AU2007205733B2 - Novel glyphosate N-acetyltransferase (GAT) genes - Google Patents

Description

AUG 2007

AUSTRALIA

Patents Act 1990

CIA

SPECIFICATION

Name of Applicants: Actual Inventors: Address for Service: Invention Title: Pioneer Hi-Bred International, Inc. and E.I. Du Pont Nemours and Company and Verdia, Inc Linda A. Castle, Dan Siehl, Lorraine J. Giver, Jeremy Minshull, Christina Ivy, Yong Hong Chen, Nicholas B. Duck Baldwins Intellectual Property 16 Chisholm Street North Ryde Sydney Novel glyphosate N-acetyltransferase (GAT) genes The following statement is a full description of this invention, including the best method of performing it known to us:- 100820172 1.DOC:JC:kd NOVEL GLYPHOSATE N-ACETYLTRANSFERASE (GAT) GENES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit ofU. S. Provisional Patent Application Serial No.

60/244,385 filed October 30,2000, the disclosure of which is incorporated herein by reference in its entirety for all purposes. For clarity and completeness, the description of the invention of AU 2002220181, from which the present application was divided, is retained herein.

COPYRIGHT NOTIFICATION PURSUANT TO 37 C.F.R. 1.71 (E) A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Crop selectivity to specific herbicides can be conferred by engineering genes into crops which encode appropriate herbicide metabolizing enzymes. In some cases these enzymes, and the nucleic acids that encode them, originate in a plant. In other cases, they are derived from other organisms, such as microbes. See, e. Padgette et al. (1996) "New weed control opportunities: Development of soybeans with a Round UP Ready M gene" in Herbicide-Resistant Crops (Duke, pp54-84, CRC Press, Boca Raton; and Vasil (1996) "Phosphinothricin-resistant crops" in Herbicide-Resistant Crops (Duke, pp85-91. Indeed, transgenic plants have been engineered to express a variety of herbicide tolerance/metabolizing genes, from a variety of organisms. For example, acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, Hattori et al. (1995) Mol Gen Genet 246:419. Other genes that confer tolerance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPHcytochrome P450 oxidoreductase (Shiota et al. (1994) Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, and genes for various phosphotransferases (Datta et al. (1992) Plant Mol Biol 20:619.

-la- (followed by page 2) WO 02/36782 PCT/US01/46227 One herbicide which is the subject of much investigation in this regard is N-phosphonomethylglycine, commonly referred to as glyphosate. Glyphosate is the top selling herbicide in the world, with sales projected to reach $5 billion by 2003. It is a broad spectrum herbicide that kills both broadleaf and grass-type plants. A successful mode of commercial level glyphosate resistance in transgenic plants is by introduction of a modified Agrobacterium CP4 5-enolpyruvylshikimate-3-phosphate synthase (hereinafter referred to as EPSP synthase or EPSPS) gene. The transgene is targeted to the chloroplast where it is capable of continuing to synthesize EPSP from phosphoenolpyruvic acid (PEP) and shildmate-3-phosphate in the presence of glyphosate. In contrast, the native EPSP synthase is inhibited by glyphosate. Without the transgene, plants sprayed with glyphosate quickly die due to inhibition of EPSP synthase which halts the downstream pathway needed for aromatic amino acid, hormone, and vitamin biosynthesis. The CP4 glyphosate-resistant soybean transgenic plants are marketed, by Monsanto under the name "Round UP Ready m In the environment, the predominant mechanism by which glyphosate is degraded is through soil microflora metabolism. The primary metabolite of glyphosate in soil has been identified as aminomethylphosphonic acid (AMPA), which is ultimately converted into ammonia, phosphate and carbon dioxide. The proposed metabolic scheme that describes the degradation of glyphosate in soil through the AMPA pathway is shown in Fig. 8. An alternative metabolic pathway for the breakdown of glyphosate by certain soil bacteria, the sarcosine pathway, occurs via initial cleavage of the C-P bond to give inorganic phosphate and sarcosine, as depicted in Fig. 9.

Another successful herbicide/transgenic crop package is glufosinate (phosphinothricin) and the LibertyLink T M trait marketed, by Aventis. Glufosinate is also a broad spectrum herbicide. Its target is the glutamate synthase enzyme of the chloroplast. Resistant plants carry the bar gene from Streptomyces hygroscopicus and achieve resistance by the N-acetylation activity of bar, which modifies and detoxifies glufosinate.

An enzyme capable of acetylating the primary amine of AMPA is reported in PCT Application No. WO00/29596. The enzyme was not described as being able to acetylate a compound with a secondary amine glyphosate).

While a variety of herbicide resistance strategies are available as noted above, aditional approaches would have considerable commercial value. The present invention provides, novel polynucleotides and polypeptides for conferring herbicide tolerance, as well as numerous other benefits as will become apparent during review of the disclosure.

SUMMARY OF THE INVENTION It is an object of the present invention to provide methods and reagents for rendering an organism, such as a plant, resistant to glyphosate, or to at least provide a useful alternative to known methods and reagents for rendering an organism, such as a plant, resistant to glyphosate. This and other objects of the invention are provided by one or more of the embodiments described below.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising' and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say in the sense of "including but not limited to".

One embodiment of the invention provides novel polypeptides referred to herein as GAT polypeptides. GAT polypeptides are characterized by their structural similarity to one another, in terms of sequence similarity when the GAT polypeptides are aligned with one another. Some GAT polypeptides possess glyphosate N-acetyl transferase activity, the ability to catalyze the acetylation of glyphosate. Some GAT polypeptides are also capable of catalyzing the acetylation of glyphosate analogs and or glyphosate metabolites, aminomethylphosphonic acid.

Also provided are novel polynucleotides referred to herein as GAT polynucleotides. GAT polynucleotides are characterized by their ability to encode GAT polypeptides. In some embodiments of the invention, a GAT polynucleotide is engineered for better plant expression by replacing one or more parental codons with a synonymous codon that is preferentially used in plants relative to the parental codon. In other embodiments, a GAT polynucleotide is modified by the introduction of a nucleotide sequence encoding an N-terminal chloroplast transit peptide.

-3- 00 GAT polypeptides, GAT polynucleotides and glyphosate N-acetyl transferase

O

activity are described in more detail below. The invention further includes certain fragments of the GAT polypeptides and GAT polynucleotides described herein.

0 i The invention includes non-native variants of the polypeptides and polynucleotides described herein, wherein one or more amino acids of the encoded polypeptide have been mutated.

More specifically, the invention provides an isolated or recombinant polynucleotide, such as herein described, comprising a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO: 1, wherein said polynucleotide encodes a polypeptide having glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formamide with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at °C for 15 minutes.

Preferably, the encoded polypeptide catalyzes the acetylation of glyphosate with a kcat/Km of at least 10 mM-~min' for glyphosate. The encoded polypeptide may also catalyze the acetylation of aminomethylphosphonic acid.

The polynucleotide may further comprise a nucleotide sequence encoding an Nterminal chloroplast transit peptide.

Another embodiment of the invention provides an isolated or recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide having glyphosate N-acetyltransferase activity, the polypeptide comprising an amino acid sequence comprising at least 20 contiguous amino acids of SEQ ID NO:6.

Preferably, the polypeptide comprises an amino acid sequence comprising at least contiguous amino acids of SEQ ID NO:6. More preferably, the polypeptide comprises an amino acid sequence comprising at least 100 contiguous amino acids of SEQ ID NO:6. Still more preferably, the polypeptide comprises an amino acid sequence comprising at least 125 contiguous amino acids of SEQ ID NO:6.

The present invention also provides a nucleic acid construct comprising a polynucleotide such as herein described, comprising a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO: 1, wherein said polynucleotide encodes a polypeptide having glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formalin with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 °C for 15 minutes.

t The nucleic acid construct may also include constructs wherein the polynucleotide is operably linked to a promoter, where the promoter is heterologous with respect to the polynucleotide and effective to cause sufficient expression of the encoded polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the nucleic acid construct.

The polynucleotide, such as herein described, comprising a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO:1, wherein said polynucleotide encodes a polypeptide having glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formalin with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 °C for 15 minutes may function as a selectable marker in the nucleic acid construct of the present invention.

The nucleic acid construct may be a vector. The vector may comprise a second polynucleotide sequence encoding a second polypeptide that confers a detectable phenotypic trait upon a cell or organism expressing the second polypeptide at an effective level. The detectable phenotypic trait may function as a selectable marker. The detectable phenotypic trait may be herbicide resistance, pest resistance or a visible marker. The vector may comprise a T-DNA sequence. The vector of the present invention may be a plant transformation vector.

The present invention also comprises a composition comprising a polynucleotide, such as herein described, comprising a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO: 1, wherein said polynucleotide encodes a polypeptide having glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formalin with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 °C for 15 minutes.

Another embodiment of the present invention provides a cell comprising at least one polynucleotide, such as herein described, comprising a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO:1, wherein said polynucleotide encodes a polypeptide having glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formalin with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 °C for 15 minutes, wherein the polynucleotide is heterologous to the cell. Further, the polynucleotide may be operably linked to a regulatory sequence.

The invention also provides cells transduced by the vector of the present invention.

The cell of the present invention may be a transgenic plant cell.

Another aspect of the present invention provides an isolated or recombinant polypeptide, such as herein described, comprising an amino acid sequence encoded by a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO: 1, wherein said polypeptide has glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formalin with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 oC for minutes.

The isolated or recombinant polypeptide preferably catalyzes the acetylation of glyphosate with a kcat/Km of at least 10 mM-'min-' for glyphosate. More preferably, the isolated or recombinant polypeptide preferably catalyzes the acetylation of glyphosate with a kcat/Km of at least 100 mM-'min for glyphosate. The isolated or recombinant polypeptide may also catalyze the acetylation of aminomethylphosphonic acid.

The present invention also provides an isolated or recombinant polypeptide, such as described herein, having glyphosate N-acetyltransferase activity, the polypeptide comprising an amino acid sequence comprising at least 20 contiguous amino acids of SEQ ID NO:6.

Preferably, the isolated or recombinant polypeptide comprises an amino acid sequence comprising at least 50 contiguous amino acids of SEQ ID NO:6. More preferably, the isolated or recombinant polypeptide comprises an amino acid sequence comprising at least 100 contiguous amino acids of SEQ ID NO:6. Still more preferably, the isolated or *1 recombinant polypeptide comprises an amino acid sequence comprising at least 125 contiguous amino acids of SEQ ID NO:6.

The isolated or recombinant polypeptide may further comprise an N-terminal chloroplast transit peptide. The polypeptide may also comprise a secretion sequence or a localization sequence. The localization sequence may be chloroplast transit sequence.

The invention also provides a cell comprising at least one recombinant polynucleotide, such as herein described, wherein said polynucleotide comprises: a nucleotide sequence encoding an amino acid sequence that can be optimally aligned with a sequence selected from the group consisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457 to generate a similarity score of at least 720, using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1; or a complementary nucleotide sequence thereof.

The cell may comprise a polynucleotide that is heterologous to the cell. The cell may comprise a polynucleotide that is operably linked to a regulatory sequence. The cell may be a plant cell.

Another embodiment of the present invention provides a cell comprising a recombinant polypeptide, such as herein described, wherein said comprising an amino acid sequence that can be optimally aligned with a sequence selected from the group consisting of SEQ ID NO:300, SEQ ID NO:445 and SEQ ID NO:457 to generate a similarity score of at least 720, using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1, wherein the polypeptide has glyphosate N-acetyl transferase activity.

The cell may comprise a polynucleotide that is heterologous to the cell. The cell may be a plant cell.

-7- BRIEF DESCRIPTION OF THE FIGURES Figure I depicts the N-acetylation of glyphosate catalyzed by a glyphosate Nacetyltransferase (followed by page 9) -8- WO 02/36782 PCT/US01/46227 Figure 2 illustrates mass spectroscopic detection of N-acetylglyphosate produced by an exemplary Bacillus culture expressing a native GAT activity.

Figure 3 is a table illustrating the relative identity between GAT sequences isolated from different strains of bacteria and yitl from Bacillus subtilis.

Figure 4 is a map of the plasmid pMAXY2120 for expression and purification of the GAT enzyme from E. coli cultures.

Figure 5 is a mass spectrometry output showing increased Nacetylglyphosate production over time in a typical GAT enzyme.reaction mix.

Figure 6 is a plot of the kinetic data of a GAT enzyme from which a KM of 2.9 mM for glyphosate was calculated.

Figure 7 is a plot of the kinetic data taken from the data of Figure 6 from which a KM of 2 pM was calculated for Acetyl CoA.

Figure 8 is a scheme that describes the degradation of glyphosate in soil through the AMPA pathway.

Figure 9 is a scheme that describes the sarcosine pathway of glyphosate degradation.

Figure 10 is the BLOSUM62 matrix.

Figure 11 is a map of the plasmid pMAXY2190.

Figure 12 depicts a T-DNA construct with gat selectable marker.

Figure 13 depicts a yeast expression vector with gat selectable marker.

DETAILED DISCUSSION The present invention relates to a novel class of enzymes exhibiting Nacetyltransferase activity. In one aspect, the invention relates to a novel class of enzymes capable of acetylating glyphosate and glyphosate analogs, enzymes possessing glyphosate N-acetyltransferase activity. Such enzymes are characterized by the ability to acetylate the secondary amine of a compound. In some aspects of the invention, the compound is a herbicide, glyphosate, as illustrated schematically in Figure 1. The compound can also be a glyphosate analog or a metabolic product of glyphosate degradation, e.g, aminomethylphosphonic acid. Although the acetylation of glyphosate is a key catalytic step in one metabolic pathway for catabolism of glyphosate, the enzymatic acetylation of glyphosate by naturally-occurring, isolated, or recombinant enzymes has not been previously described. Thus, the nucleic acids and polypeptides of the invention provide a new biochemical pathway for engineering herbicide resistance.

-9- WO 02/36782 PCT/US01/46227 In one aspect, the invention provides novel genes encoding GAT polypeptides. Isolated and recombinant GAT polynucleotides corresponding to naturally occurring polynucleotides, as well as recombinant and engineered, diversified, GAT polynucleotides are a feature of the invention. GAT polynucleotides are exemplified by SEQ ID NOS: 1-5 and 11-262. Specific GAT polynucleotide and polypeptide sequences are provided as examples to help illustrate the invention, and are not intended to limit the scope of the genus of GAT polynucleotides and polypeptides described and/or claimed herein.

The invention also provides methods for generating and selecting diversified libraries to produce additional GAT polynucleotides, including polynucleotides encoding GAT polypeptides with improved and/or enhanced characteristics, altered Km for glyphosate, increased rate of catalysis, increased stability, etc., based upon selection of a polynucleotide constituent of the library for the new or improved activities described herein. Such polynucleotides are especially favorably employed in the production of glyphosate resistant transgenic plants.

The GAT polypeptides of the invention exhibit a novel enzymatic activity.

Specifically, the enzymatic acetylation of the synthetic herbicide glyphosate has not been recognized prior to the present invention. Thus, the polypeptides herein described, as exemplified by SEQ ID NOS: 6-10 and 263-514, define a novel biochemical pathway for the detoxification of glyphosate that is functional in vivo, in plants.

Accordingly, the nucleic acids and polypeptides of the invention are of significant utility in the generation of glyphosate resistant plants by providing new nucleic acids, polypeptides and biochemical pathways for the engineering of herbicide selectivity in transgenic plants.

DEFINITIONS

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a device" includes a combination of-two or more such devices, reference to "a gene fusion construct" includes mixtures of constructs, and the like.

WO 02/36782 PCT/US01/46227 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, specific examples of appropriate materials and methods are described herein.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

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. The term "glyphosate analog" refers to any structural analog of glyphostate that has the ability to inhibit EPSPS at levels such that the glyphosate analog is herbicidally effective.

As used herein, the term "glyphosate-N-acetyltransferase activity" or "GAT activity" refers to the ability to catalyze the acetylation of the secondary amine group of glyphosate, as illustrated, for example, in Figure 1. A "glyphosate -N-acetyltransferase" or "GAT' is an enzyme that catalyzes the acetylation of the amine group of glyphosate, a glyphosate analog, and/or a glyphosate primary metabolite AMPA or sarcosine). In some preferred embodiments of the invention, a GAT is able to transfer the acetyl group from AcetylCoA to the secondary amine of glyphosate and the primary amine of AMPA.

The exemplary GATs described herein are active from pH 5-9, with optimal activity in the range of pH 6.5-8.0. Activity can be quantified using various kinetic parameters well know in the art, kr, KM, and k/ KM. These kinetic parameters can be determined as described below in Example 7.

The terms "polynucleotide," "nucleotide sequence," and "nucleic acid" are used to refer to a polymer of nucleotides etc. or naturally occurring or artificial nucleotide analogues), DNA or RNA, or a representation thereof, a character string, etc, depending on the relevant context A given polynucleotide or complementary polynucleotide can be determined from any specified nucleotide sequence.

Similarly, an "amino acid sequence" is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. The terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

-11- WO 02/36782 PCT/US01/46227 A polynucleotide, polypeptide or other component is "isolated" when it is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, synthetic reagents, etc.). A nucleic acid or polypeptide is "recombinant" when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid. For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g, in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a variant of a naturally occurring gene, is recombinant The terms "glyphosate N-acetyl transferase polypeptide" and "GAT polypeptide" are used interchangeably to refer to any of a family of novel polypeptides provided herein.

The terms "glyphosate N-acetyl transferase polynucleotide" and "GAT polynucleotide" are used interchangeably to refer to a polynucleotide that encodes a GAT polypeptide.

A "subsequence" or "fragment" is any portion of an entire sequence.

Numbering of an amino acid or nucleotide polymer corresponds to numbering of a selected amino acid polymer or nucleic acid when the position of a given monomer component (amino acid residue, incorporated nucleotide, etc.) of the polymer corresponds to the same residue position in a selected reference polypeptide or polynucleotide.

A vector is a composition for facilitating cell transduction by a selected nucleic acid, or expression of the nucleic acid in the cell. Vectors include, plasmids, cosmids, viruses, YACs, bacteria, poly-lysine, chromosome integration vectors, episomal vectors, etc.

"Substantially an entire length of a polynucleotide or amino acid sequence" refers to at least about 70%, generally at least about 80%, or typically about 90% or more of a sequence.

As used herein, an "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad -12- WO 02/36782 PCT/US01/46227 immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunologv, 4 f Edition,W.E. Paul Raven Press, N.Y. (1998), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies include single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.

A "chloroplast transit peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. "Chloroplast transit sequence" refers to a nucleotide sequence that encodes a chloroplast transit peptide.

A "signal peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein -13- WO 02/36782 PCT/US01/46227 is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel, N. (1992) Plant Phys. 100:1627-1632).

The terms "diversification" and "diversity," as applied to a polynucleotide, refers to generation of a plurality of modified forms of a parental polynucleotide, or plurality of parental polynucleotides. In the case where the polynucleotide encodes a polypeptide, diversity in the nucleotide sequence of the polynucleotide can result in diversity in the corresponding encoded polypeptide, e.g. a diverse pool of polynucleotides encoding a plurality of polypeptide variants. In some embodiments of the invention, this sequence diversity is exploited by screening/selecting a library of diversified polynucleotides for variants with desirable functional attributes, a polynucleotide encoding a GAT polypeptide with enhanced functional characteristics.

The term "encoding" refers to the ability of a nucleotide sequence to code for one or more amino acids. The term does not require a start or stop codon. An amino acid sequence can be encoded in any one of six different reading frames provided by a polynucleotide sequence and its complement When used herein, the term "artificial variant" refers to a polypeptide having GAT activity, which is encoded by a modified GAT polynucleotide, a modified form of any one of SEQ ID NOS: 1-5 and 11-262, or of a naturally-occurring GAT polynucleotide isolated from an organism. The modified polynucleotide, from which an artificial variant is produced when expressed in a suitable host, is obtained through human intervention by modification of a GAT polynucleotide.

The term "nucleic acid construct" or "polynucleotide construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and -14- WO 02/36782 PCT/US01/46227 transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.

The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.

When used herein the term "coding sequence" is intended to cover a nucleotide sequence, which directly specifies the amino acid sequence of its protein product The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon. The coding sequence typically includes a DNA, cDNA, and/or recombinant nucleotide sequence.

In the present context, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, posttranscriptional modification, translation, post-translational modification, and secretion.

In the present context, the term "expression vector" covers a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of the invention, and which is operably linked to additional segments that provide for its transcription.

The term "host cell", as used herein, includes any cell type which is susceptible to transformation with a nucleic acid construct The term "plant" includes whole plants, shoot vegetative organs/structures leaves, stems and tubers), roots, flowers and floral organs/structures bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue vascular tissue, ground tissue, and the like) and cells guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

The term "heterologous" as used herein describes a relationship between two or more elements which indicates that the elemennts are not normally found in WO 02/36782 PCT/US01/46227 proximity to one another in nature. Thus, for example, a polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter a genetically engineered coding sequence or an allele from a different ecotype or variety). An example of a heterologous polypeptide is a polypeptide expressed from a recombinant polynucleotide in a transgenic organism. Heterologous polynucleotides and polypeptides are forms of recombinant molecules.

A variety of additional terms are defined or otherwise characterized herein.

GLYPHOSATE N-ACETYLTRANSFERASES In one aspect, the invention provides a novel family of isolated or recombinant enzymes referred to herein as "glyphosate N-acetyltransferases," "GATs or "GAT enzymes." GATs are enzymes that have GAT activity, preferably sufficient activity to confer some degree of glyphosate tolerance upon a transgenic plant engineered to express the GAT. Some examples of GATs include GAT polypeptides, described in more detail below.

Of course, GAT-mediated glyphosate tolerance is a complex function of GAT activity, GAT expression levels in the transgenic plant, the particular plant, the nature and timing of herbicide application, etc. One of skill in the art can determine without undue experimentation the level of GAT activity required to effect glyphosate tolerance in a particular context.

GAT activity can be characterized using the conventional kinetic parameters k1, KM, and kt KM. keat can be thought of as a measure of the rate of acetylation, particularly at high substrate concentrations, KM is a measure of the affinity of the GAT for its substrates Acetyl CoA and glyphosate), and kcat KM is a measure of catalytic efficiency that takes both substrate affinity and catalytic rate into account this parameter is particularly important in the situation where the concentration of a substrate is at least partially rate limiting. In general, a GAT with a higher kc or k.t KM is a more efficient catalyst than another GAT with lower kcat or kat KM. A GAT with a lower KM is a more efficient catalyst than another GAT with a higher KM. Thus, to determine whether one GAT is more effective than another, one can compare kinetic parameters for the two enzymes. The relative importance of kcat, kat KM and KM will vary depending upon the -16- WO 02/36782 PCT/US01/46227 context in which the GAT will be expected to function, the anticipated effective concentration of glyphosate relative to KM for glyphosate. GAT activity can also be characterized in terms of any of a number of functional characteristics, stability, susceptibility to inhibition or activation by other molecules, etc.

GLYPHOSATE N-ACETYLTRANSFERASE POLYPEPTIDES In one aspect, the invention provides a novel family of isolated or recombinant polypeptides referred to herein as "glyphosate N-acetyltransferase polypeptides" or "GAT polypeptides." GAT polypeptides are characterized by their structural similarity to a novel family of GATs. Many but not all GAT polypeptides are GATs. The distinction is that GATs are defined in terms of function, whereas GAT polypeptides are defined in terms of structure. A subset of the GAT polypeptides consists of those GAT polypeptides that have GAT activity, preferably at a level that will function to confer glyphosate resistance upon a transgenic plant expressing the protein at an effective level. Some preferred GAT polypeptides for use in conferring glyphosate tolerance have a of at least 1 min', or more preferably at least 10 min-', 100 min-' or 1000 min"'. Other preferred GAT polypeptides for use in conferring glyphosate tolerance have a KM no greater than 100 mM, or more preferably no greater than 10 mM, 1 mM, or 0.1 mM. Still other preferred GAT polypeptides for use in conferring glyphosate tolerance have a kca KM of at least 1 mM'min or more, preferably at least 10 mM'min 1 100 mM-Cmin 1000 mM-min-', or 10,000 mMl'min'.

Exemplary GAT polypeptides have been isolated and characterized from a variety of bacterial strains. One example of a monomeric GAT polypeptide that has been isolated and characterized has a molecular radius of approximately 17 kD. An exemplary GAT enzyme isolated from a strain of B. licheniformis, SEQ ID NO:7, exhibits a Km for glyphosate of approximately 2.9 mM and a Km for acetyl CoA of approximately 2 pM, with a kcat equal to 6/minute.

The term "GAT polypeptide" refers to any polypeptide comprising an amino acid sequence that can be optimally aligned with an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514 to generate a similarity score of at least 430 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence that can be optimally aligned with an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514 to generate a similarity score of at least 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, -17- WO 02/36782 PCT/US01/46227 490, 495, 500, 505, 510, 515, 520, 525,530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585,590, 595, 600, 605, 610, 615,620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675,680,685,690,695,700,705,710,715,720, 725,730, 735,740,745,750,755, or 760 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence that can be optimally aligned with SEQ ID NO. 457 to generate a similarity score of at least 430 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence that can be optimally aligned with SEQ ID NO. 457 to generate a similarity score of at least 440, 445, 450, 455, 460, 465, 470, 475,480,485,490,495,500,505,510,515, 520, 525, 530, 535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660,665,670,675,680,685,690,695,700, 705, 710, 715,720,725,730,735,740, 745, 750, 755, or 760 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence that can be optimally aligned with SEQ ID NO. 445 to generate a similarity score of at least 430 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence that can be optimally aligned with SEQ ID NO. 445 to generate a similarity score of at least 440, 445, 450, 455, 460, 465, 470, 475,480,485, 490, 495, 500,505, 510,515, 520,525, 530, 535,540,545,550,555,560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655,660,665,670,675,680,685,690,695, 700, 705, 710, 715, 720, 725,730, 735,740, 745, 750, 755, or 760 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence that can be optimally aligned with SEQ ID NO:300 to generate a similarity score of at least 430 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence that can be optimally aligned with SEQ ID NO: 300 to generate a similarity score of at least 440, 445, 450, 455, 460, 465, 470, 475,480, 485,490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, -18- WO 02/36782 PCTUS01/46227 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665,670, 675,680, 685,690, 695,700, 705,710, 715, 720, 725,730, 735, 740, 745, 750, 755, or 760 using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

Two sequences are "optimally aligned" when they are aligned for similarity scoring using a defined amino acid substitution matrix BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acids substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, in Dayhoff et al.

(1978) "A model of evolutionary change in proteins." In "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. M.O. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, DC and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919.

