed as follows:  
     [0135] (i) Cut ECE13 and pUC with XmaI and SphI.  
     [0136] (ii) Isolate 1700bp aroE fragment and 2600bp pUC118 vector fragment.  
     [0137] (iii) Ligate fragments and transform into GB100.  
     [0138] The subclone was designated pMON21133 and the PCR-derived clone was named pMON21132. Clones from both approaches were first confirmed for complementation of the aroA mutation in  E. coli  GB100. The cultures exhibited EPSPS specific activities of 0.044 U/mg and 0.71 U/mg for the subdlone (pMON21133) and PCR-derived clone (pMON21132) enzymes, respectively. These specific activities reflect the expected types of expression levels of the two vectors. The  B. subtilis  EPSPS was found to be 88% and 100% resistant to inhibition by 1 mM glyphosate under these conditions for the subcloned (pMON21133) and PCR-derived (pMON21132) enzymes, respectively. The appk m (PEP) and the appKi(glyphosate) of the subcloned  B. subtilis  EPSPS (pMON21133) were determined as described above. The data were analyzed graphically by the same methods used for the 1A2 isolate, and the results obtained were comparable to those reported above for  B. subtilis  1A2 culture.  
     [0139] Characterization of the EPSPS gene from  Staphyloccus aureus    
     [0140] The kinetic properties of the S. aureus EPSPS expressed in  E. coli  were determined, including the specific activity, the appK m (PEP), and the appK i (glyphosate). The  S. aureus  EPSPS gene has been previously described (O&#39;C.onnell et al., 1993)  
     [0141] The strategy taken for the cloning of the  S. aureus  EPSPS was polymerase chain reaction (PCR), utilizing the known nucleotide sequence of the  S. aureus  aroA gene encoding EPSPS (O° C.onnell et al., 1993). The  S. aureus  culture (ATCC 35556) was fermented in an M2 facility in three 250 mL shake flasks containing 55 mL of TYE (tryptone 5g(L, yeast extract 3 9/L, pH 6.8). The three flasks were inoculated with 1.5 mL each of a suspension made from freeze dried ATCC 35556  S. aureus  cells in 90 mL of PBS (phosphate-buffered saline) buffer. Flasks were incubated at 30° C. for 5 days while shaking at 250 rpm. The resulting cells were Iysed (boiled in TE [tris/EDTA] buffer for 8 minutes) and the DNA utilized for PCR reactions. The EPSPS gene was amplified using PCR and engineered into an E coli expression vector as follows:  
     [0142] (i) two oligonucleotides were synthesized which incorporated two restriction enzyme recognition sites (NcoI and SacI) to the sequences of the oligonucleotides:  
                          GGGGCCATGGTAAATGAACAAATCATTG   (SEQ ID NO:47)               GGGGGAGCTCATTATCCCTCATTTTGTAAAAGC   (SEQ ID NO:48)          
 
     [0143] (ii) The purified, PCR-amplfied aroa gene from  S. aureus  was digested using NcoI and Sacd enzymes.  
     [0144] (ii) DNA of pMON 5723, which contains a pRecA bacterial promoter and Gene10 leader sequence (Olins et al., 1988) was digested NcoI and SacI and the 3.5 kb digestion product was purified.  
     [0145] (iv) The  S. aureus  PCR product and the NcoI/Sacl pMON 5723 fragment were ligated and transformed into  E. coli  JM101 competent cells.  
     [0146] (v) Two spectinomycin-resistant  E. coli  JM101 clones from above (SA#2 and SA#3) were purified and transformed into a competent aroa- E. coli  strain, GB100  
     [0147] For complementation experiments SAGB#2 and SAGB#3 were utilized, which correspond to SA#2 and SA#3, respectively, transformed into  E. coli  GB100. In addition,  E. coli  GB100 (negative control) and pMON 9563 (wt petunia EPSPS, positive control) were tested for AroA complementation. The organisms were grown in minimal media plus and minus aromatic amino acids. Later analyses showed that the SA#2 and SA#3 clones were identical, and they were assigned the plasmid identifier pMON21139.  
     [0148] SAGB#2 in  E. coli  GB100 (pMON21139) was also grown in M9 minimal media and induced with nalidixic acid. A negative control,  E. coli  GB100, was grown under identical conditions except the media was supplemented with aromatic amino acids. The cells were harvested, washed with 0.9% NaCl, and frozen at −80° C., for extraction and EPSPS analysis.  
     [0149] The frozen pMON21139  E. coli  GB100 cell pellet from above was extracted and assayed for EPSPS activity as previously described. EPSPS assays were performed using 1 mM phosphoenolpyruvate (PEP), 2 mM shikimate-3-phosphate (S3P), 0. 1 mM ammonium molybdate, 5 mM potassium fluoride, pH 7.0, 25° C. The total assay volume was 50 μL, which contained 10 μL of the undiluted desalted extract.  
     [0150] The results indicate that the two clones contain a functional aroA/EPSPS gene since they were able to grow in minimal media which contained no aromatic amino acids. As expected, the GB100 culture did not grow on minimal medium without aromatic amino acids (since no functional EPSPS is present), and the pMON9563 did confer growth in minimal media. These results demonstrated the successful cloning of a functional EPSPS gene from  S. aureus.  Both clones tested were identical, and the  E. coli  expression vector was designated pMON21139.  
     [0151] The plasmid pMON21139 in  E. coli  GB100 was grown in M9 minimal media and was induced with nalidixic acid to induce EPSPS expression driven from the RecA promoter. A desalted extract of the intracellular protein was analyzed for EPSPS activity, yielding an EPSPS specific activity of 0.005 μmol/min mg. Under these assay conditions, the  S. aureus  EPSPS activity was completely resistant to inhibition by 1 mM glyphosate. Previous analysis had shown that  E. coli  GB100 is devoid of EPSPS activity.  
     [0152] The appk m (PEP) of the  S. aureus  EPSPS was determined by measuring the reaction velocity of the enzyme kin crude bacterial extracts) at varying concentrations of PEP. The results were analyzed graphically using several standard kinetic plotting methods. Data analysis using the hyperbolic. Lineweaver-Burke, and Eadie-Hofstee methods yielded appk m (PEP) constants of 7.5, 4.8, and 4.0 μM. respectively. These three data treatments are in good agreement, and vield an average value for appK,(PEP) of 5 μM.  
     [0153] Further information of the glyphosate tolerance of  S. aureus  EPSPS was obtained by determining the reaction rates of the enzyme in the presence of several concentrations of glyphosate, at a PEP concentration of 2 μM. These results were compared to the calculated maximal velocity of the EPSPS, and making the assumption that glyphosate is a competitive inhibitor versus PEP for  S. aureus  EPSPS, as it is for all other characterized EPSPSs, an appK i (glyphosate). was determined graphically. The appK i (glyphosate) for  S. aureus  EPSPS estimated using this method was found to be 0.20 raK  
     [0154] The EPSPS from  S. aureus  was found to be glyphosate-tolerant, with an appK j (glyphosate) of approximately 0.2 mM. In addition, the appk m (PEP) for the enzyme is approximately 5 μM, yielding a appK i (glyphosate)/appk m (PEP) of 40.  
     [0155] Alternative Isolation Protocols for Other Class II EPSPS Structural Genes  
     [0156] A number of Class II genes have been isolated and described here. While the cloning of the gene from CP4 was difficult due to the low degree of similarity between the Class I and Class II enzymes and genes, the identification of the other genes was greatly facilitated by the use of this first gene as a probe. In the cloning of the LBAA EPSPS gene. the CP4 gene probe allowed the rapid identification of cosmid clones and the localization of the intact gene to a small restriction fragment and some of the CP4 sequencing primers were also used to sequence the LBAA (and PG2982) EPSPS gene(s). The CP4 gene probe was also used to confirm the PG2982 gene clone. The high degree of similarity of the Class II EPSPS genes may be used to identify and clone additional genes in much the same way that Class I EPSPS gene probes have been used to clone other Class I genes. An example of the latter was in the cloning of the  A. thaliana  EPSPS gene using the  P. hybrida  gene as a probe (Klee et al., 1987).  
     [0157] Glyphosate-tolerant EPSPS activity has been reported previously for EPSP synthases from a number of sources. These enzymes have not been characterized to any extent in most cases. The use of Class I and Class II EPSPS gene probes or antibody probes provide a rapid means of initially screening for the nature of the EPSPS and provide tools for the rapid cloning and characterization of the genes for such enzymes.  
     [0158] Two of the three genes described were isolated from bacteria that were isolated from a glYphosate treatment facility (Strains CP4 and LBAA). The third (PG2982) was from a bacterium that had been isolated from a culture collection strain. This latter isolation confirms that exposure to glyphosate is not a prerequisite for the isolation of high glyphosate-tolerant EPSPS enzymes and that the screening of collections of bacteria could yield additional isolates. It is possible to enrich for glyphosate degrading or glyphosate resistant microbial populations (Quinn et al., 1988; Talbot et al., 1984) in cases where it was felt that enrichment for such microorganisms would enhance the isolation frequency of Class II EPSPS microorganisms. Additional bacteria containing class II EPSPS gene have also been identified. A bacterium called C12, isolated from the same treatment column beads as CP4 (see above) but in a medium in which glyphosate was supplied as both the carbon and phosphorus source, was shown by Southern analysis to hybridize with a probe consisting of the CP4 EPSPS coding sequence. This result, in conjunction with that for strain LBAA, suggests that this enrichment method facilitates the identification of Class II EPSPS isolates. New bacterial isolates containing Class II EPSPS genes have also been identified from environments other than glyphosate waste treatment facilities. An inoculum was prepared by extracting soil (from a recently harvested soybean field in Jerseyville, Ill.) and a population of bacteria selected by growth at 280C in Dworkin-Foster medium containing glyphosate at 10 mM as a source of carbon (and with cycloheximide at 100 ,g/ml to prevent the growth of fungi). Upon plating on L-agar media, five colony types were identified. Chromosomal DNA was prepared from 2ml L-broth cultures of these isolates and the presence of a Class II EPSPS gene was probed using a the CP4 EPSPS coding sequence probe by Southern analysis under stringent hybridization and washing conditions. One of the soil isolates, S2, was positive by this screen.  
     [0159] Class II EPSPS enzymes are identifiable by an elevated K i  for glyphosate and thus the genes for these will impart a glyphosate tolerance phenotype in heterologous hosts. Expression of the gene from recombinant plasmids or phage may be achieved through the use of a variety of expression promoters and irclude the T7 promoter and polymerase. The T7 promoter and polymerase system has been shown to work in a wide range of bacterial (and mammalian) hosts and offers the advantage of expression of many proteins that may be present on large cloned fragments. Tolerance to growth on glyphosate may be shown on minimal growth media. In some cases, other genes or conditions that may give glyphosate tolerance have been observed, including over expression of beta-lactamase, the igrA gene (Fitzgibbon and Braymer, 1990), or the gene for glyphosate oxidoreductase (PCT Pub. No. W092/00377). These are easily distinguished from Class II EPSPS by the absence of EPSPS enzyme activity.  
     [0160] The EPSPS protein is expressed from the aroA gene (also called aroE in some genera, for example, in Bacillus) and mutants in this gene have been produced in a wide variety of bacteria. Determining the identity of the donor organism (bacterium) aids in the isolation of Class II EPSPS gene—such identification may be accomplished by standard microbiological methods and could include Gram stain reaction, growth, color of culture, and gas or acid production on different substrates, gas chromatography analysis of methylesters of the fatty acids in the membranes of the microorganism, and determination of the GC% of the genome. The identity of the donor provides information that may be used to more easily isolate the EPSPS gene. An AroA-host more closely related to the donor organism could be employed to clone the EPSPS gene by complementation but this is not essential since complementation of the  E. coli  AroA mutant by the CP4 EPSPS gene was observed. In addition. the information on the GC content the genome may be used in chooosing nucleotide probes -donor sources with high GC% would preferably use the CP4 EPSPS gene or sequences as probes and those donors with low GC would preferably employ those from  Bacillus subtilis  , for example.  
     [0161] Relationships between different EPSPS genes  
     [0162] The deduced amino acid sequences of a number of Class I and the Class II EPSPS enzymes were compared using the Bestfit computer program provided in the UWGCG package (Devereux et al. 1984). The degree of similarity and identity as determined using this program is reported. The degree of similarity/identity determined within Class I and Class II protein sequences is remarkably high, for instance, comparing  E. coli  with  S. typhimurium  (similarity/identity =93%/c/88%) and even comparing  E. coli  with a plant EPSPS (Petunia hybrida; 72%1/c/55%). These data are shown in Table W. The comparison of sequences between Class I and Class II, however, shows a much lower degree of relatedness between the Classes (similarity/identity=50-53%/c23-30%). The display of the Bestfit analysis for the  E. coli  (SEQ ID NO:8) and CP4 (SEQ ID NO:3) sequences shows the positions of the conserved residues and is presented in FIG. 6. Previous analyses of EPSPS sequences had noted the high degree of conservation of sequences of the enzymes and the almost invariance of sequences in two regions—the “20-35” and “95-107” regions (Gasser et al., 1988; numbered according to the Petunia EPSPS sequence)—and these regions are less conserved in the case of CP4 and LBAA when compared to Class I bacterial and plant EPSPS sequences (see FIG. 6 for a comparison of the  E. coli  and CP4 EPSPS sequences with the  E. coli  sequence appearing as the top sequence in the Figure). The corresponding sequences in the CP4 Class II EPSPS are:  
                                      PGDKSISHRSFMFGGL   (SEQ ID NO:32) and                       LDFGNAATGCRLT   (SEQ ID NO:33).          
 
     [0163] These comparisons show that the overall relatedness of Class I and Class II is EPSPS proteins is low and that sequences in putative conserved regions have also diverged considerably.  
     [0164] In the CP4 EPSPS an alanine residue is present at the “glycine101” position. The replacement of the conserved glycine (from the “95-107” region) by an alanine results in an elevated K i  for glyphosate and in an elevation in the K m  for PEP in Class I EPSPS. In the case of the CP4 EPSPS, which contains an alanine at this position, the K m  for PEP is in the low range, indicating that the Class II enzymes differ in many aspects from the EPSPS enzymes heretofore characterized.  
     [0165] Within the Class II isolates, the degree of similarity/identity is as high as that noted for that within Class I (Table IVA). FIG. 7 displays the Bestfit computer program alignment of the CP4 (SEQ ID NO:3) and LBAA (SEQ ID NO:5) EPSPS deduced amino acid sequences with the CP4 sequence appealing as the top sequence in the Figure. The symbols used in FIGS. 6 and 7 are the standard symbols used in the Bestfit computer program to designate degrees of similarity and identity.  
               TABLE IVA 1,2                            Comparison of relatedness of EPSPS protein sequences       Comparison between Class I and Class II EPSPS protein sequences                                     similarity   identity                         S. cerevisiae  vs. CP4   54   30             A. nidulans  vs. CP4   50   25             B. napus  vs. CP4   47   22             A. thaliana  vs. CP4   48   22             N. tabacum  vs. CP4   50   24             L. esculentum  vs. CP4   50   24             P. hybrida  vs. CP4   50   23             Z. mays  vs. CP4   48   24             S. gallinarum  vs. CP4   51   25             S. typhimurium  vs. CP4   51   25             S. typhi  vs. CP4   51   25             K. pneumoniae  vs. CP4   56   28             Y. enterocolitica  vs. CP4   53   25             H. influenzae  vs. CP4   53   27             P. multocida  vs. CP4   55   30             A. salmonicida  vs. CP4   53   23             B. pertussis  vs. CP4   53   27             E. coli  vs. CP4   52   26             E. coli  vs. LBAA   52   26             E. coli  vs.  B. subtilis     55   29             E. coli  vs.  D. nodosus     55   32             E. coli  vs.  S. aureus     55   29             E. coli  vs. Synechocystis sp. PCC6803   53   30                 Comparison between Class I EPSPS protein sequences                                   E. coli  vs.  S. typhimurium     93   88             P. hybrida  vs.  E. coli     72   55                 Comparison between Class II EPSPS protein sequences                                   D. nodosus  vs. CP4   62   43           LBAA vs. CP4   90   83           PG2892 vs. CP4   90   83             S. aureus  vs. CP4   58   34             B. subtilis  vs. CP4   59   41           Synechocystis sp. PCC6803 vs. CP4   62   45                                              
 
     [0166]               TABLE IVB                          Location of Conserved Sequences in       Class II EPSP Synthases                                 Source   Seq. 1 1     Seq. 2 2     Seq. 3 3     Seq. 4 4                 CP4                       start   200   26   173   271       end   204   29   177   274       LBAA       start   200   26   173   271       end   204   29   177   274       PG2982       start   200   26   173   273       end   204   29   177   276         B. subtilis         start   190   17   164   257       end   194   20   168   260         S. aureus         start   193   21   166   261       end   197   24   170   264       Synechocystis sp. PCC6803       start   210   34   183   278       end   214   38   187   281         D. nodosus         start   195   22   168   261       end   199   25   172   264       min. start   190   17   164   257       max. end   214   38   187   281                                                    
     [0167] The domains of EPSP synthase sequence identified in this application were determined to be those important for maintenance of glyphosate resistance and productive binding of PEP. The information used in indentifying these domains included sequence alignments of numerous glyphosate-sensitive EPSPS molecules and the three-dimensional x-ray structures of  E. coli  EPSPS (Stallings, et al. 1991) and CP4 EPSPS. The structures are representative of a glyphosate-sensitive (i.e., Class I) enzyme, and a naturally-occuring glyphosate-tolerant (i.e., Class II) enzyme of the present invention. These exemplary molecules were superposed three-dimensionally and the results displayed on a computer graphics terminal. Inspection of the display allowed for structure-based fine-tuning of the sequence alignments of glyphosate-sensitive and glyphosate-resistant EPSPS molecules. The new sequence alignments were examined to determine differences between Class I and Class II EPSPS enzymes. Seven regions were identified and these regions were located in the x-ray structure of CP4 EPSPS which also contained a bound analog of the intermediate which forms catalytically between PEP and S3P.  
     [0168] The structure of the CP4 EPSPS with the bound intermediate analog was displayed on a computer graphics terminal and the seven sequence segments were examined. Important residues for glyphosate binding were identified as well as those residues which stabilized the conformations of those important residues: adjoining residues were considered necessary for maintenance of correct three-dimensional structural motifs in the context of glyphosate-sensitive EPSPS molecules. Three of the seven domains were determined not to be important for glyphosate tolerance and maintainance of productive PEP binding. The following four primary domains were determined to be characteristic of Class II EPSPS enzymes of the present invention:  
     [0169] R—Xi-H—X 2 —E (SEQ ID NO:37), in which  
     [0170] X 1  is an uncharged polar or acidic amino acid,  
     [0171] X 2  is serine or threonine,  
     [0172] The Arginine (R) reside at position 1 is important because the positive charge of its guanidium group destabilizes the binding of glyphosate. The Histidine (H) residue at position 3 stabilizes the Arginine (R) residue at position 4 of SEQ ID NO:40. The Glutamic Acid (E) residue at position 5 stabilizes the Lysine (K) residue at position 5 of SEQ ID NO:39.  
     [0173] —G—D—I—X 3  (SEQ ID NO:38), in which  
     [0174] X 3  is serme or threonie,  
     [0175] The Aspartic acid (D) residue at position 2 stabilizes the Arginine (R) residue at position 4 of SEQ ID NO:40. The Lysine (K) residue at position 3 is important because for productive PEP binding.  
     [0176] —S—A—Q—X 4 —K (SEQ ID NO:39), in which  
     [0177] X 4  is any amino acid,  
     [0178] The Alanine (A) residue at position 2 stabilizes the Arginine (R) residue at position 1 of SEQ ID NO:37. The Serine (S) residue at position 1 and the Glutamine (Q) residue at position 3 are important for productive S3P binding.  
     [0179] —N-X 5 —T—R (SEQ ID NO:40) in which  
     [0180] X 5  is any amino acid,  
     [0181] The Asparagine (N) residue at position 1 and the Threonine (T) residue at position 3 stabilize residue X 1  at position 2 of SEQ ID NO:37. The Arginine (R) residue at position 4 is important because the positive charge of its guanidium group destabilizes the binding of glyphosate.  
     [0182] Since the above sequences are only representative of the Class II EPSPSs which would be included within the generic structure of this group of EPSP synthases, the above sequences may be found within a subject EPSP synthase molecule within slightly more expanded regions. It is believed that the above-described conserved sequences would likely be found in the following regions of the mature EPSP synthases molecule:  
     [0183] —R—X 1 —H—X 2 —E—(SEQ ID NO:37) located between amino acids 175 and 230 of the mature EPSP synthase sequence;  
     [0184] —G—D—K—X 3 —(SEQ ID NO:38) located between amino acids 5 and 55 of the mature EPSP synthase sequence;  
     [0185] —S—A—Q—X 4 —K—(SEQ ID NO:39) located between amino acids 150 and 200 of the mature EPSP synthase sequence; and  
     [0186] —N—X 5 —T—R—(SEQ ID NO:40) located between amino acids 245 and 295 of the mature EPSPS synthase sequence.  
     [0187] One difference that may be noted between the deduced amino acid sequences of the CP4 and LBAA EPSPS proteins is at position 100 where an Alanine is found in the case of the CP4 enzyme and a Glycine is found in the case of the LBAA enzyme. In the Class I EPSPS enzymes a Glycine is usually found in the equivalent position, i.e Glycine96 in  E. coli  and  K pneumoniae  and Glycine101 in Petunia. In the case of these three enzymes it has been reported that converting that Glycine to an Alanine results in an elevation of the appK i for glyphosate and a concomitant elevation in the appk m  for PEP (Kishore et al., 1986; K i  shore and Shah, 1988; Sost and Amrhein, 1990), which, as discussed above, makes the enzyme less efficient especially under conditions of lower PEP concentrations. The Glycine100 of the LBAA EPSPS was converted to an Alanine and both the appk m  for PEP and the appK i  for glyphosate were determined for the variant. The Glycine100Alanine change was introduced by mutagenesis using the following primer:  
                                      CGGCAATGCCGCCACCGGCGCGCGCG   (SEQ ID NO:34)          
 
     [0188] and both the wild type and variant genes were expressed in  E. coli  in a RecA promoter expression vector (pMON17201 and pMON17264, respectively) and the appk m &#39;s and appK i &#39;s determined in crude lysates. The data indicate that the appK i (glyphosate) for the G1OOA variant is elevated about 16-fold (Table V). This result is in agreement with the observation of the importance of this G-A change in raising the appK i (glyphosate) in the Class I EPSPS enzymes. However, in contrast to the results in the Class I G-A variants, the appk m (PEP) in the Class II (LBAA) G-A variant is unaltered. This provides yet another distinction between the Class II and Class I EPSPS enzymes.  
                           TABLE V                                   appKm (PEP)   appKi (glyphosate)                                                Lysate prepared from:                 E. coli /pMON17201 (wild type)   5.3 μM    28 μM*         E. coli /pMON17264   5.5 μM   459 μM#       (G100A variant)                                          
 
     [0189] Modification and Resynthesis of the Agrobacterium sp. strain CP4 EPSPS Gene Sequence  
     [0190] The EPSPS gene from Agrobacterium sp. strain CP4 contains sequences that could be inimical to high expression of the gene in plants. These sequences include potential polyadenvlation sites that are often and A+T rich, a higher G+C% than that frequently found in plant genes (63% versus 50%), concentrated stretches of G and C residues, and codons that are not used frequently in plant genes The high G+C% in the CP4 EPSPS gene has a number of potential consequences including the following: a higher usage of G or C than that found in plant genes in the third position in codons, and the potential to form strong hair-pin structures that may affect expression or stability of the RNA. The reduction in the G+C content of the CP4 EPSPS gene, the disruption of stretches of G&#39;s and C&#39;s, the elimination of potential polyadenylation sequences, and improvements in the codon usage to that used more frequently in plant genes, could result in higher expression of the CP4 EPSPS gene in plants.  
     [0191] A synthetic CP4 gene was designed to change as completely as possible those inimical sequences discussed above. In summary, the gene sequence was redesigned to eliminate as much as possible the following sequences or sequence features (while avoiding the introduction of unnecessary restriction sites): stretches of G&#39;s and C&#39;s of 5 or greater; and A+T rich regions (predominantly) that could function as polyadenylation sites or potential RNA destabilization region The sequence of this gene is shown in FIG. 8 (SEQ ID NO:9). This coding sequence was expressed in  E. coli  from the RecA promoter and assayed for EPSPS activity and compared with that from the native CP4 EPSPS gene. The apparent K m  for PEP for the native and synthetic genes was 11.8 and 12.7, respectively, indicating that the enzyme expressed from the synthetic gene was unaltered. The N-terminus of the coding sequence was mutagenized to place an SphI site at the ATG to permit the construction of the CTP2-CP4 synthetic fusion for chloroplast import. The following primer was used to accomplish this mutagenesis:  
                              GGACGGCTGCTTGCACCGTGAAGCATGCTTAAGCTTGGCGTAATCATGG   (SEQ ID NO:35).              
 
     [0192] Eression of Cloroplast Directed CP4 EPSPS  
     [0193] The glyphosate target in plants, the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) enzyme. is located in the chloroplast. Many chloroplast-localized proteins, including EPSPS. are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP) that is removed during the import steps. Examples of other such chloroplast proteins include the small subunit (SSU) of Ribulose-1,5-bisphosphate carboxylase (RUBISCO), Ferredoxin, Ferredoxin oxidoreductase, the Light-harvesting-complex protein I and protein II, and Thioredoxin F. It has been demonstrated in vivo and in vitro that non-chloroplast proteins may be targeted to the chloroplast by use of protein fusions with a CTP and that a CTP sequence is sufficient to target a protein to the chloroplast.  
     [0194] A CTP-CP4 EPSPS fusion was constructed between the  Arabidopsis thaliana  EPSPS CTP (Klee et al., 1987) and the CP4 EPSPS coding sequences. The Arabidopsis CTP was engineered by site-directed mutagenesis to place a SphI restriction site at the CTP processing site. This mutagenesis replaced the Glu-Lys at this location with Cys-Met. The sequence of this CTP, designated as CTP2 (SEQ ID NO:10), is shown in FIG. 9. The N-terminus of the CP4 EPSPS gene was modified to place a SphI site that spans the Met codon. The second codon was converted to one for leucine in this step also. This change had no apparent effect on the in vivo activity of CP4 EPSPS in  E. coli  as judged by rate of complementation of the aroA allele. This modified N-terminus was then combined with the Sacl C-terminus and cloned downstream of the CTP2 sequences. The CTP2-CP4 EPSPS fusion was cloned into pBlueScript KS(+). This vector may be transcribed in vitro using the T7 polymerase and the RNA translated with  35 S-Methionine to provide material that may be evaluated for import into chloroplasts isolated from Lactuca sativa using the methods described hereinafter tdella-Cioppa et al., 1986, 1987). This template was transcribed in vitro using T7 polymerase and the  35 S-methionine-labeled CTP2-CP4 EPSPS material was shown to import into chloroplasts with an efficiency comparable to that for the control Petunia EPSPS (control= 35 S labeled PreEPSPS [pMON6140: della-Cioppa et al. 1986]).  
     [0195] In another example the Arabidopsis EPSPS CTP? designated as CTP3, was fused to the CP4 EPSPS through an EcoRI site. The sequence of this CTP3 (SEQ ID NO:12) is shown in FIG. 10. An EcoRI site was introduced into the Arabidopsis EPSPS mature region around amino acid 27, replacing the sequence -Arg-Ala-Leu-Leu-with -Arg-Ee-Leu-Leu-in the process. The primer of the following sequence was used to modify the N-terminus of the CP4 EPSPS gene to add an EcoRI site to effect the fusion to the  
                              CTP3:GGAAGACGCCCA GAATTC ACGGTGCAAGCAGCCGG   (SEQ ID NO:36) (the EcaRI site is underlined.              
 
     [0196] This CTP3-CP4 EPSPS fusion was also cloned into the pBlueScript vector and the T7 expressed fusion was found to also import into chloroplasts with an efficiency comparable to that for the control Petunia EPSPS (pMON6140).  
     [0197] A related series of CTPs, designated as CTP4 (SphI) and CTP5 (EcoRI), based on the Petunia EPSPS CTP and gene were also fused to the SphI-and EcoRI-modified CP4 EPSPS gene sequences. The SphI site was added by site-directed mutagenesis to place this restriction site (and change the amino acid sequence to -Cys-Met-) at the chloroplast processing site. All of the CTP-CP4 EPSPS fusions were shown to import into chloroplasts with approximately equal efficiency. The CTP4 (SEQ ID NO:14) and CTP5 (SEQ ID NO:16) sequences are shown in FIGS. 11 and 12.  
     [0198] A CTP2-LBAA EPSPS fusion was also constructed following the modification of the N-terminus of the LBAA EPSPS gene by the addition of a SphI site. This fusion was also found to be imported efficiently into chloroplasts.  
     [0199] By similar approaches, the CTP2-CP4 EPSPS and the CTP4-CP4 EPSPS fusion have also been shown to import efficiently into chloroplasts prepared from the leaf sheaths of corn. These results indicate that these CTP-CP4 fusions could also provide useful genes to impart glyphosate tolerance in monocot species.  
     [0200] The use of CTP2 or CTP4 is preferred because these transit peptide constructions yield mature EPSPS enzymes upon import into the chloroplat which are closer in composition to the native EPSPSs not containing a transit peptide signal. Those skilled in the art will recognize that various chimeric constructs can be made which utilize the functionality of a particular CTP to import a Class II EPSPS enzyme into the plant cell chloroplast. The chloroplast import of the Class II EPSPS can be determined using the following assay.  
     [0201] Chloroplast Uptake Assay  
     [0202] Intact chloroplasts are isolated from lettuce ( Latuca, sativa,  var. longifolia) by centrifugation in Percoll/ficoll gradients as modified from Bartlett et al., (1982). The final pellet of intact chloroplasts is suspended in 0.5 ml of sterile 330 mM sorbitol in 50 mM Hepes-KOH, pH 7.7, assayed for chlorophyll (Arnon, 1949), and adjusted to the final chlorophyll concentration of 4 mg/ml (using sorbitol/Hepes). The yield of intact chloroplasts from a single head of lettuce is 3-6mg chlorophyll.  
     [0203] A typical 300 μl uptake experiment contained 5 mM ATP, 8.3 mM unlabeled methionine, 322 mM sorbitol, 58.3 mM Hepes-KOH (pH 8.0), 50 μl reticulocyte lysate translation products, and intact chloroplasts from  L. sativa  (200 μg chlorophyll). The uptake mixture is gently rocked at room temperature (in 10×75 mm glass tubes) directly in front of a fiber optic illuminator set at maximum light intensity (150 Watt bulb). Aliquot samples of the uptake mix (about 50 μl) are removed at various times and fractionated over 100 μl silicone-oil gradients (in 150μl polyethylene tubes) by centrifugation at 11,000 X g for 30 seconds. Under these conditions, the intact chloroplasts form a pellet under the silicone-oil layer and the incubation medium (containing the reticulocyte lysate floats on the surface. After centrifugation, the silicone-oil gradients are immediately frozen in dry ice. The chloroplast pellet is then resuspended in 50-100 41 of lysis buffer (10mM Hepes-KOH pH 7.5, 1 mM PMSF, 1 mM benzamidine, 5 mM e-amino-n-caproic acid, and 30 μg/ml aprotinin) and centrifuged at 15,000 X g for 20 minutes to pellet the thylakoid membranes. The clear supernatant (stromal proteins) from this spin, and an aliquot of the reticulocyte lysate incubation medium from each uptake experiment, are mixed with an equal volume of 2X SDS-PAGE sample buffer for electrophoresis (Laemmli, 1970).  
     [0204] SDS-PAGE is carried out according to Laemmli (1970) in 3-17% (w/v) acrylamide slab gels (60 mm X 1.5 mm) with 3% (w/v) acrylamide stacking gels (5 mm×1.5 mm). The gel is fixed for 20-30 min in a solution with 40% methanol and 10% acetic acid. Then, the gel is soaked in EN 3 HANCE™ (DuPont) for 20-30 minutes, followed by drying the gel on a gel dryer. The gel is imaged by autoradiography, using an intensifying screen and an overnight exposure to determine whether the CP4 EPSPS is imported into the isolated chloroplasts.  
     [0205] Plant Transformation  
     [0206] Plants which can be made glyphosate-tolerant by practice of the present invention include, but are not limited to, soybean, cotton, corn, canola, oil seed rape, flax. sugarbeet, sunflower, potato, tobacco. tomato, wheat, rice, alfalfa and lettuce as well as various tree, nut and vine species.  
     [0207] A double-stranded DNA molecule of the present invention (“chimeric gene”) can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella (1983), Bevan (1984), K i  ee (1985) and EPO publication 120,516 (Schilperoort et aI.). In addition to plant transformation vectors derived from the Ti or root-inducing R 1  plasmids of Agrobacterium. alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may Involye. for example. the use of liposomes, electroporation. chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen.  
     [0208] Class II EPSPS Plant transformation vectors  
     [0209] Class II EPSPS DNA sequences may be engineered into vectors capable of transforming plants by using known techniques The following description is meant to be illustrative and not to be read in a limiting sense. One of ordinary skill in the art would know that other plasmids, vectors, markers, promoters, etc. would be used with suitable results. The CTP2-CP4 EPSPS fusion was cloned as a BglII-EcoRI fragment into the plant vector pMON979 (described below) to form pMON17110, a map of which is presented in FIG. 13. In this vector the CP4 gene is expressed from the enhanced CaMV 35 S promoter (E 35 S; Kay et al. 1987). A FMV 35 S promoter construct (pMON17116) was completed in the following way: The SalI—NotI and the NotI-BglII fragments from pMON979 containing the Spc(AAC(3)-III/oriV and the pBR322/Right Border/NOS 3′/CP4 EPSPS gene segment from pMON17110 were ligated with the XhoI-BglII FMV 35 S promoter fragment from pMON981. These vectors were introduced into tobacco, cotton and canola.  
     [0210] A series of vectors was also completed in the vector pMON977 in which the CP4 EPSPS gene, the CTP2-CP4 EPSPS fusion, and the CTP3-CP4 fusion were cloned as BglII-SacI fragments to form pMON17124, pMON17119, and pMON17120, respectively. These plasmids were introduced into tobacco. A pMON977 derivative containing the CTP2-LBAA EPSPS gene was also completed (pMON17206) and introduced into tobacco.  
     [0211] The pMON979 plant transformation/expression vector was derived from pMON886 (described below by replacing the neomycin phosphotransferase typeII (KAN) gene in pMON886 with the 0.89 kb fragment containing the bacterial gentamicin-3—N-acetvltransferase type III (AAC(3)-III) gene (Hayford et al., 1988). The chimeric P- 35 S/AA(3)-III/NOS 3′ gene encodes gentamicin resistance which permits selection of transformed plant cells. pMON979 also contains a 0.95 kb expression cassette consisting of the enhanced CaMV  35 S promoter (Kay et al., 1987), several unique restriction sites, and the NOS 3′ end (P-En-CaMV 35 S/NOS 3′). The rest of the pMON979 DNA segments are exactly the same as in pMON886.  
     [0212] Plasmid pM ON886 is made up of the following segments of DNA The first is a 0.93 kb Aval to engineered-EcoRV fragment isolated from transposon Tn7 that encodes bacterial spectinomycin/streptomycin resistance (Spc/Str), which is a determinant for selection in  E. coli  and  Agrobacterium tumefaciens.  This is joined to the 1.61 kb segment of DNA encoding a chimeric kanamycin resistance which permits selection of transformed plant cells. The chimeric gene (P- 35 S/KAN/NOS 3′) consists of the cauliflower mosaic virus (CaMV)  35 S promoter, the neomycin phosphotransferase typeII (KAN) gene, and the 3′-nontranslated region of the nopaline synthase gene (NOS 3′) (Fraley et al., 1983). The next segment is the 0.75 kb oriV containing the origin of replication from the RK2 plasmid. It is joined to the 3.1 kb SailI to PvuI segment of pBR322 (ori322) which provides the origin of replication for maintenance in  E. coli  and the bom site for the conjugational transfer into the  Agrobacterium tumefaciens  cells. The next segment is the 0.36 kb PvuI to BciI from pTiT37 that carries the nopaline-type T-DNA right border (Fraley et al., 1985).  
     [0213] The pMON977 vector is the same as pMON981 except for the presence of the P-En-CaMV 3 5S promoter in place of the FMV 35 S promoter (see below).  
     [0214] The pMON981 plasmid contains the following DNA segments: the 0.93 kb fragment isolated from transposon Tn7 encoding bacterial spectinomycin/streptomycin resistance (Spc/Str; a determinant for selection in  E. coli  and  Agrobacteriurn tumefaciens  (Fling et al., 1985)); the chimeric kanamycin resistance gene engineered for plant expression to allow selection of the transformed tissue. consisting of the 0.35 kb cauliflower mosaic virus  35 S promoter (P-35S) (Odell et al., 1985), the 0.83 kb neomycin phosphotransferase typeli gene (KAN), and the 0.26 kb 3′-nontranslated region of the nopaline synthase gene (NOS 3′) (Fraley et al., 1983); the 0.75 kb origin of replication from the RK2 plasmid (oriV) (Stalker et al., 1981); the 3.1 kb SalI to PuuI segment of pBR322 which provides the origin of replication for maintenance in  E. coli  (ori-322) and the bom site for the conjugational transfer into the  Agrobacterium tumefaciens  cells, and the 0.36 kb PvuI to BclI fragment from the pTiT37 plasmid containing the nopaline-type T-DNA right border region (Fraley et al., 1985). The expression cassette consists of the 0.6 kb 35S promoter from the flgwort mosaic virus (P-FMV35S) (Gowda et al., 1989) and the 0.7 kb 3′ non-translated region of the pea rbcS-E9 gene (E9 3′) (Coruzzi et al., 1984, and Morelli et al., 1985). The 0.6 kb SspI fragment containing the FMV 35 S promoter (FIG. 1) was engineered to place suitable cloning sites downstream of the transcriptional start site. The CTP2-CP4syn gene fusion was introduced into plant expression vectors (including pMON981, to form pMON17131; FIG. 14) and transformed into tobacco, canola, potato, tomato, sugarbeet, cotton, lettuce, cucumber, oil seed rape, poplar, and Arabidopsis.  
     [0215] The plant vector containing the Class II EPSPS gene may be mobilized into any suitable Agrobacterium strain for transformation of the desired plant species. The plant vector may be mobilized into an ABI Agrobacterium strain. A suitable ABI strain is the A208  Agrobacterium tumefaciens  carrying the disarmed Ti plasmid pTiC58 (pMP9ORK) (Koncz and Schell, 1986). The Ti plasmid does not carry the T-DNA phytohormone genes and the strain is therefore unable to cause the crown gall disease. Mating of the plant vector into ABI was done by the triparental conjugation system using the helper plasmid pRK2013 (Ditta et al., 1980). When the plant tissue is incubated with the ABI::plant vector conjugate. the vector is transferred to the plant cells by the vir functions encoded by the disarmed pTiC58 plasmid. The vector opens at the T-DNA right border region. and the entire plant vector sequence may be inserted into the host plant chromosome. The pTiC58 Ti plasmid does not transfer to the plant cells but remains in the Agrobacterium.  
     [0216] Class II EPSPS free DNA vectors  
     [0217] Class II EPSPS genes may also be introduced into plants through direct delivery methods. A number of direct delivery vectors were completed for the CP4 EPSPS gene. The vector pMON13640, a map of which is presented in FIG. 15, is described here. The plasmid vector is based on a pUC plasmid (Vieira and Messing, 1987) containing, in this case, the nptfl gene (kanamycin resistance; KAN) from Tn903 to provide a selectable marker in  E. coli  . The CTP4-EPSPS gene fusion is expressed from the P-FMV35S promoter and contains the NOS 3′ polyadenylation sequence fragment and from a second cassette consisting of the E 35 S promoter, the CTP4-CP4 gene fusion and the NOS 3′ sequences. The scoreable GUS marker gene (Jefferson et al., 1987) is expressed from the mannopine synthase promoter (P-MAS; Velten et al., 1984) and the soybean 7S storage protein gene 3′ sequences (Schuler et al., 1982). Similar plasmids could also be made in which CTP-CP4 EPSPS fusions are expressed from the enhanced CaMV 35 S promoter or other plant promoters. Other vectors could be made that are suitable for free DNA delivery into plants and such are within the skill of the art and contemplated to be within the scope of this disclosure.  
     [0218] Plastid transformation  
     [0219] While transformation of the nuclear genome of plants is much more developed at this time. a rapidly advancing alternative is the transformation of plant organelles. The transformation of plastids of land plants and the regeneration of stable transformants has been demonstrated (Svab et al., 1990; Maliga et al., 1993). Transformants are selected, following double cross-over events into the plastid genome, on the basis of resistance to spectinomycin conferred through rRNA changes or through the introduction of an aminoglycoside 3′-adenyltransferase gene (Svab et al. 1990: Svab and Maliga, 1993), or resistance to kanamycin through the neomycin phosphotransferase NptII (Carrer et al., 1993). DNA is introduced by biolistic means (Svab et al, 1990; Maliga et al., 1993) or by using polyethylene glycol (O&#39;Neill et al., 1993). This transformation route results in the production of 500-10,000 copies of the introduced sequence per cell and high levels of expression of the introduced gene have been reported (Carrer et al., 1993; Maliga et al., 1993). The use of plastid transformation offers the adavantages of not requiring the chloroplast transit peptide signal sequence to result in the localization of the heterologous Class II EPSPS in the chloroplast and the potential to have many copies of the heterologous plant-expressible Class II EPSPS gene in each plant cell since at least one copy of the gene would be in each plastid of the cell.  
     [0220] Plant Regeneration  
     [0221] When expression of the Class II EPSPS gene is achieved in transformed cells (or protoplasts), the cells (or protoplasts) are regenerated into whole .plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, rice. corn, etc.), Solanaceae (potato, tobacco, tomato, peppers), various floral crops as well as various trees such as poplar or apple, nut crops or vine plants such as grapes. See, e.g., Ammirato, 1984; Shimamoto, 1989; Fromm, 1990; Vasil, 1990.  
     [0222] The following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications, truncations, etc. can be made to the methods and genes described herein while not departing from the spirit and scope of the present invention.  
     [0223] In the examples that follow, EPSPS activity in plants is assayed by the following method. Tissue samples were collected and immediately frozen in liquid nitrogen. One gram of young leaf tissue was frozen in a mortar with liquid nitrogen and ground to a fine powder with a pestle. The powder was then transferred to a second mortar, extraction buffer was added (1 ml /gram), and the sample was ground for an additional 45 seconds. The extraction buffer for canola consists of 100 mM Tris, 1 mM EDTA, 10 % glycerol, 5 mM DTr, 1 mM BAM, 5 mM ascorbate, 1.0 mg/ml BSA, pH 7.5 (4° C.). The extraction buffer for tobacco consists of 100 mM Tris, 10 mM EDTA, 35 mM KCI, 20 % glycerol, 5 mM DTT, 1 mM BAM, 5 mM ascorbate, 1.0 mg/ml BSA, pH 7.5 (4° C.). The mixture was transferred to a microfuge tube and centrifiged for 5 minutes. The resulting supernatants were desalted on spin G-50 (Pharmacia) columns, previously equilibrated with extraction buffer (without BSA), in 0.25 ml aliquots. The desalted extracts were assayed for EPSP synthase activity by radioactive HPLC assay. Protein concentrations in samples were determined by the BioRad microprotein assay with BSA as the standard.  
     [0224] Protein concentrations were determined using the BioRad Microprotein method. BSA was used to generate a standard curve ranging from 2 -24 μg. Either 800 μl of standard or diluted sample was mixed with 200 μl of concentrated BioRad Bradford reagent. The samples were vortexed and read at A(595) after 5 minutes and compared to the standard curve.  
     [0225] EPSPS enzyme assays contained HEPES (50 mM), shikimate-3-phosphate (2 mM), NH4 molybdate (0.1 mM) and KF (5 mM), with or without glyphosate (0.5 or 1.0 mM). The assay mix (30 RI) and plant extract (10 μl) were preincubated for 1 minute at 25° C. and the reactions were initiated by adding 14C-PEP (1 mM). The reactions were quenched after 3 minutes with 50 μl of 90% EtOH/0.lM HOAc, pH 4.5. The samples were spun at 6000 rpm and the resulting supernatants were analyzed for 14C-EPSP production by HPLC. Percent resistant EPSPS is calculated from the EPSPS activities with and without glyphosate.  
     [0226] The percent conversion of  14 C labeled PEP to  14 C EPSP was determined by HPLC radioassay using a C18 guard column (Brownlee) and an AX 100  HPLC column (0.4×25 cm, Synchropak) with 0.28 M isocratic potassium phosphate eluant, pH 6.5, at 1 ml/min. Initial velocities were calculated by multiplying fractional turnover per unit time by the initial concentration of the labeled substrate (1 mM). The assay was linear with time up to ˜3 minutes and 30% turnover to EPSPS. Samples were diluted with 10 mM Tris, 10% glycerol, 10 mM DTT, pH 7.5 (4° C.) if necessary to obtain results within the linear range.  
     [0227] In these assays DLdithiotheitol (DTT), benzamidine (BAM), and bovine serum albumin (BSA, essentially globulin free) were obtained from Sigma. Phosphoenolpyruvate (PEP) was from Boehringer Mannheim and phosphoenol-[1- 14 C]pyruvate (28 mCi/mmol) was from Amersham. 
    
    
     EXAMPLES  
     Example 1  
     [0228] Transformed tobacco plants have been generated with a number of the Class II EPSPS gene vectors containing the CP4 EPSPS DNA sequence as described above with suitable expression of the EPSPS. These transformed plants exhibit glyphosate tolerance imparted by the Class II CP4 EPSPS.  
     [0229] Transformation of tobacco employs the tobacco leaf disc transformation protocol which utilizes healthy leaf tissue about 1 month old. After a 15-20 minutes surface sterilization with 10% Clorox plus a surfactant, the leaves are rinsed 3 times in sterile water. Using a sterile paper punch, leaf discs are punched and placed upside down on MS104 media (MS salts 4.3 g/l, sucrose 30 g/l, B5 vitamins 500X 2 mill, NAA 0.1 mgtl, and BA 1.0 mg/l) for a 1 day preculture.  
     [0230] The discs are then inoculated with an overnight culture of a disarmed Agrobacternum ABI strain containing the subject vector that had been diluted  {fraction (1/5)} (i.e.: about  0.6 OD). The inoculation is done by placing the discs in centrifuge tubes with the culture. After 30 to 60 seconds, the liquid is drained off and the discs were blotted between sterile filter paper. The discs are then placed upside down on MS104 feeder plates with a filter disc to cture.  
     [0231] After 2-3 days of co-culture, the discs are transferred, still upside down, to selection plates with MS104 media. After 2-3 weeks, callus tissue formed, and individual clumps are separated from the leaf discs. Shoots are cleanly cut from the callus when they are large enough to be distinguished from stems. The shoots are placed on hormone-free rooting media (MSO: MS salts 4.3 g/l, sucrose 30 g/l, and B5 vitamins 500X 2 ml/l) with selection for the appropriate antibiotic resistance. Root formation occurred in 1-2 weeks. Any leaf callus assays are preferably done on rooted shoots while still sterile. Rooted shoots are then placed in soil and kept in a high humidity environment (i.e.: plastic containers or bags). The shoots are hardened off by gradually exposing them to ambient humidity conditions.  
     [0232] Expression of CP4 EPSPS protein in transformed plants  
     [0233] Tobacco cells were transformed with a number of plant vectors containing the native CP4 EPSPS gene, and using different promoters andlor CTP&#39;s. Preliminary evidence for expression of the gene was given by the ability of the leaf tissue from antibiotic selected transformed shoots to recallus on glyphosate. In some cases, glyphosate-tolerant callus was selected directly following transformation. The level of expression of the CP4 EPSPS was determined by the level of glyphosate-tolerant EPSPS activity (assayed in the presence of 0.5 mM glyphosate) or by Western blot analysis using a goat anti-CP4 EPSPS antibody. The Western blots were quantitated by densitometer tracing and comparison to a standard curve established using purified CP4 EPSPS. These data are presented as % soluble leaf protein. The data from a number of transformed plant lines and transformation vectors are presented in Table VI below.  
               TABLE VI                          Expression of CP4 EPSPS in transformed tobacco tissue                                         CP4 EPSPS **           Vector   Plant #   (% leaf protein)                       pMON17110   25313   0.02           pMON17110   25329   0.04           pMON17116   25095   0.02           pMON17119   25106   0.09           pMON17119   25762   0.09           pMON17119   25767   0.03                                  
 
     [0234] Glyphosate tolerance has also been demonstrated at the whole plant level in transformed tobacco plants. In tobacco, R 0  transformants of CTP2-CP4 EPSPS were sprayed at 0.4 lb/acre (0.448 kg/hectare), a rate sufficient to kill control non-transformed tobacco plants corresponding to a rating of 3. 1 and 0 at days 7, 14 and 28. respectively, and were analyzed vegetatively and reproductively (Table VII).  
               TABLE VII                          Glyphosate tolerance in R o  tobacco CP4 transformants                         Score**                             Vegetative                                             Vector/Plant #   day 7   day 14   day 28   Fertile                       pMON17110/25313   6   4    2   no           pMON17110/25329   9   10    10   yes           pMON17119/25106   9   9   10   yes                                              
 
     Example 2A  
     [0235] Canola plants were transformed with the pMON17110, pMON17116, and pMON17131 vectors and a number of plant lines of the transformed canola were obtained which exhibit glyphosate tolerance.  
     [0236] Plant Material  
     [0237] Seedlings of Brassica napus cv Westar were established in 2 inch (˜5 cm) pots containing Metro Mix 350. They were grown in a growth chamber at 24° C., 16/8 hour photoperiod, light intensity of 400 uEm- 2 sec-i (HID lamps) They were fertilized with Peters 20-10-20 General Purpose Special. After 2½ weeks they were transplanted to 6 inch (˜15 cm) pots and grown in a growth chamber at 15/10° C. day/night temperature. 16/8 hour photoperiod, light intensity of 800 uEm- 2 see-I (HID lamps). They were fertilized with Peters 15-30-15 Hi-Phos Special.  
     [0238] Transformation/Selection/Regeneration  
     [0239] Four terminal internodes from plants just prior to bolting or in the process of bolting but before flowering were removed and surfaced sterilized in 70% v/v ethanol for 1 minute, 2% w/v sodium hypochlorite for 20 minutes and rinsed 3 times with sterile deionized water. Stems with leaves attached could be refrigerated in moist plastic bags for up to 72 hours prior to sterilization. Six to seven stem segments were cut into 5mm discs with a Redco Vegetable Slicer 200 maintaining orientation of basal end.  
     [0240] The Agrobacterium was grown overnight on a rotator at 24° C. in 2 mls of Luria Broth containing 50 mg/l kanamycin, 24mg/l chloramphenicol and 100 mg/l spectinomycin. A 1:10 dilution was made in MS (Murashige and Skoog) media giving approximately 9xlO 8  cells per ml. This was confirmed with optical density readings at 660 mu. The stem discs (explants) were inoculated with 1.0 ml of Agrobacterium and the excess was aspirated from the explants.  
     [0241] The explants were placed basal side down in petri plates containing 1/10X standard MS salts. B5 vitamins. 3% sucrose. 0.8% agar, pH 5.7, 1.0 mg/l 6-benzyladenine (BA). The plates were layered with 1.5 ml of media containing MS salts, B5 vitamins, 3% sucrose, pH 5.7, 4.0 mg/i p-chlorophenoxyacetic acid, 0.005 mg/l kinetin and covered with sterile filter paper.  
     [0242] Following a 2 to 3 day co-culture, the explants were transferred to deep dish petri plates containing MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 1 mg/l BA. 500 mg/l carbenicillin, 50mg/l cefotaxime, 200 mWi kanamycin or 175 mg/l gentamicin for selection. Seven explants were placed on each plate. After 3 weeks they were transferred to fresh media. 5 explants per plate. The explants were cultured in a growth room at 25° C. continuous light (Cool White).  
     [0243] Expression Assay  
     [0244] After 3 weeks shoots were excised from the explants. Leaf recallusing assays were initiated to confirm modification of Re shoots. Three tiny pieces of leaf tissue were placed on recallusing media containing MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 5.0mg/l BA, 0.5 mg/l naphthalene acetic acid (NAA), 500 mg/l carbenicillin, 50mg/l cefotaxime and 200 mg/l kanamycin or gentamicin or 0.5 mM glyphosate. The leaf assays were incubated in a growth room under the same conditions as explant culture. After 3 weeks the leaf recallusing assays were scored for herbicide tolerance (callus or green leaf tissue) or sensitivity (bleaching).  
     [0245] Trnpantation  
     [0246] At the time of excision, the shoot stems were dipped in Rootone® and placed in 2 inch (˜5 cm) pots containing Metro-Mix 350 and placed in a closed humid environment. They were placed in a growth chamber at 24° C., 16/8 hour photoperiod, 400 uEm- 1 sec- 2 (HID lamps) for a hardening-off period of approximately 3 weeks.  
     [0247] The seed harvested from R 0 , plants is R 1  seed which gives rise to R 1  plants. To evaluate the glyphosate tolerance of an Ro plant, its progeny are evaluated. Because an R 0  plant is assumed to be hemizygous at each insert location, selfing results in maximum genotypic segregation in the R 1 . Because each insert acts as a dominant allele, in the absence of linkage and assuming only one hemizygous insert is required for tolerance expression. one insert would segregate 3:1, two inserts, 15:1, three inserts 63:1, etc. Therefore, relatively few R 1  plants need be grown to find at least one resistant phenotype.  
     [0248] Seed from an R 0  plant is harvested, threshed, and dried before planting in a glyphosate spray test. Various techniques have been used to grow the plants for R 1  spray evaluations. Tests are conducted in both greenhouses and growth chambers. Two planting systems are used; ˜10 cm pots or plant trays containing 32 or 36 cells. Soil used for planting is either Metro 350 plus three types of slow release fertilizer or plant Metro 350. Irrigation is either overhead in greenhouses or sub-irrigation in growth chambers. Fertilizer is applied as required in irrigation water. Temperature regimes appropriate for canola were maintained. A sixteen hour photoperiod was maintained. At the onset of flowering, plants ore transplanted to ˜15 cm pots for seed production.  
     [0249] A spray “batch” consists of several sets of R 1  progenies all sprayed on the same date. Some batches may also include evaluations of other than R 1  plants. Each batch also includes sprayed and unsprayed non-transgenic genotypes representing the genotypes in the particular batch which were putatively transformed. Also included in a batch is one or more non-segregating transformed genotypes previously identified as having some resistance.  
     [0250] Two-six plants from each individual Ro progeny are not sprayed and serve as controls to compare and measure the glyphosate tolerance, as well as to assess any variability not induced by the glyphosate. When the other plants reach the 2-4 leaf stage, usually 10 to 20 days after planting, glyphosate is applied at rates varying from 0.28 to 1.12 kg/ha, depending on objectives of the study. Low rate technology using low volumes has been adopted. A laboratory track sprayer has been calibrated to deliver a rate equivalent to field conditions.  
     [0251] A scale of 0 to 10 is used to rate the sprayed plants for vegetative resistance. The scale is relative to the unsprayed plants from the same R 0  plant. A 0 is death, while a 10 represents no visible difference from the unsprayed plant. A higher number between 0 and 10 represents progressively less damage as compared to the unsprayed plant. Plants are scored at 7, 14, and 28 days after treatment (DAT), or until bolting, and a line is given the average score of the sprayed plants within an R 0  plant family.  
     [0252] Six integers are used to qualitatively describe the degree of reproductive damage from glyphosate:  
     [0253] 0: No floral bud development  
     [0254] 2: Floral buds present, but aborted prior to opening  
     [0255] 4: Flooers open, but no anthers, or anthers fail to extrude past petals  
     [0256] 6: Sterile anthers  
     [0257] 8: Partially sterile anthers  
     [0258] 10: Fully fertile flowers  
     [0259] Plants are scored using this scale at or shortly after initiation of flowering, depending on the rate of floral structure development.  
     [0260] Expression of EPSPS in Canola  
     [0261] After the 3 week period, the transformed canola plants were assayed for the presence of glyphosate-tolerant EPSPS activity (assayed in the presence of glyphosate at 0.5 mM). The results are shown in Table VIII.  
               TABLE VIII                          Expression of CP4 EPSPS in transformed Canola plants                                 % resistant EPSPS activity               of Leaf extract           Plant #   (at 0.5 mM glyphosate)                                             Vector Control        0           pMON17110   41   47           pMON17110   52   28           pMON17110   71   82           pMON17110   104   75           pMON17110   172   84           pMON17110   177   85           pMON17110   252    29*           pMON17110   350   49           pMON17116   40   25           pMON17116   99   87           pMON17116   175   94           pMON17116   178   43           pMON17116   182   18           pMON17116   252   69           pMON17116   298    44*           pMON17116   332   89           pMON17116   383   97           pMON17116   395   52                                  
 