The BLOSUM62 matrix (Fig. 10) is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (http://www.ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments, can be prepared using, PSI-BLAST, available through http://www.ncbi.nlm.nih.gov and described by Altschul et' al, (1997) Nucleic Acids Res.

25:3389-3402.

With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position in the reference sequence with which the residue is paired in the alignment. The "position" is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. For example, in SEQ ID NO:300 position 1 is M, position 2 is 1, position 3 is E, etc. When a test sequence is optimally aligned with SEQ 1ID NO:300, a residue in the test sequence that aligns with the E at position 3 is said to -19- WO 02/36782 PCT/US01/46227 "correspond to position 3" of SEQ ID NO:300. Owing to deletions, insertion, truncations, fusions, etc., that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence as determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

The term "GAT polypeptide" further refers to any polypeptide comprising an amino acid sequence having at least 40% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with SEQ ID NO. 457. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO. 457.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with SEQ ID NO. 445. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO. 445.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with SEQ ID NO. 300. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO. 300.

WO 02/36782 PCT/US01/46227 The term "GAT polypeptide" further refers to any polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 1-96 of an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263- 514. Some aspects of the invention pertain to polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 1-96 of an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514.

One aspect of the invention pertains to a polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 1-96 of SEQ ID NO.

457. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 1-96 of SEQ ID NO. 457.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 1-96 of SEQ ID NO. 445. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 1-96 of SEQ ID NO. 445.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 1-96 of SEQ ID NO. 300. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 1-96 of SEQ ID NO. 300.

The term "GAT polypeptide" further refers to any polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 51-146 of an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263- 514. Some aspects of the invention pertain to polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 51-146 of an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514.

One aspect of the invention pertains to a polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 51-146 of SEQ ID NO.

457. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 51-146 of SEQ ID NO. 457.

-21 WO 02/36782 PCT/US01/46227 One aspect of the invention pertains to a GAT polypepti be'comp i~ amino acid sequence having at least 40% sequence identity with residues 51-146 of SEQ ID NO. 445. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 51-146 of SEQ ID NO. 445.

One aspect of the invention pertains to a GAT polypeptide comprising an amino acid sequence having at least 40% sequence identity with residues 51-146 of SEQ ID NO. 300. Some aspects of the invention pertain to GAT polypeptides comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with residues 51-146 of SEQ ID NO. 300.

As used herein, the term "identity" or "percent identity" when used with respect to a particular pair of aligned amino acid sequences, refers to the percent amino acid sequence identity that is obtained by ClustalW analysis (version W 1.8 available from European Bioinformatics Institute, Cambridge, UK), counting the number of identical matches in the alignment and dividing such number of identical matches by the greater of the length of the aligned sequences, and (ii) 96, and using the following default ClustalW parameters to achieve slow/accurate pairwise alignments Gap Open Gap Extension Penalty:0.10; Protein weight matrix:Gonnet series; DNA weight matrix: IUB; Toggle Slow/Fast pairwise alignments SLOW or FULL Alignment.

In another aspect, the invention provides an isolated or recombinant polypeptide that comprises at least 20, or alternatively, 50, 75, 100, 125 or 140 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514.

In another aspect, the invention provides an isolated or recombinant polypeptide that comprises at least 20, or alternatively, 50, 100 or 140 contiguous amino acids of SEQ ID NO:457.

In another aspect, the invention provides an isolated or recombinant polypeptide that comprises at least 20, or alternatively, 50, 100 or 140 contiguous amino acids of SEQ ID NO:445.

In another aspect, the invention provides an isolated or recombinant polypeptide that comprises at least 20, or alternatively, 50, 100 or 140 contiguous amino acids of SEQ ID NO:300.

22 WO 02/36782 WO 0236782PCTfUSOI/46227 In another aspect, the invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ D NOS: 6-10 and 263- 514.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the group consisting of SEQ IID NO:6-10 and 263-5 14, at least 90% of the amino acid residues in the polypeptide that correspond to the following positions conform to the following restrictions: at positions 2, 4, 15, 19, 26, 28, 31, 45, 51, 54, 86, 90, 91, 97, 103, 105, 106, 114, 123, 129, 139, and/or 145 the amino acid residue is Bi1; and at positions 3, 8, 10, 11, 14, 17, 18, 24, 27, 32, 37, 38, 47, 48, 49, 52, 57, 58, 61, 62, 63, 68, 69, 79, 82, 83, 89, 92, 100, 101, 104, 119, 120, 124, 125, 126, 128, 131, 143, and/or 144 the amino acid residue is B 2; wherein B 1 is an amino acid selected from the group consisting of A, L, L, M, F, W, Y, and V; and B2 is an amino acid selected from the group consisting ofP, N, D, C,Q, E,G, H, K,P, S,and T. When used to specify an amino acid or amino acid residue, the single letter designations A, C, D, E, F, G, H, IL K, L, MK N, P, Q, R, S, T, V, W, and Y have their standard meaning as used in the art and as provided in Table 2 herein.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the group consisting of SEQ ID NO:6-10 and 263-514, at least 80% of the amino acid residues in the polypeptide that correspond to the following positions conform to the following restrictions: at positions 2, 4, 15, 19, 26, 28, 5 1, 54, 86, 90, 91, 97, 103, 105, 106, 114, 129, 139, and/or 145 the amino acid residue is Z1; at positions 31 and/or 45 the amino acid residue is Z2; at positions 8 and/or 89 the amino acid residue is Z3; at positions 82, 92, 101 and/or 120 the amino acid residue is Z4; at positions 3, 11, 27 and/or 79 the amino acid residue is Z.5; at position 123 the amino acid residue is Z1 or Z2; at positions 12, 33, 35, 39, 53, 59, 112, 132, 135, 140, and/or 146 the amino acid residue is Z1 or Z3; at position 30 the amino acid residue is Z1 or Z4; at position 6 the amino acid residue is Z1 or Z6; at positions 81 and/or 113 the amino acid residue is Z2 or Z3; at positions 138 and/or 142 the amino acid residue is Z2 or Z4; at positions 5, 17, 24, 57, 61, 124 and/or 126 the amino acid residue is Z3 or Z4; (in) at position 104 the amino acid residue is Z3 or Z5; at positions 38, 52, 62 and/or 69 the amino acid residue is Z3 or Z6; at positions 14, 119 and/or 144 the amino acid residue is Z4 or 75; at position 18 the amino acid residue is Z4 or Z6; at positions 10, 32, 23 WO 02136782 WO 0236782PCTJUSOI/46227 48, 63, 80 and/or 83 the amino acid residue is Z5 or Z6; at position 40 the amino acid residue is Z1, Z2 or Z3; at positions 65 and/or 96 the amino acid residue is Z1, Z3 or at positions 84 and/or 115 the amino acid residue is Z1, Z3 or Z4; at position 93 the amino acid residue is Z2, Z3 or Z4; at position 130 the amino acid residue is Z2, Z4 or Z6; at positions 47 and/or 58 the amino acid residue is Z3, Z4 or Z6; at positions 49, 68, 100 and/or 143 the amino acid residue is Z3, Z4 or Z5; at position 131 the amino acid residue is Z3, Z5 or Z6; (aa) at positions 125 and/or 128 the amino acid residue is Z4, Z5 or Z6; (ab) at position 67 the amino acid residue is Z1, Z3, Z4 or Z5; (ac) at position 60 the amino acid residue is Zi, Z4, Z5 or Z6; and(ad) at position 37 the amino acid residue is Z3, Z4, Z5 or Z6; wherein Z1 is an amino acid selected from the group consisting of A, L, L, KA and V; Z2 is an amino acid selected from the group consisting of F, W, and Y; Z3 is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is an amino acid selected from the group consisting of D and E; and Z6 is an amino acid selected from the group consisting of C, G3, and P.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the group consisting of SEQ ID NO:6-10 and 263-514, at least 90% of the amino acid residues in the polypeptide that correspond to the following positions conform to the following restrictions: at positions 1, 7, 9, 13, 20, 36, 42, 46, 50, 56, 64, 70, 72, 75, 76, 78, 94, 98, 107, 110, 117, 118, 121, and/or 141 the amino acid residue is Bi1; and at positions 16, 21, 22, 23, 25, 29, 34, 41, 43, 44, 55, 66, 71, 73, 74, 77, 85, 87, 88, 95, 99, 102, 108, 10)9, 111, 116, 122, 127, 133, 134, 136, and/or 137 the amino acid residue is B2; wherein B1 is an amino acid selected from the group consisting of A, L, L, M, F, W, Y, and V; and B2 is an amino acid selected from the group consisting of R, N, D, C, Q, E, G3,H, K, P, S, and T.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the group consisting of SEQ ID) NO:6-10 and 263-5 14, at least 90% of the amino acid residues in the polypeptide that correspond to the following positions conform. to the following restrictions: at positions 1, 7, 9, 20, 36, 42, 50, 64, 72, 75, 76, 78, 94, 98, 110, 121, and/or 141 the amino acid residue is Z1; at positions 13, 46, 56, 70, 107, 117, and/or 118 the amino acid residue is Z2; at positions 23, 55, 71, 77, 88, and/or 109 the amino acid residue is Z3; at positions 16, 21, 41, 73, 85, 99, and/or 111 the amino acid residue is Z4; at positions 34 and/or 95 the amino acid residue is Z5; at position 22, 24 WO 02/36782 PCT/US01/46227 29, 43, 44, 66, 74, 87, 102, 108, 116, 122, 127, 133, 134, 136, and/or 137 the amino acid residue is Z6; wherein Z1 is an amino acid selected from the group consisting of A, I, L, M, and V; Z2 is an amino acid selected from the group consisting of F, W, and Y; Z3 is an amino acid selected from the group consisting of N, Q, S, and T; Z4 is an amino acid selected from the group consisting of R, H, and K; Z5 is an amino acid selected from the group consisting of D and E; and Z6 is an amino acid selected from the group consisting of C, G, and P.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the group consisting of SEQ ID NO:6-10 and 263-514, at least 80% of the amino acid residues in the polypeptide that correspond to the following positions conform to the following restrictions: at position 2 the amino acid residue is I or L; at position 3 the amino acid residue is E or D; at position 4 the amino acid residue is V, A or I; at position the amino acid residue is K, R or N; at position 6 the amino acid residue is P or L; at position 8 the amino acid residue is N, S or T; at position 10 the amino acid residue is E or G; at position 11 the amino acid residue is D or E; at position 12 the amino acid residue is T or A; at position 14 the amino acid residue is E or K; at position the amino acid residue is I or L; at position 17 the amino acid residue is H or Q; at position 18 the amino acid residue is R, C or K; at position 19 the amino acid residue is I or V; at position 24 the amino acid residue is Q or R; at position 26 the amino acid residue is L or I; at position 27 the amino acid residue is E or D; at position 28 the amino acid residue is A or V; at position 30 the amino acid residue is K, M or R; (t) at position 31 the amino acid residue is Y or F; at position 32 the amino acid residue is E or G; at position 33 the amino acid residue is T, A or S; at position 35 the amino acid residue is L, S or M; at position 37 the amino acid residue is R, G, E or Q; at position 38 the amino acid residue is G or S; at position 39 the amino acid residue is T, A or S; (aa) at position 40 the amino acid residue is F, L or S; (ab) at position 45 the amino acid residue is Y.or F; (ac) at position 47 the amino acid residue is R, Q or G; (ad) at position 48 the amino acid residue is G or D; (ae) at position 49 the amino acid residue is K, R, E or Q; (af) at position 51 the amino acid residue is I or V; (ag) at position 52 the amino acid residue is S, C or G; (ah) at position 53 the amino acid residue is I or T; (ai) at position 54 the amino acid residue is A or V; (aj) at position 57 the amino acid residue is H or N; (ak) at position 58 the amino acid residue is Q, K, N or P; (al) at position 59 the amino acid residue is A or S; (am) at position 60 the amino acid residue is E, K, G, V or WO 02/36782 PCT/US01/46227 D; (an) at position 61 the amino acid residue is H or Q; (ao) at position 62 the amino acid residue is P, S or T; (ap) at position 63 the amino acid residue is E, G or D; (aq) at position the amino acid residue is E, D, V or Q; (ar) at position 67 the amino acid residue is Q, E, R, L, H or K; (as) at position 68 the amino acid residue is K, R, E, or N; (at) at position 69 the amino acid residue is Q or P; (au) at position 79 the amino acid residue is E or D; (av) at position 80 the amino acid residue is G or E; (aw) at position 81 the amino acid residue is Y, N or F; (ax) at position 82 the amino acid residue is R or H; (ay) at position 83 the amino acid residue is E, G or D; (az) at position 84 the amino acid residue is Q, R or L; (ba) at position 86 the amino acid residue is A or V; (bb) at position 89 the amino acid residue is T or S; (bc) at position 90 the amino acid residue is L or I; (bd) at position 91 the amino acid residue is I or V; (be) at position 92 the amino acid residue is R or K; (bf) at position 93 the amino acid residue is H, Y or Q; (bg) at position 96 the amino acid residue is E, A or Q; (bh) at position 97 the amino acid residue is L or I; (bi) at position 100 the amino acid residue is K, R, N or E; (bj) at position 101 the amino acid residue is K or R; (bk) at position 103 the amino acid residue is A or V; (bl) at position 104 the amino acid residue is D or N; (bm) at position 105 the amino acid residue is L or M; (bn) at position 106 the amino acid residue is L or I; (bo) at position 112 the amino acid residue is T or I; (bp) at position 113 the amino acid residue is S, T or F; (bq) at position 114 the amino acid residue is A or V; (br) at position 115 the amino acid residue is S, R or A; (bs) at position 119 the amino acid residue is K, E or R; (bt) at position 120 the amino acid residue is K or R; (bu) at position 123 the amino acid residue is F or L; (bv) at position 124 the amino acid residue is S or R; (bw) at position 125 the amino acid residue is E, K, G or D; (bx) at position 126 the amino acid residue is Q or H; (by) at position 128 the amino acid residue is E, G or K; (bz) at position 129 the amino acid residue is V, I or A; (ca) at position 130 the amino acid residue is Y, H, F or C; (cb) at position 131 the amino acid residue is D, G, N or E; (cc) at position 132 the amino acid residue is I, T, A, M, V or L; (cd) at position 135 the amino acid residue is V, T, A or I; (ce) at position 138 the amino acid residue is H or Y; (cf) at position 139 the amino acid residue is I or V; (cg) at position 140 the amino acid residue is L or S; (ch) at position 142 the amino acid residue is Y or H; (ci) at position 143 the amino acid residue is K, T or E; (cj) at position 144 the amino acid residue is K, E or R; (ck) at position 145 the amino acid residue is L or I; and (cl) at position 146 the amino acid residue is T or A.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the -26- WO 02/36782 PCT/US01/46227 group consisting of SEQ ID NO:6-10 and 263-514, at least 80% of the amino acid residues in the polypeptide that correspond to the following positions conform to the following restrictions: at position 9, 76, 94 and 110 the amino acid residue is A; at position 29 and 108 the amino acid residue is C; at position 34 the amino acid residue is D; at position 95 the amino acid residue is E; at position 56 the amino acid residue is F; at position 43, 44, 66, 74, 87, 102, 116, 122, 127 and 136 the amino acid residue is G; at position 41 the amino acid residue is H; at position 7 the amino acid residue is I; at position 85 the amino acid residue is K; at position 20, 36, 42, 50, 72, 78, 98 and 121 the amino acid residue is L; at position 1, 75 and 141 the amino acid residue is M; at position 23, 64 and 109 the amino acid residue is N; at position 22, 25, 133, 134 and 137 the amino acid residue is P; at position 71 the amino acid residue is Q; at position 16, 21, 73, 99 and 111 the amino acid residue is R; at position 55 and 88 the amino acid residue is S; at position 77 the amino acid residue is T; at position 107 the amino acid residue is W; and at position 13, 46, 70, 117 and 118 the amino acid residue is Y.

Some preferred GAT polypeptides of the invention are characterized as follows. When optimally aligned with a reference amino acid sequence selected from the group consisting of SEQ ID NO:6-10 and 263-514, the amino acid residue in the polypeptide that correspond to position 28 is V or A. Valine at the 28 position generally correlates with reduced KM, while alanine at that position generally correlates with increased kt. Other preferred GAT polypeptides are characterized by having 127 an I at position 27), M30, S35, R37, S39, G48, K49, N57, Q58, P62, Q65, Q67, K68, E83, S89, A96, E96, R101, T112, A114, K119, K120, E128, V129, D131, T131, V134, R144, 1145, or T146, or any combination thereof.

Some preferred GAT polypeptides of the invention comprise an amino acid sequence selected from the group consisting of SEQ ID NOS:6-10 and 263-514.

The invention further provides preferred GAT polypeptides that are characterized by a combination of the foregoing amino acid residue position restrictions.

In addition, the invention provides GAT polynucleotides encoding the preferred GAT polypeptides described above, and complementary nucleotide sequences thereof.

Some aspects of the invention pertain particularly to the subset of any of the above-described categories of GAT polypeptides having GAT activity, as described herein. These GAT polypeptides are preferred, for example, for use as agents for -27- WO 02/36782 PCT/US01/46227 conferring glyphosate resistance upon a plant. Examples of desired levels of GAT activity are described herein.

In one aspect, the GAT polypeptides comprise an amino acid sequence encoded by a recombinant or isolated form of naturally occurring nucleic acids isolated from a natural source, a bacterial strain. Wild-type polynucleotides encoding such GAT polypeptides may be specifically screened for by standard techniques known in the art. The polypeptides defined by SEQ ID NO:6 to SEQ ID NO:10, for example, were discovered by expression cloning of sequences from Bacillus strains exhibiting GAT activity, as described in more detail below.

The invention also includes isolated or recombinant polypeptides which are encoded by an isolated or recombinant polynucleotide comprising a nucleotide sequence which hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-5 and 11-262, their complements, and nucleotide sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 6-10 and 263-514, including their complements.

The invention further includes any polypeptide having GAT activity that is encoded by a fragment of any of the GAT-encoding polynucleotides described herein.

The invention also provides fragments of GAT polypeptides that can be spliced together to form a functional GAT polypeptide. Splicing can be accomplished in vitro or in vivo, and can involve cis or trans intramolecular or intermolecular) splicing. The fragments themselves can, but need not, have GAT activity. For example, two or more segments of a GAT polypeptide can be separated by inteins; removal of the intein sequence by cis-splicing results in a functional GAT polypeptide. In another example, an encrypted GAT polypeptide can be expressed as two or more separate fragments; trans-splicing of these segments results in recovery of a functional GAT polypeptide. Various aspects of cis and trans splicing, gene encryption, and introduction of intervening sequences are described in more detail in US patent application Nos.

09/517,933 and 09/710,686, both of which are incorporated by reference herein in their entirety.

In general, the invention includes any polypeptide encoded by a modified GAT polynucleotide derived by mutation, recursive sequence recombination, and/or diversification of the polynucleotide sequences described herein. In some aspects of the invention, a GAT polypeptide is modified a by single or multiple amino acid substitution, -28- WO 02/36782 PCT/US01/46227 a deletion, an insertion, or a combination of one or more of these types of modifications.

Substitutions can be conservative, or non-conservative, can alter function or not, and can add new function. Insertions and deletions can be substantial, such as the case of a truncation of a substantial fragment of the sequence, or in the fusion of additional sequence, either internally or at N or C terminal. In some embodiments of the invention, a GAT polypeptide is part of a fusion protein comprising a functional addition such as, for example, a secretion signal, a chloroplast transit peptide, a purification tag, or any of numerous other functional groups that will be apparent to the skilled artisan, and which are described in more detail elsewhere in this specification.

Polypeptides of the invention may contain one or more modified amino acid. The presence of modified amino acids may be advantageous in, for example, (a) increasing polypeptide in vivo half-life, reducing or increasing polypeptide antigenicity, increasing polypeptide storage stability. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production Nlinked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means.

Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ.

Recombinant methods for producing and isolating GAT polypeptides of the invention are described herein. In addition to recombinant production, the polypeptides may be produced by direct peptide synthesis using solid-phase techniques Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J. Am. Chem. Soc. 85:2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. For example, subsequences may be chemically synthesized separately and combined using chemical methods to provide full-length GAT polypeptdides. Peptides can also be ordered from a variety of sources.

-29- WO 02/36782 PCT/US01/46227 0 In another aspect of the invention, a GAT polypeptide of the invention is jused to produce antibodies which have, diagnostic uses, for example, related to the activity, distribution, and expression of GAT polypeptides, for example, in various tissues Sof a transgenic plant.

GAT homologue polypeptides for antibody induction do not.require S biological activity; however, the polypeptide or oligopeptide must be antigenic. Peptides Cr used to induce specific antibodies may have an amino acid sequence consisting of at least amino acids, preferably at least 15 or 20 amino acids. Short stretches of a GAT Spolypeptide may be fused with another protein, such as keyhole limpet hemocyanin, and antibody produced against the chimeric molecule.

"1 Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art, and many antibodies are available. See, Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 pM, preferably at least about 0.01 pM or better, and most typically and preferably, 0.001 pM or better.

Additional details antibody production and engineering techniques can be found in Borrebaeck (ed) (1995) Antibody Engineering, 2 n d Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ (Paul).

Sequence Variations GAT polypeptides of the present invention include conservatively modified variations of the sequences disclosed herein as SEQ ID NOS: 6-10 and 263-514. Such conservatively modified variations comprise substitutions, additions or deletions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less WO 02/36782 PCT/US01/46227 than about more typically less than about or in any of SEQ ID NOS: 6and 263-514.

For example, a conservatively modified variation deletion) of the 146 amino acid polypeptide identified herein as SEQ ID NO:6 will have a length of at least 140 amino acids, preferably at least 141 amino acids, more preferably at least 144 amino acids, and still more preferably at least 146 amino acids, corresponding to a deletion of less than about 2% or about or less of the polypeptide sequence.

Another example of a conservatively modified variation a "conservatively substituted variation") of the polypeptide identified herein as SEQ ID NO:6 will contain "conservative substitutions", according to the six substitution groups set forth in Table 2 (infra), in up to about 7 residues less than about of the 146 amino acid polypeptide.

The GAT polypeptide sequence homologues of the invention, including conservatively substituted sequences, can be present as part of larger polypeptide sequences such as occur in a GAT polypeptide, in a GAT fusion with a signal sequence, a chloraplast targeting sequence, or upon the addition of one or more domains for purification of the protein poly his segments, FLAG tag segments, etc.). In the latter case, the additional functional domains have little or no effect on the activity of the GAT portion of the protein, or where the additional domains can be removed by post synthesis processing steps such as by treatment with a protease.

Defining Polvpeptides by Immunoreactivity Because the polypeptides of the invention provide a new class of enzymes with a defined activity, the acetylation of glyphosate, the polypeptides also provide new structural features which can be recognized, in immunological assays. The generation of antisera which specifically binds the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are a feature of the invention.

The invention includes GAT polypeptides that specifically bind to or that are specifically immunoreactive with an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of SEQ ID NO:6 to SEQ ID NO:10. To eliminate cross-reactivity with other GAT homologues, the antibody or antisera is subtracted with available related proteins, such as those represented by the proteins or peptides corresponding to GenBank accession numbers available as of the filing date of this application, and exemplified by CAA70664, Z99109 and Y09476.

-31- WO 02/36782 PCT/US01/46227 O Where the accession number corresponds to a nucleic acid, a polypeptide encoded by the 0j2) nucleic acid is generated and used for antibody/antisera subtraction purposes. Figure 3 tabulates the relative identity between exemplary GAT polypeptides and the most closely Srelated sequence available in Genbank, YitL The function of native YitI has yet to be elucidated, but the enzyme has been shown to possess detectable GAT activity.

In one typical format, the immunoassay uses a polyclonal antiserum which c was raised against one or more polypeptide comprising one or more of the sequences corresponding to one or more of SEQ ID NOS: 6-10 and 263-514, or a substantial subsequence thereof at least about 30% of the full length sequence provided). The full set of potential polypeptide immunogens derived from SEQ ID NOS: 6-10 and 263- C 514 are collectively referred to below as "the immunogenic polypeptides." The resulting antisera is optionally selected to have low cross-reactivity against other related sequences and any such cross-reactivity is removed by immunoabsorbtion with one or more of the related sequences, prior to use of the polyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more of the immunogenic polypeptides is produced and purified as described herein. For example, recombinant protein may be produced in a bacterial cell line. An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic protein(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity).

Alternatively, one or more synthetic or recombinant polypeptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.

Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with one or more of the immunogenic proteins immobilized on a solid support. Polyclonal antisera with a titer of 106 or greater are selected, pooled and subtracted with related polypeptides, those identified from GENBANK as noted, to produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for cross reactivity against the related polypeptides. Preferably at least two of the immunogenic GATs are used in this determination, preferably in conjunction with at least two of related -32- WO 02/36782 PCT/US01/46227 polypeptides, to identify antibodies which are specifically bound by the immunogenic protein(s).

In this comparative assay, discriminatory binding conditions are determined for the subtracted titered polyclonal antisera which result in at least about a 5-10 fold higher signal to noise ratio for binding of the titered polyclonal antisera to the immunogenic GAT polypeptides as compared to binding to the related polypeptides. That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or non-fat dry milk, or by adjusting salt conditions, temperature, or the like. These binding conditions are used in subsequent assays for determining whether a test polypeptide is specifically bound by the pooled subtracted polyclonal antisera. In particular, test polypeptides which show at least a 2-5x higher signal to noise ratio than the control polypeptides under discriminatory binding conditions, and at least about a 1/2 signal to noise ratio as compared to the immunogenic polypeptide(s), shares substantial structural similarity with the immunogenic polypeptide as compared to known GAT, and is, therefore a polypeptide of the invention.

In another example, immunoassays in the competitive binding format are used for detection of a test polypeptide. For example, as noted, cross-reacting antibodies are removed from the pooled antisera mixture by immunoabsorbtion with the control GAT polypeptides. The immunogenic polypeptide(s) are then immobilized to a solid support which is exposed to the subtracted pooled antisera. Test proteins are added to the assay to compete for binding to the pooled subtracted antisera. The ability of the test protein(s) to compete for binding to the pooled subtracted antisera as compared to the immobilized protein(s) is compared to the ability of the immunogenic polypeptide(s) added to the assay to compete for binding (the immunogenic polypeptides compete effectively with the immobilized immunogenic polypeptides for binding to the pooled antisera). The percent cross-reactivity for the test proteins is calculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete for binding to the pooled subtracted antisera is optionally determined as compared to the ability of the immunogenic polypeptide(s) to compete for binding to the antisera. Again, the percent cross-reactivity for the control polypeptides is calculated, using standard calculations.