     [0262] R 1  transformants of canola were then grown in a growth chamber and sprayed with glyphosate at 0.56 kg/ha tkilogram/hectare) and rated vegetatively. These results are shown in Table IXA-IXC. It is to be noted that expression of glyphosate resistant EPSPS in all tissues is preferred to observe optimal glyphosate tolerance phenotype in these transgenic plants. In the Tables below, only expression results obtained with leaf tissue are described.  
               TABLE IXA                          Glyphosate tolerance in Class II EPSPS       canola R 1  transformants       (pMON17110 = P-E35S; pMON17116 = P-FMV35S; R1 plants;       Spray rate 0.56 kg/ha)                                     Vegetative               % resistant   Score**                                     Vector/Plant No.   EPSPS*   day 7   day 14                       Control Westar    0   5   3           pMON17110/41   47   6   7           pMON17110/71   82   6   7           pMON17110/177   85   9   10            pMON17116/40   25   9   9           pMON17116/99   87   9   10            pMON17116/175   94   9   10            pMON17116/178   43   6   3           pMON17116/182   18   9   10            pMON17116/383   97   9   10                       
 
     [0263]               TABLE IXB                          Glyphosate tolerance in Class II EPSPS       canola R 1  transformants       (pMON17131 = P-FMV35S; R1 plants; Spray rate = 0.84 kg/ha)                                     Vegetative score**   Reproductive score           Vector/Plant No.   day 14   day 28                       17131/78   10    10           17131/102   9   10           17131/115   9   10           17131/116   9   10           17131/157   9   10           17131/169   10    10           17131/255   10    10           control Westar   1    0                        
     [0264]               TABLE IXC                          Glyphosate tolerance in Class I EPSPS       canola transformants       (P-E35S: R2 Plants; Spray rate = 0.28 kg/ha)                                     Vegetative               % resistant   Score**                                     Vector/Plant No.   EPSPS*   day 7   day 14                       Control Westar    0   4   2           pMON899/715   96   5   6           pMON899/744   95   8   8           pMON899/794   86   6   4           pMON899/818   81   7   8           pMON899/885   57   7   6                                                
     [0265] The data obtained for the Class II EPSPS transformants may be compared to glyphosate-tolerant Class I EPSP transformants in which the same promoter is used to express the EPSPS genes and in which the level of glyphosate-tolerant EPSPS activity was comparable for the two types of transformants. A comparison of the data of pMON17110 [in Table IXA] and pMON17131 [Table IXB] with that for pMON899 [in Table IX]; the Class I gene in pMON899 is that from  A. thaliana  [Klee et al., 1987] in which the glycine at position 101 was changed to an alaninej illustrates that the Class II EPSPS is at least as good as that of the Class I EPSPS. An improvement in vegetative tolerance of Class II EPSPS is apparent when one takes into account that the Class II plants were sprayed at twice the rate and were tested as R 1  plants.  
     Example 2B  
     [0266] The construction of two plant transformation vectors and the transformation procedures used to produce glyphosate-tolerant canola plants are described in this example The vectors, pMON17209 and pMON17237, were used to generate transgenic glyphosate-tolerant canola lines. The vectors each contain the gene encoding the 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4. The vectors also contain either the gox gene encoding the glyphosate oxidoreductase enzyme (GOX) from Achromobacter sp. strain LBAA (Barry et al., 1992) or the gene encoding a variant of GOX (GOX v.247) which displays improved catalytic properties. These enzymes convert glyphosate to aminomethylphosphonic acid and glyoxvlate and protect the plant from damage by the metabolic inactivation of glyphosate. The combined result of providing an alternative, resistant EPSPS enzyme and the metabolism of glyphosate produces transgenic plants with enhanced tolerance to glyphosate  
     [0267] Molecular biology techniques. In general. standard molecular biology and microbial genetics approaches were employed (Maniatis et al., 1982). Site-directed mutageneses were carried out as described by Kunkel et al. (1987). Plant-preferred genes were synthesized and the sequence confirmed.  
     [0268] Plant transformation vectors. The following describes the general features of the plant transformation vectors that were modified to form vectors pMON17209 and pMON17237. The Agrobacterium mediated plant transformation vectors contain the following well-characterized DNA segments which are required for replication and function of the plasmids (Rogers and Klee, 1987; KIee and Rogers, 1989). The first segment is the 0.45 kb Clal-Dral fragment from the pTi15955 octopine Ti plasmid which contains the T-DNA left border region (Barker et al., 1983). It is joined to the 0.75 kb origin of replication (oriV) derived from the broad-host range plasmid RK2 (Stalker et al., 1981). The next segment is the 3.1 kb Sall-PvuI segment of pBR322 which provides the origin of replication for maintenance in  E. coli  and the bom site for the conjugational transfer into the  Agrobacterium tumefaciens  cells (Bolivar et al., 1977). This is fused to the 0.93 kb fragment isolated from transposon Tn7 which encodes bacterial spectinomycin and streptomycin resistance (Fling et al., 1985), a determinant for the selection of the plasmids in  E. coli  and Agrobacterium. It is fused to the 0.36 kb PvuI-BclI fragment from the pTiT37 plasmid which contains the nopaline-tvpe T-DNA right border region (Fraley et al., 1985). Several chimeric genes engineered for plant expression can be introduced between the Ti right and left border regions of the vector. In addition to the elements described above, this vector also includes the 35S promoter/NPTII/NOS 3′ cassette to enable selection of transformed plant tissues on kanamycin (Klee and Rogers, 1989; Fraley et al., 1983; and Odell, et al. 1985) within the borders. An “empty” expression cassette is also present between the borders and consists of the enhanced E35S promoter (Kay et al. 1987), the 3′ region from the small subunit of RUBPcarboxylase of pea (E9) (Coruzzi et al . 1984; Morelli et al., 1986), and a number of restriction enzyme sites that may be used for the cloning of DNA sequences for expression in plants. The plant transformation system based on  Agrobacterium tumefaciens  delivery has been reviewed (Klee and Rogers, 1989; Fraley et al., 1986). The Agrobacterium mediated transfer and integration of the vector T-DNA into the plant chromosome results in the expression of the chimeric genes conferring the desired phenotype in plants.  
     [0269] Bacterial Inoculum. The binary vectors are mobilized into  Agrobacterium tumefaciens  strain ABI by the triparental conjugation system using the helper plasmid pRK2013 (Ditta et al. 1980). The ABI strain contains the disarmed pTiC58 plasmid pMP9ORK (Koncz and Schell, 1986) in the chloramphenicol resistant derivative of the Agrobacterium tumefaciens strain A208.  
     [0270] Transformation procedure. Agrobacterium inocula were grown overnight at 28° C. in 2 ml of LBSCK (LBSCK is made as follows: LB liquid medium [1 liter volume] =10 g NaCl; 5 g Yeast Extract;10 g tryptone; pH 7.0, and autoclave for 22 minutes. After autoclaving, add spectinomycin (50 mg/ml stock)—2 ml, kanamycin (50 mg/ml stock)—1 ml, and chloramphenicol (25 mg/ml stock)—1 ml.). One day prior to inoculation, the Agrobacterium was subcultured by inoculating 200ul into 2 ml of fresh LBSCK and grown overnight. For inoculation of plant material, the culture was diluted with MSO liquid medium to an A 660  range of 0.2-0.4.  
     [0271] Seedlings of Brassica napus cv. Westar were grown in Metro Mix 350 (Hummert Seed Co., St. Louis, Mo.) in a growth chamber with a day/night temperature of 15/10° C., relative humidity of 50%, 16h/8h photoperiod, and at a light intensity of 500 ;mol m  2  sec- 1 . The plants were watered daily (via sub-irngation) and fertilized every other day with Peter&#39;s 15:30:15 (Fogelsville, Pa.).  
     [0272] In general, all media recipes and the transformation protocol follow those in Fry et. aL (1987). Five to six week-old Westar plants were harvested when the plants had bolted (but prior to flowering), the leaves and buds were removed, and the 4-5 inches of stem below the flower buds were used as the explant tissue source. Following sterilization with 70% ethanol for 1 min and 38% Clorox for 20 min, the stems were rinsed three times with sterile water and cut into 5 mm-long segments (the orientation of the basal end of the stem segments was noted). The plant material was incubated for 5 minutes with the diluted Agrobacterium culture at a rate of 5 ml of culture per 5 stems. The suspension of bacteria was removed by aspiration and the explants were placed basal side down -for an optimal shoot regeneration response—onto co-culture plates (1/10 MSO solid medium with a 1.5 ml IXD (tobacco xanthi diploid) liquid medium overlay and covered with a sterile 8.5 cm filter paper). Fifty-to-sixty stem explants were placed onto each co-culture plate.  
     [0273] After a 2 day co-culture period, stem explants were moved onto MS medium containing 750 mg/l carbenicillin, 50 mg/l cefotaxime, and 1 mg/l BAP (benzylaminopurine) for 3 days. The stem explants were then placed for two periods of three weeks each, again basal side down and with 5 explants per plate, onto an MS/0.1 mM glyphosate, selection medium (also containing carbenicillin, cefotaxime, and BAP (The glyphosate stock [0.5M] is prepared as described in the following: 8.45 g glyphosate [analytical grade] is dissolyed in 50 ml deionized water, adding KOH pellets to dissolye the glyphosate, and the volume is brought to 100 ml following adjusting the pH to 5.7. The solution is filter-sterilized and stored at 4° C). After 6 weeks on this glyphosate selection medium, green. normally developing shoots were excised from the stem explants and were placed onto fresh MS medium containing 750 mg/l carbenicillin. 50 mgil cefotaxime, and 1 mg/l BAP, for further shoot development. When the shoots were 2-3 inches tall, a fresh cut at the end of the stem was made, the cut end was dipped in Root-tone, and the shoot was placed in Metro Mix 350 soil and allowed to harden-off for 2-3 weeks.  
     [0274] Construction of Canola transformation vector pMON17209.  
     [0275] The EPSPS gene was isolated originally from Agrobacterium sp. strain CP4 and expresses a highly tolerant enzyme. The original gene contains sequences that could be inimical to high expression of the gene in some plants. These sequences include potential polyadenylation sites that are often A+T rich, a higher G+C% than that frequently found in dicotyledonous plant genes (63% versus ˜50%), concentrated stretches of G and C residues, and codons that may not used frequently in dicotyledonous plant genes. The high G+C% in the CP4 EPSPS gene could also result in the formation of strong hairpin structures that may affect expression or stability of the RNA. A plant preferred version of the gene was synthesized and used for these vectors. This coding sequence was expressed in  E. coli  from a PRerA-genelOL vector (Olins et al., 1988) and the EPSPS activity was compared with that from the native CP4 EPSPS gene. The appk m for PEP for the native and synthetic genes was 11.8 μM and 12.7 μM, respectively, indicating that the enzyme expressed from the synthetic gene was unaltered. The N-terminus of the coding sequence was then mutagenized to place an SphI site (GCATGC) at the ATG to permit the construction of the CTP2-CP4 synthetic fusion for chloroplast import. This change had no apparent effect on the in vivo activity of CP4 EPSPS in  E. coli  as judged by complementation of the aroA mutant. A CTP-CP4 EPSPS fusion was constructed between the Arabidopsis thaliana EPSPS CTP (Klee et al., 1987) and the CP4 EPSPS coding sequences. The Arabidopsis CTP was engineered by site-directed mutagenesis to place a SphI restriction site at the CTP processing site. This mutagenesis replaced the Glu-Lys at this location with Cys-Met. The CTP2-CP4 EPSPS fusion was tested for import into chloroplasts isolated from  Lactuca sativa  using the methods described previously (della-Cioppa et al., 1986: 1987).  
     [0276] The GOX gene that encodes the glyphosate metabolizing enzyme glyphosate oxidoreductase (GOX) was cloned originally from Achromobacter sp. strain LBAA (Hallas et al., 1988; Barry et al., 1992). The gox gene from strain LBAA was also resynthesized in a plant-preferred sequence version and in which many of the restriction sites were removed (PCT Appin. No. WO 92100377). The GOX protein is targeted to the plastids by a fusion between the C-terminus of a CTP and the N-terminus of GOM A CTP, derived from the SSUIA gene from Arabidopsis thaliana (Timko et al., 1988) was used. This CTP (CTP1) was constructed by a combination of site-directed mutageneses. The CTP1 is made up of the SSULA CTP (amino acids 1-55), the first 23 amino acids of the mature SSUIA protein (56-78), a serine residue (amino acid 79), a new segment that repeats amino acids 50 to 56 from the CTP and the first two from the mature protein (amino acids 80-87), and an alanine and methionine residue (amino acid 88 and 89). An NcoI restriction site is located at the 3′ end (spans the Met89 codon) to facilitate the construction of precise fusions to the 5′ of GOX. At a later stage, a BglII site was introduced upstream of the N-terminus of the SSU1A sequences to facilitate the introduction of the fusions into plant transformation vectors. A fusion was assembled between CTP1 and the synthetic GOX gene.  
     [0277] The CP4 EPSPS and GOX genes were combined to form pMON17209 as described in the following. The CTP2-CP4 EPSPS fusion was assembled and inserted between the constitutive FMV 35 S promoter (Gowda et al., 1989; Richins et al., 1987) and the E9 3′ region (Coruzzi et al., 1984; Morelli et al., 1985) in a pUC vector (Yannisch-Perron et al. 1985; Vieira and Messing, 1987) to form pMON17190; this completed element may then be moved easily as a NotI—NotI fragment to other vectors. The CTP1-GOX fusion was also assembled in a pUC vector with the FMV 35 S promoter. This element was then moved as a HindlII-BamHI fragment into the plant transformation vector pMON10098 and joined to the E9 3′ region in the process. The resultant vector pMON17193 has a single NotI site into which the FMV  35 SICTP2-CP4 EPSPSIE9 3′ element from pMON17190 was cloned to form pMON17194. The kanamycin plant transformation selection cassette (Fraley et al., 1985) was then deleted from pMON17194, by cutting with XhoI and religating, to form the pMON17209 vector (FIG. 24).  
     [0278] Construction of Canola transformation vector pMON17237.  
     [0279] TheGOX enzyme has an apparent K m  for glyphosate [appk m (glyphosate)] of −25 mM. In an effort to improve the effectiveness of the glyphosate metabolic rate in planta, a variant of GOX has been identified in which the appk m (glyphosate) has been reduced approximately 10-fold; this variant is referred to as GOX v.247 and the sequence differences between it and the original plant-preferred GOX are illustrated in PCT Appln. No. WO 92100377. The GOX v.247 coding sequence was combined with CTP1 and assembled with the FMV35S promoter and the E9 3′ by cloning into the pMON17227 plant transformation vector to form pMON17241. In this vector, effectively, the CP4 EPSPS was replaced by GOX v.247. The pMON17227 vector had been constructed by replacing the CTP1-GOX sequences in pMON17193 with those for the CTP2-CP4 EPSPS, to form pMON17199 and followed by deleting the kanamycin cassette (as described above for pMON17209). The pMON17237 vector (FIG. 25) was then completed by cloning the FMV 35 S/CTP2-CP4 EPSPS/E9 3′ element as a NotI—NotI fragment into pMON17241.  
     Example 8  
     [0280] Soybean plants were transformed with the pMON13640 (FIG. 15) vector and a number of plant lines of the transformed soybean were obtained which exhibit glyphosate tolerance.  
     [0281] Soybean plants are transformed with pMON13640 by the method of microprojectile injection using particle gun technology as described in Christou et al. (1988). The seed harvested from R 0  plants is R 1  seed which gives rise to R 1  plants. To evaluate the glyphosate tolerance of an R 0  plant, its progeny are evaluated. Because an R 0  plant is assumed to be hemizygous at each insert location, selfing results in maximum genotypic segregation in the RI. Because each insert acts as a dominant allele, in the absence of linkage and assuming only one hemizygous insert is required for tolerance expression, one insert would segregate 3:1, two inserts, 15:1, three inserts 63:1, etc. Therefore, relatively few R 1  plants need be grown to find at least one resistant phenotype.  
     [0282] Seed from an R 0  soybean plant is harvested, and dried before planting in a glyphosate spray test. Seeds are planted into 4 inch (˜5 cm) square pots containing Metro 350. Twenty seedlings from each Ro plant is considered adequate for testing. Plants are maintained and grown in a greenhouse environment. A 12.5-14 hour photoperiod and temperatures of 30° C. day and 24° C. night is regulated. Water soluble Peters Pete Lite fertilizer is applied as needed.  
     [0283] A spray “batch” consists of several sets of R 1  progenies all sprayed on the same date. Some batches may also include evaluations of other than R 1  plants. Each batch also includes sprayed and unsprayed non-transgenic genotypes representing the genotypes in the particular batch which were putatively transformed. Also included in a batch is one or more non-segregating transformed genotypes previously identified as having some resistance.  
     [0284] One to two plants from each individual R 0  progeny are not sprayed and serve as controls to compare and measure the glyphosate tolerance, as well as to assess any variability not induced by the glyphosate. When the other plants reach the first trifoliate leaf stage, usually 2-3 weeks after planting, glyphosate is applied at a rate equivalent of 128 oz./acre (8.895 kg/ha) of Roundup®. A laboratory track sprayer has been calibrated to deliver a rate equivalent to those conditions.  
     [0285] A vegetative score of 0 to 10 is used. The score is relative to the unsprayed progenies from the same R 0  plant. A 0 is death, while a 10 represents no visible difference from the unsprayed plant. A higher number between 0 and 10 represents progressively less damage as compared to the unsprayed plant. Plants are scored at 7, 14, and 28 days after treatment (DAT). The data from the analysis of one set of transformed and control soybean plants are described on Table X and show that the CP4 EPSPS gene imparts glyphosate tolerance in soybean also.  
               TABLE X                          Glyphosate tolerance in Class II EPSPS soybean       transformants       (P-E35S, P-FMV35S; RO plants; Spray rate = 128 oz./acre)                         Vegetative score                                     Vector/Plant No.   day 7   day 14   day 28                       13640/40-11   5   6   7           13640140-3   9   10    10            13640/40-7   4   7   7           control A54032   1   0           control A54031   1   0                      
 
     Example 4  
     [0286] The CP4 EPSPS gene may be used to select transformed plant material directly on media containing glyphosate. The ability to select and to identify transformed plant material depends, in most cases, on the use of a dominant selectable marker gene to enable the preferential and continued growth of the transformed tissues in the presence of a normally inhibitory substance. Antibiotic resistance and herbicide tolerance genes have been used almost exclusively as such dominant selectable marker genes in the presence of the corresponding antibiotic or herbicide. The nptll/kanamycin selection scheme is probably the most frequently used. It has been demonstrated that CP4 EPSPS is also a useful and perhaps superior selectable marker/selection scheme for producing and identifying transformed plants.  
     [0287] A plant transformation vector that may be used in this scheme is pMON17227 (FIG. 16). This plasmid resembles many of the other plasmids described infra and is essentially composed of the previously described bacterial replicon system that enables this plasmid to replicate in  E. coli  and to be introduced into and to replicate in Agrobacterium, the bacterial selectable marker gene (Spc/Str), and located between the T-DNA right border and left border is the CTP2-CP4 synthetic gene in the FMV35S promoter-E9 3′cassette. This plasmid also has single sites for a number of restriction enzymes, located within the borders and outside of the expression cassette. This makes it possible to easily add other genes and genetic elements to the vector for introduction into plants.  
     [0288] The protocol for direct selection of transformed plants on glyphosate is outlined for tobacco. Explants are prepared for pre-culture as in the standard procedure as described in Example 1: surface sterilization of leaves from 1 month old tobacco plants (15 minutes in 10% clorox+surfactant; 3X dH 2 O washes); explants are cut in 0.5×0.5 cm squares, removing leaf edges, mid-rib, tip, and petiole end for uniform tissue type; explants are placed in single layer, upside down, on MS104 plates+2 ml 4COO5K media to moisten surface; pre-culture 1-2 days. Explants are inoculated using overnight culture of Agrobacterium containing the plant transformation plasmid that is adjusted to a titer of 1.2 X 109 bacteria/ml with 4C005K media. Explants are placed into a centrifuge tube. the Agrobacterium suspension is added and the mixture of bacteria and explants is “Vortexed” on maximum setting for 25 seconds to ensure even penetration of bacteria. The bacteria are poured off and the explants are blotted between layers of dry sterile filter paper to remove excess bacteria. The blotted explants are placed upside down on MS104 plates+2 ml 4COO5K media+filter disc. Co-culture is 2-3 days. The explants are transferred to MS104+Carbenicillin 1000 mg/l +cefotaxime 100 mg/l for 3 days (delayed pbase). The explants are then transferred to MS104+glyphosate 0.05 mM +Carbenicillin 1000 mg/ +cefotaxime 100 mg/l for selection phase. At 4-6 weeks shoots are cut from callus and placed on MSO+Carbenicillin 500 mgll rooting media. Roots form in 3-5 days, at which time leaf pieces can be taken from rooted plates to confirm glyphosate tolerance and that the material is transformed.  
     [0289] The presence of the CP4 EPSPS protein in these transformed tissues has been confirmed by immunoblot analysis of leaf discs. The data from one experiment with pMON17227 is presented in the following: 139 shoots formed on glyphosate from 400 explants inoculated with Agrobacterium ABI/pMON17227; 97 of these were positive on recallusing on glyphosate. These data indicate a transformation rate of 24 per 100 explants, which makes this a highly efficient and time saving transformation procedure for plants. Similar transformation frequencies have been obtained with pMON17131 and direct selection of transformants on glyphosate with the CP4 EPSPS genes has also been shown in other plant species, including, Arabidopsis, soybean, corn, wheat, potato, tomato, cotton, lettuce, and sugarbeet.  
     [0290] The pMON17227 plasmid contains single restriction enzyme recognition cleavage sites (NotI, XhoI, and BstX 1  ) between the CP4 glyphosate selection region and the left border of the vector for the cloning of additional genes and to facilitate the introduction of these genes into plants.  
     Example 5A  
     [0291] The CP4 EPSPS gene has also been introduced into Black Mexican Sweet (BMS) corn cells with expression of the protein and glyphosate resistance detected in callus.  
     [0292] The backbone for this plasmid was a derivative of the high copy plasmid pUC119 (Viera and Messing, 1987). The 1.3 Kb FspI-DraI pUC119 fragment containing the origin of replication was fused to the 1.3 Kb Smal-HindIH filled fragment from pKC7 (Rao and Rogers, 1979) which contains the neomycin phosphotransferase type II gene to confer bacterial kanamycin resistance. This plasmid was used to construct a monocot expression cassette vector containing the 0.6 kb cauliflower mosaic virus (CaMV) 35S RNA promoter with a duplication of the -90 to -300 region (Kay et al., 1987), an 0.8 kb fragment containing an intron from a maize gene in the 5′ untranslated leader region, followed by a polylinker and the 3′ termination sequences from the nopaline synthase (NOS) gene (Fraley et al., 1983). A 1.7 Kb fragment containing the 300 bp chloroplast transit peptide from the Arabidopsis EPSP synthase fused in frame to the 1.4 Kb coding sequence for the bacterial CP4 EPSP synthase was inserted into the monocot expression cassette in the polylinker between the intron and the NOS termination sequence to form the plasmid pMON19653 (FIG. 17).  
     [0293] pMON19653 DNA was introduced into Black Mexican Sweet (BMS) cells by co-bombardment with EC9, a plasmid containing a sulfonylurea-resistant form of the maize acetolactate synthase gene. 2.5 mg of each plasmid was coated onto tungsten particles and introduced into log-phase BMS cells using a PDS-1000 particle gun essentially as described (Klein et al., 1989). Transformants are selected on MS medium containing 20 ppb chlorsulfuron. After initial selection on chlorsulfuron, the calli can be assayed directly by Western blot. Glyphosate tolerance can be assessed by transferring the calli to medium containing 5 mM glyphosate. As shown in Table XI, CP4 EPSPS erance to corn callus.  
               TABLE XI                          Expression of CP4 in BMS Corn Callus - pMON 19653                                 CP expression           Line   (% extracted protein)                       284      0.006%           287   0.036           290   0.061           295   0.073           299   0.113           309   0.042           313   0.003                      
 
     [0294] To measure CP4 EPSPS expression in corn callus, the following procedure was used: BMS callus (3 g wet weight) was dried on filter paper (Whatman#1) under vacuum, reweighed, and extraction buffer (500 μl/g dry weight; 100 mM Tris, 1 mm EDTA, 10% glycerol) was added. The tissue was homogenized with a Wheaton overhead stirrer for 30 seconds at 2.8 power setting. After centrifugation (3 minutes, Eppendorf microfuge), the supernatant was removed and the protein was quantitated (BioRad Protein Assay). Samples (50 μg/well) were loaded on an SDS PAGE gel (Jule, 3-17%) along with CP4 EPSPS standard (10 ng), electrophoresed, and transferred to nitrocellulose similarly to a previously described method (Padgette, 1987). The nitrocellulose blot was probed with goat anti-CP4 EPSPS IgG, and developed with I-125 Protein G. The radioactive blot was visualized by autoradiography. Results were quantitated by densitometry on an LKB UltraScan XL laser densitomer and are tabulated below in Table X.  
               TABLE XII                          Glyphosate resistance in BMS Corn Callus       using pMON 19653                                     # chlorsulfuron-   # cross-resistant       Vector   Experiment   resistant lines   to Glyphosate               19653   253   120   81/120 = 67.5%       19653   254    80    37/80 = 46%       EC9 control   253/254    8     0/8 = 0%                  
 
     [0295] Improvements in the expression of Class II EPSPS could also be achieved by expressing the gene using stronger plant promoters, using better 3′ polyadenylation signal sequences, optimizing the sequences around the initiation codon for ribosome loading and translation initiation, or by combination of these or other expression or regulatory sequences or factors.  
     Example 5B  
     [0296] The plant-expressible genes encoding the CP4 EPSPS and a glyphosate oxidoreductasease enzyme (PCT Pub. No. W092/00377) were introduced into embryogenic corn callus through particle bombardment. Plasmid DNA was prepared using standard procedures (Ausubel et al., 1987), cesium-chloride purified, and re-suspended at 1 mg/ml in TE buffer. DNA was precipitated onto M10 tungsten or 1.0 μgold particles (BioRad) using a calcium chloride/spermidine precipitation protocol, essentially as described by Klein et al. (1987). The PDS1000® gunpowder gun (BioRad) was used. Callus tissue was obtained by isolating 1-2 mm long immature embryos from the “Hi-II” genotype (Armstrong et al., 1991), or Hi-II X B73 crosses, onto a modified N6 medium (Armstrong and Green, 1985; Songstad et al., 1991). Embryogenic callus (“type-II”; Armstrong and Green, 1985) initiated from these embryos was maintained by subculturing at two week intervals, and was bombarded when less than two months old. Each plate of callus tissue was bombarded from 1 to 3 times with either tungsten or gold particles coated with the plasmid DNA(s) of interest. Callus was transferred to a modified N6 medium containing an appropriate selective agent (either glyphosate, or one or more of the antibiotics kanamycin, G418, or paromomycin) 1-8 days following bombardment, and then re-transferred to fresh selection media at 2-3 week intervals. Glyphosate-resistant calli first appeared approximately 6-12 weeks post-bombardment. These resistant calli were propagated on selection medium, and samples were taken for assays gene expression. Plant regeneration from resistant calli was accomplished essentially as described by Petersen et al. (1992).  
     [0297] In some cases, both gene(s) were covalently linked together on the same plasmid DNA molecule. In other instances, the genes were present on separate plasmids, but were introduced into the same plant through a process termed “co-transformation”. The 1 mg/ml plasmid preparations of interest were mixed together in an equal ratio, by volume, and then precipitated onto the tungsten or gold particles. At a high frequency, as described in the literature (e.g., Schocher et al., 1986), the different plasmid molecules integrate into the genome of the same plant cell. Generally the integration is into the same chromosomal location in the plant cell, presumably due to recombination of the plasmids prior to integration. Less frequently, the different plasmids integrate into separate chromosomal locations. In either case, there is integration of both DNA molecules into the same plant cell, and any plants produced from that cell.  
     [0298] Transgenic corn plants were produced as decribed above which contained a plant-expressible CP4 gene and a plant-expressible gene encoding a glyphosate oxidoreductase enzyme.  
     [0299] The plant-expressible CP4 gene comprised a structural DNA sequence encoding a CTP2/CP4 EPSPS fusion protein. The CTP2/CP4 EPSPS is a gene fusion composed of the N-terminal 0.23 Kb chloroplast transit peptide sequence from the Arabidopsis thaiana EPSPS gene (KIlee et al. 1987, referred to herein as CTP2), and the C-terminal 1.36 Kb 5-enolpyruvylshikimate-3-phosphate synthase gene (CP4) from an Agrobacterium species. Plant expression of the gene fusion produces a pre-protein which is rapidly imported into chloroplasts where the CTP is cleaved and degraded (della-Cioppa et al., 1986) releasing the mature CP4 protein.  
     [0300] The plant-expressible gene expressing a glyphosate oxidoreductase enzyme comprised a structual DNA sequence comprising CTPIJGOXsyn gene fusion composed of the N-terminal 0.26 Kb chloroplast transit peptide sequence derived from the Arabidopsis thaliana SSU la gene (Timko et al., 1988 referred to herein as CTP1), and the C-terminal 1.3 Kb synthetic gene sequence encoding a glyphosate oxidoreductase enzyme (GOXsyn, as descibed in PCT Pub. No. W092100377 previously incorporated by reference). The GOXsyn gene encodes the enzyme glyphosate oxidoreductase from an Achromobacter sp. strain LBAA which catalyzes the conversion of glyphosate to herbicidally inactive products, aminomethylphosphonate and glyoxylate. Plant expression of the gene fusion produces a pre-protein which is rapidly imported into chloroplasts where the CTP is cleaved and degraded (della- Cioppa et al., 1986) releasing the mature GOX protein.  
     [0301] Both of the above described genes also include the following regulatory sequences for plant expression: (i) a promoter region comprising a 0.6 Kb 35S cauliflower mosaic virus (CaMV) promoter (Odell et al., 1985) with the duplicated enhancer region (Kay et al., 1987) which also contains a 0.8 Kb fragment containing the first intron from the maize heat shock protein 70 gene (Shah et al., 1985 and PCT Pub. No. W093/19189, the disclosure of which is hereby incorporated by reference); and (ii) a 3′ non-translated region comprising a 0.3 Kb fragment of the 3′ non-translated region of the nopaline synthase gene (Fraley et al., 1983 and Depicker, et al. 1982) which functions to direct polyadenylation of the mRNA.  
     [0302] The above described transgenic corn plants exhibit tolerance to glyphosate herbicide in greenhouse and field trials.  
     Example 6  
     [0303] The LBAA Class II EPSPS gene has been introduced into plants and also imparts glyphosate tolerance. Data on tobacco transformed with pMON17206 (infra) are presented in Table XIII.  
               TABLE XIII                          Tobacco Glyphosate Spray Test       (pMON17206: E35S - CTP2-LBAA EPSPS: 0.4 lbs/ac)                             Line   7 Day Rating                       33358   9           34586   9           33328   9           34606   9           33377   9           34611   10            34607   10            34601   9           34589   9           Samsun (Control)   4                      
 
     [0304] From the foregoing, it will be recognized that this invention is one well adapted to attain all the ends and objects hereinabove set forth together with advantages which are obvious and which are inherent to the invention. It will be further understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.  
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     [0385] Schulz, A., Sost, D. and Amrhein, D. (1984)  Arch. Microbiol.  137: 121-123.  
     [0386] Shah, D., Horsch, R., KIee, H., Kishore, G., Winter, J., Tumer, N., Hironaka, C., Sanders, P., Gasser, C., Aykent, S. Siegal, N., Rogers, S., and Fraley, R. (1986). Engineering herbicide tolerance in transgenic plants.  Science  233, 478-481.  
     [0387] Shah, D. M., Rochester, D. E., Krivi, G., Hironaka, C., Mozer, T. J., Fraley, R. T., and D. C. Tiemeier. 1985. Structure and expression of the maize hsp70gene.  Cell. and Mol. Biol. of Plant Stress.  Alan R. Liss, Inc. pp. 181-200.  
     [0388] Shimamoto, K. et al. (1989)  Nature  338:274-276.  
     [0389] Sost, D., Schulz. A. and Amrhein, N (1984)  FEBS Lett.  173: 238-241.  
     [0390] Sost, D. and Amrhein, N. (1990) Substitution of Gly-96 to Ala in the 5-enolpyruvylshikimate 3-phosphate synthase of Klebsiella pneumoniae results in greatly reduced affty for the herbicide glyphosate.  Arch. Biochem. Biophys.  282: 433-436.  
     [0391] Stalker, D. M., Thomas, C. M., and Helinski, D.R. (1981). Nudeotide sequence of the region of the origin of replication of the broad host range plasmid RK2.  Mol Gen Genet  181: 8-12.  
     [0392] Stalker, D. M., Hiatt, W. R. and Comai, L. (1985) A single amino acid substitution in the enzyme 5-enolpyruvylshikimate 3-phosphate synthase confers resistance to glyphosate.  J. Biol. Chem.  260: 4724-4728.  
     [0393] Stallings, W. C., Abdel-Meguid, S. S., Lim, L. W., Shieh, Huey-Sheng, Dayringer, H. E., Leimgruber, N. K., Stegeman, R. A., Anderson, K. S., Sikorski, J. A., Padgette S. R., Kishore, G. M. (1991). Structure and Topological Symmetry of the Glyphosate Target 5-enol-pyruvylshikindate- 3 -phosphate synthase.  Proc. Natl. Acad. Sci. USA  88. 5046-5050.  
     [0394] Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Stable transformation of plastids in higher plants.  Proc. Natl. Acad. Sci. USA  87: 8526-8530.  
     [0395] Svab, Z. and Maliga, P. (1993) High frequency plastid transformation in tobacco by selection for a chimeric aadA gene.  Proc. Natl. Acad Sci. USA  90:913-917.  
     [0396] Tabor, S. and Richardson. C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.  Proc. Natl. Acad. Sci. USA  82: 1074-1078.  
     [0397] Talbot, H. W., Johnson, L. M. and Munnecke, D. M. (1984) Glyphosate utilization by Pseudomonas sp. and Alcaligenes sp. isolated from environmental sources.  Current Microbiol.  10: 255-260.  
     [0398] Talmadge, K, and Gilbert, W., (1980) “Construction of plasmid vectors with unique PstI cloning sites in the signal sequence coding region”  Gene,  12: 235-241.  
     [0399] Timko, M. P., Herdies, L., de Almeida, E., Cashmore, A.R., Leemans, J., and Krebbers, E. 1988. Genetic Engineering of Nuclear-Encoded Components of the Photosynthetic Apparatus in Arabidopsis in “The Impact of Chemistry on Biotechnology,” ACS Books, 279-295.  
     [0400] Vasil, V., F. Redway and I. Vasil. (1990),  Bio/Technology  8:429434.  
     [0401] Vieira, J. and Messing J. (1987) Production of single-stranded plasmid DNA.  Methods Enzvmol.  153: 3-11.  
     [0402] Yanisch-Perron, C., Vieira, J. and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.  Gene  33, 103-119  
    
     
       
         1 
         
           
             70  
           
           
             1  
             597  
             DNA  
             Figwort mosaic virus  
           
            1 

tcatcaaaat atttagcagc attccagatt gggttcaatc aacaaggtac gagccatatc     60 

actttattca aattggtatc gccaaaacca agaaggaact cccatcctca aaggtttgta    120 

aggaagaatt ctcagtccaa agcctcaaca aggtcagggt acagagtctc caaaccatta    180 

gccaaaagct acaggagatc aatgaagaat cttcaatcaa agtaaactac tgttccagca    240 

catgcatcat ggtcagtaag tttcagaaaa agacatccac cgaagactta aagttagtgg    300 

gcatctttga aagtaatctt gtcaacatcg agcagctggc ttgtggggac cagacaaaaa    360 

aggaatggtg cagaattgtt aggcgcacct accaaaagca tctttgcctt tattgcaaag    420 

ataaagcaga ttcctctagt acaagtgggg aacaaaataa cgtggaaaag agctgtcctg    480 

acagcccact cactaatgcg tatgacgaac gcagtgacga ccacaaaaga attccctcta    540 

tataagaagg cattcattcc catttgaagg atcatcagat actaaccaat atttctc       597 

 
           
             2  
             1982  
             DNA  
             Agrobacterium sp.  
             
               CDS  
               (62)..(1426)  
             
           
            2 

aagcccgcgt tctctccggc gctccgcccg gagagccgtg gatagattaa ggaagacgcc     60 

c atg tcg cac ggt gca agc agc cgg ccc gca acc gcc cgc aaa tcc tct    109 
  Met Ser His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser 
  1               5                   10                  15 

ggc ctt tcc gga acc gtc cgc att ccc ggc gac aag tcg atc tcc cac      157 
Gly Leu Ser Gly Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 
            20                  25                  30 

cgg tcc ttc atg ttc ggc ggt ctc gcg agc ggt gaa acg cgc atc acc      205 
Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 
        35                  40                  45 

ggc ctt ctg gaa ggc gag gac gtc atc aat acg ggc aag gcc atg cag      253 
Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 
    50                  55                  60 

gcc atg ggc gcc agg atc cgt aag gaa ggc gac acc tgg atc atc gat      301 
Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp 
65                  70                  75                  80 

ggc gtc ggc aat ggc ggc ctc ctg gcg cct gag gcg ccg ctc gat ttc      349 
Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe 
                85                  90                  95 

ggc aat gcc gcc acg ggc tgc cgc ctg acc atg ggc ctc gtc ggg gtc      397 
Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly Val 
            100                 105                 110 

tac gat ttc gac agc acc ttc atc ggc gac gcc tcg ctc aca aag cgc      445 
Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu Thr Lys Arg 
        115                 120                 125 

ccg atg ggc cgc gtg ttg aac ccg ctg cgc gaa atg ggc gtg cag gtg      493 
Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val 
    130                 135                 140 

aaa tcg gaa gac ggt gac cgt ctt ccc gtt acc ttg cgc ggg ccg aag      541 
Lys Ser Glu Asp Gly Asp Arg Leu Pro Val Thr Leu Arg Gly Pro Lys 
145                 150                 155                 160 

acg ccg acg ccg atc acc tac cgc gtg ccg atg gcc tcc gca cag gtg      589 
Thr Pro Thr Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 
                165                 170                 175 

aag tcc gcc gtg ctg ctc gcc ggc ctc aac acg ccc ggc atc acg acg      637 
Lys Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 
            180                 185                 190 

gtc atc gag ccg atc atg acg cgc gat cat acg gaa aag atg ctg cag      685 
Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 
        195                 200                 205 

ggc ttt ggc gcc aac ctt acc gtc gag acg gat gcg gac ggc gtg cgc      733 
Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg 
    210                 215                 220 

acc atc cgc ctg gaa ggc cgc ggc aag ctc acc ggc caa gtc atc gac      781 
Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly Gln Val Ile Asp 
225                 230                 235                 240 

gtg ccg ggc gac ccg tcc tcg acg gcc ttc ccg ctg gtt gcg gcc ctg      829 
Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu 
                245                 250                 255 

ctt gtt ccg ggc tcc gac gtc acc atc ctc aac gtg ctg atg aac ccc      877 
Leu Val Pro Gly Ser Asp Val Thr Ile Leu Asn Val Leu Met Asn Pro 
            260                 265                 270 

acc cgc acc ggc ctc atc ctg acg ctg cag gaa atg ggc gcc gac atc      925 
Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 
        275                 280                 285 

gaa gtc atc aac ccg cgc ctt gcc ggc ggc gaa gac gtg gcg gac ctg      973 
Glu Val Ile Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 
    290                 295                 300 

cgc gtt cgc tcc tcc acg ctg aag ggc gtc acg gtg ccg gaa gac cgc     1021 
Arg Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg 
305                 310                 315                 320 

gcg cct tcg atg atc gac gaa tat ccg att ctc gct gtc gcc gcc gcc     1069 
Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala 
                325                 330                 335 

ttc gcg gaa ggg gcg acc gtg atg aac ggt ctg gaa gaa ctc cgc gtc     1117 
Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu Leu Arg Val 
            340                 345                 350 

aag gaa agc gac cgc ctc tcg gcc gtc gcc aat ggc ctc aag ctc aat     1165 
Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly Leu Lys Leu Asn 
        355                 360                 365 

ggc gtg gat tgc gat gag ggc gag acg tcg ctc gtc gtg cgc ggc cgc     1213 
Gly Val Asp Cys Asp Glu Gly Glu Thr Ser Leu Val Val Arg Gly Arg 
    370                 375                 380 

cct gac ggc aag ggg ctc ggc aac gcc tcg ggc gcc gcc gtc gcc acc     1261 
Pro Asp Gly Lys Gly Leu Gly Asn Ala Ser Gly Ala Ala Val Ala Thr 
385                 390                 395                 400 

cat ctc gat cac cgc atc gcc atg agc ttc ctc gtc atg ggc ctc gtg     1309 
His Leu Asp His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 
                405                 410                 415 

tcg gaa aac cct gtc acg gtg gac gat gcc acg atg atc gcc acg agc     1357 
Ser Glu Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 
            420                 425                 430 

ttc ccg gag ttc atg gac ctg atg gcc ggg ctg ggc gcg aag atc gaa     1405 
Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 
        435                 440                 445 

ctc tcc gat acg aag gct gcc tgatgacctt cacaatcgcc atcgatggtc        1456 
Leu Ser Asp Thr Lys Ala Ala 
    450                 455 

ccgctgcggc cggcaagggg acgctctcgc gccgtatcgc ggaggtctat ggctttcatc   1516 

atctcgatac gggcctgacc tatcgcgcca cggccaaagc gctgctcgat cgcggcctgt   1576 

cgcttgatga cgaggcggtt gcggccgatg tcgcccgcaa tctcgatctt gccgggctcg   1636 

accggtcggt gctgtcggcc catgccatcg gcgaggcggc ttcgaagatc gcggtcatgc   1696 

cctcggtgcg gcgggcgctg gtcgaggcgc agcgcagctt tgcggcgcgt gagccgggca   1756 

cggtgctgga tggacgcgat atcggcacgg tggtctgccc ggatgcgccg gtgaagctct   1816 

atgtcaccgc gtcaccggaa gtgcgcgcga aacgccgcta tgacgaaatc ctcggcaatg   1876 

gcgggttggc cgattacggg acgatcctcg aggatatccg ccgccgcgac gagcgggaca   1936 

tgggtcgggc ggacagtcct ttgaagcccg ccgacgatgc gcactt                  1982 

 
           
             3  
             455  
             PRT  
             Agrobacterium sp.  
           
            3 

Met Ser His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser 
1               5                   10                  15 

Gly Leu Ser Gly Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 
            20                  25                  30 

Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 
        35                  40                  45 

Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 
    50                  55                  60 

Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp 
65                  70                  75                  80 

Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe 
                85                  90                  95 

Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly Val 
            100                 105                 110 

Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu Thr Lys Arg 
        115                 120                 125 

Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val 
    130                 135                 140 

Lys Ser Glu Asp Gly Asp Arg Leu Pro Val Thr Leu Arg Gly Pro Lys 
145                 150                 155                 160 

Thr Pro Thr Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 
                165                 170                 175 

Lys Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 
            180                 185                 190 

Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 
        195                 200                 205 

Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg 
    210                 215                 220 

Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly Gln Val Ile Asp 
225                 230                 235                 240 

Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu 
                245                 250                 255 

Leu Val Pro Gly Ser Asp Val Thr Ile Leu Asn Val Leu Met Asn Pro 
            260                 265                 270 

Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 
        275                 280                 285 

Glu Val Ile Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 
    290                 295                 300 

Arg Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg 
305                 310                 315                 320 

Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala 
                325                 330                 335 

Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu Leu Arg Val 
            340                 345                 350 

Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly Leu Lys Leu Asn 
        355                 360                 365 

Gly Val Asp Cys Asp Glu Gly Glu Thr Ser Leu Val Val Arg Gly Arg 
    370                 375                 380 

Pro Asp Gly Lys Gly Leu Gly Asn Ala Ser Gly Ala Ala Val Ala Thr 
385                 390                 395                 400 

His Leu Asp His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 
                405                 410                 415 

Ser Glu Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 
            420                 425                 430 

Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 
        435                 440                 445 

Leu Ser Asp Thr Lys Ala Ala 
    450                 455 

 
           
             4  
             1673  
             DNA  
             Agrobacterium sp.  
             
               CDS  
               (86)..(1432)  
             
           
            4 

gtagccacac ataattacta tagctaggaa gcccgctatc tctcaatccc gcgtgatcgc     60 

gccaaaatgt gactgtgaaa aatcc atg tcc cat tct gca tcc ccg aaa cca      112 
                            Met Ser His Ser Ala Ser Pro Lys Pro 
                            1               5 

gca acc gcc cgc cgc tcg gag gca ctc acg ggc gaa atc cgc att ccg      160 
Ala Thr Ala Arg Arg Ser Glu Ala Leu Thr Gly Glu Ile Arg Ile Pro 
10                  15                  20                  25 

ggc gac aag tcc atc tcg cat cgc tcc ttc atg ttt ggc ggt ctc gca      208 
Gly Asp Lys Ser Ile Ser His Arg Ser Phe Met Phe Gly Gly Leu Ala 
                30                  35                  40 

tcg ggc gaa acc cgc atc acc ggc ctt ctg gaa ggc gag gac gtc atc      256 
Ser Gly Glu Thr Arg Ile Thr Gly Leu Leu Glu Gly Glu Asp Val Ile 
            45                  50                  55 

aat aca ggc cgc gcc atg cag gcc atg ggc gcg aaa atc cgt aaa gag      304 
Asn Thr Gly Arg Ala Met Gln Ala Met Gly Ala Lys Ile Arg Lys Glu 
        60                  65                  70 

ggc gat gtc tgg atc atc aac ggc gtc ggc aat ggc tgc ctg ttg cag      352 
Gly Asp Val Trp Ile Ile Asn Gly Val Gly Asn Gly Cys Leu Leu Gln 
    75                  80                  85 

ccc gaa gct gcg ctc gat ttc ggc aat gcc gga acc ggc gcg cgc ctc      400 
Pro Glu Ala Ala Leu Asp Phe Gly Asn Ala Gly Thr Gly Ala Arg Leu 
90                  95                  100                 105 

acc atg ggc ctt gtc ggc acc tat gac atg aag acc tcc ttt atc ggc      448 
Thr Met Gly Leu Val Gly Thr Tyr Asp Met Lys Thr Ser Phe Ile Gly 
                110                 115                 120 

gac gcc tcg ctg tcg aag cgc ccg atg ggc cgc gtg ctg aac ccg ttg      496 
Asp Ala Ser Leu Ser Lys Arg Pro Met Gly Arg Val Leu Asn Pro Leu 
            125                 130                 135 

cgc gaa atg ggc gtt cag gtg gaa gca gcc gat ggc gac cgc atg ccg      544 
Arg Glu Met Gly Val Gln Val Glu Ala Ala Asp Gly Asp Arg Met Pro 
        140                 145                 150 

ctg acg ctg atc ggc ccg aag acg gcc aat ccg atc acc tat cgc gtg      592 
Leu Thr Leu Ile Gly Pro Lys Thr Ala Asn Pro Ile Thr Tyr Arg Val 
    155                 160                 165 

ccg atg gcc tcc gcg cag gta aaa tcc gcc gtg ctg ctc gcc ggt ctc      640 
Pro Met Ala Ser Ala Gln Val Lys Ser Ala Val Leu Leu Ala Gly Leu 
170                 175                 180                 185 

aac acg ccg ggc gtc acc acc gtc atc gag ccg gtc atg acc cgc gac      688 
Asn Thr Pro Gly Val Thr Thr Val Ile Glu Pro Val Met Thr Arg Asp 
                190                 195                 200 

cac acc gaa aag atg ctg cag ggc ttt ggc gcc gac ctc acg gtc gag      736 
His Thr Glu Lys Met Leu Gln Gly Phe Gly Ala Asp Leu Thr Val Glu 
            205                 210                 215 

acc gac aag gat ggc gtg cgc cat atc cgc atc acc ggc cag ggc aag      784 
Thr Asp Lys Asp Gly Val Arg His Ile Arg Ile Thr Gly Gln Gly Lys 
        220                 225                 230 

ctt gtc ggc cag acc atc gac gtg ccg ggc gat ccg tca tcg acc gcc      832 
Leu Val Gly Gln Thr Ile Asp Val Pro Gly Asp Pro Ser Ser Thr Ala 
    235                 240                 245 

ttc ccg ctc gtt gcc gcc ctt ctg gtg gaa ggt tcc gac gtc acc atc      880 
Phe Pro Leu Val Ala Ala Leu Leu Val Glu Gly Ser Asp Val Thr Ile 
250                 255                 260                 265 

cgc aac gtg ctg atg aac ccg acc cgt acc ggc ctc atc ctc acc ttg      928 
Arg Asn Val Leu Met Asn Pro Thr Arg Thr Gly Leu Ile Leu Thr Leu 
                270                 275                 280 

cag gaa atg ggc gcc gat atc gaa gtg ctc aat gcc cgt ctt gca ggc      976 
Gln Glu Met Gly Ala Asp Ile Glu Val Leu Asn Ala Arg Leu Ala Gly 
            285                 290                 295 

ggc gaa gac gtc gcc gat ctg cgc gtc agg gct tcg aag ctc aag ggc     1024 
Gly Glu Asp Val Ala Asp Leu Arg Val Arg Ala Ser Lys Leu Lys Gly 
        300                 305                 310 

gtc gtc gtt ccg ccg gaa cgt gcg ccg tcg atg atc gac gaa tat ccg     1072 
Val Val Val Pro Pro Glu Arg Ala Pro Ser Met Ile Asp Glu Tyr Pro 
    315                 320                 325 

gtc ctg gcg att gcc gcc tcc ttc gcg gaa ggc gaa acc gtg atg gac     1120 
Val Leu Ala Ile Ala Ala Ser Phe Ala Glu Gly Glu Thr Val Met Asp 
330                 335                 340                 345 

ggg ctc gac gaa ctg cgc gtc aag gaa tcg gat cgt ctg gca gcg gtc     1168 
Gly Leu Asp Glu Leu Arg Val Lys Glu Ser Asp Arg Leu Ala Ala Val 
                350                 355                 360 

gca cgc ggc ctt gaa gcc aac ggc gtc gat tgc acc gaa ggc gag atg     1216 
Ala Arg Gly Leu Glu Ala Asn Gly Val Asp Cys Thr Glu Gly Glu Met 
            365                 370                 375 

tcg ctg acg gtt cgc ggc cgc ccc gac ggc aag gga ctg ggc ggc ggc     1264 
Ser Leu Thr Val Arg Gly Arg Pro Asp Gly Lys Gly Leu Gly Gly Gly 
        380                 385                 390 

acg gtt gca acc cat ctc gat cat cgt atc gcg atg agc ttc ctc gtg     1312 
Thr Val Ala Thr His Leu Asp His Arg Ile Ala Met Ser Phe Leu Val 
    395                 400                 405 

atg ggc ctt gcg gcg gaa aag ccg gtg acg gtt gac gac agt aac atg     1360 
Met Gly Leu Ala Ala Glu Lys Pro Val Thr Val Asp Asp Ser Asn Met 
410                 415                 420                 425 

atc gcc acg tcc ttc ccc gaa ttc atg gac atg atg ccg gga ttg ggc     1408 
Ile Ala Thr Ser Phe Pro Glu Phe Met Asp Met Met Pro Gly Leu Gly 
                430                 435                 440 

gca aag atc gag ttg agc ata ctc tagtcactcg acagcgaaaa tattatttgc    1462 
Ala Lys Ile Glu Leu Ser Ile Leu 
            445 

gagattgggc attattaccg gttggtctca gcgggggttt aatgtccaat cttccatacg   1522 

taacagcatc aggaaatatc aaaaaagctt tagaaggaat tgctagagca gcgacgccgc   1582 

ctaagctttc tcaagacttc gttaaaactg tactgaaatc ccggggggtc cggggatcaa   1642 

atgacttcat ttctgagaaa ttggcctcgc a                                  1673 

 
           
             5  
             449  
             PRT  
             Agrobacterium sp.  
           
            5 

Met Ser His Ser Ala Ser Pro Lys Pro Ala Thr Ala Arg Arg Ser Glu 
1               5                   10                  15 

Ala Leu Thr Gly Glu Ile Arg Ile Pro Gly Asp Lys Ser Ile Ser His 
            20                  25                  30 

Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 
        35                  40                  45 

Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Arg Ala Met Gln 
    50                  55                  60 

Ala Met Gly Ala Lys Ile Arg Lys Glu Gly Asp Val Trp Ile Ile Asn 
65                  70                  75                  80 

Gly Val Gly Asn Gly Cys Leu Leu Gln Pro Glu Ala Ala Leu Asp Phe 
                85                  90                  95 

Gly Asn Ala Gly Thr Gly Ala Arg Leu Thr Met Gly Leu Val Gly Thr 
            100                 105                 110 

Tyr Asp Met Lys Thr Ser Phe Ile Gly Asp Ala Ser Leu Ser Lys Arg 
        115                 120                 125 

Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val 
    130                 135                 140 

Glu Ala Ala Asp Gly Asp Arg Met Pro Leu Thr Leu Ile Gly Pro Lys 
145                 150                 155                 160 

Thr Ala Asn Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 
                165                 170                 175 

Lys Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Val Thr Thr 
            180                 185                 190 

Val Ile Glu Pro Val Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 
        195                 200                 205 

Gly Phe Gly Ala Asp Leu Thr Val Glu Thr Asp Lys Asp Gly Val Arg 
    210                 215                 220 

His Ile Arg Ile Thr Gly Gln Gly Lys Leu Val Gly Gln Thr Ile Asp 
225                 230                 235                 240 

Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu 
                245                 250                 255 

Leu Val Glu Gly Ser Asp Val Thr Ile Arg Asn Val Leu Met Asn Pro 
            260                 265                 270 

Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 
        275                 280                 285 

Glu Val Leu Asn Ala Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 
    290                 295                 300 

Arg Val Arg Ala Ser Lys Leu Lys Gly Val Val Val Pro Pro Glu Arg 
305                 310                 315                 320 

Ala Pro Ser Met Ile Asp Glu Tyr Pro Val Leu Ala Ile Ala Ala Ser 
                325                 330                 335 

Phe Ala Glu Gly Glu Thr Val Met Asp Gly Leu Asp Glu Leu Arg Val 
            340                 345                 350 

Lys Glu Ser Asp Arg Leu Ala Ala Val Ala Arg Gly Leu Glu Ala Asn 
        355                 360                 365 

Gly Val Asp Cys Thr Glu Gly Glu Met Ser Leu Thr Val Arg Gly Arg 
    370                 375                 380 

Pro Asp Gly Lys Gly Leu Gly Gly Gly Thr Val Ala Thr His Leu Asp 
385                 390                 395                 400 

His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Ala Ala Glu Lys 
                405                 410                 415 

Pro Val Thr Val Asp Asp Ser Asn Met Ile Ala Thr Ser Phe Pro Glu 
            420                 425                 430 

Phe Met Asp Met Met Pro Gly Leu Gly Ala Lys Ile Glu Leu Ser Ile 
        435                 440                 445 

Leu 

 
           
             6  
             1500  
             DNA  
             Pseudomonas sp.  
             
               CDS  
               (34)..(1380)  
             
           
            6  
           
             7  
             449  
             PRT  
             Pseudomonas sp.  
           