Where the percent cross-reactivity is at least 5-10x as high for the test polypeptides, the test polypeptides are said to specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide to -33- WO 02/36782 PCT/US01/46227 the immunogenic polypeptide(s). In order to make this comparison, the two polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the subtracted antisera to the immobilized protein is determined using standard techniques. If the amount of the test polypeptide required is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to specifically bind to an antibody generated to the immunogenic protein, provided the amount is at least about 5-10x as high as for a control polypeptide.

As a final determination of specificity, the pooled antisera is optionally fully immunosorbed with the immunogenic polypeptide(s) (rather than the control polypeptides) until little or no binding of the resulting immunogenic polypeptide subtracted pooled antisera to the immunogenic polypeptide(s) used in the immunosorbtion is detectable. This fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If little or no reactivity is observed no more than 2x the signal to noise ratio observed for binding of the fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.

GLYPHOSATE N-ACETYLTRANSFERASE

POLYNUCLEOTIDES

In one aspect, the invention provides a novel family of isolated or recombinant polynucleotides referred to herein as "glyphosate N-acetyltransferase polynucleotides" or "GAT polynucleotides." GAT polynucleotide sequences are characterized by the ability to encode a GAT polypeptide. In general, the invention includes any nucleotide sequence that encodes any of the novel GAT polypeptides described herein. In some aspects of the invention, a GAT polynucleotide that encodes a GAT polypeptide with GAT activity is preferred.

In one aspect, the GAT polynucleotides comprise recombinant or isolated forms of naturally occurring nucleic acids isolated from an organism, e,g, a bacterial strain. Exemplary GAT polynucleotides, SEQ ID NO:1 to SEQ ID NO:5, were discovered by expression cloning of sequences from Bacillus strains exhibiting GAT activity. Briefly, a collection of approximately 500 Bacillus and Pseudomonas strains were screened for native ability to N-acetylate glyphosate. Strains were grown in LB overnight, harvested by centrifugation, permeabilizied in dilute toluene, and then washed and resuspended in a reaction mix containing buffer, 5 mM glyphosate, and 200 pM acetyl-CoA. The cells were incubated in the reaction mix for between 1 and 48 hours, at which time an equal volume of methanol was added to the reaction. The cells were then -34- WO 02/36782 PCT/US01/46227 pelleted by centrifugation and the supernatant was filtered before analysis by parent ion mode mass spectrometry. The product of the reaction was positively identified as Nacetylglyphosate by comparing the mass spectrometry profile of the reaction mix to an Nacetylglyphosate standard as shown in Figure 2. Product detection was dependent on inclusion of both substrates (acetylCoA and glyphosate) and was abolished by heat denaturing the bacterial cells.

Individual GAT polynucleotides were then cloned from the identified strains by functional screening. Genomic DNA was prepared and partially digested with Sau3Al enzyme. Fragments of approximately 4 Kb were cloned into an E. coli expression vector and transformed into electrocompetent E. coli. Individual clones exhibiting GAT activity were identified by mass spectrometry following a reaction as described previously except that the toluene wash was replaced by permeabilization with PMBS. Genomic fragments were sequenced and the putative GAT polypeptide-encoding open reading frame identified. Identity of the GAT gene was confirmed by expression of the open reading frame in E. coli and detection of high levels of N-acetylglyphosate produced from reaction mixtures.

In another aspect of the invention, GAT polynucleotides are produced by diversifying, recombining and/or mutating one or more naturally occurring, isolated, or recombinant GAT polynucleotides. As described in more detail elsewhere herein, it is often possible to generate diversified GAT polynucleotides encoding GAT polypeptides with superior functional attributes, increased catalytic function, increased stability, higher expression level, than a GAT polynucleotide used as a substrate or parent in the diversification process.

The polynucleotides of the invention have a variety of uses in, for example: recombinant production expression) of the GAT polypeptides of the invention; as transgenes to confer herbicide resistance in transgenic plants); as selectable markers for transformation and plasmid maintenance; as immunogens; as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural GAT coding nucleic acids; as substrates for further diversity generation, recombination reactions or mutation reactions to produce new and/or improved GAT homologues, and the like.

It is important to note that certain specific, substantial and credible utilities of GAT polynucleotides do not require that the polynucleotide encode a polypeptide with substantial GAT activity. For example, GAT polynucleotides that do not encode active WO 02/36782 PCT/US01/46227 enzymes can be valuable sources of parental polynucleotides for use in diversification procedures to arrive at GAT polynucleotide variants, or non-GAT polynucleotides, with desirable functional properties high kcat or kcat/Km, low Km, high stability towards heat or other environmental factor, high transcription or translation rates, resistance to proteolytic cleavage, reducing antigenicity, etc.). For example, nucleotide sequences encoding protease variants with little or no detectable activity have been used as parent polynucleotides in DNA shuffling experiments to produce progeny encoding highly active proteases (Ness et al. (1999) Nature Biotechnology 17:893-96).

Polynucleotide sequences produced by diversity generation methods or recursive sequence recombination methods DNA shuffling) are a feature of the invention. Mutation and recombination methods using the nucleic acids described herein are a feature of the invention. For example, one method of the invention includes recursively recombining one or more nucleotide sequences of the invention as described above and below with one or more additional nucleotides. The recombining steps are optionally performed in vivo, ex vivo, in silico or in vitro. Said diversity generation or recursive sequence recombination produces at least one library of recombinant modified GAT polynucleotides. Polypeptides encoded by members of this library are included in the invention.

Also contemplated are uses of polynucleotides, also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least 20, 30, or 50 or more bases, which hybridize under stringent or highly stringent conditions to a GAT polynucleotide sequence. The polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted herein.

In accordance with the present invention, GAT polynucleotides, including nucleotide sequences that encode GAT poolypeptides, fragments of GAT polypeptides, related fusion proteins, or functional equivalents thereof, are used in recombinant DNA molecules that direct the expression of the GAT polypeptides in appropriate host cells, such as bacterial or plant cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can also be used to clone and express the GAT polynucleotides.

The invention provides GAT polynucleotides that encode transcription and/or translation product that are subsequently spliced to ultimately produce functional GAT polypeptides. Splicing can be accomplished in vitro or in vivo, and can involve cis -36- WO 02136782 PCT/US01/46227 or trans splicing. The substrate for splicing can be polynucleotides RNA transcripts) or polypeptides. An example of cis splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a GAT polypeptide encoding sequence. An example of trans splicing would be where a GAT polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the fulllength GAT encoding sequence. The use of a splicing enhancer sequence (which can be introduced into a construct of the invention) can facilitate splicing either in cis or trans.

Cis and trans splicing of polypeptides are described in more detail elsehwhere herein.

More detailed description of cis and trans splicing can be found in US patent application Nos. 09/517,933 and 09/710,686.

Thus, some GAT polynucleotides do not directly encode a full-length GAT polypeptide, but rather encode a fragment or fragments of a GAT polypeptide. These GAT polynucleotides can be used to express a functional GAT polypeptide through a mechanism involving splicing, where splicing can occur at the level of polynucleotide intron/exon) and/or polypeptide intein/extein). This can be useful, for example, in controlling expression of GAT activity, since functional GAT polypeptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product. In another example, introduction of one or more insertion sequences into a GAT polynucleotide can facilitate recombination with a low homology polynucleotide; use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms preferentially use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons (see, Zhang SP et al. (1991) Gene 105:61-72). Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called "codon optimization" or "controlling for species codon bias." Optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray, E. et al. (1989) Nuc. Acids Res. 17:477- 508) can be prepared, for example, to increase the rate of translation or to produce -37- WO 02/36782 PCT/US01/46227 recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for S. cerevisiae and mammals are UAA and UGA respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer to use UAA as the stop codon (Dalphin ME et al. (1996) Nuc. Acids Res. 24: 216-218).

Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Patent No. 6,015,891, and references cited therein.

One embodiment of the invention includes a GAT polynucleotide having optimal codons for expression in a relevant host, a transgenic plant host This is particularly desirable when a GAT polynucleotide of bacterial origin is introduced into a transgenic plant, to confer glyphosate resistance to the plant The polynucleotide sequences of the present invention can be engineered in order to alter a GAT polynucleotide for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.

For example, alterations may be introduced using techniques that are well known in the art, site-directed mutagenesis, to insert new restriction sites, alter glycosylation patterns, change codon preference, introduce splice sites, etc.

As described in more detail herein, the polynucleotides of the invention include sequences which encode novel GAT polypeptides and sequences complementary to the coding sequences, and novel fragments of coding sequence and complements thereof. The polynucleotides can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, synthetic RNA and DNA, genomic DNA and cDNA. The polynucleotides can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (anti-sense, complementary) strand. The polynucleotides optionally include the coding sequence of a GAT polypeptide in isolation, (ii) in combination with additional coding sequence, so as to encode, a fusion protein, a pre-protein, a prepro-protein, or the like, (iii) in combination with noncoding sequences, such as introns or inteins, control elements such as a promoter, an enhancer, a terminator element, or 5' and/or 3' untranslated regions effective for expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment in which the GAT polynucleotide is a heterologous gene. Sequences can also be found in combination with typical compositional formulations of nucleic acids, including in the presence of carriers, buffers, adjuvants, excipients and the like.

-38- WO 02/36782 PCT/US01/46227 Polynucleotides and oligonucleotides of the invention can be prepared by standard solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined by enzymatic or chemical ligation methods, or polymerase mediated methods) to form essentially any desired continuous sequence. For example, polynucleotides and oligonucleotides of the invention can be prepared by chemical synthesis using, the classical phosphoramidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-69, or the method described by Matthes et al. (1984) EMBO J. 3: 801-05., as is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others. Similarly, peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.

(http://www.htibio.com), BMA Biomedicals Ltd Bio.Synthesis, Inc., and many others.

Polynucleotides may also be synthesized by well-known techniques as described in the technical literature. See, Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. An. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

-31 1 -1 1. WO 02/36782 PCT/US01/46227 sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qp-replicase amplification and other RNA polymerase mediated techniques NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Patent No.

4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds.) Academic Press Inc. San Diego, CA (1990); Arnheim Levinson (October 1, 1990) Chemical and Engineering News 3647; The Journal Of NIH Research (1991) 3:81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al. (1990) Proc. Natl.

Acad. Sci. USA 87:1874; Lomell et al. (1989) J. Clin. Chem. 35:1826; Landegren et al., (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace, (1989) Gene 4:560; Barringer et al. (1990) Gene 89:117, and Sooknanan and Malek (1995) Biotechnology 13:563-564. Improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369:684-685 and the references therein, in which PCR amplicons of up to 40kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausbel, Sambrook and Berger, all supra.

Sequence Variations It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding GAT polypeptides of the invention may be produced, some of which bear substantial identity to the nucleic acid sequences explicitly disclosed herein.

WO 02/36782 PCT/US01/46227 Table 1 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine lie I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU For instance, inspection of the codon table (Table 1) shows that codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.

Thus, at every position in the nucleic acids of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.

Using, as an example, the nucleic acid sequence corresponding to nucleotides 1-15 of SEQ ID NO:1, ATG ATT GAA GTC AAA, a silent variation of this sequence includes AGT ATC GAG GTG AAG, both sequences which encode the amino acid sequence MIEVK, corresponding to amino acids 1-5 of SEQ ID NO:6.

Such "silent variations" are one species of "conservatively modified variations", discussed below. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence. The invention provides each and every possible variation of nucleic acid -41- WO 02/36782 PCT/US01/46227 sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as set forth in Table 1) as applied to the nucleic acid sequence encoding a GAT homologue polypeptide of the invention. All such variations of every nucleic acid herein are specifically provided and described by consideration of the sequence in combination with the genetic code. Any variant can be produced as noted herein.

A group of two or more different codons that, when translated in the same context, all encode the same amino acid, are referred to herein as "synonoumous codons." As described herein, in some aspects of the invention a GAT polynucleotide is engineered for optimized codon usage in a desired host organism, for example a plant host. The term "optimized" or "optimal" are not meant to be restricted to the very best possible combination of codons, but simple indicates that the coding sequence as a whole possesses an improved usage of codons relative to a precursor polynucleotide from which it was derived. Thus, in one aspect the invention provides a method for producing a GAT polynucleotide variant by replacing at least one parental codon in a nucleotide sequence with a synonomous codon that is preferentially used in a desired host organism, a plant, relative to the parental codon.

"Conservatively modified variations" or, simply, "conservative variations" of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. One of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than more typically less than 2% or or less) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar amino acids are well known in the art. Table 2 sets forth six groups which contain amino acids that are "conservative substitutions" for one another.

-42- WO 02/36782 PCT/US01/46227 L P r 1. U O I,'ltL 2 ia- 7 Table 2 Conservative Substitution Groups 1 Alanine Serine Threonine (T) 2 Aspartic acid Glutamic acid (E) 3 Asparagine Glutamine (Q) 4 Arginine Lysine (K) 5 Isoleucine Leucine Methionine Valine (V) 6 Phenylalanine Tyrosine Tryptophan (W) Cc, Cc, In Thus, "conservatively substituted variations" of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than more typically less than 2% and often less than of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.

For example, a conservatively substituted variation of the polypeptide identified herein as SEQ ID NO:6 will contain "conservative substitutions", according to the six groups defined above, in up to 7 residues 5% of the amino acids) in the 146 amino acid polypeptide.

In a further example, if four conservative substitutions were localized in the region corresponding to amino acids 21 to 30 of SEQ ID NO:6, examples of conservatively substituted variations of this region, RPN QPL EAC M, include: KPQ QPV ESC M and KPN NPL DAC V and the like, in accordance with the conservative substitutions listed in Table 2 (in the above example, conservative substitutions are underlined). Listing of a protein sequence herein, in conjunction with the above substitution table, provides an express listing of all conservatively substituted proteins.

Finally, the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence, is a conservative variation of the basic nucleic acid.

One of skill will appreciate that many conservative variations of the nucleic acid constructs which are disclosed yield a functionally identical construct. For example, as discussed above, owing to the degeneracy of the genetic code, "silent substitutions" -43- WO 02/36782 PCTfUS01/46227 substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid. Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct Such conservative variations of each disclosed sequence are a feature of the present invention.

Non-conservative modifications of a particular nucleic acid are those which substitute any amino acid not characterized as a conservative substitution. For example, any substitution which crosses the bounds of the six groups set forth in Table 2. These include substitutions of basic or acidic amino acids for neutral amino acids, Asp, Glu, Asn, or Gin for Val, Ile, Leu or Met), aromatic amino acid for basic or acidic amino acids Phe, Tyr or Trp for Asp, Asn, Glu or Gin) or any other substitution not replacing an amino acid with a like amino acid.

Nucleic Acid Hybridization Nucleic acids "hybridize" when they associate, typically in solution.

Nucleic acids hybridize due to a variety of well-characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, New York), as well as in Ausubel, supra, Hames and Higgins (1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2, IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.

"Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments, such as Southern and northern hybridizations, are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra.

For purposes of the present invention, generally, "highly stringent" hybridization and wash conditions are selected to be about 5°C or less lower than the -44- WO 02/36782 PCT/US01/46227 thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms).

The Tm is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.

The Tm of a nucleic acid duplex indicates the temperature at which the duplex is 50% denatured under the given conditions and its represents a direct measure of the stability of the nucleic acid hybrid. Thus, the Tm corresponds to the temperature corresponding to the midpoint in transition from helix to random coil; it depends on length, nucleotide composition, and ionic strength for long stretches of nucleotides.

After hybridization, unhybridized nucleic acid material can be removed by a series of washes, the stringency of which can be adjusted depending upon the desired results. Low stringency washing conditions using higher salt and lower temperature) increase sensitivity, but can product nonspecific hybridization signals and high background signals. Higher stringency conditions using lower salt and higher temperature that is closer to the hybridization temperature) lowers the background signal, typically with only the specific signal remaining. See Rapley, R. and Walker, J.M. eds., Molecular Biomethods Handbook (Humana Press, Inc. 1998) (hereinafter "Rapley and Walker"), which is incorporated herein by reference in its entirety for all purposes.

The Tm of a DNA-DNA duplex can be estimated using Equation 1 as follows: Tm 81.5 0 C 16.6 (ogloM) 0.41 C) 0.72 500/n, where M is the molarity of the monovalent cations (usually C) is the percentage of guanosine and cystosine nucleotides, is the percentage of formalize and n is the number of nucleotide bases length) of the hybrid. See Rapley and Walker, supra.

The Tm of an RNA-DNA duplex can be estimated by using Equation 2 as follows: Tm (oC) 79.8 0 C 18.5 (logloM) 0.58 C) 11.8(%G C) 2 0.56 820/n,where M is the molarity of the monovalent cations (usually C)is the percentage of guanosine (G and cystosine nucleotides, is the percentage of formamide and n is the number of nucleotide bases length) of the hybrid. Id Equations 1 and 2 are typically accurate only for hybrid duplexes longer than about 100-200 nucleotides. Id WO 02/36782 PCT/US01/46227 The Tm of nucleic acid sequences shorter than 50 nucleotides can be calculated as follows: Tm (OC) 4(G C) 2(A T), where A (adenine), C, T (thymine), and G are the numbers of the corresponding nucleotides.

An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of beparin at 42 0 C, with the hybridization being carried out overnight. An example of stringent wash conditions is a 0.2x SSC wash at 65 0 C for 15 minutes (see Sambrook, supra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40 0 C for minutes.

In general, a signal to noise ratio of 2.5x-5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Detection of at least stringent hybridization between two sequences in the context of the present invention indicates relatively strong structural similarity or homology to, the nucleic acids of the present invention provided in the sequence listings herein.

As noted, "highly stringent" conditions are selected to be about 5° C or less lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Target sequences that are closely related or identical to the nucleotide sequence of interest "probe") can be identified under highly stringent conditions.

Lower stringency conditions are appropriate for sequences that are less complementary.

See, Rapley and Walker, supra.

Comparative hybridization can be used to identify nucleic acids of the invention, and this comparative hybridization method is a preferred method of distinguishing nucleic acids of the invention. Detection of highly stringent hybridization between two nucleotide sequences in the context of the present invention indicates relatively strong structural similarity/homology to, the nucleic acids provided in the sequence listing herein. Highly stringent hybridization between two nucleotide sequences demonstrates a degree of similarity or homology of structure, nucleotide base composition, arrangement or order that is greater than that detected by stringent hybridization -46- WO 02/36782 PCT/US01/46227 conditions. In particular, detection of highly stringent hybridization in the context of the present invention indicates strong structural similarity or structural homology nucleotide structure, base composition, arrangement or order) to, the nucleic acids provided in the sequence listings herein. For example, it is desirable to identify test nucleic acids that hybridize to the exemplar nucleic acids herein under stringent conditions.

Thus, one measure of stringent hybridization is the ability to hybridize to one of the listed nucleic acids nucleic acid sequences SEQ ID NO:1 to SEQ ID and SEQ ID NO: 11 to SEQ ID NO:262, and complementary polynucleotide sequences thereof), under highly stringent conditions (or very stringent conditions, or ultra-high stringency hybridization conditions, or ultra-ultra high stringency hybridization conditions). Stringent hybridization (as well as highly stringent, ultra-high stringency, or ultra-ultra high stringency hybridization conditions) and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formalin, in the hybridization or wash), until a selected set of criteria are met. For example, the hybridization and wash conditions are gradually increased until a probe comprising one or more nucleic acid sequences selected from SEQ ID NO: 1 to SEQ ID and SEQ ID NO:11 to SEQ ID NO:262, and complementary polynucleotide sequences thereof, binds to a perfectly matched complementary target (again, a nucleic acid comprising one or more nucleic acid sequences selected from SEQ ID NO:1 to SEQ ID NO:5 and SEQ ID NO:11 to SEQ ID NO:262, and complementary polynucleotide sequences thereof), with a signal to noise ratio that is at least about 2.5x, and optionally about 5x or more as high as that observed for hybridization of the probe to an unmatched target. In this case, the unmatched target is a nucleic acid corresponding to a nucleic acid (other than those in the accompanying sequence listing) that is present in a public database such as GenBank T M at the time of filing of the subject application. Such sequences can be identified in GenBank by one of skill. Examples include Accession Nos. Z99109 and Y09476. Additional such sequences can be identified in GenBank, by one of ordinary skill in the art.

A test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least V as well to the probe as to the perfectly matched -47- WO 02/36782 PCT/US01/46227 complementary target, with a signal to noise ratio at least as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to Sthe perfectly matched complementary target with a signal to noise ratio that is at least Sabout 2x-10x, and occasionally 20x, 50x or greater than that observed for hybridization to any of the unmatched polynucleotides Accession Nos. Z99109 and Y09476.

Ultra high-stringency hybridization and wash conditions are those in which C the stringency of hybridization and wash conditions are increased until the signal to noise It ratio for binding of the probe to the perfectly matched complementary target nucleic acid C, is at least 10x as high as that observed for hybridization to any of the unmatched target nucleic acids Genbank Accession numbers Z99109 and Y09476. A target nucleic acid C1 which hybridizes to a probe under such conditions, with a signal to noise ratio of at least V that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay.

For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10x, 20X, 50X, 100X, or 500X or more as high as that observed for hybridization to any of the unmatched target nucleic acids Genbank Accession numbers Z99109 and Y09476. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least z that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.

Target nucleic acids which hybridize to the nucleic acids represented by SEQ ID NO:1 to SEQ ID NO:5 and SEQ ID NO:11 to SEQ ID NO:262 under high, ultrahigh and ultra-ultra high stringency conditions are a feature of the invention. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.

Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code, or when antisera or antiserum generated against one or more of SEQ ID NO:6 to SEQ ID NO:10 and SEQ ID NO:263 to SEQ DD NO:514, which has been subtracted using the polypeptides encoded by known -48- WO 02/36782 PCT/US01/46227 O nucleotide sequences, including Genbank Accession number CAA70664. Further details b on immunological identification of polypeptides of the invention are found below.

Additionally, for distinguishing between duplexes with sequences of less than about 100 Snucleotides, a TMAC1 hybridization procedure known to those of ordinary skill in the art can be used. See, Sorg, U. et al. 1 Nucleic Acids Res. (Sept. 11, 1991) 19(17), incorporated herein by reference in its entirety for all purposes.

c In one aspect, the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from SEQ ID NO:1 to SEQ ID NO:5 and SEQ ID NO:11 to SEQ ID NO:262. The unique subsequence is unique as compared to a nucleic acid corresponding to any of Genbank Accession numbers Z99109 and Y09476.

C

Such unique subsequences can be determined by aligning any of SEQ ID NO: 1 to SEQ ID and SEQ ID NO:11 to SEQ ID NO:262 against the complete set of nucleic acids represented by GenBank accession numbers Z99109, Y09476 or other related sequences available in public databases as of the filing date of the subject application. Alignment can be performed using the BLAST algorithm set to default parameters. Any unique subsequence is useful, as a probe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a unique subsequence in a polypeptide selected from: SEQ ID NO:6 to SEQ ID NO:10 and SEQ ID NO:263 to SEQ ID NO:514. Here, the unique subsequence is unique as compared to a polypeptide corresponding to GenBank accession number CAA70664. Here again, the polypeptide is aligned against the sequences represented by accession number CAA70664.

Note that if the sequence corresponds to a non-translated sequence such as a pseudo gene, the corresponding polypeptide is generated simply by in silico translation of the nucleic acid sequence into an amino acid sequence, where the reading frame is selected to correspond to the reading frame of homologous GAT polynucleotides.

The invention also provides for target nucleic acids which hybridizes under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from SEQ ID NO:6 to SEQ ID NO:10 and SEQ ID NO:263 to SEQ ID NO:514, wherein the unique subsequence is unique as compared to a polypeptide corresponding to any of the control polypeptides. Unique sequences are determined as noted above.

In one example, the stringent conditions are selected such that a perfectly complementary oligonucleotide to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 2.5x-10x higher, preferably at least about a 5-10x -49- WO 02/36782 PCT/US01/46227 higher signal to noise ratio than for hybridization of the perfectly complementary oligonucleotide to a control nucleic acid corresponding to any of the control polypeptides.

Conditions can be selected such that higher ratios of signal to noise are observed in the particular assay which is used, about 15x, 20x, 30x, 50x or more. In this example, the target nucleic acid hybridizes to the unique coding oligonucleotide with at least a 2x higher signal to noise ratio as compared to hybridization of the control nucleic acid to the coding oligonucleotide. Again, higher signal to noise ratios can be selected, about 5x, 10x, 20x, 30x, 50x or more. The particular signal will depend on the label used in the relevant assay, a fluorescent label, a colorimetric label, a radioactive label, or the like.

Vectors. Promoters and Expression Systems, The present invention also includes recombinant constructs comprising one or more of the nucleic acid sequences as broadly described above. The constructs comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.

Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Berger and Kimmel, Guide to Molecular Cloning Techniques. Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning A Laboratory Manual (2nd Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F.M.

Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley Sons, Inc., (supplemented through 1999) ("Ausubel").

Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qp-replicase amplification and other RNA polymerase mediated techniques NASBA), for the production of the homologous nucleic acids of the invention are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Patent No.

4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) WO 02/36782 PCT/US01/46227 Academic Press Inc. San Diego, CA (1990) (Innis); Amheim Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnolovg 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, Ausubel, Sambrook and Berger, all supra.

The present invention also relates to engineered host cells that are transduced (transformed or transfected) with a vector of the invention an invention cloning vector or an invention expression vector), as well as the production of polypeptides of the invention by recombinant techniques. The vector may be, for example, a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the GAT homologue gene. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook, Ausubel and Berger, as well as Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein.

GAT polypeptides of the invention can be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like. In addition to Sambrook, Berger and Ausubel, details regarding non-animal cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton,

FL.

-51- WO 02/36782 PCT/US01/46227 Polynucleotides of the present invention can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences, derivatives of bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others.

Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used.

When incorporated into an expression vector, a polynucleotide of the invention is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such transcription control sequences particularly suited for use in transgenic plants include the cauliflower mosaic virus (CaMV), figwort mosaic virus (FMV) and strawberry vein banding virus (SVBV) promoters, described in U.S. Provisional Application No. 60/245,354. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses and which can be used in some embodiments of the invention include SV40 promoter, E. coli lac or trp promoter, phage lambda PL promoter. An expression vector optionally contains a ribosome binding site for translation initiation, and a transcription terminator. The vector also optionally includes appropriate sequences for amplifying expression, an enhancer. In addition, the expression vectors of the present invention optionally contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

Vectors of the present invention can be employed to transform an appropriate host to permit the host to express an invention protein or polypeptide.

Examples of appropriate expression hosts include: bacterial cells, such as E. coli, B.

subtilis, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharonryces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells such as Drosophila and Spodopterafrugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma; or plant cells or explants, etc. It is understood that not all cells or cell lines need to be capable of producing fully functional GAT polypeptides; for example, antigenic fragments of a GAT polypeptide may be produced. The invention is not limited by the host cells employed.