            7 

Met Ser His Ser Ala Ser Pro Lys Pro Ala Thr Ala Arg Arg Ser Glu 
1               5                   10                  15 

Ala Leu Thr Gly Glu Ile Arg Ile Pro Gly Asp Lys Ser Ile Ser His 
            20                  25                  30 

Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 
        35                  40                  45 

Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Arg Ala Met Gln 
    50                  55                  60 

Ala Met Gly Ala Lys Ile Arg Lys Glu Gly Asp Val Trp Ile Ile Asn 
65                  70                  75                  80 

Gly Val Gly Asn Gly Cys Leu Leu Gln Pro Glu Ala Ala Leu Asp Phe 
                85                  90                  95 

Gly Asn Ala Gly Thr Gly Ala Arg Leu Thr Met Gly Leu Val Gly Thr 
            100                 105                 110 

Tyr Asp Met Lys Thr Ser Phe Ile Gly Asp Ala Ser Leu Ser Lys Arg 
        115                 120                 125 

Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val 
    130                 135                 140 

Glu Ala Ala Asp Gly Asp Arg Met Pro Leu Thr Leu Ile Gly Pro Lys 
145                 150                 155                 160 

Thr Ala Asn Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 
                165                 170                 175 

Lys Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Val Thr Thr 
            180                 185                 190 

Val Ile Glu Pro Val Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 
        195                 200                 205 

Gly Phe Gly Ala Asp Leu Thr Val Glu Thr Asp Lys Asp Gly Val Arg 
    210                 215                 220 

His Ile Arg Ile Thr Gly Gln Gly Lys Leu Val Gly Gln Thr Ile Asp 
225                 230                 235                 240 

Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu 
                245                 250                 255 

Leu Val Glu Gly Ser Asp Val Thr Ile Arg Asn Val Leu Met Asn Pro 
            260                 265                 270 

Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 
        275                 280                 285 

Glu Val Leu Asn Ala Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 
    290                 295                 300 

Arg Val Arg Ala Ser Lys Leu Lys Gly Val Val Val Pro Pro Glu Arg 
305                 310                 315                 320 

Ala Pro Ser Met Ile Asp Glu Tyr Pro Val Leu Ala Ile Ala Ala Ser 
                325                 330                 335 

Phe Ala Glu Gly Glu Thr Val Met Asp Gly Leu Asp Glu Leu Arg Val 
            340                 345                 350 

Lys Glu Ser Asp Arg Leu Ala Ala Val Ala Arg Gly Leu Glu Ala Asn 
        355                 360                 365 

Gly Val Asp Cys Thr Glu Gly Glu Met Ser Leu Thr Val Arg Gly Arg 
    370                 375                 380 

Pro Asp Gly Lys Gly Leu Gly Gly Gly Thr Val Ala Thr His Leu Asp 
385                 390                 395                 400 

His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Ala Ala Glu Lys 
                405                 410                 415 

Pro Val Thr Val Asp Asp Ser Asn Met Ile Ala Thr Ser Phe Pro Glu 
            420                 425                 430 

Phe Met Asp Met Met Pro Gly Leu Gly Ala Lys Ile Glu Leu Ser Ile 
        435                 440                 445 

Leu 

 
           
             8  
             423  
             PRT  
             Escherichia coli  
           
            8 

Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Thr Ile Asn Leu 
1               5                   10                  15 

Pro Gly Ser Lys Thr Val Ser Asn Arg Ala Leu Leu Leu Ala Ala Leu 
            20                  25                  30 

Ala His Gly Lys Thr Val Leu Thr Asn Leu Leu Asp Ser Asp Asp Val 
        35                  40                  45 

Arg His Met Leu Asn Ala Leu Thr Ala Leu Gly Val Ser Tyr Thr Leu 
    50                  55                  60 

Ser Ala Asp Arg Thr Arg Cys Glu Ile Ile Gly Asn Gly Gly Pro Leu 
65                  70                  75                  80 

His Ala Glu Gly Ala Leu Glu Leu Phe Leu Gly Asn Ala Gly Thr Ala 
                85                  90                  95 

Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Ser Asn Asp Ile Val 
            100                 105                 110 

Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His Leu Val 
        115                 120                 125 

Asp Ala Leu Arg Leu Gly Gly Ala Lys Ile Thr Tyr Leu Glu Gln Glu 
    130                 135                 140 

Asn Tyr Pro Pro Leu Arg Leu Gln Gly Gly Phe Thr Gly Gly Asn Val 
145                 150                 155                 160 

Asp Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu Leu Met 
                165                 170                 175 

Thr Ala Pro Leu Ala Pro Glu Asp Thr Val Ile Arg Ile Lys Gly Asp 
            180                 185                 190 

Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met Lys Thr 
        195                 200                 205 

Phe Gly Val Glu Ile Glu Asn Gln His Tyr Gln Gln Phe Val Val Lys 
    210                 215                 220 

Gly Gly Gln Ser Tyr Gln Ser Pro Gly Thr Tyr Leu Val Glu Gly Asp 
225                 230                 235                 240 

Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Ala Ala Ile Lys Gly Gly 
                245                 250                 255 

Thr Val Lys Val Thr Gly Ile Gly Arg Asn Ser Met Gln Gly Asp Ile 
            260                 265                 270 

Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala Thr Ile Cys Trp Gly 
        275                 280                 285 

Asp Asp Tyr Ile Ser Cys Thr Arg Gly Glu Leu Asn Ala Ile Asp Met 
    290                 295                 300 

Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr Ala Ala 
305                 310                 315                 320 

Leu Phe Ala Lys Gly Thr Thr Arg Leu Arg Asn Ile Tyr Asn Trp Arg 
                325                 330                 335 

Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu Arg Lys 
            340                 345                 350 

Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile Thr Pro 
        355                 360                 365 

Pro Glu Lys Leu Asn Phe Ala Glu Ile Ala Thr Tyr Asn Asp His Arg 
    370                 375                 380 

Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro Val Thr 
385                 390                 395                 400 

Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr Phe Glu 
                405                 410                 415 

Gln Leu Ala Arg Ile Ser Gln 
            420 

 
           
             9  
             1377  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            9 

ccatggctca cggtgcaagc agccgtccag caactgctcg taagtcctct ggtctttctg     60 

gaaccgtccg tattccaggt gacaagtcta tctcccacag gtccttcatg tttggaggtc    120 

tcgctagcgg tgaaactcgt atcaccggtc ttttggaagg tgaagatgtt atcaacactg    180 

gtaaggctat gcaagctatg ggtgccagaa tccgtaagga aggtgatact tggatcattg    240 

atggtgttgg taacggtgga ctccttgctc ctgaggctcc tctcgatttc ggtaacgctg    300 

caactggttg ccgtttgact atgggtcttg ttggtgttta cgatttcgat agcactttca    360 

ttggtgacgc ttctctcact aagcgtccaa tgggtcgtgt gttgaaccca cttcgcgaaa    420 

tgggtgtgca ggtgaagtct gaagacggtg atcgtcttcc agttaccttg cgtggaccaa    480 

agactccaac gccaatcacc tacagggtac ctatggcttc cgctcaagtg aagtccgctg    540 

ttctgcttgc tggtctcaac accccaggta tcaccactgt tatcgagcca atcatgactc    600 

gtgaccacac tgaaaagatg cttcaaggtt ttggtgctaa ccttaccgtt gagactgatg    660 

ctgacggtgt gcgtaccatc cgtcttgaag gtcgtggtaa gctcaccggt caagtgattg    720 

atgttccagg tgatccatcc tctactgctt tcccattggt tgctgccttg cttgttccag    780 

gttccgacgt caccatcctt aacgttttga tgaacccaac ccgtactggt ctcatcttga    840 

ctctgcagga aatgggtgcc gacatcgaag tgatcaaccc acgtcttgct ggtggagaag    900 

acgtggctga cttgcgtgtt cgttcttcta ctttgaaggg tgttactgtt ccagaagacc    960 

gtgctccttc tatgatcgac gagtatccaa ttctcgctgt tgcagctgca ttcgctgaag   1020 

gtgctaccgt tatgaacggt ttggaagaac tccgtgttaa ggaaagcgac cgtctttctg   1080 

ctgtcgcaaa cggtctcaag ctcaacggtg ttgattgcga tgaaggtgag acttctctcg   1140 

tcgtgcgtgg tcgtcctgac ggtaagggtc tcggtaacgc ttctggagca gctgtcgcta   1200 

cccacctcga tcaccgtatc gctatgagct tcctcgttat gggtctcgtt tctgaaaacc   1260 

ctgttactgt tgatgatgct actatgatcg ctactagctt cccagagttc atggatttga   1320 

tggctggtct tggagctaag atcgaactct ccgacactaa ggctgcttga tgagctc      1377 

 
           
             10  
             318  
             DNA  
             Arabidopsis thaliana  
             
               CDS  
               (87)..(317)  
             
           
            10 

agatctatcg ataagcttga tgtaattgga ggaagatcaa aattttcaat ccccattctt     60 

cgattgcttc aattgaagtt tctccg atg gcg caa gtt agc aga atc tgc aat     113 
                             Met Ala Gln Val Ser Arg Ile Cys Asn 
                             1               5 

ggt gtg cag aac cca tct ctt atc tcc aat ctc tcg aaa tcc agt caa      161 
Gly Val Gln Asn Pro Ser Leu Ile Ser Asn Leu Ser Lys Ser Ser Gln 
10                  15                  20                  25 

cgc aaa tct ccc tta tcg gtt tct ctg aag acg cag cag cat cca cga      209 
Arg Lys Ser Pro Leu Ser Val Ser Leu Lys Thr Gln Gln His Pro Arg 
                30                  35                  40 

gct tat ccg att tcg tcg tcg tgg gga ttg aag aag agt ggg atg acg      257 
Ala Tyr Pro Ile Ser Ser Ser Trp Gly Leu Lys Lys Ser Gly Met Thr 
            45                  50                  55 

tta att ggc tct gag ctt cgt cct ctt aag gtc atg tct tct gtt tcc      305 
Leu Ile Gly Ser Glu Leu Arg Pro Leu Lys Val Met Ser Ser Val Ser 
        60                  65                  70 

acg gcg tgc atg c                                                    318 
Thr Ala Cys Met 
    75 

 
           
             11  
             77  
             PRT  
             Arabidopsis thaliana  
           
            11 

Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 
1               5                   10                  15 

Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 
            20                  25                  30 

Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 
        35                  40                  45 

Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 
    50                  55                  60 

Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met 
65                  70                  75 

 
           
             12  
             402  
             DNA  
             Arabidopsis thaliana  
             
               CDS  
               (87)..(401)  
             
           
            12 

agatctatcg ataagcttga tgtaattgga ggaagatcaa aattttcaat ccccattctt     60 

cgattgcttc aattgaagtt tctccg atg gcg caa gtt agc aga atc tgc aat     113 
                             Met Ala Gln Val Ser Arg Ile Cys Asn 
                             1               5 

ggt gtg cag aac cca tct ctt atc tcc aat ctc tcg aaa tcc agt caa      161 
Gly Val Gln Asn Pro Ser Leu Ile Ser Asn Leu Ser Lys Ser Ser Gln 
10                  15                  20                  25 

cgc aaa tct ccc tta tcg gtt tct ctg aag acg cag cag cat cca cga      209 
Arg Lys Ser Pro Leu Ser Val Ser Leu Lys Thr Gln Gln His Pro Arg 
                30                  35                  40 

gct tat ccg att tcg tcg tcg tgg gga ttg aag aag agt ggg atg acg      257 
Ala Tyr Pro Ile Ser Ser Ser Trp Gly Leu Lys Lys Ser Gly Met Thr 
            45                  50                  55 

tta att ggc tct gag ctt cgt cct ctt aag gtc atg tct tct gtt tcc      305 
Leu Ile Gly Ser Glu Leu Arg Pro Leu Lys Val Met Ser Ser Val Ser 
        60                  65                  70 

acg gcg gag aaa gcg tcg gag att gta ctt caa ccc att aga gaa atc      353 
Thr Ala Glu Lys Ala Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile 
    75                  80                  85 

tcc ggt ctt att aag ttg cct ggc tcc aag tct cta tca aat aga att c    402 
Ser Gly Leu Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile 
90                  95                  100                 105 

 
           
             13  
             105  
             PRT  
             Arabidopsis thaliana  
           
            13 

Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 
1               5                   10                  15 

Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 
            20                  25                  30 

Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 
        35                  40                  45 

Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 
    50                  55                  60 

Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Glu Lys Ala Ser Glu 
65                  70                  75                  80 

Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly Leu Ile Lys Leu Pro 
                85                  90                  95 

Gly Ser Lys Ser Leu Ser Asn Arg Ile 
            100                 105 

 
           
             14  
             233  
             DNA  
             Petunia x hybrida  
             
               CDS  
               (14)..(232)  
             
           
            14  
           
             15  
             73  
             PRT  
             Petunia x hybrida  
           
            15 

Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 
1               5                   10                  15 

Asn Ser Asn Phe His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu 
            20                  25                  30 

Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu Val 
        35                  40                  45 

Leu Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 
    50                  55                  60 

Ser Ala Ser Val Ala Thr Ala Cys Met 
65                  70 

 
           
             16  
             352  
             DNA  
             Petunia x hybrida  
             
               CDS  
               (49)..(351)  
             
           
            16  
           
             17  
             101  
             PRT  
             Petunia x hybrida  
           
            17 

Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 
1               5                   10                  15 

Asn Ser Asn Phe His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu 
            20                  25                  30 

Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu Val 
        35                  40                  45 

Leu Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 
    50                  55                  60 

Ser Ala Ser Val Ala Thr Ala Gln Lys Pro Ser Glu Ile Val Leu Gln 
65                  70                  75                  80 

Pro Ile Lys Glu Ile Ser Gly Thr Val Lys Leu Pro Gly Ser Lys Ser 
                85                  90                  95 

Leu Ser Asn Arg Ile 
            100 

 
           
             18  
             28  
             PRT  
             Agrobacterium sp.  
             
               UNSURE  
               (1)..(18)  
               Xaa = Unknown  
             
           
            18 

Xaa His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser Gly 
1               5                   10                  15 

Leu Xaa Gly Thr Val Arg Ile Pro Gly Asp Lys Met 
            20                  25 

 
           
             19  
             13  
             PRT  
             Agrobacterium sp.  
           
            19 

Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val 
1               5                   10 

 
           
             20  
             15  
             PRT  
             Agrobacterium sp.  
           
            20 

Ile Thr Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys 
1               5                   10                  15 

 
           
             21  
             17  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            21 

atgathgayg artaycc                                                    17 

 
           
             22  
             17  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            22 

gargaygtna thaacac                                                    17 

 
           
             23  
             17  
             DNA  
             Artificial sequence  
             
               Synthetic  
             
           
            23 

gargaygtna thaatac                                                    17 

 
           
             24  
             38  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            24 

cgtggataga tctaggaaga caaccatggc tcacggtc                             38 

 
           
             25  
             44  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            25 

ggatagatta aggaagacgc gcatgcttca cggtgcaagc agcc                      44 

 
           
             26  
             35  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            26 

ggctgcctga tgagctccac aatcgccatc gatgg                                35 

 
           
             27  
             32  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            27 

cgtcgctcgt cgtgcgtggc cgccctgacg gc                                   32 

 
           
             28  
             29  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            28 

cgggcaaggc catgcaggct atgggcgcc                                       29 

 
           
             29  
             31  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            29 

cgggctgccg cctgactatg ggcctcgtcg g                                    31 

 
           
             30  
             15  
             PRT  
             Pseudomonas sp.  
             
               NON_CONS  
               (1)..(1)  
               Xaa = unknown  
             
           
            30 

Xaa His Ser Ala Ser Pro Lys Pro Ala Thr Ala Arg Arg Ser Glu 
1               5                   10                  15 

 
           
             31  
             17  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            31 

gcggtbgcsg gyttsgg                                                    17 

 
           
             32  
             16  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            32 

Pro Gly Asp Lys Ser Ile Ser His Arg Ser Phe Met Phe Gly Gly Leu 
1               5                   10                  15 

 
           
             33  
             13  
             PRT  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            33 

Leu Asp Phe Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr 
1               5                   10 

 
           
             34  
             26  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            34 

cggcaatgcc gccaccggcg cgcgcc                                          26 

 
           
             35  
             49  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            35 

ggacggctgc ttgcaccgtg aagcatgctt aagcttggcg taatcatgg                 49 

 
           
             36  
             35  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            36 

ggaagacgcc cagaattcac ggtgcaagca gccgg                                35 

 
           
             37  
             5  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            37 

Arg Xaa His Xaa Glu 
1               5 

 
           
             38  
             4  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            38 

Gly Asp Lys Xaa 
1 

 
           
             39  
             5  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            39 

Ser Ala Gln Xaa Lys 
1               5 

 
           
             40  
             4  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            40 

Asn Xaa Thr Arg 
1 

 
           
             41  
             1287  
             DNA  
             Bacillus subtilis  
             
               CDS  
               (1)..(1287)  
             
           
            41 

atg aaa cga gat aag gtg cag acc tta cat gga gaa ata cat att ccc       48 
Met Lys Arg Asp Lys Val Gln Thr Leu His Gly Glu Ile His Ile Pro 
1               5                   10                  15 

ggt gat aaa tcc att tct cac cgc tct gtt atg ttt ggc gcg cta gcg       96 
Gly Asp Lys Ser Ile Ser His Arg Ser Val Met Phe Gly Ala Leu Ala 
            20                  25                  30 

gca ggc aca aca aca gtt aaa aac ttt ctg ccg gga gca gat tgt ctg      144 
Ala Gly Thr Thr Thr Val Lys Asn Phe Leu Pro Gly Ala Asp Cys Leu 
        35                  40                  45 

agc acg atc gat tgc ttt aga aaa atg ggt gtt cac att gag caa agc      192 
Ser Thr Ile Asp Cys Phe Arg Lys Met Gly Val His Ile Glu Gln Ser 
    50                  55                  60 

agc agc gat gtc gtg att cac gga aaa gga atc gat gcc ctg aaa gag      240 
Ser Ser Asp Val Val Ile His Gly Lys Gly Ile Asp Ala Leu Lys Glu 
65                  70                  75                  80 

cca gaa agc ctt tta gat gtc gga aat tca ggt aca acg att cgc ctg      288 
Pro Glu Ser Leu Leu Asp Val Gly Asn Ser Gly Thr Thr Ile Arg Leu 
                85                  90                  95 

atg ctc gga ata ttg gcg ggc cgt cct ttt tac agc gcg gta gcc gga      336 
Met Leu Gly Ile Leu Ala Gly Arg Pro Phe Tyr Ser Ala Val Ala Gly 
            100                 105                 110 

gat gag agc att gcg aaa cgc cca atg aag cgt gtg act gag cct ttg      384 
Asp Glu Ser Ile Ala Lys Arg Pro Met Lys Arg Val Thr Glu Pro Leu 
        115                 120                 125 

aaa aaa atg ggg gct aaa atc gac ggc aga gcc ggc gga gag ttt aca      432 
Lys Lys Met Gly Ala Lys Ile Asp Gly Arg Ala Gly Gly Glu Phe Thr 
    130                 135                 140 

ccg ctg tca gtg agc ggc gct tca tta aaa gga att gat tat gta tca      480 
Pro Leu Ser Val Ser Gly Ala Ser Leu Lys Gly Ile Asp Tyr Val Ser 
145                 150                 155                 160 

cct gtt gca agc gcg caa att aaa tct gct gtt ttg ctg gcc gga tta      528 
Pro Val Ala Ser Ala Gln Ile Lys Ser Ala Val Leu Leu Ala Gly Leu 
                165                 170                 175 

cag gct gag ggc aca aca act gta aca gag ccc cat aaa tct cgg gac      576 
Gln Ala Glu Gly Thr Thr Thr Val Thr Glu Pro His Lys Ser Arg Asp 
            180                 185                 190 

cac act gag cgg atg ctt tct gct ttt ggc gtt aag ctt tct gaa gat      624 
His Thr Glu Arg Met Leu Ser Ala Phe Gly Val Lys Leu Ser Glu Asp 
        195                 200                 205 

caa acg agt gtt tcc att gct ggt ggc cag aaa ctg aca gct gct gat      672 
Gln Thr Ser Val Ser Ile Ala Gly Gly Gln Lys Leu Thr Ala Ala Asp 
    210                 215                 220 

att ttt gtt cct gga gac att tct tca gcc gcg ttt ttc ctt gct gct      720 
Ile Phe Val Pro Gly Asp Ile Ser Ser Ala Ala Phe Phe Leu Ala Ala 
225                 230                 235                 240 

ggc gcg atg gtt cca aac agc aga att gta ttg aaa aac gta ggt tta      768 
Gly Ala Met Val Pro Asn Ser Arg Ile Val Leu Lys Asn Val Gly Leu 
                245                 250                 255 

aat ccg act cgg aca ggt att att gat gtc ctt caa aac atg ggg gca      816 
Asn Pro Thr Arg Thr Gly Ile Ile Asp Val Leu Gln Asn Met Gly Ala 
            260                 265                 270 

aaa ctt gaa atc aaa cca tct gct gat agc ggt gca gag cct tat gga      864 
Lys Leu Glu Ile Lys Pro Ser Ala Asp Ser Gly Ala Glu Pro Tyr Gly 
        275                 280                 285 

gat ttg att ata gaa acg tca tct cta aag gca gtt gaa atc gga gga      912 
Asp Leu Ile Ile Glu Thr Ser Ser Leu Lys Ala Val Glu Ile Gly Gly 
    290                 295                 300 

gat atc att ccg cgt tta att gat gag atc cct atc atc gcg ctt ctt      960 
Asp Ile Ile Pro Arg Leu Ile Asp Glu Ile Pro Ile Ile Ala Leu Leu 
305                 310                 315                 320 

gcg act cag gcg gaa gga acc acc gtt att aag gac gcg gca gag cta     1008 
Ala Thr Gln Ala Glu Gly Thr Thr Val Ile Lys Asp Ala Ala Glu Leu 
                325                 330                 335 

aaa gtg aaa gaa aca aac cgt att gat act gtt gtt tct gag ctt cgc     1056 
Lys Val Lys Glu Thr Asn Arg Ile Asp Thr Val Val Ser Glu Leu Arg 
            340                 345                 350 

aag ctg ggt gct gaa att gaa ccg aca gca gat gga atg aag gtt tat     1104 
Lys Leu Gly Ala Glu Ile Glu Pro Thr Ala Asp Gly Met Lys Val Tyr 
        355                 360                 365 

ggc aaa caa acg ttg aaa ggc ggc gct gca gtg tcc agc cac gga gat     1152 
Gly Lys Gln Thr Leu Lys Gly Gly Ala Ala Val Ser Ser His Gly Asp 
    370                 375                 380 

cat cga atc gga atg atg ctt ggt att gct tcc tgt ata acg gag gag     1200 
His Arg Ile Gly Met Met Leu Gly Ile Ala Ser Cys Ile Thr Glu Glu 
385                 390                 395                 400 

ccg att gaa atc gag cac acg gat gcc att cac gtt tct tat cca acc     1248 
Pro Ile Glu Ile Glu His Thr Asp Ala Ile His Val Ser Tyr Pro Thr 
                405                 410                 415 

ttc ttc gag cat tta aat aag ctt tcg aaa aaa tcc tga                 1287 
Phe Phe Glu His Leu Asn Lys Leu Ser Lys Lys Ser 
            420                 425 

 
           
             42  
             428  
             PRT  
             Bacillus subtilis  
           
            42 

Met Lys Arg Asp Lys Val Gln Thr Leu His Gly Glu Ile His Ile Pro 
1               5                   10                  15 

Gly Asp Lys Ser Ile Ser His Arg Ser Val Met Phe Gly Ala Leu Ala 
            20                  25                  30 

Ala Gly Thr Thr Thr Val Lys Asn Phe Leu Pro Gly Ala Asp Cys Leu 
        35                  40                  45 

Ser Thr Ile Asp Cys Phe Arg Lys Met Gly Val His Ile Glu Gln Ser 
    50                  55                  60 

Ser Ser Asp Val Val Ile His Gly Lys Gly Ile Asp Ala Leu Lys Glu 
65                  70                  75                  80 

Pro Glu Ser Leu Leu Asp Val Gly Asn Ser Gly Thr Thr Ile Arg Leu 
                85                  90                  95 

Met Leu Gly Ile Leu Ala Gly Arg Pro Phe Tyr Ser Ala Val Ala Gly 
            100                 105                 110 

Asp Glu Ser Ile Ala Lys Arg Pro Met Lys Arg Val Thr Glu Pro Leu 
        115                 120                 125 

Lys Lys Met Gly Ala Lys Ile Asp Gly Arg Ala Gly Gly Glu Phe Thr 
    130                 135                 140 

Pro Leu Ser Val Ser Gly Ala Ser Leu Lys Gly Ile Asp Tyr Val Ser 
145                 150                 155                 160 

Pro Val Ala Ser Ala Gln Ile Lys Ser Ala Val Leu Leu Ala Gly Leu 
                165                 170                 175 

Gln Ala Glu Gly Thr Thr Thr Val Thr Glu Pro His Lys Ser Arg Asp 
            180                 185                 190 

His Thr Glu Arg Met Leu Ser Ala Phe Gly Val Lys Leu Ser Glu Asp 
        195                 200                 205 

Gln Thr Ser Val Ser Ile Ala Gly Gly Gln Lys Leu Thr Ala Ala Asp 
    210                 215                 220 

Ile Phe Val Pro Gly Asp Ile Ser Ser Ala Ala Phe Phe Leu Ala Ala 
225                 230                 235                 240 

Gly Ala Met Val Pro Asn Ser Arg Ile Val Leu Lys Asn Val Gly Leu 
                245                 250                 255 

Asn Pro Thr Arg Thr Gly Ile Ile Asp Val Leu Gln Asn Met Gly Ala 
            260                 265                 270 

Lys Leu Glu Ile Lys Pro Ser Ala Asp Ser Gly Ala Glu Pro Tyr Gly 
        275                 280                 285 

Asp Leu Ile Ile Glu Thr Ser Ser Leu Lys Ala Val Glu Ile Gly Gly 
    290                 295                 300 

Asp Ile Ile Pro Arg Leu Ile Asp Glu Ile Pro Ile Ile Ala Leu Leu 
305                 310                 315                 320 

Ala Thr Gln Ala Glu Gly Thr Thr Val Ile Lys Asp Ala Ala Glu Leu 
                325                 330                 335 

Lys Val Lys Glu Thr Asn Arg Ile Asp Thr Val Val Ser Glu Leu Arg 
            340                 345                 350 

Lys Leu Gly Ala Glu Ile Glu Pro Thr Ala Asp Gly Met Lys Val Tyr 
        355                 360                 365 

Gly Lys Gln Thr Leu Lys Gly Gly Ala Ala Val Ser Ser His Gly Asp 
    370                 375                 380 

His Arg Ile Gly Met Met Leu Gly Ile Ala Ser Cys Ile Thr Glu Glu 
385                 390                 395                 400 

Pro Ile Glu Ile Glu His Thr Asp Ala Ile His Val Ser Tyr Pro Thr 
                405                 410                 415 

Phe Phe Glu His Leu Asn Lys Leu Ser Lys Lys Ser 
            420                 425 

 
           
             43  
             1293  
             DNA  
             Staphylococcus aureus  
             
               CDS  
               (1)..(1293)  
             
           
            43 

atg gta aat gaa caa atc att gat att tca ggt ccg tta aag ggc gaa       48 
Met Val Asn Glu Gln Ile Ile Asp Ile Ser Gly Pro Leu Lys Gly Glu 
1               5                   10                  15 

ata gaa gtg ccg ggc gat aag tca atg aca cac cgt gca atc atg ttg       96 
Ile Glu Val Pro Gly Asp Lys Ser Met Thr His Arg Ala Ile Met Leu 
            20                  25                  30 

gcg tcg cta gct gaa ggt gta tct act ata tat aag cca cta ctt ggc      144 
Ala Ser Leu Ala Glu Gly Val Ser Thr Ile Tyr Lys Pro Leu Leu Gly 
        35                  40                  45 

gaa gat tgt cgt cgt acg atg gac att ttc cga cac tta ggt gta gaa      192 
Glu Asp Cys Arg Arg Thr Met Asp Ile Phe Arg His Leu Gly Val Glu 
    50                  55                  60 

atc aaa gaa gat gat gaa aaa tta gtt gtg act tcc cca gga tat caa      240 
Ile Lys Glu Asp Asp Glu Lys Leu Val Val Thr Ser Pro Gly Tyr Gln 
65                  70                  75                  80 

gtt aac acg cca cat caa gta ttg tat aca ggt aat tct ggt acg aca      288 
Val Asn Thr Pro His Gln Val Leu Tyr Thr Gly Asn Ser Gly Thr Thr 
                85                  90                  95 

aca cga tta ttg gca ggt ttg tta agt ggt tta ggt aat gaa agt gtt      336 
Thr Arg Leu Leu Ala Gly Leu Leu Ser Gly Leu Gly Asn Glu Ser Val 
            100                 105                 110 

ttg tct ggc gat gtt tca att ggt aaa agg cca atg gat cgt gtc ttg      384 
Leu Ser Gly Asp Val Ser Ile Gly Lys Arg Pro Met Asp Arg Val Leu 
        115                 120                 125 

aga cca ttg aaa ctt atg gat gcg aat att gaa ggt att gaa gat aat      432 
Arg Pro Leu Lys Leu Met Asp Ala Asn Ile Glu Gly Ile Glu Asp Asn 
    130                 135                 140 

tat aca cca tta att att aag cca tct gtc ata aaa ggt ata aat tat      480 
Tyr Thr Pro Leu Ile Ile Lys Pro Ser Val Ile Lys Gly Ile Asn Tyr 
145                 150                 155                 160 

caa atg gaa gtt gca agt gca caa gta aaa agt gcc att tta ttt gca      528 
Gln Met Glu Val Ala Ser Ala Gln Val Lys Ser Ala Ile Leu Phe Ala 
                165                 170                 175 

agt ttg ttt tct aag gaa ccg acc atc att aaa gaa tta gat gta agt      576 
Ser Leu Phe Ser Lys Glu Pro Thr Ile Ile Lys Glu Leu Asp Val Ser 
            180                 185                 190 

cga aat cat act gag acg atg ttc aaa cat ttt aat att cca att gaa      624 
Arg Asn His Thr Glu Thr Met Phe Lys His Phe Asn Ile Pro Ile Glu 
        195                 200                 205 

gca gaa ggg tta tca att aat aca acc cct gaa gca att cga tac att      672 
Ala Glu Gly Leu Ser Ile Asn Thr Thr Pro Glu Ala Ile Arg Tyr Ile 
    210                 215                 220 

aaa cct gca gat ttt cat gtt cct ggc gat att tca tct gca gcg ttc      720 
Lys Pro Ala Asp Phe His Val Pro Gly Asp Ile Ser Ser Ala Ala Phe 
225                 230                 235                 240 

ttt att gtt gca gca ctt atc aca cca gga agt gat gta aca att cat      768 
Phe Ile Val Ala Ala Leu Ile Thr Pro Gly Ser Asp Val Thr Ile His 
                245                 250                 255 

aat gtt gga atc aat caa aca cgt tca ggt att att gat att gtt gaa      816 
Asn Val Gly Ile Asn Gln Thr Arg Ser Gly Ile Ile Asp Ile Val Glu 
            260                 265                 270 

aaa atg ggc ggt aat atc caa ctt ttc aat caa aca act ggt gct gaa      864 
Lys Met Gly Gly Asn Ile Gln Leu Phe Asn Gln Thr Thr Gly Ala Glu 
        275                 280                 285 

cct act gct tct att cgt att caa tac aca cca atg ctt caa cca ata      912 
Pro Thr Ala Ser Ile Arg Ile Gln Tyr Thr Pro Met Leu Gln Pro Ile 
    290                 295                 300 

aca atc gaa gga gaa tta gtt cca aaa gca att gat gaa ctg cct gta      960 
Thr Ile Glu Gly Glu Leu Val Pro Lys Ala Ile Asp Glu Leu Pro Val 
305                 310                 315                 320 

ata gca tta ctt tgt aca caa gca gtt ggc acg agt aca att aaa gat     1008 
Ile Ala Leu Leu Cys Thr Gln Ala Val Gly Thr Ser Thr Ile Lys Asp 
                325                 330                 335 

gcc gag gaa tta aaa gta aaa gaa aca aat aga att gat aca acg gct     1056 
Ala Glu Glu Leu Lys Val Lys Glu Thr Asn Arg Ile Asp Thr Thr Ala 
            340                 345                 350 

gat atg tta aac ttg tta ggg ttt gaa tta caa cca act aat gat gga     1104 
Asp Met Leu Asn Leu Leu Gly Phe Glu Leu Gln Pro Thr Asn Asp Gly 
        355                 360                 365 

ttg att att cat ccg tca gaa ttt aaa aca aat gca aca gat att tta     1152 
Leu Ile Ile His Pro Ser Glu Phe Lys Thr Asn Ala Thr Asp Ile Leu 
    370                 375                 380 

act gat cat cga ata gga atg atg ctt gca gtt gct tgt gta ctt tca     1200 
Thr Asp His Arg Ile Gly Met Met Leu Ala Val Ala Cys Val Leu Ser 
385                 390                 395                 400 

agc gag cct gtc aaa atc aaa caa ttt gat gct gta aat gta tca ttt     1248 
Ser Glu Pro Val Lys Ile Lys Gln Phe Asp Ala Val Asn Val Ser Phe 
                405                 410                 415 

cca gga ttt tta cca aaa cta aag ctt tta caa aat gag gga taa         1293 
Pro Gly Phe Leu Pro Lys Leu Lys Leu Leu Gln Asn Glu Gly 
            420                 425                 430 

 
           
             44  
             430  
             PRT  
             Staphylococcus aureus  
           
            44 

Met Val Asn Glu Gln Ile Ile Asp Ile Ser Gly Pro Leu Lys Gly Glu 
1               5                   10                  15 

Ile Glu Val Pro Gly Asp Lys Ser Met Thr His Arg Ala Ile Met Leu 
            20                  25                  30 

Ala Ser Leu Ala Glu Gly Val Ser Thr Ile Tyr Lys Pro Leu Leu Gly 
        35                  40                  45 

Glu Asp Cys Arg Arg Thr Met Asp Ile Phe Arg His Leu Gly Val Glu 
    50                  55                  60 

Ile Lys Glu Asp Asp Glu Lys Leu Val Val Thr Ser Pro Gly Tyr Gln 
65                  70                  75                  80 

Val Asn Thr Pro His Gln Val Leu Tyr Thr Gly Asn Ser Gly Thr Thr 
                85                  90                  95 

Thr Arg Leu Leu Ala Gly Leu Leu Ser Gly Leu Gly Asn Glu Ser Val 
            100                 105                 110 

Leu Ser Gly Asp Val Ser Ile Gly Lys Arg Pro Met Asp Arg Val Leu 
        115                 120                 125 

Arg Pro Leu Lys Leu Met Asp Ala Asn Ile Glu Gly Ile Glu Asp Asn 
    130                 135                 140 

Tyr Thr Pro Leu Ile Ile Lys Pro Ser Val Ile Lys Gly Ile Asn Tyr 
145                 150                 155                 160 

Gln Met Glu Val Ala Ser Ala Gln Val Lys Ser Ala Ile Leu Phe Ala 
                165                 170                 175 

Ser Leu Phe Ser Lys Glu Pro Thr Ile Ile Lys Glu Leu Asp Val Ser 
            180                 185                 190 

Arg Asn His Thr Glu Thr Met Phe Lys His Phe Asn Ile Pro Ile Glu 
        195                 200                 205 

Ala Glu Gly Leu Ser Ile Asn Thr Thr Pro Glu Ala Ile Arg Tyr Ile 
    210                 215                 220 

Lys Pro Ala Asp Phe His Val Pro Gly Asp Ile Ser Ser Ala Ala Phe 
225                 230                 235                 240 

Phe Ile Val Ala Ala Leu Ile Thr Pro Gly Ser Asp Val Thr Ile His 
                245                 250                 255 

Asn Val Gly Ile Asn Gln Thr Arg Ser Gly Ile Ile Asp Ile Val Glu 
            260                 265                 270 

Lys Met Gly Gly Asn Ile Gln Leu Phe Asn Gln Thr Thr Gly Ala Glu 
        275                 280                 285 

Pro Thr Ala Ser Ile Arg Ile Gln Tyr Thr Pro Met Leu Gln Pro Ile 
    290                 295                 300 

Thr Ile Glu Gly Glu Leu Val Pro Lys Ala Ile Asp Glu Leu Pro Val 
305                 310                 315                 320 

Ile Ala Leu Leu Cys Thr Gln Ala Val Gly Thr Ser Thr Ile Lys Asp 
                325                 330                 335 

Ala Glu Glu Leu Lys Val Lys Glu Thr Asn Arg Ile Asp Thr Thr Ala 
            340                 345                 350 

Asp Met Leu Asn Leu Leu Gly Phe Glu Leu Gln Pro Thr Asn Asp Gly 
        355                 360                 365 

Leu Ile Ile His Pro Ser Glu Phe Lys Thr Asn Ala Thr Asp Ile Leu 
    370                 375                 380 

Thr Asp His Arg Ile Gly Met Met Leu Ala Val Ala Cys Val Leu Ser 
385                 390                 395                 400 

Ser Glu Pro Val Lys Ile Lys Gln Phe Asp Ala Val Asn Val Ser Phe 
                405                 410                 415 

Pro Gly Phe Leu Pro Lys Leu Lys Leu Leu Gln Asn Glu Gly 
            420                 425                 430 

 
           
             45  
             28  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            45 

ggaacatatg aaacgagata aggtgcag                                        28 

 
           
             46  
             35  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            46 

ggaattcaaa cttcaggatc ttgagataga aaatg                                35 

 
           
             47  
             28  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            47 

ggggccatgg taaatgaaca aatcattg                                        28 

 
           
             48  
             33  
             DNA  
             Artificial sequence  
             
               Oligonucleotide  
             
           
            48 

gggggagctc attatccctc attttgtaaa agc                                  33 

 
           
             49  
             480  
             PRT  
             Saccharomyces cerevisiae  
           
            49 

Leu Thr Asp Glu Thr Leu Val Tyr Pro Phe Lys Asp Ile Pro Ala Asp 
1               5                   10                  15 

Gln Gln Lys Val Val Ile Pro Pro Gly Ser Lys Ser Ile Ser Asn Arg 
            20                  25                  30 

Ala Leu Ile Leu Ala Ala Leu Gly Glu Gly Gln Cys Lys Ile Lys Asn 
        35                  40                  45 

Leu Leu His Ser Asp Asp Thr Lys His Met Leu Thr Ala Val His Glu 
    50                  55                  60 

Leu Lys Gly Ala Thr Ile Ser Trp Glu Asp Asn Gly Glu Thr Val Val 
65                  70                  75                  80 

Val Glu Gly His Gly Gly Ser Thr Leu Ser Ala Cys Ala Asp Pro Leu 
                85                  90                  95 

Tyr Leu Gly Asn Ala Gly Thr Ala Ser Arg Phe Leu Thr Ser Leu Ala 
            100                 105                 110 

Ala Leu Val Asn Ser Thr Ser Ser Gln Lys Tyr Ile Val Leu Thr Gly 
        115                 120                 125 

Asn Ala Arg Met Gln Gln Arg Pro Ile Ala Pro Leu Val Asp Ser Leu 
    130                 135                 140 

Arg Ala Asn Gly Thr Lys Ile Glu Tyr Leu Asn Asn Glu Gly Ser Leu 
145                 150                 155                 160 

Pro Ile Lys Val Tyr Thr Asp Ser Val Phe Lys Gly Gly Arg Ile Glu 
                165                 170                 175 

Leu Ala Ala Thr Val Ser Ser Gln Tyr Val Ser Ser Ile Leu Met Cys 
            180                 185                 190 

Ala Pro Tyr Ala Glu Glu Pro Val Thr Leu Ala Leu Val Gly Gly Lys 
        195                 200                 205 

Pro Ile Ser Lys Leu Tyr Val Asp Met Thr Ile Lys Met Met Glu Lys 
    210                 215                 220 

Phe Gly Ile Asn Val Glu Thr Ser Thr Thr Glu Pro Tyr Thr Tyr Tyr 
225                 230                 235                 240 

Ile Pro Lys Gly His Tyr Ile Asn Pro Ser Glu Tyr Val Ile Glu Ser 
                245                 250                 255 

Asp Ala Ser Ser Ala Thr Tyr Pro Leu Ala Phe Ala Ala Met Thr Gly 
            260                 265                 270 

Thr Thr Val Thr Val Pro Asn Ile Gly Phe Glu Ser Leu Gln Gly Asp 
        275                 280                 285 

Ala Arg Phe Ala Arg Asp Val Leu Lys Pro Met Gly Cys Lys Ile Thr 
    290                 295                 300 

Gln Thr Ala Thr Ser Thr Thr Val Ser Gly Pro Pro Val Gly Thr Leu 
305                 310                 315                 320 

Lys Pro Leu Lys His Val Asp Met Glu Pro Met Thr Asp Ala Phe Leu 
                325                 330                 335 

Thr Ala Cys Val Val Ala Ala Ile Ser His Asp Ser Asp Pro Asn Ser 
            340                 345                 350 

Ala Asn Thr Thr Thr Ile Glu Gly Ile Ala Asn Gln Arg Val Lys Glu 
        355                 360                 365 

Cys Asn Arg Ile Leu Ala Met Ala Thr Glu Leu Ala Lys Phe Gly Val 
    370                 375                 380 

Lys Thr Thr Glu Leu Pro Asp Gly Ile Gln Val His Gly Leu Asn Ser 
385                 390                 395                 400 

Ile Lys Asp Leu Lys Val Pro Ser Asp Ser Ser Gly Pro Val Gly Val 
                405                 410                 415 

Cys Thr Tyr Asp Asp His Arg Val Ala Met Ser Phe Ser Leu Leu Ala 
            420                 425                 430 

Gly Met Val Asn Ser Gln Asn Glu Arg Asp Glu Val Ala Asn Pro Val 
        435                 440                 445 

Arg Ile Leu Glu Arg His Cys Thr Gly Lys Thr Trp Pro Gly Trp Trp 
    450                 455                 460 

Asp Val Leu His Ser Glu Leu Gly Ala Lys Leu Asp Gly Ala Glu Pro 
465                 470                 475                 480 

 
           
             50  
             460  
             PRT  
             Aspergillus ridulaus  
           
            50 

Leu Ala Pro Ser Ile Glu Val His Pro Gly Val Ala His Ser Ser Asn 
1               5                   10                  15 

Val Ile Cys Ala Pro Pro Gly Ser Lys Ser Ile Ser Asn Arg Ala Leu 
            20                  25                  30 

Val Leu Ala Ala Leu Gly Ser Gly Thr Cys Arg Ile Lys Asn Leu Leu 
        35                  40                  45 

His Ser Asp Asp Thr Glu Val Met Leu Asn Ala Leu Glu Arg Leu Gly 
    50                  55                  60 

Ala Ala Thr Phe Ser Trp Glu Glu Glu Gly Glu Val Leu Val Val Asn 
65                  70                  75                  80 

Gly Lys Gly Gly Asn Leu Gln Ala Ser Ser Ser Pro Leu Tyr Leu Gly 
                85                  90                  95 

Asn Ala Gly Thr Ala Ser Arg Phe Leu Thr Thr Val Ala Thr Leu Ala 
            100                 105                 110 

Asn Ser Ser Thr Val Asp Ser Ser Val Leu Thr Gly Asn Asn Arg Met 
        115                 120                 125 

Lys Gln Arg Pro Ile Gly Asp Leu Val Asp Ala Leu Thr Ala Asn Val 
    130                 135                 140 

Leu Pro Leu Asn Thr Ser Lys Gly Arg Ala Ser Leu Pro Leu Lys Ile 
145                 150                 155                 160 

Ala Ala Ser Gly Gly Phe Ala Gly Gly Asn Ile Asn Leu Ala Ala Lys 
                165                 170                 175 

Val Ser Ser Gln Tyr Val Ser Ser Leu Leu Met Cys Ala Pro Tyr Ala 
            180                 185                 190 

Lys Glu Pro Val Thr Leu Arg Leu Val Gly Gly Lys Pro Ile Ser Gln 
        195                 200                 205 

Pro Tyr Ile Asp Met Thr Thr Ala Met Met Arg Ser Phe Gly Ile Asp 
    210                 215                 220 

Val Gln Lys Ser Thr Thr Glu Glu His Thr Tyr His Ile Pro Gln Gly 
225                 230                 235                 240 

Arg Tyr Val Asn Pro Ala Glu Tyr Val Ile Glu Ser Asp Ala Ser Cys 
                245                 250                 255 

Ala Thr Tyr Pro Leu Ala Val Ala Ala Val Thr Gly Thr Thr Cys Thr 
            260                 265                 270 

Val Pro Asn Ile Gly Ser Ala Ser Leu Gln Gly Asp Ala Arg Phe Ala 
        275                 280                 285 

Val Glu Val Leu Arg Pro Met Gly Cys Thr Val Glu Gln Thr Glu Thr 
    290                 295                 300 

Ser Thr Thr Val Thr Gly Pro Ser Asp Gly Ile Leu Arg Ala Thr Ser 
305                 310                 315                 320 

Lys Arg Gly Tyr Gly Thr Asn Asp Arg Cys Val Pro Arg Cys Phe Arg 
                325                 330                 335 

Thr Gly Ser His Arg Pro Met Glu Lys Ser Gln Thr Thr Pro Pro Val 
            340                 345                 350 

Ser Ser Gly Ile Ala Asn Gln Arg Val Lys Glu Cys Asn Arg Ile Lys 
        355                 360                 365 

Ala Met Lys Asp Glu Leu Ala Lys Phe Gly Val Ile Cys Arg Glu His 
    370                 375                 380 

Asp Asp Gly Leu Glu Ile Asp Gly Ile Asp Arg Ser Asn Leu Arg Gln 
385                 390                 395                 400 

Pro Val Gly Gly Val Phe Cys Tyr Asp Asp His Arg Val Ala Phe Ser 
                405                 410                 415 

Phe Ser Val Leu Ser Leu Val Thr Pro Gln Pro Thr Leu Ile Leu Glu 
            420                 425                 430 

Lys Glu Cys Val Gly Lys Thr Trp Pro Gly Trp Trp Asp Thr Leu Arg 
        435                 440                 445 

Gln Leu Phe Lys Val Lys Leu Glu Gly Lys Glu Leu 
    450                 455                 460 

 
           
             51  
             444  
             PRT  
             Brassica napus  
           
            51 

Lys Ala Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly Leu 
1               5                   10                  15 

Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 
            20                  25                  30 

Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn Ser 
        35                  40                  45 

Asp Asp Ile Asn Tyr Met Leu Asp Ala Leu Lys Lys Leu Gly Leu Asn 
    50                  55                  60 

Val Glu Arg Asp Ser Val Asn Asn Arg Ala Val Val Glu Gly Cys Gly 
65                  70                  75                  80 

Gly Ile Phe Pro Ala Ser Leu Asp Ser Lys Ser Asp Ile Glu Leu Tyr 
                85                  90                  95 

Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 
            100                 105                 110 

Ala Ala Gly Gly Asn Ala Ser Tyr Val Leu Asp Gly Val Pro Arg Met 
        115                 120                 125 

Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly 
    130                 135                 140 

Ala Asp Val Glu Cys Thr Leu Gly Thr Asn Cys Pro Pro Val Arg Val 
145                 150                 155                 160 

Asn Ala Asn Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 
                165                 170                 175 

Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu Ala 
            180                 185                 190 

Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Val Pro 
        195                 200                 205 

Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser Ala 
    210                 215                 220 

Glu His Ser Asp Ser Trp Asp Arg Phe Phe Val Lys Gly Gly Gln Lys 
225                 230                 235                 240 

Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala 
                245                 250                 255 

Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Glu Thr Val Thr Val 
            260                 265                 270 

Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 
        275                 280                 285 

Val Leu Glu Lys Met Gly Cys Lys Val Ser Trp Thr Glu Asn Ser Val 
    290                 295                 300 

Thr Val Thr Gly Pro Ser Arg Asp Ala Phe Gly Met Arg His Leu Arg 
305                 310                 315                 320 

Ala Val Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 
                325                 330                 335 

Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp Val 
            340                 345                 350 

Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 
        355                 360                 365 

Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Ser Asp Tyr Cys 
    370                 375                 380 

Val Ile Thr Pro Pro Ala Lys Val Lys Pro Ala Glu Ile Asp Thr Tyr 
385                 390                 395                 400 

Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Asp 
                405                 410                 415 

Val Pro Val Thr Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 
            420                 425                 430 

Asp Tyr Phe Gln Val Leu Glu Ser Ile Thr Lys His 
        435                 440 

 
           
             52  
             444  
             PRT  
             Arabidopsis thaliana  
           
            52 

Lys Ala Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly Leu 
1               5                   10                  15 

Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 
            20                  25                  30 

Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn Ser 
        35                  40                  45 

Asp Asp Ile Asn Tyr Met Leu Asp Ala Leu Lys Arg Leu Gly Leu Asn 
    50                  55                  60 

Val Glu Thr Asp Ser Glu Asn Asn Arg Ala Val Val Glu Gly Cys Gly 
65                  70                  75                  80 

Gly Ile Phe Pro Ala Ser Ile Asp Ser Lys Ser Asp Ile Glu Leu Tyr 
                85                  90                  95 

Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 
            100                 105                 110 

Ala Ala Gly Gly Asn Ala Ser Tyr Val Leu Asp Gly Val Pro Arg Met 
        115                 120                 125 

Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly 
    130                 135                 140 

Ala Asp Val Glu Cys Thr Leu Gly Thr Asn Cys Pro Pro Val Arg Val 
145                 150                 155                 160 

Asn Ala Asn Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 
                165                 170                 175 

Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ser Ala Pro Leu Ala 
            180                 185                 190 

Leu Gly Asp Val Glu Ile Glu Ile Val Asp Lys Leu Ile Ser Val Pro 
        195                 200                 205 

Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser Val 
    210                 215                 220 

Glu His Ser Asp Ser Trp Asp Arg Phe Phe Val Lys Gly Gly Gln Lys 
225                 230                 235                 240 

Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala 
                245                 250                 255 

Cys Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Glu Thr Val Thr Val 
            260                 265                 270 

Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 
        275                 280                 285 

Val Leu Glu Lys Met Gly Cys Lys Val Ser Trp Thr Glu Asn Ser Val 
    290                 295                 300 

Thr Val Thr Gly Pro Pro Arg Asp Ala Phe Gly Met Arg His Leu Arg 
305                 310                 315                 320 

Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 
                325                 330                 335 

Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp Val 
            340                 345                 350 

Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 
        355                 360                 365 

Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Ser Asp Tyr Cys 
    370                 375                 380 

Val Ile Thr Pro Pro Lys Lys Val Lys Thr Ala Glu Ile Asp Thr Tyr 
385                 390                 395                 400 

Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Asp 
                405                 410                 415 

Val Pro Ile Thr Ile Asn Asp Ser Gly Cys Thr Arg Lys Thr Phe Pro 
            420                 425                 430 

Asp Tyr Phe Gln Val Leu Glu Arg Ile Thr Lys His 
        435                 440 

 
           
             53  
             444  
             PRT  
             Nicotiana tabacum  
           
            53 

Lys Pro Asn Glu Ile Val Leu Gln Pro Ile Lys Asp Ile Ser Gly Thr 
1               5                   10                  15 

Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 
            20                  25                  30 

Ala Ala Leu Ser Lys Gly Arg Thr Val Val Asp Asn Leu Leu Ser Ser 
        35                  40                  45 

Asp Asp Ile His Tyr Met Leu Gly Ala Leu Lys Thr Leu Gly Leu His 
    50                  55                  60 

Val Glu Asp Asp Asn Glu Asn Gln Arg Ala Ile Val Glu Gly Cys Gly 
65                  70                  75                  80 

Gly Gln Phe Pro Val Gly Lys Lys Ser Glu Glu Glu Ile Gln Leu Phe 
                85                  90                  95 

Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 
            100                 105                 110 

Val Ala Gly Gly His Ser Arg Tyr Val Leu Asp Gly Val Pro Arg Met 
        115                 120                 125 

Arg Glu Arg Pro Ile Gly Asp Leu Val Asp Gly Leu Lys Gln Leu Gly 
    130                 135                 140 

Ala Glu Val Asp Cys Phe Leu Gly Thr Asn Cys Pro Pro Val Arg Ile 
145                 150                 155                 160 

Val Ser Lys Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 
                165                 170                 175 

Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu Ala 
            180                 185                 190 

Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Val Pro 
        195                 200                 205 

Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser Val 
    210                 215                 220 

Glu His Thr Ser Ser Trp Asp Lys Phe Leu Val Arg Gly Gly Gln Lys 
225                 230                 235                 240 

Tyr Lys Ser Pro Gly Lys Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala 
                245                 250                 255 

Ser Tyr Phe Leu Ala Gly Ala Ala Val Thr Gly Gly Thr Val Thr Val 
            260                 265                 270 

Glu Gly Cys Gly Thr Ser Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 
        275                 280                 285 

Val Leu Glu Lys Met Gly Ala Glu Val Thr Trp Thr Glu Asn Ser Val 
    290                 295                 300 

Thr Val Lys Gly Pro Pro Arg Asn Ser Ser Gly Met Lys His Leu Arg 
305                 310                 315                 320 

Ala Val Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 
                325                 330                 335 

Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val 
            340                 345                 350 

Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 
        355                 360                 365 

Glu Leu Arg Lys Leu Gly Ala Thr Val Val Glu Gly Ser Asp Tyr Cys 
    370                 375                 380 

Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Glu Ile Asp Thr Tyr 
385                 390                 395                 400 

Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Asp 
                405                 410                 415 

Val Pro Val Thr Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 
            420                 425                 430 

Asn Tyr Phe Asp Val Leu Gln Gln Tyr Ser Lys His 
        435                 440 

 
           
             54  
             444  
             PRT  
             Lycopersicon esculentum  
             
               UNSURE  
               (1)..(444)  
               Xaa = any  
             
           
            54 

Lys Pro His Glu Ile Val Leu Xaa Pro Ile Lys Asp Ile Ser Gly Thr 
1               5                   10                  15 

Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 
            20                  25                  30 

Ala Ala Leu Ser Glu Gly Arg Thr Val Val Asp Asn Leu Leu Ser Ser 
        35                  40                  45 

Asp Asp Ile His Tyr Met Leu Gly Ala Leu Lys Thr Leu Gly Leu His 
    50                  55                  60 

Val Glu Asp Asp Asn Glu Asn Gln Arg Ala Ile Val Glu Gly Cys Gly 
65                  70                  75                  80 

Gly Gln Phe Pro Val Gly Lys Lys Ser Glu Glu Glu Ile Gln Leu Phe 
                85                  90                  95 

Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 
            100                 105                 110 

Val Ala Gly Gly His Ser Arg Tyr Val Leu Asp Gly Val Pro Arg Met 
        115                 120                 125 

Arg Glu Arg Pro Ile Gly Asp Leu Val Asp Gly Leu Lys Gln Leu Gly 
    130                 135                 140 

Ala Glu Val Asp Cys Ser Leu Gly Thr Asn Cys Pro Pro Val Arg Ile 
145                 150                 155                 160 

Val Ser Lys Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 
                165                 170                 175 

Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu Ala 
            180                 185                 190 

Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Val Pro 
        195                 200                 205 

Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Phe Val 
    210                 215                 220 

Glu His Ser Ser Gly Trp Asp Arg Phe Leu Val Lys Gly Gly Gln Lys 
225                 230                 235                 240 

Tyr Lys Ser Pro Gly Lys Ala Phe Val Glu Gly Asp Ala Ser Ser Ala 
                245                 250                 255 

Ser Tyr Phe Leu Ala Gly Ala Ala Val Thr Gly Gly Thr Val Thr Val 
            260                 265                 270 

Glu Gly Cys Gly Thr Ser Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 
        275                 280                 285 

Val Leu Glu Lys Met Gly Ala Glu Val Thr Trp Thr Glu Asn Ser Val 
    290                 295                 300 

Thr Val Lys Gly Pro Pro Arg Asn Ser Ser Gly Met Lys His Leu Arg 
305                 310                 315                 320 

Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 
                325                 330                 335 

Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp Val 
            340                 345                 350 

Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 
        355                 360                 365 

Glu Leu Arg Lys Leu Gly Ala Thr Val Val Glu Gly Ser Asp Tyr Cys 
    370                 375                 380 

Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Glu Ile Asp Thr Tyr 
385                 390                 395                 400 

Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Asp 
                405                 410                 415 

Val Pro Val Thr Ile Lys Asn Pro Gly Cys Thr Arg Lys Thr Phe Pro 
            420                 425                 430 

Asp Tyr Phe Glu Val Leu Gln Lys Tyr Ser Lys His 
        435                 440 

 
           
             55  
             444  
             PRT  
             Petunia x hybrida  
           
            55 

Lys Pro Ser Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser Gly Thr 
1               5                   10                  15 

Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 
            20                  25                  30 

Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Ser Ser 
        35                  40                  45 

Asp Asp Ile His Tyr Met Leu Gly Ala Leu Lys Thr Leu Gly Leu His 
    50                  55                  60 

Val Glu Glu Asp Ser Ala Asn Gln Arg Ala Val Val Glu Gly Cys Gly 
65                  70                  75                  80 

Gly Leu Phe Pro Val Gly Lys Glu Ser Lys Glu Glu Ile Gln Leu Phe 
                85                  90                  95 

Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 
            100                 105                 110 

Val Ala Gly Gly Asn Ser Arg Tyr Val Leu Asp Gly Val Pro Arg Met 
        115                 120                 125 

Arg Glu Arg Pro Ile Ser Asp Leu Val Asp Gly Leu Lys Gln Leu Gly 
    130                 135                 140 

Ala Glu Val Asp Cys Phe Leu Gly Thr Lys Cys Pro Pro Val Arg Ile 
145                 150                 155                 160 

Val Ser Lys Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 
                165                 170                 175 

Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu Ala 
            180                 185                 190 

Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Val Pro 
        195                 200                 205 

Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Ile Ser Val 
    210                 215                 220 

Glu His Ser Ser Ser Trp Asp Arg Phe Phe Val Arg Gly Gly Gln Lys 
225                 230                 235                 240 

Tyr Lys Ser Pro Gly Lys Ala Phe Val Glu Gly Asp Ala Ser Ser Ala 
                245                 250                 255 

Ser Tyr Phe Leu Ala Gly Ala Ala Val Thr Gly Gly Thr Ile Thr Val 
            260                 265                 270 

Glu Gly Cys Gly Thr Asn Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 
        275                 280                 285 

Val Leu Glu Lys Met Gly Ala Glu Val Thr Trp Thr Glu Asn Ser Val 
    290                 295                 300 

Thr Val Lys Gly Pro Pro Arg Ser Ser Ser Gly Arg Lys His Leu Arg 
305                 310                 315                 320 

Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 
                325                 330                 335 

Ala Val Val Ala Leu Tyr Ala Asp Gly Pro Thr Ala Ile Arg Asp Val 
            340                 345                 350 

Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 
        355                 360                 365 

Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Pro Asp Tyr Cys 
    370                 375                 380 

Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Asp Ile Asp Thr Tyr 
385                 390                 395                 400 

Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Asp 
                405                 410                 415 

Val Pro Val Thr Ile Asn Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 
            420                 425                 430 

Asn Tyr Phe Asp Val Leu Gln Gln Tyr Ser Lys His 
        435                 440 

 
           
             56  
             444  
             PRT  
             Zea mays  
           
            56 

Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser Gly 
1               5                   10                  15 

Thr Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu 
            20                  25                  30 

Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn 
        35                  40                  45 

Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly Leu 
    50                  55                  60 

Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala Val Val Val Gly Cys 
65                  70                  75                  80 

Gly Gly Lys Phe Pro Val Glu Asp Ala Lys Glu Glu Val Gln Leu Phe 
                85                  90                  95 

Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 
            100                 105                 110 

Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg Met 
        115                 120                 125 

Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly 
    130                 135                 140 

Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg Val 
145                 150                 155                 160 

Asn Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 
                165                 170                 175 

Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu Pro 
            180                 185                 190 

Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile Pro 
        195                 200                 205 

Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala 
    210                 215                 220 

Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys 
225                 230                 235                 240 

Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala 
                245                 250                 255 

Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr Val 
            260                 265                 270 

Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 
        275                 280                 285 

Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu Thr Ser Val 
    290                 295                 300 

Thr Val Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys His Leu Lys 
305                 310                 315                 320 

Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 
                325                 330                 335 

Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val 
            340                 345                 350 

Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg Thr 
        355                 360                 365 

Glu Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp Tyr Cys 
    370                 375                 380 

Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr Tyr 
385                 390                 395                 400 

Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Glu 
                405                 410                 415 

Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 
            420                 425                 430 

Asp Tyr Phe Asp Val Leu Ser Thr Phe Val Lys Asn 
        435                 440 

 
           
             57  
             427  
             PRT  
             Salmonella gallinarum  
           
            57 

Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Ala Ile 
1               5                   10                  15 

Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu Ala 
            20                  25                  30 

Ala Leu Ala Cys Gly Lys Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 
        35                  40                  45 

Asp Val Arg His Met Leu Asn Ala Leu Ser Ala Leu Gly Ile Asn Tyr 
    50                  55                  60 

Thr Leu Ser Ala Asp Arg Thr Arg Cys Asp Ile Thr Gly Asn Gly Gly 
65                  70                  75                  80 

Pro Leu Arg Ala Pro Gly Ala Leu Glu Leu Phe Leu Gly Asn Ala Gly 
                85                  90                  95 

Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Gln Asn Glu 
            100                 105                 110 

Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His 
        115                 120                 125 

Leu Val Asp Ser Leu Arg Gln Gly Gly Ala Asn Ile Asp Tyr Leu Glu 
    130                 135                 140 

Gln Glu Asn Tyr Pro Pro Leu Arg Leu Arg Gly Gly Phe Ile Gly Gly 
145                 150                 155                 160 

Asp Ile Glu Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 
                165                 170                 175 

Leu Met Thr Ala Pro Leu Ala Pro Lys Asp Thr Ile Ile Arg Val Lys 
            180                 185                 190 

Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met 
        195                 200                 205 

Lys Thr Phe Gly Val Glu Ile Ala Asn His His Tyr Gln Gln Phe Val 
    210                 215                 220 

Val Lys Gly Gly Gln Gln Tyr His Ser Pro Gly Arg Tyr Leu Val Glu 
225                 230                 235                 240 

Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Ala Ile Lys 
                245                 250                 255 

Gly Gly Thr Val Lys Val Thr Gly Ile Gly Arg Lys Ser Met Gln Gly 
            260                 265                 270 

Asp Ile Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala Thr Ile Thr 
        275                 280                 285 

Trp Gly Asp Asp Phe Ile Ala Cys Thr Arg Gly Glu Leu His Ala Ile 
    290                 295                 300 

Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr 
305                 310                 315                 320 

Thr Ala Leu Phe Ala Lys Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn 
                325                 330                 335 

Trp Arg Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 
            340                 345                 350 

Arg Lys Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile 
        355                 360                 365 

Thr Pro Pro Ala Lys Leu Gln His Ala Asp Ile Gly Thr Tyr Asn Asp 
    370                 375                 380 

His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro 
385                 390                 395                 400 

Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 
                405                 410                 415 

Phe Glu Gln Leu Ala Arg Met Ser Thr Pro Ala 
            420                 425 

 
           
             58  
             427  
             PRT  
             Salmonella typhimurium  
           
            58 

Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Ala Ile 
1               5                   10                  15 

Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu Ala 
            20                  25                  30 

Ala Leu Ala Cys Gly Lys Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 
        35                  40                  45 

Asp Val Arg His Met Leu Asn Ala Leu Ser Ala Leu Gly Ile Asn Tyr 
    50                  55                  60 

Thr Leu Ser Ala Asp Arg Thr Arg Cys Asp Ile Thr Gly Asn Gly Gly 
65                  70                  75                  80 

Pro Leu Arg Ala Ser Gly Thr Leu Glu Leu Phe Leu Gly Asn Ala Gly 
                85                  90                  95 

Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Gln Asn Glu 
            100                 105                 110 

Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His 
        115                 120                 125 

Leu Val Asp Ser Leu Arg Gln Gly Gly Ala Asn Ile Asp Tyr Leu Glu 
    130                 135                 140 

Gln Glu Asn Tyr Pro Pro Leu Arg Leu Arg Gly Gly Phe Ile Gly Gly 
145                 150                 155                 160 

Asp Ile Glu Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 
                165                 170                 175 

Leu Met Thr Ala Pro Leu Ala Pro Glu Asp Thr Ile Ile Arg Val Lys 
            180                 185                 190 

Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met 
        195                 200                 205 

Lys Thr Phe Gly Val Glu Ile Ala Asn His His Tyr Gln Gln Phe Val 
    210                 215                 220 

Val Lys Gly Gly Gln Gln Tyr His Ser Pro Gly Arg Tyr Leu Val Glu 
225                 230                 235                 240 

Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Gly Ile Lys 
                245                 250                 255 

Gly Gly Thr Val Lys Val Thr Gly Ile Gly Gly Lys Ser Met Gln Gly 
            260                 265                 270 

Asp Ile Arg Phe Ala Asp Val Leu His Lys Met Gly Ala Thr Ile Thr 
        275                 280                 285 

Trp Gly Asp Asp Phe Ile Ala Cys Thr Arg Gly Glu Leu His Ala Ile 
    290                 295                 300 

Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr 
305                 310                 315                 320 

Thr Ala Leu Phe Ala Lys Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn 
                325                 330                 335 

Trp Arg Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 
            340                 345                 350 

Arg Lys Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile 
        355                 360                 365 

Thr Pro Pro Ala Lys Leu Gln His Ala Asp Ile Gly Thr Tyr Asn Asp 
    370                 375                 380 

His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro 
385                 390                 395                 400 

Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 
                405                 410                 415 

Phe Glu Gln Leu Ala Arg Met Ser Thr Pro Ala 
            420                 425 

 
           
             59  
             427  
             PRT  
             Klebsiella pneumoniae  
           
            59 

Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Thr Val 
1               5                   10                  15 

Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu Ala 
            20                  25                  30 

Ala Leu Ala Arg Gly Thr Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 
        35                  40                  45 

Asp Val Arg His Met Leu Asn Ala Leu Ser Ala Leu Gly Val His Tyr 
    50                  55                  60 

Val Leu Ser Ser Asp Arg Thr Arg Cys Glu Val Thr Gly Thr Gly Gly 
65                  70                  75                  80 

Pro Leu Gln Ala Gly Ser Ala Leu Glu Leu Phe Leu Gly Asn Ala Gly 
                85                  90                  95 

Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Ser Asn Asp 
            100                 105                 110 

Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His 
        115                 120                 125 

Leu Val Asp Ala Leu Arg Gln Gly Gly Ala Gln Ile Asp Tyr Leu Glu 
    130                 135                 140 

Gln Glu Asn Tyr Pro Pro Leu Arg Leu Arg Gly Gly Phe Thr Gly Gly 
145                 150                 155                 160 

Asp Val Glu Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 
                165                 170                 175 

Leu Met Ala Ser Pro Leu Ala Pro Gln Asp Thr Val Ile Ala Ile Lys 
            180                 185                 190 

Gly Glu Leu Val Ser Arg Pro Tyr Ile Asp Ile Thr Leu His Leu Met 
        195                 200                 205 

Lys Thr Phe Gly Val Glu Val Glu Asn Gln Ala Tyr Gln Arg Phe Ile 
    210                 215                 220 

Val Arg Gly Asn Gln Gln Tyr Gln Ser Pro Gly Asp Tyr Leu Val Glu 
225                 230                 235                 240 

Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Ala Ile Lys 
                245                 250                 255 

Gly Gly Thr Val Lys Val Thr Gly Ile Gly Arg Asn Ser Val Gln Gly 
            260                 265                 270 

Asp Ile Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala Thr Val Thr 
        275                 280                 285 

Trp Gly Glu Asp Tyr Ile Ala Cys Thr Arg Gly Glu Leu Asn Ala Ile 
    290                 295                 300 

Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr 
305                 310                 315                 320 

Ala Ala Leu Phe Ala Arg Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn 
                325                 330                 335 

Trp Arg Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 
            340                 345                 350 

Arg Lys Val Gly Ala Glu Val Glu Glu Gly Glu Asp Tyr Ile Arg Ile 
        355                 360                 365 

Thr Pro Pro Leu Thr Leu Gln Phe Ala Glu Ile Gly Thr Tyr Asn Asp 
    370                 375                 380 

His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro 
385                 390                 395                 400 

Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 
                405                 410                 415 

Phe Gly Gln Leu Ala Arg Ile Ser Thr Leu Ala 
            420                 425 

 
           
             60  
             427  
             PRT  
             Yersinia enterocolitica  
           
            60 

Met Leu Glu Ser Leu Thr Leu His Pro Ile Ala Leu Ile Asn Gly Thr 
1               5                   10                  15 

Val Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu 
            20                  25                  30 

Ala Ala Leu Ala Glu Gly Thr Thr Gln Leu Asn Asn Leu Leu Asp Ser 
        35                  40                  45 

Asp Asp Ile Arg His Met Leu Asn Ala Leu Gln Ala Leu Gly Val Lys 
    50                  55                  60 

Tyr Arg Leu Ser Ala Asp Arg Thr Arg Cys Glu Val Asp Gly Leu Gly 
65                  70                  75                  80 

Gly Lys Leu Val Ala Glu Gln Pro Leu Glu Leu Phe Leu Gly Asn Ala 
                85                  90                  95 

Gly Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Lys Asn 
            100                 105                 110 

Asp Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly 
        115                 120                 125 

His Leu Val Asp Ala Leu Arg Gln Gly Gly Ala Gln Ile Asp Tyr Leu 
    130                 135                 140 

Glu Gln Glu Asn Tyr Arg Arg Cys Ile Ala Gly Gly Phe Arg Gly Gly 
145                 150                 155                 160 

Lys Leu Thr Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 
                165                 170                 175 

Leu Met Thr Ala Pro Leu Ala Glu Gln Asp Thr Glu Ile Gln Ile Gln 
            180                 185                 190 

Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu His Leu Met 
        195                 200                 205 

Lys Ala Phe Gly Val Asp Val Val His Glu Asn Tyr Gln Ile Phe His 
    210                 215                 220 

Ile Lys Gly Gly Gln Thr Tyr Arg Ser Pro Gly Ile Tyr Leu Val Glu 
225                 230                 235                 240 

Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Ala Ala Ile Lys 
                245                 250                 255 

Gly Gly Thr Val Arg Val Thr Gly Ile Gly Lys Gln Ser Val Gln Gly 
            260                 265                 270 

Asp Thr Lys Phe Ala Asp Val Leu Glu Lys Met Gly Ala Lys Ile Ser 
        275                 280                 285 

Trp Gly Asp Asp Tyr Ile Glu Cys Ser Arg Gly Glu Leu Gln Gly Ile 
    290                 295                 300 

Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr 
305                 310                 315                 320 

Thr Ala Leu Phe Ala Asp Gly Pro Thr Val Ile Arg Asn Ile Tyr Asn 
                325                 330                 335 

Trp Arg Val Lys Glu Thr Asp Arg Leu Ser Ala Met Ala Thr Glu Leu 
            340                 345                 350 

Arg Lys Val Gly Ala Glu Val Glu Glu Gly Gln Asp Tyr Ile Arg Val 
        355                 360                 365 

Val Pro Pro Ala Gln Leu Ile Ala Ala Glu Ile Gly Thr Tyr Asn Asp 
    370                 375                 380 

His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro 
385                 390                 395                 400 

Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 
                405                 410                 415 

Phe Glu Gln Leu Ala Arg Leu Ser Gln Ile Ala 
            420                 425 

 
           
             61  
             432  
             PRT  
             Haemophilus influenzae  
           
            61 

Met Glu Lys Ile Thr Leu Ala Pro Ile Ser Ala Val Glu Gly Thr Ile 
1               5                   10                  15 

Asn Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ala Leu Leu Leu Ala 
            20                  25                  30 

Ala Leu Ala Lys Gly Thr Thr Lys Val Thr Asn Leu Leu Asp Ser Asp 
        35                  40                  45 

Asp Ile Arg His Met Leu Asn Ala Leu Lys Ala Leu Gly Val Arg Tyr 
    50                  55                  60 

Gln Leu Ser Asp Asp Lys Thr Ile Cys Glu Ile Glu Gly Leu Gly Gly 
65                  70                  75                  80 

Ala Phe Asn Ile Gln Asp Asn Leu Ser Leu Phe Leu Gly Asn Ala Gly 
                85                  90                  95 

Thr Ala Met Arg Pro Leu Thr Ala Ala Leu Cys Leu Lys Gly Asn His 
            100                 105                 110 

Glu Val Glu Ile Ile Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro 
        115                 120                 125 

Ile Leu His Leu Val Asp Ala Leu Arg Gln Ala Gly Ala Asp Ile Arg 
    130                 135                 140 

Tyr Leu Glu Asn Glu Gly Tyr Pro Pro Leu Ala Ile Arg Asn Lys Gly 
145                 150                 155                 160 

Ile Lys Gly Gly Lys Val Lys Ile Asp Gly Ser Ile Ser Ser Gln Phe 
                165                 170                 175 

Leu Thr Ala Leu Leu Met Ser Ala Pro Leu Ala Glu Asn Asp Thr Glu 
            180                 185                 190 

Ile Glu Ile Ile Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr 
        195                 200                 205 

Leu Ala Met Met Arg Asp Phe Gly Val Lys Val Glu Asn His His Tyr 
    210                 215                 220 

Gln Lys Phe Gln Val Lys Gly Asn Gln Ser Tyr Ile Ser Pro Asn Lys 
225                 230                 235                 240 

Tyr Leu Val Glu Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala 
                245                 250                 255 

Gly Ala Ile Lys Gly Lys Val Lys Val Thr Gly Ile Gly Lys Asn Ser 
            260                 265                 270 

Ile Gln Gly Asp Arg Leu Phe Ala Asp Val Leu Glu Lys Met Gly Ala 
        275                 280                 285 

Lys Ile Thr Trp Gly Glu Asp Phe Ile Gln Ala Glu His Ala Glu Leu 
    290                 295                 300 

Asn Gly Ile Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr 
305                 310                 315                 320 

Ile Ala Thr Thr Ala Leu Phe Ser Asn Gly Glu Thr Val Ile Arg Asn 
                325                 330                 335 

Ile Tyr Asn Trp Arg Val Lys Glu Thr Asp Arg Leu Thr Ala Met Ala 
            340                 345                 350 

Thr Glu Leu Arg Lys Val Gly Ala Glu Val Glu Glu Gly Glu Asp Phe 
        355                 360                 365 

Ile Arg Ile Gln Pro Leu Ala Leu Asn Gln Phe Lys His Ala Asn Ile 
    370                 375                 380 

Glu Thr Tyr Asn Asp His Arg Met Ala Met Cys Phe Ser Leu Ile Ala 
385                 390                 395                 400 

Leu Ser Asn Thr Pro Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys 
                405                 410                 415 

Thr Phe Pro Thr Phe Phe Asn Glu Phe Glu Lys Ile Cys Leu Lys Asn 
            420                 425                 430 

 
           
             62  
             441  
             PRT  
             Pasteurella multocida  
           
            62 

Val Ile Lys Asp Ala Thr Ala Ile Thr Leu Asn Pro Ile Ser Tyr Ile 
1               5                   10                  15 

Glu Gly Glu Val Arg Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ala 
            20                  25                  30 

Leu Leu Leu Ser Ala Leu Ala Lys Gly Lys Thr Thr Leu Thr Asn Leu 
        35                  40                  45 

Leu Asp Ser Asp Asp Val Arg His Met Leu Asn Ala Leu Lys Glu Leu 
    50                  55                  60 

Gly Val Thr Tyr Gln Leu Ser Glu Asp Lys Ser Val Cys Glu Ile Glu 
65                  70                  75                  80 

Gly Leu Gly Arg Ala Phe Glu Trp Gln Ser Gly Leu Ala Leu Phe Leu 
                85                  90                  95 

Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Leu Cys Leu 
            100                 105                 110 

Ser Thr Pro Asn Arg Glu Gly Lys Asn Glu Ile Val Leu Thr Gly Glu 
        115                 120                 125 

Pro Arg Met Lys Glu Arg Pro Ile Gln His Leu Val Asp Ala Leu Cys 
    130                 135                 140 

Gln Ala Gly Ala Glu Ile Gln Tyr Leu Glu Gln Glu Gly Tyr Pro Pro 
145                 150                 155                 160 

Ile Ala Ile Arg Asn Thr Gly Leu Lys Gly Gly Arg Ile Gln Ile Asp 
                165                 170                 175 

Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu Leu Met Ala Ala Pro 
            180                 185                 190 

Met Ala Glu Ala Asp Thr Glu Ile Glu Ile Ile Gly Glu Leu Val Ser 
        195                 200                 205 

Lys Pro Tyr Ile Asp Ile Thr Leu Lys Met Met Gln Thr Phe Gly Val 
    210                 215                 220 

Glu Val Glu Asn Gln Ala Tyr Gln Arg Phe Leu Val Lys Gly His Gln 
225                 230                 235                 240 

Gln Tyr Gln Ser Pro His Arg Phe Leu Val Glu Gly Asp Ala Ser Ser 
                245                 250                 255 

Ala Ser Tyr Phe Leu Ala Ala Ala Ala Ile Lys Gly Lys Val Lys Val 
            260                 265                 270 

Thr Gly Val Gly Lys Asn Ser Ile Gln Gly Asp Arg Leu Phe Ala Asp 
        275                 280                 285 

Val Leu Glu Lys Met Gly Ala His Ile Thr Trp Gly Asp Asp Phe Ile 
    290                 295                 300 

Gln Val Glu Lys Gly Asn Leu Lys Gly Ile Asp Met Asp Met Asn His 
305                 310                 315                 320 

Ile Pro Asp Ala Ala Met Thr Ile Ala Thr Thr Ala Leu Phe Ala Glu 
                325                 330                 335 

Gly Glu Thr Val Ile Arg Asn Ile Tyr Asn Trp Arg Val Lys Glu Thr 
            340                 345                 350 

Asp Arg Leu Thr Ala Met Ala Thr Glu Leu Arg Lys Val Gly Ala Glu 
        355                 360                 365 

Val Glu Glu Gly Glu Asp Phe Ile Arg Ile Gln Pro Leu Asn Leu Ala 
    370                 375                 380 

Gln Phe Gln His Ala Glu Leu Asn Ile His Asp His Arg Met Ala Met 
385                 390                 395                 400 

Cys Phe Ala Leu Ile Ala Leu Ser Lys Thr Ser Val Thr Ile Leu Asp 
                405                 410                 415 

Pro Ser Cys Thr Ala Lys Thr Phe Pro Thr Phe Leu Ile Leu Phe Thr 
            420                 425                 430 

Leu Asn Thr Arg Glu Val Ala Tyr Arg 
        435                 440 

 
           
             63  
             426  
             PRT  
             Aeromonas salmonicida  
           
            63 

Asn Ser Leu Arg Leu Glu Pro Ile Ser Arg Val Ala Gly Glu Val Asn 
1               5                   10                  15 

Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu Ala Ala 
            20                  25                  30 

Leu Ala Arg Gly Thr Thr Arg Leu Thr Asn Leu Leu Asp Ser Asp Asp 
        35                  40                  45 

Ile Arg His Met Leu Ala Ala Leu Thr Gln Leu Gly Val Lys Tyr Lys 
    50                  55                  60 

Leu Ser Ala Asp Lys Thr Glu Cys Thr Val His Gly Leu Gly Arg Ser 
65                  70                  75                  80 

Phe Ala Val Ser Ala Pro Val Asn Leu Phe Leu Gly Asn Ala Gly Thr 
                85                  90                  95 

Ala Met Arg Pro Leu Cys Ala Ala Leu Cys Leu Gly Ser Gly Glu Tyr 
            100                 105                 110 

Met Leu Gly Gly Glu Pro Arg Met Glu Glu Arg Pro Ile Gly His Leu 
        115                 120                 125 

Val Asp Cys Leu Ala Leu Lys Gly Ala His Ile Gln Tyr Leu Lys Lys 
    130                 135                 140 

Asp Gly Tyr Pro Pro Leu Val Val Asp Ala Lys Gly Leu Trp Gly Gly 
145                 150                 155                 160 

Asp Val His Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Phe 
                165                 170                 175 

Leu Met Ala Ala Pro Ala Met Ala Pro Val Ile Pro Arg Ile His Ile 
            180                 185                 190 

Lys Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu His Ile 
        195                 200                 205 

Met Asn Ser Ser Gly Val Val Ile Glu His Asp Asn Tyr Lys Leu Phe 
    210                 215                 220 

Tyr Ile Lys Gly Asn Gln Ser Ile Val Ser Pro Gly Asp Phe Leu Val 
225                 230                 235                 240 

Glu Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Ala Ile 
                245                 250                 255 

Lys Gly Lys Val Arg Val Thr Gly Ile Gly Lys His Ser Ile Gly Asp 
            260                 265                 270 

Ile His Phe Ala Asp Val Leu Glu Arg Met Gly Ala Arg Ile Thr Trp 
        275                 280                 285 

Gly Asp Asp Phe Ile Glu Ala Glu Gln Gly Pro Leu His Gly Val Asp 
    290                 295                 300 

Met Asp Met Asn His Ile Pro Asp Val Gly His Asp His Ser Gly Gln 
305                 310                 315                 320 

Ser His Cys Leu Pro Arg Val Pro Pro His Ser Gln His Leu Gln Leu 
                325                 330                 335 

Ala Val Arg Asp Asp Arg Cys Thr Pro Cys Thr His Gly His Arg Arg 
            340                 345                 350 

Ala Gln Ala Gly Val Ser Glu Glu Gly Thr Thr Phe Ile Thr Arg Asp 
        355                 360                 365 

Ala Ala Asp Pro Ala Gln Ala Arg Arg Asp Arg His Leu Gln Arg Ser 
    370                 375                 380 

Arg Ile Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Ile Ala Val 
385                 390                 395                 400 

Thr Ile Asn Asp Pro Gly Cys Thr Ser Lys Thr Phe Pro Asp Tyr Phe 
                405                 410                 415 

Asp Lys Leu Ala Ser Val Ser Gln Ala Val 
            420                 425 

 
           
             64  
             442  
             PRT  
             Bacillus pertussis  
           
            64 

Met Ser Gly Leu Ala Tyr Leu Asp Leu Pro Ala Ala Arg Leu Ala Arg 
1               5                   10                  15 

Gly Glu Val Ala Leu Pro Gly Ser Lys Ser Ile Ser Asn Arg Val Leu 
            20                  25                  30 

Leu Leu Ala Ala Leu Ala Glu Gly Ser Thr Glu Ile Thr Gly Leu Leu 
        35                  40                  45 

Asp Ser Asp Asp Thr Arg Val Met Leu Ala Ala Leu Arg Gln Leu Gly 
    50                  55                  60 

Val Ser Val Gly Glu Val Ala Asp Gly Cys Val Thr Ile Glu Gly Val 
65                  70                  75                  80 

Ala Arg Phe Pro Thr Glu Gln Ala Glu Leu Phe Leu Gly Asn Ala Gly 
                85                  90                  95 

Thr Ala Phe Arg Pro Leu Thr Ala Ala Leu Ala Leu Met Gly Gly Asp 
            100                 105                 110 

Tyr Arg Leu Ser Gly Val Pro Arg Met His Glu Arg Pro Ile Gly Asp 
        115                 120                 125 

Leu Val Asp Ala Leu Arg Gln Phe Gly Ala Gly Ile Glu Tyr Leu Gly 
    130                 135                 140 

Gln Ala Gly Tyr Pro Pro Leu Arg Ile Gly Gly Gly Ser Ile Arg Val 
145                 150                 155                 160 

Asp Gly Pro Val Arg Val Glu Gly Ser Val Ser Ser Gln Phe Leu Thr 
                165                 170                 175 

Ala Leu Leu Met Ala Ala Pro Val Leu Ala Arg Arg Ser Gly Gln Asp 
            180                 185                 190 

Ile Thr Ile Glu Val Val Gly Glu Leu Ile Ser Lys Pro Tyr Ile Glu 
        195                 200                 205 

Ile Thr Leu Asn Leu Met Ala Arg Phe Gly Val Ser Val Arg Arg Asp 
    210                 215                 220 

Gly Trp Arg Ala Phe Thr Ile Ala Arg Asp Ala Val Tyr Arg Gly Pro 
225                 230                 235                 240 

Gly Arg Met Ala Ile Glu Gly Asp Ala Ser Thr Ala Ser Tyr Phe Leu 
                245                 250                 255 

Ala Leu Gly Ala Ile Gly Gly Gly Pro Val Arg Val Thr Gly Val Gly 
            260                 265                 270 

Glu Asp Ser Ile Gln Gly Asp Val Ala Phe Ala Ala Thr Leu Ala Ala 
        275                 280                 285 

Met Gly Ala Asp Val Arg Tyr Gly Pro Gly Trp Ile Glu Thr Arg Gly 
    290                 295                 300 

Val Arg Val Ala Glu Gly Gly Arg Leu Lys Ala Phe Asp Ala Asp Phe 
305                 310                 315                 320 

Asn Leu Ile Pro Asp Ala Ala Met Thr Ala Ala Thr Leu Ala Leu Tyr 
                325                 330                 335 

Ala Asp Gly Pro Cys Arg Leu Arg Asn Ile Gly Ser Trp Arg Val Lys 
            340                 345                 350 

Glu Thr Asp Arg Ile His Ala Met His Thr Glu Leu Glu Lys Leu Gly 
        355                 360                 365 

Ala Gly Val Gln Ser Gly Ala Asp Trp Leu Glu Val Ala Pro Pro Glu 
    370                 375                 380 

Pro Gly Gly Trp Arg Asp Ala His Ile Gly Thr Trp Asp Asp His Arg 
385                 390                 395                 400 

Met Ala Met Cys Phe Leu Leu Ala Ala Phe Gly Pro Ala Ala Val Arg 
                405                 410                 415 

Ile Leu Asp Pro Gly Cys Val Ser Lys Thr Phe Pro Asp Tyr Phe Asp 
            420                 425                 430 

Val Tyr Ala Gly Leu Leu Ala Ala Arg Asp 
        435                 440 

 
           
             65  
             427  
             PRT  
             Salmonella typhimurium  
           
            65 

Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Ala Ile 
1               5                   10                  15 

Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu Ala 
            20                  25                  30 

Ala Leu Ala Cys Gly Lys Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 
        35                  40                  45 

Asp Val Arg His Met Leu Asn Ala Leu Ser Ala Leu Gly Ile Asn Tyr 
    50                  55                  60 

Thr Leu Ser Ala Asp Arg Thr Arg Cys Asp Ile Thr Gly Asn Gly Gly 
65                  70                  75                  80 

Pro Leu Arg Ala Ser Gly Thr Leu Glu Leu Phe Leu Gly Asn Ala Gly 
                85                  90                  95 

Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Gln Asn Glu 
            100                 105                 110 

Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His 
        115                 120                 125 

Leu Val Asp Ser Leu Arg Gln Gly Gly Ala Asn Ile Asp Tyr Leu Glu 
    130                 135                 140 

Gln Glu Asn Tyr Pro Pro Leu Arg Leu Arg Gly Gly Phe Ile Gly Gly 
145                 150                 155                 160 

Asp Ile Glu Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 
                165                 170                 175 

Leu Met Thr Ala Pro Leu Ala Pro Glu Asp Thr Ile Ile Arg Val Lys 
            180                 185                 190 

Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met 
        195                 200                 205 

Lys Thr Phe Gly Val Glu Ile Ala Asn His His Tyr Gln Gln Phe Val 
    210                 215                 220 

Val Lys Gly Gly Gln Gln Tyr His Ser Pro Gly Arg Tyr Leu Val Glu 
225                 230                 235                 240 

Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Gly Ile Lys 
                245                 250                 255 

Gly Gly Thr Val Lys Val Thr Gly Ile Gly Gly Lys Ser Met Gln Gly 
            260                 265                 270 

Asp Ile Arg Phe Ala Asp Val Leu His Lys Met Gly Ala Thr Ile Thr 
        275                 280                 285 

Trp Gly Asp Asp Phe Ile Ala Cys Thr Arg Gly Glu Leu His Ala Ile 
    290                 295                 300 

Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr 
305                 310                 315                 320 

Thr Ala Leu Phe Ala Lys Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn 
                325                 330                 335 

Trp Arg Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 
            340                 345                 350 

Arg Lys Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile 
        355                 360                 365 

Thr Pro Pro Ala Lys Leu Gln His Ala Asp Ile Gly Thr Tyr Asn Asp 
    370                 375                 380 

His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro 
385                 390                 395                 400 

Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 
                405                 410                 415 

Phe Glu Gln Leu Ala Arg Met Ser Thr Pro Ala 
            420                 425 

 
           
             66  
             1894  
             DNA  
             Synechocystis sp.  
             