-52- WO 02/36782 PCT/US01/46227 In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the GAT polypeptide. For example, when large quantities of GAT polypeptide or fragments thereof are needed for commercial production or for induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be desirable. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the GAT polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke Schuster (1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison WI); and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used for production of the GAT polypeptides of the invention. For reviews, see Ausubel et al. (supra) and Grant et al. (1987; Methods in Enzvmologv 153:516-544).

In mammalian host cells, a variety of expression systems, including viralbased systems, may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence, of a GAT polypeptide, is optionally ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion of a GAT polypeptide coding region into a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing a GAT in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659). In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Similarly, in plant cells, expression can be driven from a transgene integrated into a plant chromosome, or cytoplasmically from an episomal or viral nucleic acid. In the case of stably integrated transgenes, it is often desirable to provide sequences capable of driving constitutive or inducible expression of the GAT polynucleotides of the invention, for example, using viral, CaMV, or plant derived regulatory sequences.

Numerous plant derived regulatory sequences have been described, including sequences which direct expression in a tissue specific manner, TobRB7, patatin B33, GRP gene promoters, the rbcS-3A promoter, and the like. Alternatively, high level expression can be achieved by transiently expressing exogenous sequences of a plant viral vector, TMV, BMV, etc. Typically, transgenic plants constitutively expressing a GAT polynucleotide of -53- WO 02/36782 PCT/US01/46227 the invention will be preferred, and the regulatory sequences selected to insure constitutive stable expression of the GAT polypeptide.

In some embodiments of the present invention, a GAT polynucleotide construct suitable for transformation of plant cells is prepared. For example, a desired GAT polynucleotide can be incorporated into a recombinant expression cassette to facilitate introduction of the gene into a plant and subsequent expression of the encoded polypeptide. An expression cassette will typically comprise a GAT polynucleotide, or functional fragment thereof, operably linked to a promoter sequence and other transcriptional and translational initiation regulatory sequences which will direct expression of the sequence in the intended tissues entire plant, leaves, seeds) of the transformed plant.

For example, a strongly or weakly constitutive plant promoter can be employed which will direct expression of the GAT polypeptide all tissues of a plant. Such promoters are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the or promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. In situations in which overexpression of a GAT poynucleotide is detrimental to the plant or otherwise undesirable, one of skill, upon review of this disclosure, will recognize that weak constitutive promoters can be used for low-levels of expression. In those cases where high levels of expression is not harmful to the plant, a strong promoter, a t-RNA or other pol II promoter, or a strong pol II promoter, such as the cauliflower mosaic virus promoter, can be used.

Alternatively, a plant promoter may be under environmental control. Such promoters are referred to here as "inducible" promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.

The promoters used in the present invention can be "tissue-specific" and, as such, under developmental control in that the polynucleotide is expressed only in certain tissues, such as leaves and seeds. In embodiments in which one or more nucleic acid sequences endogenous to the plant system are incorporated into the construct, the endogenous promoters (or variants thereof) from these genes can be employed for directing expression of the genes in the transfected plant. Tissue-specific promoters can also be used to direct expression of heterologous polynucleotides.

-54- WO 02/36782 PCT/US01/46227 In general, the particular promoter used in the expression cassette in plants depends on the intended application. Any of a number of promoters which direct transcription in plant cells are suitable. The promoter can be either constitutive or inducible. In addition to the promoters noted above, promoters of bacterial origin which operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids (see, Herrara-Estrella et al. (1983) Nature 303:209-213). Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812). Other plant promoters include the ribulose-l,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer (1988) EMBO J. 7:3315-3327.

To identify candidate promoters, the 5' portions of a genomic clone is analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) as described by Messing et al. (1983) Genetic Engineering in Plants, Kosage, et al. pp. 221-227.

In preparing polyucleotide constructs, vectors, of the invention, sequences other than the promoter and the cojoined polynucleotide can also be employed.

If normal polypeptide expression is desired, a polyadenylation region at the 3'-end of a GAT-encoding region can be included. The polyadenylation region can be derived, for example, from a variety of plant genes, or from T-DNA.

The construct can also include a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide tolerance, particularly antibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, such as tolerance to chlorosluforon, or phosphinothricin (the active ingredient in the herbicides bialaphos and Basta).

Specific initiation signals can aid in efficient translation of a GAT polynucleotide-encoding sequence of the present invention. These signals can include, the ATG initiation codon and adjacent sequences. In cases where a GAT polypeptide-encoding sequence, its initiation codon and upstream sequences are inserted into an appropriate expression vector, no additional translational control signals may be WO 02/36782 PCT/US01/46227 needed. However, in cases where only coding sequence a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf D et al. (1994) Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods in Enzymol 153:516-544).

Secretion/Localization Sequences Polynucleotides of the invention can also be fused, for example, in-frame to nucleic acids encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle of a mammalian cell, or to direct polypeptide secretion to the periplasmic space or into the cell culture media. Such sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences stop transfer sequences, GPI anchor sequences), and the like.

In a preferred embodiment, a polynucleotide of the invention is fused in frame with an N-terminal chloroplast transit sequence (or chloroplast transit peptide sequence) derived from a gene encoding a polypeptide that is normally targeted to the chloroplast. Such sequences are typically rich in serine and threonine; are deficient in aspartate, glutamate, and tyrosine; and generally have a central domain rich in positively charged amino acids.

Expression Hosts In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a eukaryotic cell, such as a mammalian cell, a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, or other common techniques (Davis, Dibner, and Battey, I. (1986) Basic Methods in Molecular Biology).

A host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired -56- WO 02/36782 PCT/US01/46227 fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves a "pre" or a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as E. coli, Bacillus sp., yeast or mammalian cells such as CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms, for post-translational activities and may be chosen to ensure the desired modification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression systems can be used. For example, plant cells, explants or tissues, e.g. shoots, leaf discs, which stably express a polypeptide of the invention are transduced using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for a period determined to be appropriate for the cell type, 1 or more hours for bacterial cells, 1-4 days for plant cells, 2-4 weeks for some plant explants, in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. For example, transgenic plants expressing the polypeptides of the invention can be selected directly for resistance to the herbicide, glyphosate. Resistant embryos derived from stably transformed explants can be proliferated, using tissue culture techniques appropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing GAT polynuclebtides of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.

Additional Polypeptide Sequences Polynucleotides of the present invention may also comprise a coding sequence fused in-frame to a marker sequence that, facilitates purification of the encoded polypeptide. Such purification facilitating domains include, but are not limited -57- WO 02/36782 PCT/US01/46227 to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; Wilson et al. (1984) Cell 37:767), maltose binding protein sequences, the FLAG epitope utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA), and the like. The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and the GAT homologue sequence is useful to facilitate purification. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site.

The histidine residues facilitate purification on IMDAC (immobilized metal ion affinity chromatography, as described in Porath et al. (1992) Protein Expression and Purification 3:263-281) while the enterokinase cleavage site provides a means for separating the GAT homologue polypeptide from the fusion protein. pGEX vectors (Promega; Madison, WI) may also be used to express foreign polypeptides as fusion proteins with glutathione Stransferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.

Polvpeptide Production and Recovery Following transduction of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well known to those skilled in the art.

As noted, many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin. See Sambrook, Ausubel, and Berger (all supra), as well as Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman and Company; and -58- WO 02/36782 PCT/US01/46227 Ricciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. For plant cell culture and regeneration, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); Jones, ed. (1984) Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, New Jersey and Plant Molecular Biolgy (1993) R.R.D.Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in general are set forth in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma- Aldrich, Inc (St Louis, MO) ("Sigma- LSRCCC") and, The Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, MO) ("Sigma-PCCS"). Further details regarding plant cell transformation and transgenic plant production are found below.

Polypeptides of the invention can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography using any of the tagging systems noted herein), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing the configuration of the mature protein.

Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps. In addition to the references noted supra, a variety of purification methods are well known in the art, including, those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al. (1996) Protein Methods, 2 nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3 d Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles. High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

-59- WO 02/36782 PCT/US01/46227 In some cases, it is desirable to produce the GAT polypeptide of the invention in a large scale suitable for industrial and/or commercial applications. In such cases bulk fermentation procedures are employed. Briefly, a GAT polynucleotide, a polynucleotide comprising any one of SEQ ID NOS: 1-5 and 11-262. or other nucleic acids encoding GAT polypeptides of the invention can be cloned into an expression vector. For example, U.S. Patent No. 5,955,310 to Widner et al. "METHODS FOR PRODUCING A POLYPEPTIDE IN A BACILLUS CELL," describes a vector with tandem promoters, and stabilizing sequences operably linked to a polypeptide encoding sequence. After inserting the polynucleotide of interest into a vector, the vector is tranformed into a bacterial, a Bacillus subtilis strain PL1801IIE (amyE, apr, npr, spoflE::Tn917) host. The introduction of an expression vector into a Bacillus cell may, for instance, be effected by protoplast transformation (see, Chang and Cohen (1979) Molecular General Genetics 168:111), by using competent cells (see, Young and Spizizin (1961) Journal of Bacteriology 81:823, or Dubnau and Davidoff-Abelson (1971) Journal of Molecular Biology 56:209), by electroporation (see, Shigekawa and Dower (1988) Biotechniques 6:742), or by conjugation (see, Koehler and Thome (1987) Journal of Bacteriology 169:5271), also Ausubel, Sambrook and Berger, all supra.

The transformed cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods that are known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions in catalogues of the American Type Culture Collection). The secreted polypeptide can be recovered directly from the medium.

The resulting polypeptide may be isolated by methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), WO 02/36782 PCT/US01/46227 electrophoretic procedures preparative isoelectric focusing), differential solubility ammonium sulfate precipitation), or extraction (see, Bollag et al. (1996) Protein Methods. 2 n d Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ; Bollag et al. (1996) Protein Methods. 2 n d Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ).

Cell-free transcription/translation systems can also be employed to produce polypeptides using DNAs or RNAs of the present invention. Several such systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tymms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology Volume 37, Garland Publishing, NY.

SUBSTRATES AND FORMATS FOR SEQUENCE RECOMBINATION The polynucleotides of the invention are optionally used as substrates for a variety of diversity generating procedures, mutation, recombination and recursive recombination reactions, in addition to their use in standard cloning methods as set forth in, Ausubel, Berger and Sambrook, to produce additional GAT polynucleotides and polypeptides with desired properties. A variety of diversity generating protocols are available and described in the art. The procedures can be used separately, and/or in combination to produce one or more variants of a polynucleotide or set of polynucleotides, as well variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified polynucleotides and sets of polynucleotides (including, polynucleotide libraries) useful, for the engineering or rapid evolution of polynucleotides, proteins, pathways, cells and/or organisms with new and/or improved characteristics. The process of altering the sequence can result in, for example, single nucleotide substitutions, multiple nucleotide substitutions, and insertion or deletion of regions of the nucleic acid sequence.

While distinctions and classifications are made in the course of the ensuing discussion for clarity, it will be appreciated that the techniques are often not mutually exclusive. Indeed, the various methods can be used singly or in combination, in parallel or in series, to access diverse sequence variants.

The result of any of the diversity generating procedures described herein can be the generation of one or more polynucleotides, which can be selected or screened for polynucleotides that encode proteins with or which confer desirable properties.

Following diversification by one or more of the methods herein, or otherwise available to -61- WO 02/36782 PCT/US01/46227 one of skill, any polynucleotides that are produced can be selected for a desired activity or property, e.g. altered Km for glyphosate, altered Km for acetyl CoA, use of alternative cofactors propionyl CoA) increased kcat, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable format, by any of the assays in the art. For example, GAT homologs with increased specific activity can be detected by assaying the conversion of glyphosate to N-acetylglyphosate, by mass spectrometry. Alternatively, improved ability to confer resistance to glyphosate can be assayed by growing bacteria transformed with a nucleic acid of the invention on agar containing increasing concentrations of glyphosate or by spraying transgenic plants incorporating a nucleic acid of the invention with glyphosate. A variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner. Additional details regarding recombination and selection for herbicide tolerance can be found, in "DNA SHUFFLING TO PRODUCE HERBICIDE RESISTANT CROPS" (USSN 09/373,333) filed August 12,1999.

Descriptions of a variety of diversity generating procedures, including family shuffling and methods for generating modified nucleic acid sequences encoding multiple enzymatic domains, are found the following publications and the references cited therein: Soong, N. et al. (2000) "Molecular breeding of viruses" Nat Genet 25(4):436-39; Stemmer, et al. (1999) "Molecular breeding of viruses for targeting and other clinical properties" Tumor Targeting 4:1-4; Ness et al. (1999) "DNA Shuffling of subgenomic sequences of subtilisin" Nature Biotechnology 17:893-896; Chang et al. (1999) "Evolution of a cytokine using DNA family shuffling" Nature Biotechnology 17:793-797; Minshull and Stemmer (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3:284-290; Christians et al. (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17:259-264; Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291; Crameri et al. (1997) "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotechnology 15:436-438; Zhang et al. (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad. Sci.

USA 94:4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling" Nature Medicine 2:100-103; Crameri et al. (1996) "Improved green fluorescent protein by -62- WO 02/36782 PCT/US01/46227 molecular evolution using DNA shuffling" Nature Biotechnology 14:315-319; Gates et al.

(1996) "Affinity selective isolation of ligands from peptide libraries through display on a lac repressor headpiece dimer"' Journal of Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology.

VCH Publishers, New York. pp.447-457; Crameri and Stemmer (1995) "Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioTechniques 18:194-195; Stemmer et al., (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxy-ribonucleotides" Gene, 164:49-53; Stemmer (1995) "The Evolution of Molecular Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370:389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution." Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example, sitedirected mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178; Dale et al. (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods Mol. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462; Botstein Shortle (1985) "Strategies and applications of in vitro mutagenesis" Science 229:1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987) 'The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids Molecular Biology (Eckstein, F. and Lilley, D.M.J. eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant Trp repressors with new DNA-binding specificities" Science 242:240-245); oligonucleotidedirected mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol.

154: 329-350 (1987); Zoller Smith (1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller Smith (1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors" Methods in Enzymol. 100:468-500; and Zoller Smith (1987) "Oligonucleotide- -63- WO 02/36782 PCT/US01/46227 directed mutagenesis: a simple method using two oligonucleotide primers and a singlestranded DNA template" Methods in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye Eckstein (1986) "Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14: 9679-9698; Sayers et al. (1988) "Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16:791- 802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide" Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al.

(1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids Res. 12: 9441-9456; Kramer Fritz (1987) Methods in Enzymol. "Oligonucleotide-directed construction of mutations via gapped duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations" Nucl. Acids Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro" Nucl. Acids Res.

16: 6987-6999).

Additional suitable methods include point mismatch repair (Kramer et al.

(1984) "Point Mismatch Repair" Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using M13 vectors" Methods in Enzymol. 154: 382- 403), deletion mutagenesis (Eghtedarzadeh Henikoff (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res. 14: 5115), restriction-selection and restrictionselection and restriction-purification (Wells et al. (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin)" Nucl. Acids Res. 14: -64- WO 02/36782 PCT/US01/46227 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites" Gene 34:315-323; and Grundstr6m et al. (1985) "Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis" Nucl.

Acids Res. 13: 3305-3316), double-strand break repair (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4:450- 455. "Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis" Proc. Natl. Acad. Sci. USA, 83:7177-7181).

Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can be found in the following U.S. patents, PCT publications, and EPO publications: U.S. Pat No. 5,605,793 to Stemmer (February 25, 1997), "Methods for In Vitro Recombination;" U.S. Pat No. 5,811,238 to Stemmer et al. (September 22, 1998) "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et al. (November 3, 1998), "DNA Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (November 10, 1998) "End-Complementary Polymerase Reaction;" U.S.

Pat. No. 5,837,458 to Minshull, et al. (November 17, 1998), "Methods and Compositions for Cellular and Metabolic Engineering;" WO 95/22625, Stemmer and Crameri, "Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods and Compositions for Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al. 'Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et al. "Antigen Library Immunization;" WO 99/41369 by Punnonen et al.

"Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et al. "Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al., "Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of Whole Cells and Organisms by WO 02/36782 PCT/US01/46227 Recursive Sequence Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/13487 by Stemmer et al., "Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling and Selection," WO 00/00632, "Methods for Generating Highly Diverse Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination of Polynucleotide Sequences Using Random or Defined Primers," WO 99/29902 by Arnold et al., "Method for Creating Polynucleotide and Polypeptide Sequences," WO 98/41653 by Vind, "An in Vitro Method for Construction of a DNA Library," WO 98/41622 by Borchert et al., "Method for Constructing a Library Using DNA Shuffling," and WO 98/42727 by Pati and Zarling, "Sequence Alterations using Homologous Recombination," WO 00/18906 by Patten et al., "Shuffling of Codon-Altered Genes;" WO 00/04190 by del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive Recombination;" WO 00/42561 by Crameri et al., "Oligonucleotide Mediated Nucleic Acid Recombination;" WO 00/42559 by Selifonov and Stemmer "Methods of Populating Data Structures for Use in Evolutionary Simulations;" WO 00/42560 by Selifonov et al., "Methods for Making Character Strings, Polynucleotides Polypeptides Having Desired Characteristics;" WO 01/23401 by Welch et al., "Use of Codon-Varied Oligonucleotide Synthesis for Synthetic Shuffling;" and PCT/US01/06775 "Single-Stranded Nucleic Acid Template-Mediated Recombination and Nucleic Acid Fragment Isolation" by Affholter.

Certain U.S. applications provide additional details regarding various diversity generating methods, including "SHUFFLING OF CODON ALTERED GENES" by Patten et al. filed September 28, 1999, (USSN 09/407,800); "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION", by del Cardayre et al. filed July 15, 1998 (USSN 09/166,188), and July 15, 1999 (USSN 09/354,922); "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., filed September 28, 1999 (USSN 09/408,392), and "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., filed January 18, 2000 (PCT/US00/01203); "USE OF CODON-BASED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al., filed September 28, 1999 (USSN 09/408,393); "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed January 18, 2000, (PCT/US00/01202) and, "METHODS FOR MAKING -66- WO 02/36782 PCT/US01/46227 CHARACTER STRINGS, POLYNUCLEOTIDES POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed July 18, 2000 (USSN 09/618,579); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer (PCT/US00/01138), filed January 18, 2000; and "SINGLE-STRANDED NUCLEIC ACID TEMPLATE- MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION" by Affholter (USSN 60/186,482, filed March 2, 2000).

In brief, several different general classes of sequence modification methods, such as mutation, recombination, etc. are applicable to the present invention and set forth, in the references above. That is, alterations to the component nucleic acid sequences to produced modified gene fusion constructs can be performed by any number of the protocols described, either before cojoining of the sequences, or after the cojoining step. The following exemplify some of the different types of preferred formats for diversity generation in the context of the present invention, including, certain recombination based diversity generation formats.

Nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids. For example, sexual PCR mutagenesis can be used in which random (or pseudo random, or even non-random) fragmentation of the DNA molecule is followed by recombination, based on sequence similarity, between DNA molecules with different but related DNA sequences, in vitro, followed by fixation of the crossover by extension in a polymerase chain reaction. This process and many process variants is described in several of the references above, in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751.

Similarly, nucleic acids can be recursively recombined in vivo, by allowing recombination to occur between nucleic acids in cells. Many such in vivo recombination formats are set forth in the references noted above. Such formats optionally provide direct recombination between nucleic acids of interest, or provide recombination between vectors, viruses, plasmids, etc., comprising the nucleic acids of interest, as well as other formats. Details regarding such procedures are found in the references noted above.

Whole genome recombination methods can also be used in which whole genomes of cells or other organisms are recombined, optionally including spiking of the genomic recombination mixtures with desired library components genes -67- WO 02/36782 PCT/US01/46227 O corresponding to the pathways of the present invention). These methods have many applications, including those in which the identity of a target gene is not known. Details on such methods are found, in WO 98/31837 by del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;" and in, e.g., PCT/US99/15972 by del Cardayre et al., also entitled "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination." Thus, any of these processes and Stechniques for recombination, recursive recombination, and whole genome recombination, alone or in combination, can be used to generate the modified nucleic acid sequences CI and/or modified gene fusion constructs of the present invention.

Synthetic recombination methods can also be used, in which C. oligonucleotides corresponding to targets of interest are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which correspond to more than one parental nucleic acid, thereby generating new recombined nucleic acids.

Oligonucleotides can be made by standard nucleotide addition methods, or can be made, by tri-nucleotide synthetic approaches. Details regarding such approaches are found in the references noted above, including, WO 00/42561 by Crameri et al., "Olgonucleotide Mediated Nucleic Acid Recombination;" WO 01/23401 by Welch et al., "Use of Codon-Varied Oligonucleotide Synthesis for Synthetic Shuffling;" WO 00/42560 by Selifonov et al., "Methods for Making Character Strings, Polynucleotides and Polypeptides Having Desired Characteristics;" and WO 00/42559 by Selifonov and Stemmer "Methods of Populating Data Structures for Use in Evolutionary Simulations." In silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which correspond to homologous (or even non-homologous) nucleic acids. The resulting recombined sequence stings are optionally converted into nucleic acids by synthesis of nucleic acids which correspond to the recombined sequences, in concert with oligonucleotide synthesis/ gene reassembly techniques. This approach can generate random, partially random or designed variants. Many details regarding in silico recombination, including the use of genetic algorithms, genetic operators and the like in computer systems, combined with generation of corresponding nucleic acids (and/or proteins), as well as combinations of designed nucleic acids and/or proteins based on cross-over site selection) as well as designed, pseudo-random or random recombination methods are described in WO 00/42560 by Selifonov et al., 'Methods for Making Character Strings, Polynucleotides and Polypeptides Having Desired Characteristics" and WO 00/42559 by Selifonov and -68- WO 02/36782 PCT/US01/46227 Stemmer "'Methods of Populating Data Structures for Use in Evolutionary Simulations." Extensive details regarding in silico recombination methods are found in these applications. This methodology is generally applicable to the present invention in providing for recombination of nucleic acid sequences and/or gene fusion constructs encoding proteins involved in various metabolic pathways (such as, for example, carotenoid biosynthetic pathways, ectoine biosynthetic pathways, polyhydroxyalkanoate biosynthetic pathways, aromatic polyketide biosynthetic pathways, and the like) in silico and/ or the generation of corresponding nucleic acids or proteins.

Many methods of accessing natural diversity, by hybridization of diverse nucleic acids or nucleic acid fragments to single-stranded templates, followed by polymerization and/or ligation to regenerate full-length sequences, optionally followed by degradation of the templates and recovery of the resulting modified nucleic acids can be similarly used. In one method employing a single-stranded template, the fragment population derived from the genomic library(ies) is annealed with partial, or, often approximately full length ssDNA or RNA corresponding to the opposite strand. Assembly of complex chimeric genes from this population is then mediated by nuclease-base removal of non-hybridizing fragment ends, polymerization to fill gaps between such fragments and subsequent single stranded ligation. The parental polynucleotide strand can be removed by digestion if RNA or uracil-containing), magnetic separation under denaturing conditions (if labeled in a manner conducive to such separation) and other available separation/purification methods. Alternatively, the parental strand is optionally co-purified with the chimeric strands and removed during subsequent screening and processing steps. Additional details regarding this approach are found, in "Single- Stranded Nucleic Acid Template-Mediated Recombination and Nucleic Acid Fragment Isolation" by Affholter, PCT/US01/06775.

In another approach, single-stranded molecules are converted to doublestranded DNA (dsDNA) and the dsDNA molecules are bound to a solid support by ligandmediated binding. After separation of unbound DNA, the selected DNA molecules are released from the support and introduced into a suitable host cell to generate a library enriched sequences which hybridize to the probe. A library produced in this manner provides a desirable substrate for further diversification using any of the procedures described herein.

Any of the preceding general recombination formats can be practiced in a reiterative fashion one or more cycles of mutation/recombination or other diversity -69- WO 02/36782 PCT/US01/46227 generation methods, optionally followed by one or more selection methods) to generate a more diverse set of recombinant nucleic acids.

Mutagenesis employing polynucleotide chain termination methods have also been proposed (see U.S. Patent No. 5,965,408, "Method of DNA reassembly by interrupting synthesis" to Short, and the references above), and can be applied to the present invention. In this approach, double stranded DNAs corresponding to one or more genes sharing regions of sequence similarity are combined and denatured, in the presence or absence of primers specific for the gene. The single stranded polynucleotides are then annealed and incubated in the presence of a polymerase and a chain terminating reagent ultraviolet, gamma or X-ray irradiation; ethidium bromide or other intercalators; DNA binding proteins, such as single strand binding proteins, transcription activating factors, or histones; polycyclic aromatic hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated polymerization mediated by rapid thermocycling; and the like), resulting in the production of partial duplex molecules. The partial duplex molecules, containing partially extended chains, are then denatured and reannealed in subsequent rounds of replication or partial replication resulting in polynucleotides which share varying degrees of sequence similarity and which are diversified with respect to the starting population of DNA molecules. Optionally, the products, or partial pools of the products, can be amplified at one or more stages in the process. Polynucleotides produced by a chain termination method, such as described above, are suitable substrates for any other described recombination format.

Diversity also can be generated in nucleic acids or populations of nucleic acids using a recombinational procedure termed "incremental truncation for the creation of hybrid enzymes" ('TCHY") described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology" Nature Biotech 17:1205.

This approach can be used to generate an initial a library of variants which can optionally serve as a substrate for one or more in vitro or in vivo recombination methods. See, also, Ostermeier et al. (1999) "Combinatorial Protein Engineering by Incremental Truncation," Proc. Natl. Acad. Sci. USA, 96: 3562-67; Ostermeier et al. (1999), "Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts," Biological and Medicinal Chemistry, 7: 2139-44.

Mutational methods which result in the alteration of individual nucleotides or groups of contiguous or non-contiguous nucleotides can be favorably employed to introduce nucleotide diversity into the nucleic acid sequences and/or gene fusion WO 02/36782 PCT/US01/46227 constructs of the present invention. Many mutagenesis methods are found in the abovecited references; additional details regarding mutagenesis methods can be found in following, which can also be applied to the present invention.

For example, error-prone PCR can be used to generate nucleic acid variants. Using this technique, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Examples of such techniques are found in the references above and, in Leung et al. (1989) Technique 1:11-15 and Caldwell et al.

(1992) PCR Methods Applic. 2:28-33. Similarly, assembly PCR can be used, in a process which involves the assembly of a PCR product from a mixture of small DNA fragments.

A large number of different PCR reactions can occur in parallel in the same reaction mixture, with the products of one reaction priming the products of another reaction.