               CDS  
               (275)..(1618)  
             
           
            66 

acgggctgta acggtagtag gggtcccgag cacaaaagcg gtgccggcaa gcagaactaa     60 

tttccatggg gaataatggt atttcattgg tttggcctct ggtctggcaa tggttgctag    120 

gcgatcgcct gttgaaatta acaaactgtc gcccttccac tgaccatggt aacgatgttt    180 

tttacttcct tgactaaccg aggaaaattt ggcggggggc agaaatgcca atacaattta    240 

gcttggtctt ccctgcccct aatttgtccc ctcc atg gcc ttg ctt tcc ctc aac    295 
                                      Met Ala Leu Leu Ser Leu Asn 
                                      1               5 

aat cat caa tcc cat caa cgc tta act gtt aat ccc cct gcc caa ggg      343 
Asn His Gln Ser His Gln Arg Leu Thr Val Asn Pro Pro Ala Gln Gly 
        10                  15                  20 

gtc gct ttg act ggc cgc cta agg gtg ccg ggg gat aaa tcc att tcc      391 
Val Ala Leu Thr Gly Arg Leu Arg Val Pro Gly Asp Lys Ser Ile Ser 
    25                  30                  35 

cat cgg gcc ttg atg ttg ggg gcg atc gcc acc ggg gaa acc att atc      439 
His Arg Ala Leu Met Leu Gly Ala Ile Ala Thr Gly Glu Thr Ile Ile 
40                  45                  50                  55 

gaa ggg cta ctg ttg ggg gaa gat ccc cgt agt acg gcc cat tgc ttt      487 
Glu Gly Leu Leu Leu Gly Glu Asp Pro Arg Ser Thr Ala His Cys Phe 
                60                  65                  70 

cgg gcc atg gga gca gaa atc agc gaa cta aat tca gaa aaa atc atc      535 
Arg Ala Met Gly Ala Glu Ile Ser Glu Leu Asn Ser Glu Lys Ile Ile 
            75                  80                  85 

gtt cag ggt cgg ggt ctg gga cag ttg cag gaa ccc agt acc gtt ttg      583 
Val Gln Gly Arg Gly Leu Gly Gln Leu Gln Glu Pro Ser Thr Val Leu 
        90                  95                  100 

gat gcg ggg aac tct ggc acc acc atg cgc tta atg ttg ggc ttg cta      631 
Asp Ala Gly Asn Ser Gly Thr Thr Met Arg Leu Met Leu Gly Leu Leu 
    105                 110                 115 

gcc ggg caa aaa gat tgt tta ttc acc gtc acc ggc gat gat tcc ctc      679 
Ala Gly Gln Lys Asp Cys Leu Phe Thr Val Thr Gly Asp Asp Ser Leu 
120                 125                 130                 135 

cgt cac cgc ccc atg tcc cgg gta att caa ccc ttg caa caa atg ggg      727 
Arg His Arg Pro Met Ser Arg Val Ile Gln Pro Leu Gln Gln Met Gly 
                140                 145                 150 

gca aaa att tgg gcc cgg agt aac ggc aag ttt gcg ccg ctg gca gtc      775 
Ala Lys Ile Trp Ala Arg Ser Asn Gly Lys Phe Ala Pro Leu Ala Val 
            155                 160                 165 

cag ggt agc caa tta aaa ccg atc cat tac cat tcc ccc att gct tca      823 
Gln Gly Ser Gln Leu Lys Pro Ile His Tyr His Ser Pro Ile Ala Ser 
        170                 175                 180 

gcc cag gta aag tcc tgc ctg ttg cta gcg ggg tta acc acc gag ggg      871 
Ala Gln Val Lys Ser Cys Leu Leu Leu Ala Gly Leu Thr Thr Glu Gly 
    185                 190                 195 

gac acc acg gtt aca gaa cca gct cta tcc cgg gat cat agc gaa cgc      919 
Asp Thr Thr Val Thr Glu Pro Ala Leu Ser Arg Asp His Ser Glu Arg 
200                 205                 210                 215 

atg ttg cag gcc ttt gga gcc aaa tta acc att gat cca gta acc cat      967 
Met Leu Gln Ala Phe Gly Ala Lys Leu Thr Ile Asp Pro Val Thr His 
                220                 225                 230 

agc gtc act gtc cat ggc ccg gcc cat tta acg ggg caa cgg gtg gtg     1015 
Ser Val Thr Val His Gly Pro Ala His Leu Thr Gly Gln Arg Val Val 
            235                 240                 245 

gtg cca ggg gac atc agc tcg gcg gcc ttt tgg tta gtg gcg gca tcc     1063 
Val Pro Gly Asp Ile Ser Ser Ala Ala Phe Trp Leu Val Ala Ala Ser 
        250                 255                 260 

att ttg cct gga tca gaa ttg ttg gtg gaa aat gta ggc att aac ccc     1111 
Ile Leu Pro Gly Ser Glu Leu Leu Val Glu Asn Val Gly Ile Asn Pro 
    265                 270                 275 

acc agg aca ggg gtg ttg gaa gtg ttg gcc cag atg ggg gcg gac att     1159 
Thr Arg Thr Gly Val Leu Glu Val Leu Ala Gln Met Gly Ala Asp Ile 
280                 285                 290                 295 

acc ccg gag aat gaa cga ttg gta acg ggg gaa ccg gta gca gat ctg     1207 
Thr Pro Glu Asn Glu Arg Leu Val Thr Gly Glu Pro Val Ala Asp Leu 
                300                 305                 310 

cgg gtt agg gca agc cat ctc cag ggt tgc acc ttc ggc ggc gaa att     1255 
Arg Val Arg Ala Ser His Leu Gln Gly Cys Thr Phe Gly Gly Glu Ile 
            315                 320                 325 

att ccc cga ctg att gat gaa att ccc att ttg gca gtg gcg gcg gcc     1303 
Ile Pro Arg Leu Ile Asp Glu Ile Pro Ile Leu Ala Val Ala Ala Ala 
        330                 335                 340 

ttt gca gag ggc act acc cgc att gaa gat gcc gca gaa ctg agg gtt     1351 
Phe Ala Glu Gly Thr Thr Arg Ile Glu Asp Ala Ala Glu Leu Arg Val 
    345                 350                 355 

aaa gaa agc gat cgc ctg gcg gcc att gct tcg gag ttg ggc aaa atg     1399 
Lys Glu Ser Asp Arg Leu Ala Ala Ile Ala Ser Glu Leu Gly Lys Met 
360                 365                 370                 375 

ggg gcc aaa gtc acc gaa ttt gat gat ggc ctg gaa att caa ggg gga     1447 
Gly Ala Lys Val Thr Glu Phe Asp Asp Gly Leu Glu Ile Gln Gly Gly 
                380                 385                 390 

agc ccg tta caa ggg gcc gag gtg gat agc ttg acg gat cat cgc att     1495 
Ser Pro Leu Gln Gly Ala Glu Val Asp Ser Leu Thr Asp His Arg Ile 
            395                 400                 405 

gcc atg gcg ttg gcg atc gcc gct tta ggt agt ggg ggg caa aca att     1543 
Ala Met Ala Leu Ala Ile Ala Ala Leu Gly Ser Gly Gly Gln Thr Ile 
        410                 415                 420 

att aac cgg gcg gaa gcg gcc gcc att tcc tat cca gaa ttt ttt ggc     1591 
Ile Asn Arg Ala Glu Ala Ala Ala Ile Ser Tyr Pro Glu Phe Phe Gly 
    425                 430                 435 

acg cta ggg caa gtt gcc caa gga taa agttagaaaa actcctgggc           1638 
Thr Leu Gly Gln Val Ala Gln Gly 
440                 445 

ggtttgtaaa tgttttacca aggtagtttg gggtaaaggc cccagcaagt gctgccaggg   1698 

taatttatcc gcaattgacc aatcggcatg gaccgtatcg ttcaaactgg gtaattctcc   1758 

ctttaattcc ttaaaagctc gcttaaaact gcccaacgta tctccgtaat ggcgagtgag   1818 

tagaagtaat ggggccaaac ggcgatcgcc acgggaaatt aaagcctgca tcactgacca   1878 

cttataactt tcggga                                                   1894 

 
           
             67  
             447  
             PRT  
             Synechocystis sp.  
           
            67 

Met Ala Leu Leu Ser Leu Asn Asn His Gln Ser His Gln Arg Leu Thr 
1               5                   10                  15 

Val Asn Pro Pro Ala Gln Gly Val Ala Leu Thr Gly Arg Leu Arg Val 
            20                  25                  30 

Pro Gly Asp Lys Ser Ile Ser His Arg Ala Leu Met Leu Gly Ala Ile 
        35                  40                  45 

Ala Thr Gly Glu Thr Ile Ile Glu Gly Leu Leu Leu Gly Glu Asp Pro 
    50                  55                  60 

Arg Ser Thr Ala His Cys Phe Arg Ala Met Gly Ala Glu Ile Ser Glu 
65                  70                  75                  80 

Leu Asn Ser Glu Lys Ile Ile Val Gln Gly Arg Gly Leu Gly Gln Leu 
                85                  90                  95 

Gln Glu Pro Ser Thr Val Leu Asp Ala Gly Asn Ser Gly Thr Thr Met 
            100                 105                 110 

Arg Leu Met Leu Gly Leu Leu Ala Gly Gln Lys Asp Cys Leu Phe Thr 
        115                 120                 125 

Val Thr Gly Asp Asp Ser Leu Arg His Arg Pro Met Ser Arg Val Ile 
    130                 135                 140 

Gln Pro Leu Gln Gln Met Gly Ala Lys Ile Trp Ala Arg Ser Asn Gly 
145                 150                 155                 160 

Lys Phe Ala Pro Leu Ala Val Gln Gly Ser Gln Leu Lys Pro Ile His 
                165                 170                 175 

Tyr His Ser Pro Ile Ala Ser Ala Gln Val Lys Ser Cys Leu Leu Leu 
            180                 185                 190 

Ala Gly Leu Thr Thr Glu Gly Asp Thr Thr Val Thr Glu Pro Ala Leu 
        195                 200                 205 

Ser Arg Asp His Ser Glu Arg Met Leu Gln Ala Phe Gly Ala Lys Leu 
    210                 215                 220 

Thr Ile Asp Pro Val Thr His Ser Val Thr Val His Gly Pro Ala His 
225                 230                 235                 240 

Leu Thr Gly Gln Arg Val Val Val Pro Gly Asp Ile Ser Ser Ala Ala 
                245                 250                 255 

Phe Trp Leu Val Ala Ala Ser Ile Leu Pro Gly Ser Glu Leu Leu Val 
            260                 265                 270 

Glu Asn Val Gly Ile Asn Pro Thr Arg Thr Gly Val Leu Glu Val Leu 
        275                 280                 285 

Ala Gln Met Gly Ala Asp Ile Thr Pro Glu Asn Glu Arg Leu Val Thr 
    290                 295                 300 

Gly Glu Pro Val Ala Asp Leu Arg Val Arg Ala Ser His Leu Gln Gly 
305                 310                 315                 320 

Cys Thr Phe Gly Gly Glu Ile Ile Pro Arg Leu Ile Asp Glu Ile Pro 
                325                 330                 335 

Ile Leu Ala Val Ala Ala Ala Phe Ala Glu Gly Thr Thr Arg Ile Glu 
            340                 345                 350 

Asp Ala Ala Glu Leu Arg Val Lys Glu Ser Asp Arg Leu Ala Ala Ile 
        355                 360                 365 

Ala Ser Glu Leu Gly Lys Met Gly Ala Lys Val Thr Glu Phe Asp Asp 
    370                 375                 380 

Gly Leu Glu Ile Gln Gly Gly Ser Pro Leu Gln Gly Ala Glu Val Asp 
385                 390                 395                 400 

Ser Leu Thr Asp His Arg Ile Ala Met Ala Leu Ala Ile Ala Ala Leu 
                405                 410                 415 

Gly Ser Gly Gly Gln Thr Ile Ile Asn Arg Ala Glu Ala Ala Ala Ile 
            420                 425                 430 

Ser Tyr Pro Glu Phe Phe Gly Thr Leu Gly Gln Val Ala Gln Gly 
        435                 440                 445 

 
           
             68  
             1479  
             DNA  
             Dichelobacter nodosus  
             
               CDS  
               (107)..(1438)  
             
           
            68 

tttaaaaaca atgagttaaa aaattatttt tctggcacac gcgctttttt tgcatttttt     60 

ctcccatttt tccggcacaa taacgttggt tttataaaag gaaatg atg atg acg       115 
                                                   Met Met Thr 
                                                   1 

aat ata tgg cac acc gcg ccc gtc tct gcg ctt tcc ggc gaa ata acg      163 
Asn Ile Trp His Thr Ala Pro Val Ser Ala Leu Ser Gly Glu Ile Thr 
    5                   10                  15 

ata tgc ggc gat aaa tca atg tcg cat cgc gcc tta tta tta gca gcg      211 
Ile Cys Gly Asp Lys Ser Met Ser His Arg Ala Leu Leu Leu Ala Ala 
20                  25                  30                  35 

tta gca gaa gga caa acg gaa atc cgc ggc ttt tta gcg tgc gcg gat      259 
Leu Ala Glu Gly Gln Thr Glu Ile Arg Gly Phe Leu Ala Cys Ala Asp 
                40                  45                  50 

tgt ttg gcg acg cgg caa gca ttg cgc gca tta ggc gtt gat att caa      307 
Cys Leu Ala Thr Arg Gln Ala Leu Arg Ala Leu Gly Val Asp Ile Gln 
            55                  60                  65 

aga gaa aaa gaa ata gtg acg att cgc ggt gtg gga ttt ctg ggt ttg      355 
Arg Glu Lys Glu Ile Val Thr Ile Arg Gly Val Gly Phe Leu Gly Leu 
        70                  75                  80 

cag ccg ccg aaa gca ccg tta aat atg caa aac agt ggc act agc atg      403 
Gln Pro Pro Lys Ala Pro Leu Asn Met Gln Asn Ser Gly Thr Ser Met 
    85                  90                  95 

cgt tta ttg gca gga att ttg gca gcg cag cgc ttt gag agc gtg tta      451 
Arg Leu Leu Ala Gly Ile Leu Ala Ala Gln Arg Phe Glu Ser Val Leu 
100                 105                 110                 115 

tgc ggc gat gaa tca tta gaa aaa cgt ccg atg cag cgc att att acg      499 
Cys Gly Asp Glu Ser Leu Glu Lys Arg Pro Met Gln Arg Ile Ile Thr 
                120                 125                 130 

ccg ctt gtg caa atg ggg gca aaa att gtc agt cac agc aat ttt acg      547 
Pro Leu Val Gln Met Gly Ala Lys Ile Val Ser His Ser Asn Phe Thr 
            135                 140                 145 

gcg ccg tta cat att tca gga cgc ccg ctg acc ggc att gat tac gcg      595 
Ala Pro Leu His Ile Ser Gly Arg Pro Leu Thr Gly Ile Asp Tyr Ala 
        150                 155                 160 

tta ccg ctt ccc agc gcg caa tta aaa agt tgc ctt att ttg gca gga      643 
Leu Pro Leu Pro Ser Ala Gln Leu Lys Ser Cys Leu Ile Leu Ala Gly 
    165                 170                 175 

tta ttg gct gac ggt acc acg cgg ctg cat act tgc ggc atc agt cgc      691 
Leu Leu Ala Asp Gly Thr Thr Arg Leu His Thr Cys Gly Ile Ser Arg 
180                 185                 190                 195 

gac cac acg gaa cgc atg ttg ccg ctt ttt ggt ggc gca ctt gag atc      739 
Asp His Thr Glu Arg Met Leu Pro Leu Phe Gly Gly Ala Leu Glu Ile 
                200                 205                 210 

aag aaa gag caa ata atc gtc acc ggt gga caa aaa ttg cac ggt tgc      787 
Lys Lys Glu Gln Ile Ile Val Thr Gly Gly Gln Lys Leu His Gly Cys 
            215                 220                 225 

gtg ctt gat att gtc ggc gat ttg tcg gcg gcg gcg ttt ttt atg gtt      835 
Val Leu Asp Ile Val Gly Asp Leu Ser Ala Ala Ala Phe Phe Met Val 
        230                 235                 240 

gcg gct ttg att gcg ccg cgc gcg gaa gtc gtt att cgt aat gtc ggc      883 
Ala Ala Leu Ile Ala Pro Arg Ala Glu Val Val Ile Arg Asn Val Gly 
    245                 250                 255 

att aat ccg acg cgg gcg gca atc att act ttg ttg caa aaa atg ggc      931 
Ile Asn Pro Thr Arg Ala Ala Ile Ile Thr Leu Leu Gln Lys Met Gly 
260                 265                 270                 275 

gga cgg att gaa ttg cat cat cag cgc ttt tgg ggc gcc gaa ccg gtg      979 
Gly Arg Ile Glu Leu His His Gln Arg Phe Trp Gly Ala Glu Pro Val 
                280                 285                 290 

gca gat att gtt gtt tat cat tca aaa ttg cgc ggc att acg gtg gcg     1027 
Ala Asp Ile Val Val Tyr His Ser Lys Leu Arg Gly Ile Thr Val Ala 
            295                 300                 305 

ccg gaa tgg att gcc aac gcg att gat gaa ttg ccg att ttt ttt att     1075 
Pro Glu Trp Ile Ala Asn Ala Ile Asp Glu Leu Pro Ile Phe Phe Ile 
        310                 315                 320 

gcg gca gct tgc gcg gaa ggg acg act ttt gtg ggc aat ttg tca gaa     1123 
Ala Ala Ala Cys Ala Glu Gly Thr Thr Phe Val Gly Asn Leu Ser Glu 
    325                 330                 335 

ttg cgt gtg aaa gaa tcg gat cgt tta gcg gcg atg gcg caa aat tta     1171 
Leu Arg Val Lys Glu Ser Asp Arg Leu Ala Ala Met Ala Gln Asn Leu 
340                 345                 350                 355 

caa act ttg ggc gtg gcg tgc gac gtt ggc gcc gat ttt att cat ata     1219 
Gln Thr Leu Gly Val Ala Cys Asp Val Gly Ala Asp Phe Ile His Ile 
                360                 365                 370 

tat gga aga agc gat cgg caa ttt tta ccg gcg cgg gtg aac agt ttt     1267 
Tyr Gly Arg Ser Asp Arg Gln Phe Leu Pro Ala Arg Val Asn Ser Phe 
            375                 380                 385 

ggc gat cat cgg att gcg atg agt ttg gcg gtg gca ggt gtg cgc gcg     1315 
Gly Asp His Arg Ile Ala Met Ser Leu Ala Val Ala Gly Val Arg Ala 
        390                 395                 400 

gca ggt gaa tta ttg att gat gac ggc gcg gtg gcg gcg gtt tct atg     1363 
Ala Gly Glu Leu Leu Ile Asp Asp Gly Ala Val Ala Ala Val Ser Met 
    405                 410                 415 

ccg caa ttt cgc gat ttt gcc gcc gca att ggt atg aat gta gga gaa     1411 
Pro Gln Phe Arg Asp Phe Ala Ala Ala Ile Gly Met Asn Val Gly Glu 
420                 425                 430                 435 

aaa gat gcg aaa aat tgt cac gat tga tggtcctagc ggtgttggaa           1458 
Lys Asp Ala Lys Asn Cys His Asp 
                440 

aaggcacggt ggcgcaagct t                                             1479 

 
           
             69  
             443  
             PRT  
             Dichelobacter nodosus  
           
            69 

Met Met Thr Asn Ile Trp His Thr Ala Pro Val Ser Ala Leu Ser Gly 
1               5                   10                  15 

Glu Ile Thr Ile Cys Gly Asp Lys Ser Met Ser His Arg Ala Leu Leu 
            20                  25                  30 

Leu Ala Ala Leu Ala Glu Gly Gln Thr Glu Ile Arg Gly Phe Leu Ala 
        35                  40                  45 

Cys Ala Asp Cys Leu Ala Thr Arg Gln Ala Leu Arg Ala Leu Gly Val 
    50                  55                  60 

Asp Ile Gln Arg Glu Lys Glu Ile Val Thr Ile Arg Gly Val Gly Phe 
65                  70                  75                  80 

Leu Gly Leu Gln Pro Pro Lys Ala Pro Leu Asn Met Gln Asn Ser Gly 
                85                  90                  95 

Thr Ser Met Arg Leu Leu Ala Gly Ile Leu Ala Ala Gln Arg Phe Glu 
            100                 105                 110 

Ser Val Leu Cys Gly Asp Glu Ser Leu Glu Lys Arg Pro Met Gln Arg 
        115                 120                 125 

Ile Ile Thr Pro Leu Val Gln Met Gly Ala Lys Ile Val Ser His Ser 
    130                 135                 140 

Asn Phe Thr Ala Pro Leu His Ile Ser Gly Arg Pro Leu Thr Gly Ile 
145                 150                 155                 160 

Asp Tyr Ala Leu Pro Leu Pro Ser Ala Gln Leu Lys Ser Cys Leu Ile 
                165                 170                 175 

Leu Ala Gly Leu Leu Ala Asp Gly Thr Thr Arg Leu His Thr Cys Gly 
            180                 185                 190 

Ile Ser Arg Asp His Thr Glu Arg Met Leu Pro Leu Phe Gly Gly Ala 
        195                 200                 205 

Leu Glu Ile Lys Lys Glu Gln Ile Ile Val Thr Gly Gly Gln Lys Leu 
    210                 215                 220 

His Gly Cys Val Leu Asp Ile Val Gly Asp Leu Ser Ala Ala Ala Phe 
225                 230                 235                 240 

Phe Met Val Ala Ala Leu Ile Ala Pro Arg Ala Glu Val Val Ile Arg 
                245                 250                 255 

Asn Val Gly Ile Asn Pro Thr Arg Ala Ala Ile Ile Thr Leu Leu Gln 
            260                 265                 270 

Lys Met Gly Gly Arg Ile Glu Leu His His Gln Arg Phe Trp Gly Ala 
        275                 280                 285 

Glu Pro Val Ala Asp Ile Val Val Tyr His Ser Lys Leu Arg Gly Ile 
    290                 295                 300 

Thr Val Ala Pro Glu Trp Ile Ala Asn Ala Ile Asp Glu Leu Pro Ile 
305                 310                 315                 320 

Phe Phe Ile Ala Ala Ala Cys Ala Glu Gly Thr Thr Phe Val Gly Asn 
                325                 330                 335 

Leu Ser Glu Leu Arg Val Lys Glu Ser Asp Arg Leu Ala Ala Met Ala 
            340                 345                 350 

Gln Asn Leu Gln Thr Leu Gly Val Ala Cys Asp Val Gly Ala Asp Phe 
        355                 360                 365 

Ile His Ile Tyr Gly Arg Ser Asp Arg Gln Phe Leu Pro Ala Arg Val 
    370                 375                 380 

Asn Ser Phe Gly Asp His Arg Ile Ala Met Ser Leu Ala Val Ala Gly 
385                 390                 395                 400 

Val Arg Ala Ala Gly Glu Leu Leu Ile Asp Asp Gly Ala Val Ala Ala 
                405                 410                 415 

Val Ser Met Pro Gln Phe Arg Asp Phe Ala Ala Ala Ile Gly Met Asn 
            420                 425                 430 

Val Gly Glu Lys Asp Ala Lys Asn Cys His Asp 
        435                 440 

 
           
             70  
             455  
             PRT  
             Artificial sequence  
             
               Synthetic  
             
           
            70 

Met Leu His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser 
1               5                   10                  15 

Gly Leu Ser Gly Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 
            20                  25                  30 

Arg Ser Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 
        35                  40                  45 

Gly Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 
    50                  55                  60 

Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp 
65                  70                  75                  80 

Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe 
                85                  90                  95 

Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly Val 
            100                 105                 110 

Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu Thr Lys Arg 
        115                 120                 125 

Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val 
    130                 135                 140 

Lys Ser Glu Asp Gly Asp Arg Leu Pro Val Thr Leu Arg Gly Pro Lys 
145                 150                 155                 160 

Thr Pro Thr Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 
                165                 170                 175 

Lys Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 
            180                 185                 190 

Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 
        195                 200                 205 

Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg 
    210                 215                 220 

Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly Gln Val Ile Asp 
225                 230                 235                 240 

Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu 
                245                 250                 255 

Leu Val Pro Gly Ser Asp Val Thr Ile Leu Asn Val Leu Met Asn Pro 
            260                 265                 270 

Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 
        275                 280                 285 

Glu Val Ile Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 
    290                 295                 300 

Arg Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg 
305                 310                 315                 320 

Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala 
                325                 330                 335 

Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu Leu Arg Val 
            340                 345                 350 

Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly Leu Lys Leu Asn 
        355                 360                 365 

Gly Val Asp Cys Asp Glu Gly Glu Thr Ser Leu Val Val Arg Gly Arg 
    370                 375                 380 

Pro Asp Gly Lys Gly Leu Gly Asn Ala Ser Gly Ala Ala Val Ala Thr 
385                 390                 395                 400 

His Leu Asp His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 
                405                 410                 415 

Ser Glu Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 
            420                 425                 430 

Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 
        435                 440                 445 

Leu Ser Asp Thr Lys Ala Ala 
    450                 455