Oligonucleotide directed mutagenesis can be used to introduce site-specific mutations in a nucleic acid sequence of interest. Examples of such techniques are found in the references above and, in Reidhaar-Olson et al. (1988) Science, 241:53-57.

Similarly, cassette mutagenesis can be used in a process that replaces a small region of a double stranded DNA molecule with a synthetic oligonucleotide cassette that differs from the native sequence. The oligonucleotide can contain, completely and/or partially randomized native sequence(s).

Recursive ensemble mutagenesis is a process in which an algorithm for protein mutagenesis is used to produce diverse populations of phenotypically related mutants, members of which differ in amino acid sequence. This method uses a feedback mechanism to monitor successive rounds of combinatorial cassette mutagenesis.

Examples of this approach are found in Arkin Youvan (1992) Proc. Natl. Acad. Sci.

USA 89:7811-7815.

Exponential ensemble mutagenesis can be used for generating combinatorial libraries with a high percentage of unique and functional mutants. Small groups of residues in a sequence of interest are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Examples of such procedures are found in Delegrave Youvan (1993) Biotechnology Research 11:1548- 1552.

In vivo mutagenesis can be used to generate random mutations in any cloned DNA of interest by propagating the DNA, in a strain of E. coli that carries mutations in one or more of the DNA repair pathways. These "mutator" strains have a -71- WO 02136782 WO 026782PUS01146227 higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA. Such procedures are described in the references noted above.

Other procedures for introducing diversity into a genome, e.g. a bacterial, fungal, animal or plant genome can be used in conjunction with the above described and/or referenced methods. For example, in addition to the methods above, techniques have been proposed which produce nucleic acid multimers suitable for transformation into a variety of species (see, Schellenberger U.S. Patent No. 5,756,316 and the references above). Transformation of a suitable host with such multimers, consisting of genes that are divergent with respect to one another, derived from natural diversity or through application of site directed mutagenesis, error prone PCR, passage through mutagenic bacterial strains, and the like), provides a source of nucleic acid diversity for DNA diversification, by an in vivo recombination process as indicated above.

Alternatively, a multiplicity of monomeric polynucleotides sharing regions of partial sequence similarity can be transformed into a host species and recombined in vivo by the host cell. Subsequent rounds of cell division can be used to generate libraries, members of which, include a single, homogenous population, or pool of monomeric polynucleotides. Alternatively, the monomeric nucleic acid can be recovered by standard techniques, PCR and/or cloning, and recombined in any of the recombination formats, including recursive recombination formats, described above.

Methods for generating multispecies expression libraries, have been described (in addition to the reference noted above, see, Peterson et al. (1998) U.S.

Pat. No. 5,783,431 "METHODS FOR GENERATING AND SCREENING NOVEL MEABOLIC PATHWAYS," and Thompson, et al. (1998) U.S. Pat. No. 5,824,485 METhiODS FOR GENERATING AND SCREENING NOVEL METABOLIC PATHWAYS) and their use to identify protein activities of interest has been proposed (In addition to the references noted above, see, Short (1999) U.S. Pat. No. 5,958,672 "PROTEIN ACTIVITY SCREENING OF CLONES HAVING DNA FROM UNCULTIVATED MICROORGANISMS"). Multispecies expression libraries include, in general, libraries comprising cDNA or genomnic sequences from a plurality of species or strains, operably linked to appropriate regulatory sequences, in an expression cassette.

The cDNA and/or genomic sequences are optionally randomly ligated to further enhance diversity. The vector can be a shuttle vector suitable for transformation and expression in more than one species of host organism, bacterial species, eukaryotic cells. In some 72 WO 02/36782 PCT/US01/46227 cases, the library is biased by preselecting sequences which encode a protein of interest, or which hybridize to a nucleic acid of interest. Any such libraries can be provided as substrates for any of the methods herein described.

The above described procedures have been largely directed to increasing nucleic acid and/ or encoded protein diversity. However, in many cases, not all of the diversity is useful, functional, and contributes merely to increasing the background of variants that must be screened or selected to identify the few favorable variants. In some applications, it is desirable to preselect or prescreen libraries an amplified library, a genomic library, a cDNA library, a normalized library, etc.) or other substrate nucleic acids prior to diversification, by recombination-based mutagenesis procedures, or to otherwise bias the substrates towards nucleic acids that encode functional products. For example, in the case of antibody engineering, it is possible to bias the diversity generating process toward antibodies with functional antigen binding sites by taking advantage of in vivo recombination events prior to manipulation by any of the described methods. For example, recombined CDRs derived from B cell cDNA libraries can be amplified and assembled into framework regions Jirholt et al. (1998) "Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework" Gene 215: 471) prior to diversifying according to any of the methods described herein.

Libraries can be biased towards nucleic acids which encode proteins with desirable enzyme activities. For example, after identifying a clone from a library which exhibits a specified activity, the clone can be mutagenized using any known method for introducing DNA alterations. A library comprising the mutagenized homologues is then screened for a desired activity, which can be the same as or different from the initially specified activity. An example of such a procedure is proposed in Short (1999) U.S.

Patent No. 5,939,250 for "PRODUCTION OF ENZYMES HAVING DESIRED ACTIVITIES BY MUTAGENESIS." Desired activities can be identified by any method known in the art. For example, WO 99/10539 proposes that gene libraries can be screened by combining extracts from the gene library with components obtained from metabolically rich cells and identifying combinations which exhibit the desired activity. It has also been proposed WO 98/58085) that clones with desired activities can be identified by inserting bioactive substrates into samples of the library, and detecting bioactive fluorescence corresponding to the product of a desired activity using a fluorescent analyzer, a flow cytometry device, a CCD, a fluorometer, or a spectrophotometer.

73 WO 02/36782 PCT/US01/46227 Libraries can also be biased towards nucleic acids which have specified characteristics, hybridization to a selected nucleic acid probe. For example, application WO 99/10539 proposes that polynucleotides encoding a desired activity an enzymatic activity, for example: a lipase, an esterase, a protease, a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, a hydratase, a nitrilase, a transaminase, an amidase or an acylase) can be identified from among genomic DNA sequences in the following manner. Single stranded DNA molecules from a population of genomic DNA are hybridized to a ligand-conjugated probe. The genomic DNA can be derived from either a cultivated or uncultivated microorganism, or from an environmental sample. Alternatively, the genomic DNA can be derived from a multicellular organism, or a tissue derived therefrom. Second strand synthesis can be conducted directly from the hybridization probe used in the capture, with or without prior release from the capture medium or by a wide variety of other strategies known in the art. Alternatively, the isolated single-stranded genomic DNA population can be fragmented without further cloning and used directly in, a recombination-based approach, that employs a single-stranded template, as described above.

"Non-Stochastic" methods of generating nucleic acids and polypeptides are alleged in Short "Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344. These methods, including proposed non-stochastic polynucleotide reassembly and site-saturation mutagenesis methods be applied to the present invention as well.

Random or semi-random mutagenesis using doped or degenerate oligonucleotides is also described in, Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10:297-300; Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods Enzymol. 208:564-86; Lim and Sauer (1991) "The role of internal packing interactions in determining the structure and stability of a protein" J. Mol. Biol. 219:359-76; Breyer and Sauer (1989) "Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambda repressor" J. Biol. Chem.

264:13355-60); and "Walk-Through Mutagenesis" (Crea, R; US Patents 5,830,650 and 5,798,208, and EP Patent 0527809 B1.

It will readily be appreciated that any of the above described techniques suitable for enriching a library prior to diversification can also be used to screen the products, or libraries of products, produced by the diversity generating methods. Any of -74- WO 02/36782 PCT/US01/46227 the above described methods can be practiced recursively or in combination to alter nucleic acids, GAT encoding polynucleotides.

Kits for mutagenesis, library construction and other diversity generation methods are also commercially available. For example, kits are available from, e.g., Stratagene QuickChange M site-directed mutagenesis kit; and Chameleon T doublestranded, site-directed mutagenesis kit), Bio/Can Scientific, Bio-Rad using the Kunkel method described above), Boehringer Mannheim Corp., Clonetech Laboratories, DNA Technologies, Epicentre Technologies 5 prime 3 prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs, Pharmacia Biotech, Promega Corp., Quantum Biotechnologies, Amersham International plc using the Eckstein method above), and Anglian Biotechnology Ltd using the Carter/Winter method above).

The above references provide many mutational formats, including recombination, recursive recombination, recursive mutation and combinations or recombination with other forms of mutagenesis, as well as many modifications of these formats. Regardless of the diversity generation format that is used, the nucleic acids of the present invention can be recombined (with each other, or with related (or even unrelated) sequences) to produce a diverse set of recombinant nucleic acids for use in the gene fusion constructs and modified gene fusion constructs of the present invention, including, e.g., sets of homologous nucleic acids, as well as corresponding polypeptides.

Many of the above-described methodologies for generating modified polynucleotides generate a large number of diverse variants of a parental sequence or sequences. In some preferred embodiments of the invention the modification technique some form of shuffling) is used to generate a library of variants that is then screened for a modified polynucleotide or pool of modified polynucleotides encoding some desired functional attribute, improved GAT activity. Exemplary enzymatic activities that can be screened for include catalytic rates (conventionally characterized in terms of kinetic constants such as k1 and KM, substrate specificity, and susceptibility to activation or inhibition by substrate, product or other molecules inhibitors or activators).

One example of selection for a desired enzymatic activity entails growing host cells under conditions that inhibit the growth and/or survival of cells that do not sufficiently express an enzymatic activity of interest, e.g. the GAT activity. Using such a selection process can eliminate from consideration all modified polynucleotides except those encoding a desired enzymatic activity. For example, in some embodiments of the WO 02136782 PCT/US01/46227 O invention host cells are maintained under conditions that inhibit cell growth or survival in Jthe absence of sufficient levels of GAT, a concentration of glyphosate that is lethal or inhibits the growth of a wild-type plant of the same variety that lack does not express GAT polynucleotide. Under these conditions, only a host cell harboring a modified nucleic acid that encodes enzymatic activity or activities able to catalyze production of sufficient levels of the product will survive and grow. Some embodiments of the invention employ c multiples rounds of screening at increasing concentrations of glyphosate or a glyphosate analog.

C In some embodiments of the invention, mass spectrometry is used to detect the acetylation of glyphosate, or a glyphosate analog or metabolite. The used of mass C spectrometry is described in more detail in the Examples below.

For convenience and high throughput it will often be desirable to screen/select for desired modified nucleic acids in a microorganism, a bacteria such as E. coli. On the other hand, screening in plant cells or plants can will in some cases be preferable where the ultimate aim is to generate a modified nucleic acid for expression in a plant system.

In some preferred embodiments of the invention throughput is increased by screening pools of host cells expressing different modified nucleic acids, either alone or as part of a gene fusion construct Any pools showing significant activity can be deconvoluted to identify single clones expressing the desirable activity.

The skilled artisan will recognize that the relevant assay, screening or selection method will vary depending upon the desired host organism, etc. It is normally advantageous to employ an assay that can be practiced in a high-throughput format.

In high through put assays, it is possible to screen up to several thousand different variants in a single day. For example, each well of a microtiter plate can be used to run a separate assay, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single variant.

In addition to fluidic approaches, it is possible, as mentioned above, simply to grow cells on media plates that select for the desired enzymatic or metabolic function.

This approach offers a simple and high-throughput screening method.

A number of well known robotic systems have also been developed for solution phase chemistries useful in assay systems. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate -76-

I

WO 02/36782 PCT/US01/46227 II, Zymark Corporation, Hopkinton, MA.; Orca, Hewlett-Packard, Palo Alto, CA) which mimic the manual synthetic operations performed by a scientist. Any of the above devices are suitable for application to the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein with reference to the integrated system will be apparent to persons skilled in the relevant art.

High throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization.

The manufacturers of such systems provide detailed protocols for the various high throughput devices. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. Microfluidic approaches to reagent manipulation have also been developed, by Caliper Technologies (Mountain View, CA).

Optical images viewed (and, optionally, recorded) by a camera or other recording device a photodiode and data storage device) are optionally further processed in any of the embodiments herein, by digitizing the image and/or storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, using PC (Intel x86 or pentium chip compatible DOS T M

OS

T M

WINDOWS

T M WINDOWS NT T m or WINDOWS 95 TM based machines), MACINTOSHFM, or UNIX based SUN T M work station) computers.

One conventional system carries light from the assay device to a cooled charge-coupled device (CCD) camera, a common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD.

Particular pixels corresponding to regions of the specimen individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g. by fluorescent or dark field microscopic techniques.

-77- WO 02136782 PCT/US01/46227 OTHER POLYNUCLEOTIDE COMPOSITIONS The invention also includes compositions comprising two or more polynucleotides of the invention as substrates for recombination). The composition can comprise a library of recombinant nucleic acids, where the library contains at least 2, 3, 5, 10, 20, or 50 or more polynucleotides. The polynucleotides are optionally cloned into expression vectors, providing expression libraries.

The invention also includes compositions produced by digesting one or more polynucleotide of the invention with a restriction endonuclease, an RNAse, or a DNAse as is performed in certain of the recombination formats noted above); and compositions produced by fragmenting or shearing one or more polynucleotide of the invention by mechanical means sonication, vortexing, and the like), which can also be used to provide substrates for recombination in the methods above. Similarly, compositions comprising sets of oligonucleotides corresponding to more than one nucleic acid of the invention are useful as recombination substrates and are a feature of the invention. For convenience, these fragmented, sheared, or oligonucleotide synthesized mixtures are referred to as fragmented nucleic acid sets.

Also included in the invention are compositions produced by incubating one or more of the fragmented nucleic acid sets in the presence of ribonucleotide- or deoxyribonucelotide triphosphates and a nucleic acid polymerase. This resulting composition forms a recombination mixture for many of the recombination formats noted above. The nucleic acid polymerase may be an RNA polymerase, a DNA polymerase, or an RNA-directed DNA polymerase a "reverse transcriptase"); the polymerase can be, a thermostable DNA polymerase (such as, VENT, TAQ, or the like).

INTEGRATED SYSTEMS The present invention provides computers, computer readable media and integrated systems comprising character strings corresponding to the sequence information herein for the polypeptides and nucleic acids herein, including, those sequences listed herein and the various silent substitutions and conservative substitutions thereof.

For example, various methods and genetic algorithms (GAs) known in the art can be used to detect homology or similarity between different character strings, or can be used to perform other desirable functions such as to control output files, provide the basis for making presentations of information including the sequences and the like.

Examples include BLAST, discussed supra.

-78- WO 02/36782 WO 0236782PCT[USOI146227 Thus, different types of homology and similarity of various stringency and length can be detected and recognized in the integrated systems herein. For example, many homology determination methods have been designed for comparative analysis of sequences of biopolyiners, for spell-checking in word processing, and for data retrieval from various databases. With an understanding of double-helix pair-wise complement interactions among 4 principal nucleobases in natural polynucleotides, models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings corresponding to the sequences herein word-processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.). An example of a software package with GAs for calculating sequence similarity is BLAST, which can be adapted to the present invention by inputting character strings corresponding to the sequences herein.

Similarly, standard desktop applications such as word processing software Microsoft Word-1 or Corel WordPerfectT4) and database software spreadsheet software such as Microsoft ExcelPm, Corel Quattro Prom, or database programs such as Microsoft Accessm or Paradox~m) can be adapted to the present invention by inputting a character string corresponding to the GAT homologues of the invention (either nucleic acids or proteins, or both). For example, the integrated systems can include the foregoing software having the appropriate character string information, used in conjunction with a user interface a GUI in a standard operating system such as a Windows, Macintosh or LINUX system) to manipulate strings of characters. As noted, specialized alignment programs such as BLAST can also be incorporated into the systems of the invention for alignment of nucleic acids or proteins (or corresponding character strings).

Integrated systems for analysis in the present invention typically include a digital computer with GA software for aligning sequences, as well as data sets entered into the software system comprising any of the sequences herein. The computer can be, a PC (Intel x86 or Pentium chip- compatible DOS~m, OS2Tm WINDOWS'm WINDOWS NT~m, WlNDOWS95Tm, WINDOWS98Tm LINUX based machine, a MACINTOSHTm, Power PC, or a UNIX based SUN~m work station) machine) or other commercially common computer which is known to one of skill. Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, Fortran, Basic, Java, or the like.

79 WO 02/36782 PCT/US01/46227 Any controller or computer optionally includes a monitor which is often a cathode ray tube display, a flat panel display active matrix liquid crystal display, liquid crystal display), or others. Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements. Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.

The computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, in a GUI, or in the form of preprogrammed instructions, preprogrammed for a variety of different specific operations. The software then converts these instructions to appropriate language for instructing the operation of the fluid direction and transport controller to carry out the desired operation.

The software can also include output elements for controlling nucleic acid synthesis based upon a sequence or an alignment of a sequences herein) or other operations which occur downstream from an alignment or other operation performed using a character string corresponding to a sequence herein. Nucleic acid synthesis equipment can, accordingly, be a component in one or more integrated systems herein.

In an additional aspect, the present invention provides kits embodying the methods, composition, systems and apparatus herein. Kits of the invention optionally comprise one or more of the following: an apparatus, system, system component or apparatus component as described herein; instructions for practicing the methods described herein, and/or for operating the apparatus or apparatus components herein and/or for using the compositions herein; one or more GAT composition or component; a container for holding components or compositions, and, packaging materials.

In a further aspect, the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of.any apparatus or kit to practice any assay or method herein.

80 WO 02/36782 PCT/US01/46227 HOST CELLS AND ORGANISMS The host cell can be eukaryotic, for example, a eukaryotic cell, a plant cell, an animal cell, a protoplast, or a tissue culture. The host cell optionally comprises a plurality of cells, for example, an organism. Alternatively, the host cell can be prokaryotic including, but not limited to, bacteria gram positive bacteria, purple bacteria, green sulfur bacteria, green non-sulfur bacteria, cyanobacteria, spirochetes, thermatogales, flavobacteria, and bacteroides) and archaebacteria Korarchaeota, Thermoproteus, Pyrodictium, Thermococcales, methanogens, Archaeoglobus, and extreme halophiles).

Transgenic plants, or plant cells, incorporating the GAT nucleic acids, and/or expressing the GAT polypeptides of the invention are a feature of the invention.

The transformation of plant cells and protoplasts can be carried out in essentially any of the various ways known to those skilled in the art of plant molecular biology, including, but not limited to, the methods described herein. See, in general, Methods in Enzymolovy, Vol. 153 (Recombinant DNA Part D) Wu and Grossman (eds.) 1987, Academic Press, incorporated herein by reference. As used herein, the term "transformation" means alteration of the genotype of a host plant by the introduction of a nucleic acid sequence, a "heterologous" or "foreign" nucleic acid sequence. The heterologous nucleic acid sequence need not necessarily originate from a different source but it will, at some point, have been external to the cell into which is introduced.

In addition to Berger, Ausubel and Sambrook, useful general references for plant cell cloning, culture and regeneration include Jones (ed) (1995) Plant Gene Transfer and Expression Protocols- Methods in Molecular Biology, Volume 49 Humana Press Towata NJ; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley Sons, Inc. New York, NY (Payne); and Gamborg and Phillips (eds) (1995) Plant Cell. Tissue and Organ Culture: Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) (Gamborg). A variety of cell culture media are described in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL (Atlas). Additional information for plant cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma- Aldrich, Inc (St Louis, MO) (Sigma-LSRCCC) and, the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, MO) (Sigma-PCCS). Additional details regarding plant cell culture are found in Croy, (ed.) (1993) Plant Molecular Biology Bios Scientific Publishers, Oxford, U.K -81- WO 02/36782 PCT/US01/46227 0 In an embodiment of this invention, recombinant vectors including one or more GAT polynucleotides, suitable for the transformation of plant cells are prepared. A ;Z DNA sequence encoding for the desired GAT polypeptide, selected from among SEQ SID NOS: 1-5 and 11-262, is conveniently used to construct a recombinant expression cassette which can be introduced into the desired plant In the context of the present -C invention, an expression cassette will typically comprise a selected GAT polynucleotide c operably linked to a promoter sequence and other transcriptional and translational initiation regulatory sequences which are sufficient to direct the transcription of the GAT sequence in the intended tissues entire plant, leaves, roots, etc.) of the transformed plant.

For example, a strongly or weakly constitutive plant promoter that directs expression of a GAT nucleic acid in all tissues of a plant can be favorably employed.

Such promoters are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the or promoter of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Where overexpression of a GAT polypeptide of the invention is detrimental to the plant, one of skill, will recognize that weak constitutive promoters can be used for low-levels of expression. In those cases where high levels of expression is not harmful to the plant, a strong promoter, a t-RNA, or other pol II promoter, or a strong pol II promoter, the cauliflower mosaic virus promoter, CaMV, promoter) can be used.

Alternatively, a plant promoter can be under environmental control. Such promoters are referred to as "inducible" promoters. Examples of environmental conditions that may alter transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. In some cases, it is desirable to use promoters that are "tissue-specific" and/or are under developmental control such that the GAT polynucleotide is expressed only in certain tissues or stages of development, e.g., leaves, roots, shoots, etc. Endogenous promoters of genes related to herbicide tolerance and related phenotypes are particularly useful for driving expression of GAT nucleic acids, P450 monooxygenases, glutathione-S-transferases, homoglutathione-S-transferases, glyphosate oxidases and 5-enolpyruvylshikimate-2-phosphate synthases.

Tissue specific promoters can also be used to direct expression of heterologous structural genes, including the GAT polynucleotides described herein. Thus the promoters can be used in recombinant expression cassettes to drive expression of any -82- WO 02/36782 PCT/US01/46227 gene whose expression is desirable in the transgenic plants of the invention, GAT and/or other genes conferring herbicide resistance or tolerance, genes which influence other useful characteristics, heterosis. Similarly, enhancer elements, derived from the 5' regulatory sequences or intron of a heterologous gene, can also be used to improve expression of a heterologous structural gene, such as a GAT polynucleotide.

In general, the particular promoter used in the expression cassette in plants depends on the intended application. Any of a number of promoters which direct transcription in plant cells can be suitable. The promoter can be either constitutive or inducible. In addition to the promoters noted above, promoters of bacterial origin which operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids. See, Herrera-Estrella et al. (1983) Nature 303:209. Viral promoters include the 35S and 19S RNA promoters of CaMV. See, Odell et al., (1985) Nature 313:810. Other plant promoters include the ribulose-1,3bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene (see, Deikman and Fischer (1988) EMBO J 7:3315) and other genes are also favorably used. Promoters specific for monocotyledonous species are also considered (McElroy Brettell R.LS. 1994. Foreign gene expression in transgenic cereals. Trends Biotech., 12:62-68.) Alternatively, novel promoters with useful characteristics can be identified from any viral, bacterial, or plant source by methods, including sequence analysis, enhancer or promoter trapping, and the like, known in the art.

In preparing expression vectors of the invention, sequences other than the promoter and the GAT encoding gene are also favorably used. If proper polypeptide expression is desired, a polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. Signal/localization peptides, which, e.g., facilitate translocation of the expressed polypeptide to internal organelles chloroplasts) or extracellular secretion, can also be employed.

The vector comprising the GAT polynucleotide also can include a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide tolerance, particularly antibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, such as tolerance to chlorosulfuron, or phophinothricin. Reporter genes, which are used to monitor gene expression and protein localization via visualizable reaction products beta-glucuronidase, beta- -83- WO 02/36782 PCT/US01/46227 galactosidase, and chloramphenicol acetyltransferase) or by direct visualization of the gene product itself green fluorescent protein, GFP; Sheen et al. (1995) The Plant Journal 8:777) can be used for, monitoring transient gene expression in plant cells.

Transient expression systems can be employed in plant cells, for example, in screening plant cell cultures for herbicide tolerance activities.

PLANT TRANSFORMATION Protoplasts Numerous protocols for establishment of transformable protoplasts from a variety of plant types and subsequent transformation of the cultured protoplasts are available in the art and are incorporated herein by reference. For examples, see, Hashimoto et al. (1990) Plant Physiol. 93:857; Fowke and Constabel (eds)(1994) Plant Protoplasts; Saunders et al. (1993) Applications of Plant In Vitro Technology Symposium, UPM 16-18; and Lyznik et al. (1991) BioTechniaues 10:295, each of which is incorporated herein by reference.

Chloroplasts Chloroplasts are a site of action of some herbicide tolerance activities, and, in some instances, the GAT polynucleotide is fused to a chloroplast transit sequence peptide to facilitate translocation of the gene products into the chloroplasts. In these cases, it can be advantageous to transform the GAT polynucleotide into the chloroplasts of the plant host cells. Numerous methods are available in the art to accomplish chloroplast transformation and expression Daniell et al. (1998) Nature Biotechnology 16:346; O'Neill et al. (1993) The Plant Journal 3:729; Maliga (1993) TIBTECH 11:1). The expression construct comprises a transcriptional regulatory sequence functional in plants operably linked to a polynucleotide encoding the GAT polypeptide. Expression cassettes that are designed to function in chloroplasts (such as an expression cassette including a GAT polynucleotide) include the sequences necessary to ensure expression in chloroplasts. Typically, the coding sequence is flanked by two regions of homology to the chloroplastid genome to effect a homologous recombination with the chloroplast genome; often a selectable marker gene is also present within the flanking plastid DNA sequences to facilitate selection of genetically stable transformed chloroplasts in the resultant transplastonic plant cells (see, Maliga (1993) and Daniell (1998), and references cited therein).

-84- WO 02/36782 PCT/US01/46227 General transformation methods DNA constructs of the invention can be introduced into the genome of the desired plant host by a variety of conventional techniques. Techniques for tranforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, Payne, Gamborg, Croy, Jones, etc. all supra, as well as, e.g., Weising et al. (1988) Ann. Rev. Genet. 22:421.

For example, DNAs can be introduced directly into the genomic DNA of a plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA constructs can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the plant cell is infected by the bacteria.

Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al (1984) EMBO J 3:2717.

Electroporation techniques are described in Fromm et al. (1985) Proc Nat'l Acad Sci USA 82:5824. Ballistic transformation techniques are described in Klein et al. (1987) Nature 327:70; and Weeks et al. Plant Physiol 102:1077.

In some embodiments, Agrobacterium mediated transformation techniques are used to transfer the GAT sequences of the invention to transgenic plants.

Agrobacterium-mediated transformation is widely used for the transformation of dicots, however, certain monocots can also be transformed by Agrobacterium. For example, Agrobacterium transformation of rice is described by Hiei et al. (1994) Plant J. 6:271; US Patent No. 5,187,073; US Patent No. 5,591,616; Li et al. (1991) Science in China34:54; and Raineri et al. (1990) Bio/Technology 8:33. Transformed maize, barley, triticale and asparagus by Agrobacterium mediated transformation have also been described (Xu et al.

(1990) Chinese J Bot 2:81).

Agrobacterium mediated transformation techniques take advantage of the ability of the tumor-inducing (Ti) plasmid of A tumefaciens to integrate into a plant cell genome, to co-transfer a nucleic acid of interest into a plant cell. Typically, an expression vector is produced wherein the nucleic acid of interest, such as a GAT polynucleotide of the invention, is ligated into an autonomously replicating plasmid which also contains T- WO 02/36782 PCT/US01/46227 DNA sequences. T-DNA sequences typically flank the expression casssette nucleic acid of interest and comprise the integration sequences of the plasmid. In addition to the expression cassette, T-DNA also typically include a marker sequence, antibiotic resistance genes. The plasmid with the T-DNA and the expression cassette are then transfected into Agrobacterium cells. Typically, for effective tranformation of plant cells, the A. tumefaciens bacterium also possesses the necessary vir regions on a plasmid, or integrated into its chromosome. For a discussion of Agrobacterium mediated transformation, see, Firoozabady and Kuehnle, (1995) Plant Cell Tissue and Organ Culture Fundamental Methods, Gamborg and Phillips (eds.).

Regeneration of Transgenic Plants Transformed plant cells which are derived by plant transformation techniques, including those discussed above, can be cultured to regenerate a whole plant which possesses the transformed genotype a GAT polynucleotide), and thus the desired phenotype, such as acquired resistance tolerance) to glyphosate or a glyphosate analog. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Alternatively, selection for glyphosate resistance conferred by the GAT polynucleotide of the invention can be performed. Plant regeneration from cultured protoplasts is described in Evans et al. (1983) Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp 124-176, Macmillan Publishing Company, New York; and Binding (1985) Regeneration of Plants, Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann Rev of Plant Phys 38:467. See also, Payne and Gamborg. After transformation with Agrobacterium, the explants typically are transferred to selection medium. One of skill will realize that the selection medium depends on the selectable marker that was cotransfected into the explants. After a suitable length of time, transformants will begin to form shoots. After the shoots are about 1-2 cm in length, the shoots should be transferred to a suitable root and shoot medium. Selection pressure should be maintained in the root and shoot medium.

Typically, the transformants will develop roots in about 1-2 weeks and form plantlets. After the plantlets are about 3-5 cm in height, they are placed in sterile soil in fiber pots. Those of skill in the art will realize that different acclimation procedures are 86 WO 02/36782 PCT/US01/46227 used to obtain transformed plants of different species. For example, after developing a root and shoot, cuttings, as well as somatic embryos of transformed plants, are transferred to medium for establishment of plantlets. For a description of selection and regeneration of transformed plants, see, Dodds and Roberts (1995) Experiments in Plant Tissue Culture, 3 r Ed., Cambridge University Press.

There are also methods for Agrobacterium transformation of Arabidopsis using vacuum infiltration (Bechtold Ellis J. and Pelletier 1993, In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.

CR Acad Sci Paris Life Sci 316:1194-1199) and simple dipping of flowering plants (Desfeux, Clough and Bent 2000, Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123:895-904). Using these methods, transgenic seed are produced without the need for tissue culture.

There are plant varieties for which effective Agrobacterium-mediated transformation protocols have yet to be developed. For example, successful tissue transformation coupled with regeneration of the transformed tissue to produce a transgenic plant has not been reported for some of the most commercially relevant cotton cultivars.

Nevertheless, an approach that can be used with these plants involves stably introducing the polynucleotide into a related plant variety via Agrobacterium-mediated transformation, confirming operability, and then transferring the transgene to the desired commercial strain using standard sexual crossing or back-crossing techniques. For example, in the case of cotton, Agrobacterium can be used to transform a Coker line of Gossypiumn hirustum Coker lines 310, 312, 5110 Deltapine 61 or Stoneville 213), and then the transgene can be introduced into another more commercially relevant G. hirustum cultivar by back-crossing.

The transgenic plants of this invention can be characterized either genotypically or phenotypically to determine the presence of the GAT polynucleotide of the invention. Genotypic analysis can be performed by any of a number of well-known techniques, including PCR amplification of genomic DNA and hybridization of genomic DNA with specific labeled probes. Phenotypic analysis includes, survival of plants or plant tissues exposed to a selected herbicide such as glyphosate.

Essentially any plant can be transformed with the GAT polynucleotides of the invention. Suitable plants for the transformation and expression of the novel GAT polynucleotides of this invention include agronomically and horticulturally important -87- WO 02/36782 PCT/US01/46227 species. Such species include, but are not restricted to members of the families: Graminae (including corn, rye, triticale, barley, millet, rice, wheat, oats, etc.); Leguminosae S(including pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, Salfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea); Compositae (the largest family of vascular plants, including at least 1,000 genera, including important commercial crops such as sunflower) and Rosaciae (including raspberry, apricot, almond, peach, rose, C etc.), as well as nut plants (including, walnut, pecan, hazelnut, etc.), and forest trees I¢n (including Pinus, Quercus, Pseutotsuga, Sequoia, Populus,etc.) 0 C1 Additional targets for modification by the GAT polynucleotides of the 0 10 invention, as well as those specified above, include plants from the genera: Agrostis, C Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena oats), Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Gossypium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum barley), Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza rice), Panicum, Pelargonium, Pennisetum millet), Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale rye), Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum wheat), Vicia, Vigna, Vitis, Zea corn), and the Olyreae, the Pharoideae and many others. As noted, plants in the family Graminae are a particularly target plants for the methods of the invention.

Common crop plants which are targets of the present invention include corn, rice, triticale, rye, cotton, soybean, sorghum, wheat, oats, barley, millet, sunflower, canola, peas, beans, lentils, peanuts, yam beans, cowpeas, velvet beans, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweetpea and nut plants walnut, pecan, etc).

In one aspect, the invention provides a method for producing a crop by growing a crop plant that is glyphosate-tolerant as a result of being transformed with a gene encoding a glyphosate N-acteyltransferase, under conditions such that the crop plant produces a crop, and harvesting the crop. Preferably, glyphosate is applied to the plant, or in the vicinity of the plant, at a concentration effective to control weeds without preventing the transgenic crop plant from growing and producing the crop. The application of -88- WO 02/36782 PCT/US01/46227 glyphosate can be before planting, or at any time after planting up to and including the time of harvest. Glyphosate can be applied once or multiple times. The timing of glyphosate application, amount applied, mode of application, and other parameters will vary based upon the specific nature of the crop plant and the growing environment, and can be readily determined by one of skill in the art. The invention further provides the crop produced by this method.

The invention provides for the propagation of a plant containing a GAT polynucleotide transgene. The plant can be, for example, a monocot or a dicot. In one aspect, propagation entails crossing a plant containing a GAT polynucleotide transgene with a second plant, such that at least some progeny of the cross display glyphosate tolerance.

In one aspect, the invention provides a method for selectively controlling weeds in a field where a crop is being grown. The method involves planting crop seeds or plants that are glyphosate-tolerant as a result of being transformed with a gene encoding a GAT, a GAT polynucleotide, and applying to the crop and any weeds a sufficient amount of glyphosate to control the weeds without a significant adverse impact on the crops. It is important to note that it is not necessary for the crop to be totally insensitive to the herbicide, so long as the benefit derived from the inhibition of weeds outweighs any negative impact of the glyphosate or glyphosate analog on the crop or crop plant.

In another aspect, the invention provides for use of a GAT polynucleotide as a selectable marker gene. In this embodiment of the invention, the presence of the GAT polynucleotide in a cell or organism confers upon the cell or organism the detectable phenotypic trait of glyphosate resistance, thereby allowing one to select for cells or organisms that have been transformed with a gene of interest linked to the GAT polynucleotide. Thus, for example, the GAT polynucleotide can be introduced into a nucleic acid construct, a vector, thereby allowing for the identification of a host a cell or transgenic plant) containing the nucleic acid construct by growing the host in the presence of glyphosate and selecting for the ability to survive and/or grow at a rate that is discernibly greater than a host lacking the nucleic acid construct would survive or grow.

A GAT polynucleotide can be used as a selectable marker in a wide variety of hosts that are sensitive to glyphosate, including plants, most bacteria (including E. coli), actinomycetes, yeasts, algae and fungi. One benefit of using herbicide resistance as a marker in plants, as opposed to conventional antibiotic resistance, is that it obviates the concern of some members of the public that antibiotic resistance might escpe into the -89- WO 02/36782 PCT/US01/46227 environment. Some experimental data from experiments demonstrating the use of a GAT polynucleotide as a selectable marker in diverse host systems are described in the Examples section of this specification.

Selection of gat polvnucleotides conferring enhanced glvphosate resistance in transgenic plants.

Libraries of GAT encoding nucleic acids diversified according to the methods described herein can be selected for the ability to confer resistance to glyphosate in transgenic plants. Following one or more cycles of diversification and selection, the modified GAT genes can be used as a selection marker to facilitate the production and evaluation of transgenic plants and as a means of conferring herbicide resistance in experimental or agricultural plants. For example, after diversification of any one or more of SEQ ID NO: 1 to SEQ ID NO:5 to produce a library of diversified GAT polynucleotides, an initial functional evaluation can be performed by expressing the library of GAT encoding sequences in E. coli. The expressed GAT polypeptides can be purified, or partially purified as described above, and screened for improved kinetics by mass spectrometry. Following one or more preliminary rounds of diversification and selection, the polynucleotides encoding improved GAT polypeptides are cloned into a plant expression vector, operably linked to, a strong constitutive promoter, such as the CaMV 35S promoter. The expression vectors comprising the modified GAT nucleic acids are transformed, typically by Agrobacterium mediated transformation, into Arabidopsis thaliana host plants. For example, Arabidopsis hosts are readily transformed by dipping inflorescences into solutions of Agrobacterium and allowing them to grow and set seed.

Thousands of seeds are recovered in approximately 6 weeks. The seeds are then collected in bulk from the dipped plants and germinated in soil. In this manner it is possible to generate several thousand independently transformed plants for evaluation, constituting a high throughput (HTP) plant transformation format. Bulk grown seedlings are sprayed with glyphosate and surviving seedlings exhibiting glyphosate resistance survive the selection process, whereas non-transgenic plants and plants incorporating less favorable modified GAT nucleic acids are damaged or killed by the herbicide treatment Optionally, the GAT encoding nucleic acids conferring improved resistance to glyphosate are recovered, by PCR amplification using T-DNA primers flanking the library inserts, and used in further diversification procedures or to produce additional transgenic plants of the same or different species. If desired, additional rounds of diversification and selection WO 02/36782 PCT/US01/46227 can be performed using increasing concentrations of glyphosate in each subsequent selection. In this manner, GAT polynucleotides and polypeptides conferring resistance to concentrations of glyphosate useful in field conditions can be obtained.

Herbicide Resistance The mechanism of glyphosate resistance of the present invention can be combined with other modes of glyphosate resistance known in the art to produce plants and plant explants with superior glyphosate resistance. For example, glyphosate-tolerant plants can be produced by inserting into the genome of the plant the capacity to produce a higher level of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) as more fully described in U.S. Patent Nos. 6,248,876 B1; 5,627,061; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 Bl; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and international publications WO 97/04103; WO 00/66746; WO 01/66704; and WO 00/66747, which are incorporated herein by reference in their entireties for all purposes. Glyphosate resistance is also imparted to plants that express a gene that encodes a glyphosate oxido-reductase enzyme as described more fully in U.S. Patent Nos.

5,776,760 and 5,463,175, which are incorporated herein by reference in their entireties for all purposes.

Further, the mechanism of glyphosate resistance of the present invention may be combined with other modes of herbicide resistance to provide plants and plant explants that are resistant to glyphosate and one or more other herbicides. For example, the hydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. Molecules which inhibit this enzyme, and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate are useful as herbicides. Plants more resistant to certain herbicides are described in U.S Patent Nos. 6,245,968 B1; 6,268,549; and 6,069,115; and international publication WO 99/23886, which are incorporated herein by reference in their entireties for all purposes.

Sulfonylurea and imidazolinone herbicides also inhibit growth of higher plants by blocking acetolactate synthase (ALS) or acetohydroxy acid synthase (AHAS).

The production of sulfonylurea and imidazolinone tolerant plants is described more fully in U.S Patent Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270, which are incorporated herein by reference in their entireties for all purposes.

-91- WO 02/36782 PCT/US01/46227 Glutamine synthetase (GS) appears to be an essential enzyme necessary for the development and life of most plant cells. Inhibitors of GS are toxic to plant cells.

Glufosinate herbicides have been developed based on the toxic effect due to the inhibition of GS in plants. These herbicides are non-selective. They inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing an exogenous phosphinothricin acetyl transferase is described in U.S.

Patent Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 Bl; and 5,879,903, which are incorporated herein by reference in their entireties for all purposes.

Protoporphyrinogen oxidase (protox) is necessary for the production of chlorophyll, which is necessary for all plant survival. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in U.S. Patent Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and international publication WO 01/12825, which are incorporated herein by reference in their entireties for all purposes.

EXAMPLES

The following examples are illustrative and not limiting. One of skill will recognize a variety of non-critical parameters that can be altered to achieve essentially similar results.

EXAMPLE 1: ISOLATING NOVEL NATIVE GAT POLYNUCLEOTIDES Five native GAT polynucleotides GAT polynucleotides that occur naturally in a non-genetically modified organism) were discovered by expression cloning of sequences from Bacillus strains exhibiting GAT activity. Their nucleotide sequences were determined and are provided herein as SEQ ID NO: 1 to SEQ ID NO:5. Briefly, a collection of approximately 500 Bacillus and Pseudomonas strains were screened for native ability to N-acetylate glyphosate. Strains were grown in LB overnight, harvested by centrifugation, permeabilizied in dilute toluene, and then washed and resuspended in a reaction mix containing buffer, 5 mM glyphosate, and 200 lM acetyl-CoA. The cells were incubated in the reaction mix for between 1 and 48 hours, at which time an equal volume of methanol was added to the reaction. The cells were then pelleted by centrifugation and the supernatant was filtered before analysis by parent ion mode mass -92- WO 02/36782 PCT/US01/46227 O spectrometry. The product of the reaction was positively identified as N-acetylglyphosate by comparing the mass spectrometry profile of the reaction mix to an N-acetylglyphosate standard as shown in Figure 2. Product detection was dependent on inclusion of both substrates (acetylCoA and glyphosate) and was abolished by heat denaturing the bacterial cells.

Individual GAT polynucleotides were then cloned from the identified strains by functional screening. Genomic DNA was prepared and partially digested with l Sau3A1 enzyme. Fragments of approximately 4 Kb were cloned into an E. coli expression C vector and transformed into electrocompetent E. coli. Individual clones exhibiting GAT S 10 activity were identified by mass spectrometry following a reaction as described previously Cl except that the toluene wash was replaced by permeabilization with PMBS. Genomic fragments were sequenced and the putative GAT polypeptide-encoding open reading frame identified. Identity of the GAT gene was confirmed by expression of the open reading frame in E. coli and detection of high levels of N-acetylglyphosate produced from reaction mixtures.

EXAMPLE 2: CHARACTERIZATION OF A GAT POLYPEPTIDE ISOLATED FROM B.LICHENIFORMIS STRAIN B6.

Genomic DNA from B. licheniformis strain B6 was purified, partially digested with Sau3Al and fragments of 1-10 Kb were cloned into an E. coli expression vector. A clone with a 2.5 kb insert conferred the glyphosate N-acetyltransferase (GAT) activity on the E. coli host as determined with mass spectrometry analysis. Sequencing of the insert revealed a single complete open reading frame of 441 base pairs. Subsequent cloning of this open reading frame confirmed that it encoded the GAT enzyme. A plasmid, pMAXY2120, shown in figure 4, with the gene encoding the GAT enzyme of B6 was transformed into E. coli strain XL1 Blue. A 10% innoculum of a saturated culture was added to Luria broth, and the culture was incubated at 370 C for 1 hr. Expression of GAT was induced by the addition of IPTG at a concentration of 1 mM. The culture was incubated a further 4 hrs, following which, cells were harvested by centrifugation and the cell pellet stored at -80° C.

Lysis of the cells was effected by the addition of 1 ml of the following buffer to 0.2 g of cells: 25 mM HEPES, pH 7.3, 100 mM KC1 and 10% methanol (HKM) plus 0.1 mM EDTA, 1 mM DTT, 1 mg/ml chicken egg lysozyme, and a protease inhibitor -93- WO 02/36782 PCT/US01/46227 cocktail obtained from Sigma and used according to the manufacturer's recommendations.

After 20 minutes incubation at room temperature 22-250 lysis was completed Swith brief sonication. The lysate was centrifuged and the supernatant was desalted by Spassage through Sephadex G25 equilibrated with HKM. Partial purification was obtained by affinity chromatography on CoA Agarose (Sigma). The column was equilibrated with HKM and the clarified extract allowed to pass through under hydrostatic pressure. Nonr< binding proteins were removed by washing the column with HKM, and GAT was eluted In with HKM containing 1 mM Coenzyme A. This procedure provided 4-fold purification.

Ci At this stage, approximately 65% of the protein staining observed on an SDS 0 10 polyacrylamide gel loaded with crude lysate was due to GAT, with another 20% due to chloramphenicol acetyltransferase encoded by the vector.

Purification to homogeneity was obtained by gel filtration of the partially purified protein through Superdex 75 (Pharmacia). The mobile phase was HKM, in which GAT activity eluted at a volume corresponding to a molecular radius of 17 kD. This material was homogeneous as judged by Coomassie staining of a 3 tLg sample of GAT subjected to SDS polyacrylamide gel electrophoresis on a 12% acrylamide gel, 1 mm thickness. Purification was achieved with a 6-fold increase in specific activity.

The apparent KM for glyphosate was determined on reaction mixtures containing saturating (200 pM) Acetyl CoA, varying concentrations of glyphosate, and 1 M purified GAT in buffer containing 5 mM morpholine adjusted to pH 7.7 with acetic acid and 20 ethylene glycol. Initial reaction rates were determined by continuous monitoring of the hydrolysis of the thioester bond of Acetyl CoA at 235 nm (E 3.4 OD/mM/cm).

Hyperbolic saturation kinetics were observed (Figure from which an apparent KM of 2.9 0.2 (SD) mM was obtained.

The apparent KM for AcCoA was determined on reaction mixtures containing 5 mM glyphosate, varying concentrations of Acetyl CoA, and 0.19 pM GAT in buffer containing 5 mM morpholine adjusted to pH 7.7 with acetic acid and methanol. Initial reaction rates were determined using mass spectrometric detection of Nacetyl glyphosate. Five pl were repeatedly injected to the instrument and reaction rates were obtained by plotting reaction time vs area of the integrated peak (Figure 6).

Hyperbolic saturation kinetics were observed (Figure from which an apparent KM of 2 pM was derived. From values for Vmax obtained at a known concentration of enzyme, a kcat of 6/min was calculated.

-94- WO 02/36782 PCT/US01/46227 EXAMPLE 3: MASS SPECTROMETRY (MS) SCREENING PROCESS Sample (5 ul) is drawn from a 96-well microtiter plate at a speed of one sample every 26 seconds and injected into the mass spectrometer (Micromass Quattro LC, triple quadrupole mass spectrometer) without any separation. The sample is carried into the mass spectrometer by a mobile phase of water/methanol (50:50) at a flow rate of 500 Ul/min. Each injected sample is ionized by negative electrospray ionization process (needle voltage, -3.5 KV; cone voltage, 20 V; source temperature, 120 C; desolvation temperature, 250 C; cone gas flow, 90 IJHr; and desolvation gas flow, 600 LJHr). The molecular ions (m/z 210) formed during this process arre selected by the first quadrupole for performing collison induced dissociation (CID) in the second quadrupole, where the pressure is set at 5 x 10 4 mBar and the collision energy is adjusted to 20 Ev. The third quadrupole is set for only allowing one of the daughter ions (m/z 124) produced from the parent ions (m/z 210) to get into the detector for signal recording. The first and third quadupoles are set at unit resolution, while the photomultiplier is operated at 650 V. Pure N-acetylglyphosate standards are used for comparison and peak integration used to estimate concentrations. It is possible to detect less than 200 Nm N-acetylglyphosate by this method.

EXAMPLE 4: DETECTION OF NATIVE OR LOW ACTIVITY GAT ENZYMES Native or low activity GAT enzymes typically have Kcat of approximately 1 min' 1 and KM for glyphosate of 1.5-10 Mm. KM for acetylCoA is typically less than pM.

Bacterial cultures are grown in rich medium in deep 96-well plates and ml stationary phase cells are harvested by centrifugation, washed with 5 mM morpholine acetate pH 8, and resuspended in 0.1 ml reaction mix containing 200 iM ammonium acetylCoA, 5 mM ammonium glyphosate, and 5 pig/ml PMBS (Sigma) in 5 mM morpholine acetate, pH 8. The PMBS permeabilizes the cell membrane allowing the substrates and products to move from the cells to the buffer without releasing the entire cellular contents. Reactions are carried out at 25-37 0 C for 1-48 hours. The reactions are quenched with an equal volume of 100% ethanol and the entire mixture is filtered on a 0.45 pm MAHV Multiscreen filter plate (Millipore). Samples are analyzed using a mass spectrometer as desribed above and compared to synthetic N-acetylglyphosate standards.

WO 02/36782 PCT/US01/46227 EXAMPLE 5: DETECTION OF HIGH ACTTVITY GAT ENZYMES High activity GAT enzymes typically have kcat up to 400 min and KM below 0.1 mM glyphosate.

Genes coding for GAT enzymes are cloned into E. coli expression vectors such as pQE80 (Qiagen) and introduced into E. coli strains such as XL1 Blue (Stratagene).

Cultures are grown in 150 ul rich medium (such as LB with 50 ug/ml carbenicllin) in shallow U-bottom 96-well polystyrene plates to late-log phase and diluted 1:9 with fresh medium containing 1 mM IPTG (USB). After 4-8 hours induction, cells are harvested, washed with 5mM morpholine acetate pH 6.8 and resuspended in an equal volume of the same morpholine buffer. Reactions are carried out with up to 10 ul of washed cells. At higher activity levels, the cells are first diluted up to 1:200 and 5 ul is added tol00 ul reaction mix. To measure GAT activity, the same reaction mix as described for low activity can be used. However, for detecting highly active GAT enzymes the glyphosate concentration is reduced to 0.15 0.5 mM, the pH is reduced to 6.8, and reactions are carried out for 1 hour at 37 0 C. Reaction workup and MS detection are as described herein.

EXAMPLE 6: PURIFICATION OF GAT ENZYMES Enzyme purification is achieved by affinity chromatography of cell lysates on CoA-agarose and gel-filtration on Superdex-75. Quantities of purified GAT enzyme up to 10 mg are obtained as follows: A 100-ml culture of E. coli carrying a GAT polynucleotide on a pQE80 vector and grown overnight in LB containing 50 ug/ml carbenicillin is used to inoculate 1 L of LB plus 50 ug/ml carbenicillin. After 1 hr, IPTG is added to 1 mM, and the culture is grown a further 6 hr. Cells are harvested by centrifugation. Lysis is effected by suspending the cells in 25 mM HEPES (pH 100 mM KC1, 10% methanol (termed HKM), 0.1 mM EDTA, 1 mM DTT, protease inhibitor cocktail supplied by Sigma-Aldrich and 1 mg/ml of chicken egg lysozyme. After minutes at room temperature, the cells are briefly sonicated. Particulate material is removed by centrifugation, and the lysate is passed through a bed of coenzyme A- Agarose. The column is washed with several bed volumes of HKM and GAT is eluted in 1.5 bed volumes of HKM containing 1 mM acetyl-coenzyme A. GAT in the eluate is concentrated by its retention above a Centricon YM 50 ultrafiltration membrane. Further purification is obtained by passing the protein through a Superdex 75 column through a series of 0.6-ml injections. The peak of GAT activity elutes at a volume corresponding to -96- WO 02/36782 PCT/US01/46227 a molecular weight of 17 kD. This method results in purification of GAT enzyme to homogeneity with >85% recovery. A similar procedure is used to obtain 0.1 to 0.4 mg quantities of up to 96 shuffled variants at a time. The volume of induced culture is reduced to 1 to 10 ml, coenzyme A-Agarose affinity chromatography is performed in 0.15ml columns packed in an MAHV filter plate (Millipore) and Superdex 75 chromatography is omitted.

EXAMPLE 7: STANDARD PROTOCOL FOR DETERMINATION OF KcAT AND KM K.t and KM for glyphosate of purified protein are determined using a continuous spectrophotometric assay, in which hydrolysis of the sulfoester bond of AcCoA is monitored at 235 nm. Reactions are performed at ambient temperature (about 23 0 C) in the wells of a 96-well assay plate, with the following components present in a final volume of 0.3 ml: 20 mM HEPES, pH 6.8, 10% ethylene glycol, 0.2 mM acetyl coenzyme A, and various concentration of ammonium glyphosate. In comparing the kinetics of two GAT enzymes, both enzymes should be assayed under the same condition, both at 23 0 C. Kat is calculated from Vm and the enzyme concentration, determined by Bradford assay. KM is calculated from the initial reaction rates obtained from concentrations of glyphosate ranging from 0.125 to 10 mM, using the Lineweaver-Burke transformation of the Michaelis-Menten equation. Kca/KM is determined by dividing the value determined for Ka by the value determined for Ku.

Using this methodology, kinetic parameters for a number of GAT polypeptides exemplified herein have been determined. For example, the Kt, KM and Kca/KM for the GAT polypeptide corresponding to SEQ ID NO:445 have been determined to be 322 min-', 0.5 mM and 660 mMTmin' 1 respectively, using the assay conditions described above. The Kt, KM and KI,/KM for the GAT polypeptide corresponding to SEQ ID NO:457 have been determined to be 118 min-', 0.1 mM and 1184 mM'min' 1 respectively, using the assay conditions described above. The Kat, KM and KCEt/KM for the GAT polypeptide corresponding to SEQ ID NO:300 have been determined to be 296 min- 1 0.65 mM and 456 mM'min 1 respectively, using the assay conditions described above. One of skill in the art can use these numbers to confirm that a GAT activity assay is generating kinetic parameters for a GAT suitable for comparison with the values given herein. For example, the conditions used to compare the activity of GATs should yield the same kinetic constants for SEQ ID NOS: 300, 445 and 457 (within normal experimental -97- WO 02136782 PCTUSO1/46227 variance) as those reported herein, if the conditions are going to be used to compare a test GAT with the GAT polypeptides exemplified herein. Kinetic parameters for a number of GAT polypeptide variants were determined according to this methodology and are provided in Tables 3, 4 and Table 3. GAT polypeptide values SEQ ID NO. Clone ID K,(min") SEQ ID NQ:263 13 10F6 48.6 SEQ ID NO:264 13_12G6 52.1 SEQ ID NO:265 14 2A5 280.8 SEQ ID NO:266 14 2C1 133.4 SEQ -ID NO:267 14 2F11 136.9 SEQ ID NO.268 CHIMERA 155.4 SEQ ID NO:269 10_12D7 77.3 SEQ ID NO:270 10 15F4 37.6 SEQ ID NO:271 10 17D1 176.2 SEQ ID NO:272 10 17F6 47.9 SEQ ID NO:273 10 18G9 24 SEQ ID N0274 10 1H3 76.2 SEQ ID NO:275 1 20D10 86.2 SEQ ID NO:276 10_23F2 101.3 SEQ ID NO:277 10 2B8 108.4 SEQ ID NO:278 10 207 135 SEQ ID NO:279 10 3G5 87.4 SEQ ID NO:280 10 4H7 112 SEQ ID NO:281 10 6D1 1 62.4 SEQ ID NO:282 10 8C6 21.7 SEQ ID NO:283 11C3 2.8 SEQ ID NO:284 11G3 15.6 SEQ ID NO:285 11H3 1.2 SEQ ID NO:286 12 1F9 80.4 SEQ ID NO:287 122G9 151.4 SEQ ID NO:288 12 3F1 44.1 SEQ ID NO:289 12-5010 89.6 SEQ ID NO:290 12SAlO0 54.7 SEQ ID NO:291 12 6D1 49 SEQ ID NO:292 12 6F9 89.1 SEQ ID NO:293 12 6H6 90.5 SEQ ID NO:294 12 7D6 53.9 SEQ ID NO295 12 7G11 234.5 SEQ ID NO:296 12F5 3.1 SEQ ID NO:297 12G7 2.3 SEQ ID NO:298 1 2H6 9.3 SEQ ID NO:299 13_12G12 36.1 SEQ ID NO:300 13_6D10 296.5 SEQ ID NO:301 13 7A7 117 SEQ ID NO:302 13 7B12 68.9 SEQ ID NQ:303 13_7C1 48.1 SEQ ID NO:304 13_8G6 33.7 SEQ ID NQ:305 13 9F6 59 SEQ ID NO:306 14_1 09 127 SEQ ID NO:307 14_10H3 105.2 SEQ ID NO:308 141 0H9 127.2 -98- WO 02/36782 PCTIUSOI/46227 SEQ ID NQ:309 14 1102 108.7 SEQ ID NO:310 14 1208 62.1 SEQ ID NO:311 14-12H6 91.1 SEQ ID NO:312 14 2B6 34.2 SEQ ID NO:313 14 2G11 69.4 SEQ ID NO:314 14 3B2 68.7 SEQ ID NO:315 14 4H8 198.8 SEQ ID NQ:316 14 6A8 43.7 SEQ ID NO:317 14 6B10 134.7 SEQ ID NO:318 14-604 256 SEQ ID NO:319 14 7A11 197.2 SEQ ID NO:320 14 7A1 155.8 SEQ ID NO:321 14 7A9 245.9 SEQ ID NO:322 14 7G1 136.7 SEQ ID NO:323 14 7H9 64.4 SEQ ID NO:324 14_8F7 90.5 SEQ ID NO:325 15 1002 69.9 SEQ ID NO:326 15 1006 67.1 SEQ ID NQ:327 15 1 F9 76.4 SEQ ID NO:328 15 11H3 61.9 SEQ ID NO:329 15 12A8 77.1 SEQ ID NO:330 15_1 206 148.6 SEQ ID NO:331 1512D8 59.7 SEQ ID NO:332 15 12D9 59.7 SEQ ID N:333 15 3F10 48.7 SEQ ID NO:334 153G11 71.5 SEQ ID NO:335 15 4F11 80.3 SEQ ID NO:336 15,4H3 93.3 SEQ ID NO:337 156D3 85.9 SEQ ID NO:338 15 6G11 36.9 SEQ ID NO:339 15_9F8 59.6 SEQ ID NO:340 15F5 SEQ ID NO:341 16A1 10.4 SEQ ID NO:342 16H3 SEQ ID NO:343 17C12 3.2 SEQ ID NO:344 1 8D6 9.6 SEQ ID NO:345 1906 2.2 SEQ ID NO:346 19D5 2.2 SEQ ID NO:347 20A12 2.8 SEQ ID NO:348 20F2 3.9 SEQ ID NQ:349 2.1 OE+12 1.1 SEQ ID NO:350 23H1 1 7.1 SEQ ID NO:351 2401 1.7 SEQ ID NO:352 2406 2.7 SEQ ID NQ:353 2.40E+08 8.9 SEQ ID NO:354 2_803 24.8 SEQ ID N:355 2H3 16.1 SEQ ID NO:356 30G8 10.2 SEQ ID NO:357 3B 1004 24.8 SEQ ID ND:358 3B 10G7 19.6 SEQ ID NO:359 3B 12B1 22.8 SEQ ID NO:360 3B_12D10 5.4 SEQ ID NO:361 3B-2E5 16.4 SEQ ID NO:362 30_10H3 33.9 SEQ ID N:363 30 12H10 9.1 SEQ ID N:364 30 9H8 11.7 SEQ ID NO:365 4A iBli 23.2 SEQ ID NO:366 4A 102 20.4 -99- WO 02/36782 PCTIUS01/46227 SEQ ID NO:367 4B 13E1 37.2 SEQ ID NQ:368 4B 13G10 34.9 SEQ ID NO:369 4B 16E1 17 SEQ ID NQ:370 4B 17A1 19.1 SEQ ID NQ:371 4B 18F11 14.6 SEQ ID NO:372 4B 19GB 15.9 SEQ ID NQ:373 4B 1G4 3.7 SEQ ID NQ:374 4B 2106 11.8 SEQ ID NO:375 4B 2H7 27 SEQ ID NQ:376 4B 2H8 38.3 SEQ ID N:377 4B 6D8 22.7 SEQ ID N:378 4B 7E8 20.5 SEQ ID NO:379 4C 809 9 SEQ ID N:380 4H1 1.3 SEQ ID NO:381 614D10 42.2 SEQ ID N:382 615G7 48.4 SEQ ID N:383 6 16A5 43.8 SEQ ID NO:3B4 6 16F5 35.2 SEQ ID NO:385 6 1705 35.2 SEQ ID N:386 6 1807 32.2 SEQ ID N:387 6 18D7 43 SEQ ID N:388 6 19A10 86.8 SEQ ID NO:389 6 19B6 23.9 SEQ ID NQ:390 6 1903 23.1 SEQ ID NO:391 61908 74.8 SEQ ID NO:392 6 20A7 40.4 SEQ ID NO:393 6 20A9 45.1 SEQ ID NO:394 6 20H5 19.5 SEQ ID NQ:395 621F4 24.3 SEQ ID NQ:396 6 22C9 47.4 SEQ ID NQ:397 6 22D9 43.9 SEQ ID NQ:398 622H9 17.4 SEQ ID NO:399 6 23H3 43.9 SEQ ID NO:400 6.23H7 46.2 SEQ ID NQ:401 6_2H1 26.6 SEQ ID NO:402 6 3D6 41.7 SEQ ID N:403 6 3G3 51.9' SEQ ID NO:404 6.3H2 57.2 SEQ ID NO:405 6 4A10 SEQ ID N:406 6 4B1 27 SEQ ID NO:407 65D11 15.2 SEQ ID NO:408 65SF11 40.1 SEQ ID NO:409 6 5G9 35.8 SEQ ID NQ:410 6 6D5 55.3 SEQ ID NO:411 6 7D1 19.7 SEQ ID NO:412 6 8H3 44.7 SEQ ID NO:413 6 9G11 78.4 SEQ ID NQ:414 6F1 10.1 SEQ ID NQ:415 7 104 17.4 SEQ ID NO:416 7 2A10 14.5 SEQ ID NO:417 7 2A11 46.8 SEQ ID NQ:418 7 2D7 54.9 SEQ ID NQ:419 7_5C7 44.7 SEQ ID NO:420 7-909 SEQ ID NO:421 9 3F10 34.7 SEQ ID N:422 9.j3F1 31.6 SEQ ID NO:423 91 5D5 27.6 SEQ ID N:424 915D8 107.3 -100- WO 02/36782 WO 0236782PCTfUS01/46227 SEQ ID NO:425 1 15H3 68.7 CISEQ ID NO:426 9 18H2 t)SEQ ID NO:427 9 20F12 37.8 ;ZSEQ ID NQ:428 9-210C8 28.6 SEQ ID NO:429 9 22Bl 50.1 SEQ ID NO:430 9_23A1 0 21 SEQ ID NO:431 9_24F6 52.5 SEQ ID NO:432 9_4H 10 101.3 SEQ ID NO:433 9_4H8 47.1 SEQ ID NO:434 9 8H1 74.8 SEQ ID NO:435 9 9H7 28 SEQ ID NO:436 906 13 knSEQ ID NO:437 9HI1 4 SEQ ID NO:438 0_4B1 0 190 CISEQ ID NO:439 05SB1 1 219 SEQ ID NQ:440 0_5B3 143 SEQ ID NO:441 0_5B4 180 SEQ ID NQ:442 0_5B8 143 SE DNO43I-C 0 SEQ ID NO:443 0 5041 205 SEQ ID NO:445 0 503 322 SEQ ID NO:446 0 507 244 SEQ ID NO:447 0 6B4 252 SEQ ID NO:448 0 601 0 ill SEQ ID NO:449 0 601 1 212 SEQ ID NO:450 0_6F2 175 SEQ ID NO:451 0 6H9 228 SEQ ID NQ:452 1L4C1 0 69.6 SEQ ID NO:453 10 4D5 82.72 SEQ ID NO:454 1 0_..4F2 231.04 SEQ ID NO:455 10 4F9 55.39 SEQ ID NO:456 1 0-.4G5 176.65 SEQ ID NO:457 10 4H4 118.36 SEQ ID NO:458 11-3A1 1155.66 SEQ ID NO:459 11 3B1 219.97 SEQ ID NO:460 1!13B5 194.61 SEQ ID ND:461 11_3012 49.07 SEQ ID NO:462 113C3 214.02 SEQ ID NO:463 11 306 184.44 SEQ ID ND:464 11 3D6 55.3 SEQ ID NO:465 1 1G12 58.48 SEQ ID NO:466 1ilHi 291 SEQ ID NO:467 1-1H2 164 SEQ ID NO:46B 1 1H5 94 SEQ ID NO:469 1_2A12 229 SEQ ID NO:470 1 2B6 138 SEQ ID NO:471 1- 204 193 SEQ ID NO:472 1 202 124 SEQ ID NO:473 1-204 182 SEQ ID NO:474 i 2F8 161 SEQ ID NO:475 1 2H8 141 SEQ ID NO:476 1_A2 181 SEQ ID NO:477 13D6 226 SEQ ID NO:478 1 3F3 167 SEQ ID NO'479 1_3H2 128 SEQ ID NO:480 1-405 1254 SEQ ID NO:481 14AD6 1137 SEQ ID NO:482 1 4H 1 1236 WO 02/36782 WO 0236782PCT[ISOI/46227 SEQ ID NO:483 1 51-5 214 SEQ 1D NO:484 1 6F12 209 SEQ ID NO:485 1 6H6 274 SEQ ID NO:486 3 11A1O 135.41 SEQ ID NO:487 3 14F6 188.43 SEQ ID NQ:488 3 15132 10D4.13 SEQ ID NO:489 3 6A1 0 126.48 SEQ ID NO:490 3_6831 263.08 SEQ ID NO:491 37F 9 193.55 SEQ ID NO:492 3_8G1 1 99.14 SEQ ID NO:493 4 18310 77.09 SEQ ID NO:494 5 2133 55.75 SEQ ID NQ:495 521D9 75.44 SEQ ID NO:496 5 2F1 0 54.72 SEQ ID NO:497 61 All1 45.54 SEQIDNO:498 6 1 D5 42.92 SEQ ID NO:499 6 1 Fl 1 105.76 SEQ ID NO:500 61 F1 69.81 SEQIDNO:501 6 1H1O 17.01 SEQ ID NO:502 6 11H4 85.91 SEQ ID NO:503 81 F8 82.88 SEQ ID NQ:504 8 1 G2 67.47 SEQ ID NO:505 8 1G3 108.9 SEQ ID NO:506 8 1H7 101.24 SEQ ID NO:507 8_ 1H9 78.39 SEQ ID NO:508 GATi 21F12 5.4 SEQ ID NO:509 GATi 24G3 4.9 SEQ ID 140:510 GATi 29G1 6.2 SEQ ID NO:51 1 GAT1-32G1 SEQ ID NO:512 GAT2-15G8 SEQ I DNO:513 GAT2 191-8 14.1 SEQ ID NO:514 IGAT2_21 F1 14.2 Table 4. GAT polypeptide (glyphosate) Km values SEQ ID NO. Clone ID KL,(mM) SEQ ID NO:263 13_ 10F6 1.3 SEQ ID NO:264 13_ 12G6 1.2 SEQ ID NO:265 14 -2A5 1.6 SEQ ID NO:266 14_201 3.1 SEQ ID NQ:267 14 21 11 1.7 SEQ ID NO:268 CHIMERA 1.3 SEQ ID NO:269 1 0_ 2D7 1.8 SEQ ID NO:270 1 0_ 5F4 1 SEQ ID NO:271 10 17D1 2.2 SEQ ID NO:272 10 C17F6 1.4 SEQ ID NO:273 10 18G9 1.2 SEQ ID NO:274 101 H3 1.9 SEQ ID NO:275 10 Q20D1 0 1.6 SEQ ID N0:276 1 &.23F2 0.9 SEQ ID NO:277 102B38 1.1 SEQ ID NO:278 10 0_7 1.4 SEQ ID NQ:279 10 Q3G5 2 SEQ ID N0:280 1 0_4117 1.7 SEQ ID NQ:281 I10_6D1 1 1.2 SEQ D -NO:282 10 Q806 0.7 SEQ ID NO:283 1103 3.1 -102- WO 02136782 WO 0236782PCTf1US01146227 SEQ ID NO:284 11G3 1.7 C1SEQ ID NO:285 11H3 1.4 SEQ ID NO:286 12 1 F9 3 ;ZSEQ ID NO:287 12 2G9 SEQ ID NO:288 12 3F1 0.9 SEQ ID NO:289 12 5C1 0 SEQ ID NO290 12 6A1 0 1.1 SEQ ID N0:291 12061 1.2 SEQ ID NO0292 12 6F9 1.9 SEQ ID NO:293 12 6H6 1.6 SEQ ID NO:294 12 7D6 1.4 SEQ ID NO295 12 7G11 2 SEQ ID NO:296 12F5 1.8 SEQ ID NO:297 12G7 3.7 CISEQ ID NO:298 1 2H6 0.9 SEQ ID NO:299 13 12G12 0.69 SEQ ID NO:300 130610 0.65 SEQ ID NO:301 13 7A7 SEQ ID NO:302 13_7B1 2 1.7 SEQ ID NO:303 13-70 SEQ ID NQ:304 13 8G6 0.61 SEQ ID NO:305 13_9F6 1.3 SEQ iD NO:306 14 100C9 0.9 SEQ ID NO:307 14_ 10H3 0.6 SEQ ID NO:308 14 10OH9 1.1 SEQ ID ND:309 14 110C2 1 SEQ) ID NO:310 14 12D8 1 SEQ ID NO:311 14j 2H6 0.9 SEQ ID NO:312 14 2B6 0.83 SEQ ID NO:313 14 2G11 1.4 SEQ ID NO:314 14 3B2 0.85 SEQ ID NO:315 14 _4H8 2 SEQ ID NO:316 14 6A8 0.78 SEQ ID NO:317 14 6B10 1.4 SEQ ID NO;31 8 14 6D4 1 SEQ ID NO:319 14M 7A1 3.7 SEQ ID NO:320 142AM 1.6 SEQ ID NO:321 14 7A9 3.2 SEQ ID NO:322 14 7G1 0.66 SEQ ID NQ:323 14 7H9 1.3 SEQ ID NQ:324 14_8F7 1.8 SEQ ID NO:325 15_1002 0.8 SEQ ID NO:326 15_10D6 1 SEQ ID NO:327 15 11 F9 I SEQ ID NO:328 15 11 H3 SEQ ID NO:329 15_.1 ZA 1.6 SEQ ID NO:330 15.12DB 0.74 SEQ ID NO:331 15_12D8 1.3 SEQ ID NO:332 1512D9 1.4 SEQ ID NQ:333 15 3F10 0.9 SEQ ID NQ:334 15 3G 111.

SEQ ID NO:335 15 _4F1 1 0.9 SEQ ID NO:336 15--.4H31 SEQ ID NO:337 15063 1.4 SEQ ID NO:338 115-6G 110.

SEQ ID NO:339 15 9F61.

SEQ ID N:340 15F5 2.9 SEQ ID NO:341 16A1 2.9 103 WO 02136782 WO 0236782PCTfUSOI/46227 SEQ ID NO:342 16H3 2.9 C1SEQ ID NO:343 17C12 1.4 SEQ ID NO:344 1 8D6 1.2 ;ZSEQ ID NO:345 1906 1.1 SEQ ID NQ:346 19gD5 1.7 SEQ ID NQ:347 20A12 1.1 __SEQ ID NO:348 20F2 1.9 SEQ ID NO:349 2.1 OE+1 2 0.7 SEQ ID NO:350 23H1 1 2.2 SEQ ID NO:351 2401 0.9 SEQ ID NO:352 2406 1.3 SEQ ID ND:353 2.40E+08 0.9 SEQ ID NO:354 2_803 SEQ ID NO:355 2H3 0.9 SEQ ID ND:356 30GB 1.6 SEQ ID NO:357 3B 1004 1.6 SEQ ID NO:358 3B_10OG7 I SEQ ID NQ:359 3B_12B1 1.2 SEQ ID NO:360 3B -12D10 0.9 SEQ ID NO:361 3B 2E5 1.3 SEQ ID NO:362 30- 10H3 1.1 SEQ ID NO:363 30- 12H1 0 1.2 SEQ ID NO:364 30 9H8 1 SEQ ID NO:365 4A 1 Bi 11.6 SEQ ID NO:366 4Al 1021.

SEQ ID NO:367 4B 13E1 SEQ ID NO:368 4B13G10 7.6 SEQ ID NO:369 4B 16E1 SEQ ID NO:370 4B 17A1 1.1 SEQ ID NO:371 4B- 18F1 1 1.7 SEQ ID NO:372 4B 1908 1.2 SEQ ID NO:373 4B- 1G4 SEQ ID NO:374 4B -210C60.

SEQ ID NO:375 4B-2H7 6.2 SEQ ID NO:376 4B 2H81.

SEQ ID NO:377 4B 6DB1.

SEQ ID NO:378 4B 7E81.

SEQ ID NO:379 40 8090.

SEQ ID NO:380 4H1 SEQ ID NO:381 6 14D1 01.

SEQ ID NO:382 6 15G71.

SEQ ID NO:383 06A 1.A1 SEQ ID NO:384 6 16F5 1 SEQ ID NO:3B5 6 1705 1.3 SEQ ID NO:386 6 1807 1.2 SEQ ID NO:387 618D7 1.2 SEQ ID NO:388 6_ 19A1 0 1.9 SEQ ID NO:389 6..19B6 0.7 SEQ ID NO:390 6_ 1903 1.4 SEQ ID NO:391 6 1908 2 SEQ ID NO:392 6.20A7 1 SEQ ID NO:393 8_20A9 1.3 SEQ ID NO:394 6_20H5 0.8 SEQ ID NO:395 6 21 F4 0.7 SEQ ID NQ:396 622C9 3.2 SEQ ID NO:397 6 22D9 1.3 SEQ I D NO:39B 622H9 1.1 SEQ ID N:399 16_23H31.

-104- WO 02/36782 WO (i236782PCTJUSOI/46227

;Z

SEQ ID NO:400 6_23H7 1.2 SEQ ID NQ:401 6 2H1 0.9 SEQ 10 NO:402 6 3D6 1 SEQ ID NO:403 6 3G3 1 SEQ ID NO:404 6 3H2 SEQ ID NO:405 6 4A10 1.1 SEQ ID NO:406 6_4B1 1 SEQ ID NO:407 6 5D11 1 SEQ ID NO:408 6_5F1 1 1.9 SEQ ID NO:409 6 5G9 1.4 SEQ ID NO:410 6 6D5 1 SEQ ID NO:411 6L7D1 SEQ ID NO:412 6_-8H3 1 SEQ ID NQ:413 6-9G 11 1.3 SEQ ID NO:414 6P1 1.8 SEQ ID NO:415 7 104 1.1 SEQ ID NO:416 7_2A10 0.8 SEQ ID NO:417 7 _2A 11 1.1 SEQ ID NO:418 7 2D71.

SEQ ID NO:419 7 507 SEQ ID NO:420 7 909 SEQ ID NO:421 9 13F10 0.7 SEQ ID NO:422 9-13FI 1.1 SEQ ID NO:423 915D5 1.2 SEQ ID NQ:424 9 15D8 1.1 SEQ ID NO:425 9 _15H3 1.9 SEQ ID NO:426 9 18H2 1.1 SEQ ID NQ:427 9_20F 12 1 SEQ ID NO:428 9_21C8 1.2 SEQ ID NO:429 922B1 1.4 SEQ ID NQ:430 9_23A1 0 1 SEQ ID NO:431 9_24F6 0.9 SEQ ID NO:432 9_4H 10 SEQ ID NO:433 9_4H8 0.6 SEQ ID NO:434 9 8Hl 1.7 SEQ ID NO:435 9_9H7 0.7 SEQ ID ND:436 9C6 SEQ ID NO:437 9H1 1 2.3 SEQ ID NO:438 0 4AB1 0 0.68 SEQ ID NO:439 0..5Bl 1 0.54 SEQ ID NO:440 0_5B3 0.39 SEQ ID NQ:441 0_5B4 0.6 SEQ ID NO.442 0_5B8 0.27 SEQ ID NO:443 0_504 0.67 SEQ ID NO:4.44 0 5D1 1 0.67 SEQ ID NO:445 0 5D3 SEQ ID NO:446 0_5D7 1.1 SEQ ID NO:447 0_6B4 0.8 SEQ ID NO:448 0 6D1 0 0.1 SEQ ID NQ:449 0_6D1 1 0.44 SEQ ID NO:450 0_SF2 0.34 SEQ ID NO:451 0 6H9 0.47 SEQ ID NO:452 10 4C10 0.1 SEQ ID NQ:453 10 Q4D5 0.1 SEQ ID NO:454 10 4F2 0.2 SEQ ID NO:455 1 0_4F9 0.1 SEQ ID NO:456 104-65 0.58 SEQ ID NO:457 10 4H4 0.1 105 WO 02136782 WO 0236782PCT[USOI/46227 SEQ ID NO:458 11 3A1 1 0.1 SEQ ID NO:459 11 3B1 0.63 SEQ ID NO:460 11 3B5 0.26 SEQ ID NQ:461 11 3012 0.1 SEQ ID NO:462 11 .3C3 0.22 SEQ ID NQ:463 11 306 0.21 SEQ ID NO:464 11 3D6 0.1 SEQ ID NO:465 1 1G12 0.1 SEQ ID ND:466 1-lHl 1.8 SEQ ID NO:467 1 1H2 0.44 SEO ID NO:468 1 1H5 1.5 SEQ ID NO:469 1-2Al2 1.3 SEQ ID NO:470 1 2B6 SEQ ID NO:471 1 2C4 0.8 SEQ ID NO:472 1 2D2 1.2 SEQ ID NO:473 1 2D4 1.2 SEQ ID NO:474 1 2F8 1.9 SEQ ID NO:475 1 _2H8 0.48 SEQ ID NO:476 1 3A2 0.8 SEQ ID NO:477 1 3D6 SEQ ID NO:478 13F3 SEQ ID NO:479 13H2 0.7 SEQ ID NO:480 1 4C5 0.93 SEQ ID NO:481 1 4D6 1.4 SEQ ID NQ:482 1_4Hl 1.2 SEQ ID NO:483 1 5H5 0.51 SEQ ID NO:484 1 6F12 14.7 SEQ ID NO:485 1 6H6 1.05 SEQ ID NO:486 311A1O 0.17 SEQ ID NO:487 3 14F6 0.25 SEQ ID NQ:488 3 15B2 0.1 SEQ ID NO:489 3_6A1 0.66 SEQ ID NQ:490 3 _6B1 0.43 SEQ ID NO:491 3_7F9 0.29 SEQ ID NO:492 3_8G1 1 0.1 SEQ ID NO:493 4 ilBlO 0.1 SEQ ID NO:494 5 2B3 0.1 SEQ ID NO:495 5 _2D9 0.1 SEQ ID NQ:496 5 2F 10 0.1 SEQ ID NO:497 6 _lAll 0.1 SEQ ID NO:498 6 1D5 0.1 SEQ ID NQ:499 61 Fl1 0.1 SEQ ID NO:500 6_1 l0.1 SEQ ID NO:501 61 Hl10 0.1 SEQ ID NO:502 61 H4 0.1 SEQ 10 NO:503 81 F8 0.1 SEQ ID NO:504 81 G2 0.1 SEQ ID NO:505 8 1G3 0.1 SEQ ID NO:506 81 H7 0.1 SEQ ID NO:507 81 H9 0.1 SEQ ID NO:508 GATi 21 F12 4.6 SEQ ID NO:509 GATi 24G3 3.8 SEQ ID NO:51 0 GAT1L29G1 4 SEQ ID NO:51 1 GAT-32G1 3.3 SEQ ID NO:51 2 GAT2-1.i5G8 2.8 SEQ ID NO:513 GAT2 19HB 2.8 SEQ IDNO:514 GAT2 21Fl 13 106 WO 02/36782 PCT[US01146227 Table 5. GAT polypeptide ki/ KM values SEQ ID NO. Clone ID mM-' min- SEQ ID NO:263 13_10F6 37.4 SEQ ID NO:264 13_12G6 43.4 SEQ ID NO:265 14_2A5 175.5 SEQ ID NO:266 14_2C1 43 SEQ ID NO:267 142F11 80.6 SEQ ID N0268 CHIMERA 119.6 SEQ ID NO:269 10 1207 43 SEQ ID N0270 10_1 5F4 37.6 SEQ ID NO:271 10_17D1 80.1 SEQ ID NO:272 10 17F6 34.2 SEQ ID NO:273 10 8G9 SEQ ID NO:274 10 1H3 40.1 SEQ ID N0275 10 20D10 53.9 SEQ ID N:276 1023F2 112.5 SEQ ID NO:277 10 2B8 98.5 SEQ ID NO:278 10 207 96.4 SEQ ID NO:279 10 3G5 43.7 SEQ ID N0280 1D 4H7 65.9 SEQ ID NO:281 10-6D11 52 SEQ ID NO:282 10 806 31 SEQ ID NO:283 1103 0.9 SEQ ID NO:284 11G3 8.9 SEQ ID NO:285 11H3 0.9 SEQ ID NO:286 12 1F9 26.8 SEQ ID NO:287 12 2G9 101 SEQ ID NO:288 12 3F1 49 SEQ ID NQ:289 12 5C10 59.7 SEQ ID NO:290 12 6A10 49.7 SEQ ID NO:291 12 601 40.8 SEQ ID NO:292 12_6F9 46.9 SEQ ID NO:293 126H6 56.5 SEQ ID NQ:294 12 7D6 38.5 SEQ ID NO:295 12_7G11 117.2 SEQ ID NO:296 12F5 1.7 SEQ ID NO:297 12G7 0.6 SEQ ID NO:298 1 2H6 10.4 SEQ ID NO:299 13 12G12 52.4 SEQ ID NO:300 13_6D10 456.1 SEQ ID NO:301 13 7A7 234 SEQ ID NO:302 137B 12 40.5 SEQ ID N:303 13 701 32.1 SEQ ID NO:304 13 8G6 55.2 SEQ ID NO:305 13 9F6 45.3 SEQ ID NO:306 14_1009 141.1 SEQ ID NO:307 14 10H3 175.3 SEQ ID NO:308 141 0H9 115.6 SEQ ID NO:39 14 11C2 108.7 SEQ ID NO:310 14 1208 62.1 SEQ ID NO:311 14_12H6 101.3 SEQ ID NO:312 14_2B6 54.3 SEQ ID NO:313 14 2G11 49.6 SEQ ID NO:314 14_3B2 80.9 -107- WO 02/36792 WO 0236782PCTf/USOI/46227 SEQ ID NO:315 14 4H8 99.4 SEQ ID NO:316 14_6A8 56 SEQ ID NO:317 14_6B1 0 96.2 SEQ ID NQ:318 14 6D4 256 SEQ ID NQ:319 14 MA11 53.3 SEQ ID NQ:320 14 MA 97.4 SEQ ID NO:321 14_-7A9 76.9 SEQ ID NO:322 14 7G 1 207.1 SEQ ID NO:323 14_7H9 49.5 SEQ ID NO:324 14_BF7 50.3 SEQ 10 NO:325 15 1002 87.3 SEQ ID NO:326 151 ODS 67.1 SEQ ID NO:327 1 5j 1 F9 76.4 SEQ ID NO:328 15_ 11H3 61.9 SEQ ID NQ:329 15 12?A8 48.2 SEQ ID NQ:330 15 12D6 200.8 SEQ ID NO:331 15 12D8 45.9 SEQ ID NO:332 15 12D9 42.6 SEQ ID NO:333 15 3F1 0 5.4.1 SEQ ID NO:334 15_3G 11 59.6 SEQ ID NO:335 15_4F1 1 89.2 SEQ ID NO:336 15 -4H3 93.3 SEQ ID NO:337 15 6D3 61.3 SEQ ID NO:338 15 6G1 1 41 SEQ ID NO:339 15_9F6 54.2 SEQ ID NO:340 15F5 0.2 SEQ ID NO:341 16A1 3.6 SEQ ID NQ:342 16H3 1.2 SEQ ID NO:343 1701 2 2.3 SEQ ID NO:344 18D6 8 SEQ ID NQ:345 1906 2 SEQ ID NO:346 19D5 1.3 SEQ ID NO:347 20Al12 SEQ ID NQ:348 20F2 2 SEQ ID NO:349 2.1 OE+1 2 SEQ ID NO:350 23H1 1 3.2 SEQ ID NO:351 2401 1.8 SEQ ID NQ:352 2406 2.1 SEQ ID NQ:353 2.40E+08 9.8 SEQ ID NO:354 2 803 1t6.6 SEQ ID NQ:355 2H3 17.7 SEQ ID NO:356 30G8 6.4 SEQ ID NQ:357 3B 104 15.5 SEQ ID NQ:358 38- 10G7 19.6 SEQ ID NO:359 3Bl 12B1 19 SEQ lbDNQ:360 3B-12D10 6 SEQ ID NO:361 3B 2E5 12.6 SEQ ID NQ:362 30 1 0H3 30.8 SEQ ID NO:363 30 12H10 7.6 SEQ UD NO-364 '30 9H8 11.7 SEQ ID NQ:365 4A l~11 SEQ ID NO:366 4A- 102 17 SEQ ID NO:367 4B_13E1 18.6 SEQ ID NQ:36B 4B 13G10 4.6 SEQ ID NQ:369 4B 16E1 17 SEQ ID NO:370 4B 17A1 17.4 SEQ ID NO:371 14B 18F1 1 B.6 SEQ ID NQ:372 14B_1908 13.2 108 WO 02136782 PCTJUS01/46227 SEQ ID NO:373 4B_1G4 3.7 SEQ ID NO:374 4B 2106 14.8 SEQ ID NO:375 4B 2H7 4.4 SEQ ID NO:376 4B 2H8 31.9 SEQ ID NO:377 4B 6D8 15.2 SEQ ID NO:378 4B 7E8 17.1 SEQ ID NO:379 4C-8C9 15.1 SEQ ID NO:380 4H1 0.9 SEQ ID NO:381 6 14D10 28.2 SEQ ID NO:382 61 5G7 37.3 SEQ ID NO:383 616A5 39.8 SEQ ID ND:384 6 16F5 35.2 SEQ ID NO:385 61705 27.1 SEQ ID NO:386 61807 26.8 SEQ ID NO387 6 18D7 35.8 SEQ ID NO:388 6 19A10 45.7 SEQ ID NO:389 6 19B6 34.2 SEQ ID NO:390 61903 16.5 SEQ ID NO:391 6 1908 37.4 SEQ ID NO:392 6 20A7 40.4 SEQ ID NO:393 6 20A9 34.7 SEQ ID NO:394 620H5 24.3 SEQ ID NO:395 6 21F4 34.7 SEQ ID NO:396 6 22C9 14.8 SEQ ID NO:397 6 22D9 33.8 SEQ ID NO:398 6_22H9 15.9 SEQ ID NO:399 623H3 39.9 SEQ ID NO:400 6_23H7 38.5 SEQ ID NO:401 6_2H1 29.5 SEQ ID NO:402 6_3DB 41.7 SEQ ID NO:403 6_3G3 51.9 SEQ ID NO:404 6_3H2 57.2 SEQ ID NO:405 6_4A10 SEQ ID NQ:406 6 4B1 27 SEQ ID NO:407 6 5D1 1 15.2 SEQ ID NO:408 6-5Fl1 21.1 SEQ ID NO:409 65G9 25.6 SEQ ID NO:410 6 6_5 55.3 SEQ ID NO:411 6 7D1 39.5 SEQ ID NO:412 6_8H3 44.7 SEQ ID NO:413 6-9G11 60.3 SEQ ID NO:414 6F1 5.6 SEQ ID NO:415 71 4 15.9 SEQ ID NO:416 7 2A10 18.2 SEQ ID NO:417 7 2A11 42.6 SEQ ID NO418 7 2D7 49.9 SEQ ID NO:419 7 507 44.7 SEQ ID NO:420 7 909 SEQ ID NO:421 9_13F10 49.6 SEQ ID NO:422 913F1 28.7 SEQ ID NO:423 915D5 23 SEQ ID NO:424 915D8 97.6 SEQ ID NO:425 915H3 36.2 SEQ ID NO:426 918H2 22.7 SEQ ID NO:427 920F12 37.8 SEQ ID NO:428 9 2108 23.8 SEQ ID NO:429 9 22B1 35.8 SEQ ID NO:430 9 23A10 21 -109- WO 02/36782 PCTIUSO1/46227 SEQ ID NQ:431 9_24F6 58.3 SEQ ID NQ:432 9 4H10 67.5 SEQ ID NQ:433 9_4H8 78.5 SEQ ID NQ:434 9 8H1 44 SEQ ID NO:435 9_9H7 SEQ ID NQ:436 9C6 5.1 SEQ ID NO:437 9Hl1 1.7 SEQ ID NQ:438 0 4B10 279 SEQ ID NO:439 0 5B11 406 SEQ ID NO:440 0 5B3 367 SEQ ID NO:441 0 5B4 301 SEQ ID NQ:442 0 5B8 522 SEQ ID NO:443 0 5C4 306 SEQ ID NO:444 0 5D1 1 334 SEQ ID NO:445 0 5D3 660 SEQ ID NO:446 0_5D7 222 SEQ ID NO:447 0 6B4 315 SEQ ID NO:448 0 6D10 1177 SEQ ID NO:449 0_6D11 481 SEQ ID NO:450 0_6F2 516 SEQ ID NQ:451 0 6H9 486 SEQ ID NO:452 10_4C10 695.98 SEQ ID NO:453 104D5 827.16 SEQ ID NO:454 1 4F2 1155.19 SEQ ID NO:455 10 4F9 553.93 SEQ ID NO:456 10 4G5 304.57 SEQ ID NO:457 10 4H4 1183.6 SEQ ID NQ:458 11_3A11 556.62 SEQ ID NO:459 11 3B1 349.17 SEQ ID NO:46O 11 3B5 748.49 SEQ ID NO:461 11 3012 490.67 SEQ ID NQ:462 11 3C3 972.81 SEQ ID NO:463 11 306 878.27 SEQ ID NO:464 11 3D6 553.01 SEQ ID NO:465 1 1G12 584.79 SEQ ID NO:466 1 ii- 162 SEQ ID NO:467 1 1H2 366 SEQ ID NO:468 1 1H5 63 SEQ ID NQ:469 1 2A12 176 SEQ ID NO:470 1 2B6 239 SEQ ID NO:471 1 2G4 242 SEQ ID NO:472 12D2 104 SEQ ID NQ:473 1 2D4 152 SEQ ID NO:474 1 2F8 SEQ ID NO:475 1 2H8 294 SEQ ID NO:476 1 3A2 227 SEQ ID NO:477 1 3D6 64 SEQ ID NO:478 1 3F3 112 SEQ ID NO:479 1 3H2 183 SEQ ID NO:480 1 405 273 SEQ ID NQ:481 1 4D6 98 SEQ ID NO:482 1 4H1 196 SEQ ID NO:483 i 5H5 419 SEQ ID NO:484 1 6F12 14 SEQ ID NO:485 1 6H6 259 SEQ ID NO:486 3 11 Al 0 796.55 SEQ ID NO:487 3-14F6 753.73 SEQ ID NO:488 3-15B2 1041.32 -110- WO 02/36782 WO 0236782PCTIUS01/46227 SEQ ID ND:489 3_6AI 0 191.64 SEQ ID NO:490 3 6B1 611.81 SEQ ID NO:491 3-7F9 667.4 SEQ ID NO:492 3 8G1 1 991.44 SEQ ID NO:493 4 1B1O 770.91 SEQ ID NO:494 5 2B3 567.5 SEQ ID NO:495 5 2D9 754.36 SEQ ID NO:496 5 2F1 0 547.22 SEQ ID NO:497 6_ lAll1 455.41 SEQ ID ND:498 6 1D5 429.16 SEQ ID NO:499 6 1F11 1057.6 SEQ ID NO:500 6 1F1 698.15 SEQ ID NO:501 6_iHlO 170.11 SEQ ID NO:502 61 H4 859.12 SEQ ID ND:503 81 F8 828.78 SEQ ID NO:504 8 1G2 674.73 SEQ ID NO:505 81 G3 1088.97 SEQ ID NO:506 81 H7 1012.4 SEQ ID NO:507 8 1 H9 783.89 SEQ ID NO:508 GAT1-21F12 1.2 SEQ ID NO:509 GATi 24G3 1.3 SEQ ID NQ:510 GATi 29G1 SEQ ID NO:51 1 GATi 32G1 1.4 SEQ ID NO:512 GAT2_1 5G8 1.6 SEQ ID NO:513 GAT2-19H8 SEQ ID NO:514 IGAT2 21 F1 11.4 Km for AcCoA is measured using the mass spectrometry method with repeated sampling during the reaction. Acetyl.-coenzyme A and glyphosate (aminonium salts) are placed as 50-fold-concentrated stock solutions into a well of a mass spectrometry sample plate. Reactions are initiated with the addition of enzyme appropriately diluted in a volatile buffer such as morpholine acetate or ammonium carbonate, pH 6.8 or 7.7. The sample is repeatedly injected into the instrument and initial rates are calculated from plots of retention time and peak area. KM is calculated as for glyphosate.

EXAMPLE 8: SELECTION OF TRANSFORNM E. COUI An evolved gat gene (a chimera with a native B. licheniformis ribosome binding site (AACTGAAGGAGGAATCTC; SEQ IID NO:515) attached directly to the end of the GAT coding sequence) was cloned into the expression vector pQE8O (Qiagen) between the EcoRI and Hindfl sites, resulting in the plasinid pMAXY2l9O (Figure 11).

This eliminated the His tag domain from the plasmid and retained the B-lactamase gene conferring resistance to the antibiotics ampicillin and carbenicillin. pMAXY2l9O was electroporated (BioRad Gene Pulser) into XLL Blue (Stratagene) coli cells. The cells were suspended in SOC rich medium and allowed to recover for one hour. The cells were ill WO 02/36782 PCT/US01/46227 then gently pelleted, washed one time with M9 minimal media lacking aromatic amino acids (12.8 g/L Na2HPO4.7 H20, 3.0 g/L KH2PO4, 0.5 g/L NaCI, 1.0 g/L NH4C1, 0.4% glucose, 2 mM MgSO4, 0.1 mM CaC12, 10 mg/L thiamine, 10 mg/L proline, 30 mg/L carbenicillin), and resuspended in 20 ml of the same M9 medium. After overnight growth at 37 0 C at 250 rpm, equal volumes of cells were plated on either M9 medium or M9 plus 1 mM glyphosate medium. pQE80 vector with no gat gene was similarly introduced into E.

coli cells and plated for single colonies for comparison. The results are summarized in Table 6 and clearly demonstrate that GAT activity allows selection and growth of transformed E. coli cells with less than 1% background. Note that no IPTG induction was necessary for sufficient GAT activity to allow growth of transformed cells.

Transformation was verified by re-isolation of pMAXY2190 from the E. coli cells grown in the presence of glyphosate.

Table 6. Glyphosate selection of pMAXY2190 in E. coli Number of colonies Plasmid M9 glyphosate M9 1 mM glyphosate pMAXY2190 568 512 324 3 EXAMPLE 9: SELECTION OF TRANSFORMED PLANT CELLS Agrobacterium-mediated transformation of plant cells occurs at low efficiencies. To allow propagation of transformed cells while inhibiting proliferation of non-transformed cells, a selectable marker is needed. Antibiotic markers for kanamycin and hygromycin and the herbicide modifying gene bar, which detoxifies the herbicidal compound phosphinothricin, are examples of selectable markers used in plants (Methods in Molecular Biology, 1995, 49:9-18). Here we demonstrate that GAT activity serves as an efficient selectable marker for plant transformation. An evolved gat gene (0_5B8) was cloned between a plant promoter (enhanced strawberry vein banded virus) and a ubiquinone terminator and introduced into the T-DNA region of the binary vector pMAXY3793 suitable for transformation of plant cells via Agrobacterium twnefaciens EHA105 as shown in Figure 12. A screenable GUS marker was present in the T-DNA to allow confirmation of transformation. Transgenic tobacco shoots were generated using glyphosate as the only selecting agent.

Axillary buds of Nicotiana tabacum L. Xanthi were subcultured on halfstrength MS medium with sucrose (1.5 and Gelrite (0.3 under 16-h light (35-42 -112- WO 02/36782 PCT/US01/46227 pEinsteins m 2 s 1 cool white fluorescent lamps) at 24 °C every 2-3 weeks. Young leaves were excised from plants after 2-3 weeks subculture and were cut into 3 x 3 mm segments.

A tunefaciens EHA105 was inoculated into LB medium and grown overnight to a density of A600= 1.0. Cells were pelleted at 4,000 rpm for 5 minutes and resuspended in 3 volumes of liquid co-cultivation medium composed of Murashige and Skoog (MS) medium (pH 5.2) with 2 mg/L N6-benzyladenine 1% glucose and 400 uM acetysyringone. The leaf pieces were then fully submerged in 20 ml of A. tumefaciens in 100 x 25 mm Petri dishes for 30 min, blotted with autoclaved filter paper, then placed on solid co-cultivation medium Gelrite) and incubated as described above. After 3 days of co-cultivation, 20-30 segments were transferred to basal shoot induction (BSI) medium composed of MS solid medium (pH 5.7) with 2 mg/L BA, 3% sucrose, 0.3% Gelrite, 0- 200 uM glyphosate, and 400 ug/ml Timentin.

After 3 weeks, shoots were clearly evident on the explants placed on media with no glyphosate regardless of the presence or absence of the gat gene. T-DNA transfer from both constructs was confirmed by GUS histochemical staining of leaves from regenerated shoots. Glyphosate concentrations greater than 20 uM completely inhibited any shoot formation from the explants lacking a gat gene. Explants infected with A tumefaciens with the gat construct regenerated shoots at glyphosate concentrations up to 200 uM (the highest level tested). Transformation was confirmed by GUS histochemical staining and by PCR fragment amplification of the gat gene using primers annealing to the promoter and 3' regions. The results are summarized in Table 7.

Table 7. Tobacco shoot regeneration with glyphosate selection.

Glyphosate concentration Shoot Regeneration Transferred 0 uM 20 uM 40 uM 80 uM 200 uM genes GUS 100 0 0 0 0 gat and 100 60 30 5 3

GUS

-113- WO 02/36782 PCT/US01/46227 0 EXAMPLE 10: GLYPHOSATE SELECTION OF TRANSFORMED YEAST CELLS Selection markers for yeast transformation are usually auxotrophic genes Sthat allow growth of transformed cells on a medium lacking the specific amino acid or nucleotide. Because Saccharomyces cerevisiae is sensitive to glyphosate, GAT can also be used as a selectable marker. To demonstrate this, an evolved gat gene (0_6D10) is cloned from the T-DNA vector pMAXY3793 (as shown in Example 9) as a PstI-ClaI c fragment containing the entire coding region and ligated into PstI-ClaI digested p424TEF I (Gene, 1995, 156:119-122) as shown in Figure 13. This plasmid contains an E. coli origin ,1 of replication and a gene conferring carbenicillin resistance as well as a TRP1, tryptophan S 10 auxotroph selectable marker for yeast transformation.

C1 The gat containing construct is transformed into E. coli XL1 Blue (Statagene) and plated on LB carbenicillin (50 ug/ml) agar medium. Plasmid DNA is prepared and used to transform yeast strain YPH499 (Stratagene) using a transformation kit (Bioll01). Equal amounts of transformed cells are plated on CSM-YNB-glucose medium (BiolOl) lacking all aromatic amino acids (tryptophan, tyrosine, and phenylalanine) with added glyphosate. For comparison, p424TEF lacking the gat gene is also introduced into YPH499 and plated as described. The results demonstrate that GAT activity function will as an efficient selectable marker. The presence of the gat containing vector in glyphosate selected colonies can be confirmed by re-isolation of the plasmid and restriction digest analysis.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques, methods, compositions, apparatus and systems described above may be used in various combinations. The invention is intended to include all methods and reagents described herein, as well as all polynculeotides, polypeptides, cells, organisms, plants, crops, etc., that are the products of these novel methods and reagents.

All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

-114- WO 02136782 WO 0236782PCTUSOI/46227 SEQ IOD NO. Clone ID Sequence SEQ ED NO: 1 ST401 gat ATGATTGAAGTCAAACCAATAAA.,CGCGGAAGATACGTA

TGAGATCAGGCACCGCATT'CTCCGGCCGAATCAGCCGC

2FrGAAGCATGTATGTATGAAACCGA M GCTCGGGK3T 000f TACCTCGGTGGATATTACCGOGGCAAGCTGATC AGCATCGC~rCC2=CATAAAGCCGAACATrCAGAGCTT

GAAGGCGAAGAACAGTATCAGCTGAGAOGGATGGCGA

CGCTrGAAGGATACCGTGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAGG

GGOCAGACCITEATGGTGCAATGCCAGGACATCTGTG

AGCGGCTACTATGAAAAGCTCGGCLTCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATA'r~

________GATGTATAAGAAAITGACGTAA

SEQ ID NO:2 B6 gat ATGA2FIGAAGTCAAACCTATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATETCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAITPGCTCGGGGGC

ACG'TTCACCTCGGCGGATATTATCGGGACAGGCTGATC

AGCATCGCCTCcFICATCAAGCCGAACATTCAGAGCITT GAAGGCCAAAAACAGTATCAGCTfGAGAGGGATGGCGA

CACTTGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG

CTTATCCGCATGCCGAAGAGCTTCTTCGGAAAAAAGG

CGCAGACC2IFATGGTGCAACGCCAGGACATCTGTGA GCGGGTACTATAAAAAGCTCGGC1TCAGCGAACAAGGC

GGGGTCTACGATATACCGCCGATCGGACCTCATATTIG

ATGTATAAGAAAITGACATAA

SEQ lID NO:3 DS3 ATGA1TGAAGTCAAACCAATAAACGCGGAAGATACGTA TGAGATCAGGCACCGCATTCTCCGcICCGAATCAGCCGC FPGAAGCATGTATGTATGAAACCGA M GCTCGOGGGT ACG1TTCACCTCGGTGGATAFrACCGGGGCAAGCTGAT)C AGCATCGCTrCCTITATAATGCCGAACATrCAGAGCTr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTrGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCTTATCCGCCATGCCGAAGAGCITTTCGAAAAAAAG

GCGCGGACcIIrATGGTGCAACGCCAGGATATCTGTG AGCGGCTACTATGAAAAGCTCGC=rCAGCGAACAAGG CGGGATCTACGACATACCGCCGATCGGACCTCATA=1I _____GATGTATAAGAAATrGGCATAA SEQ ID NO:4 NHA-2 ATGATTGAAGTCAAACCAATAAACOCGGAAGATACGTA

TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

ITGAAGCATGTATGTATGAAACCGATTTGCTCGGGGGT

ACGT1CACCTCGGTGGATA'ITACCGGGGCAAGCTGATC AGCATCGCITCC'=CATAATGCCc3AACA1rCAGAc3CTr

GAAGGCCAAAAACAGTATCAGCTGAGAGOGATGOGGA

CGCTTGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GC'TTATCCGCCATGCCGAAGAGC'TCTrCGAAAAAAAG

GCGCGGACCITIATGGTGCAACGCCAGGATATCTGTG

AGC000TACTATGAAAAGCTCGGCCTCAGCGAACAAGG _____CGGGATCTACGACATACCGCCGATCGGACCTCATA=rT 115 WO 02/36782 WO 0236782PCTfUS01/46227

IGATGTATAAGAAATTGGCATAA

SEQIDNO:5JINH5-2 Il ATGATGAAGTCAA.ACCAATAAACGCGGAAGATACGTA TGAGATCAGGCACCGCA17CTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATITGCTCGGGGGT

GCGITCACCTCGGTGGATATrACCAGGGCAAGCTGATC AGCATCGC1TCC7ICATAAAGCCGAACA1TCAGAGCTT GAGGGCGAAGAACAc3TATCAGCTGAGAGGGATGGCGA

CGGTTGAAGGATACCGTGAGCAAAAAGCGOGAAGCAC

GCTCATCCGCCATGCCGAAGAGCT-1CTTCGGAAAAAGG

GGGCAGACC'IIATGGTGCAATGCCAGGACATCTGTG

AGCGG%2ACTATGAAAAGCTCGGCTCAGCGAACAGOG CGAAGTCTACGACATACCGCCGATCGGACCTCATA TfTT

GATGTATAAGAAATTGACGTAA

SEQ ED NO:6 ST401 MIEVKPLNAEDTYEaRIRNQPLEFACMYETDLLGGAFH GAT LGGYYRGKLISLASFKEHSELEO-EEQYQLRGMAT.EGY

REQKAGSTIJRHAEEULRKKGADLLWCNARTSVSGYYEK

LGFSEQGEVYDIPPIGPHILMYKKLT

SEQ ID NO:7 B6 GAT MEVKPINAEDTYE]RHFJLRPNQPLEACKYETDLLGGTFH LGGYYRDRLLSIASFI{QAEHSELEC3QKQYQLRGMAThEGY REQKAGSThTRHAEELLRKKGADLLWCNARTSVSGYYXK

_____LGFSEQGGVYDIPPIGPHILMYKKLT

SEQ ID NO:8 DS3 GAT AMVKPINAEDTYEHIRRJRPNQPL-A CMYETLLOGTFH

LGGYYRGKUISIASFHNAEHSELEG;QKQYQL-RGMA

TLEGYRPEQKAGSThIRHAEELLRKKGADLLWCNARdSVSG

_____________YYEKLGFSEQGGIYDIPPIGPHJLMYKKLA

SEQ ID NO:9 NHA-2 MIEVX<PINAEDTYEaRHRLRPNQPLEACMYETIDLLGGTFH GAT LGGYYRGKLISIASFHNAEHfSELEGQKQYQLROMATLEGY

RBQKAGSTLUIAEELLRKKGADLLWCNARISVSGYYEK-L

OGIYDIPPIGPHIIMYKK[LA

SEQ H) NH5-2 MIEVKPINAEDTYEIRHPJLRPNQPLEACMYETDLLGGAFH NO: 10 GAT LGGYYQGKLISIASF~KAEHSELEGEEQYQLRGMATLEGY

REQKAGSTLJRHEELLRKKGADLLWCNARTSVSGYYEK

________LGFSEQGEVYDIPPIGPHILMYKKLT

SEQ ID 13_10F6 ATGATI7GAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 11 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCciC

TGGAAGCATGCAAGTATGAAACCGATTGCTCAGGGGT

ACGTICACCTCGGTGGATA1TrACCGGGGCAAGCTGATC AGCATCGCCTCC2=CATCAAGCCGAACATCCAGAOCTT

GAAGGCCAAAAACAGTATCAGCT'GAGAGGGATGOCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTrC'rrCGGAAAAAAG GCGCGGACcTITT1'ATGGTGCAACGCCAGGACGTCGCG AGCGGGTACTATAAAAAGCTCGGC1TCAGCGAACAGGG CGAAGTCTACGACATACCGCCGGTCGGACCTCATA TT

ATGTATAAGAAATTGACGTAA

SEQ U) NO:12 13_12G6 ATGATT'OAAGTCAAACCAATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCOC

TGGAAOCATOCAAGTATGAAACCGATITCTCAOGGGT

GCG=ICACCTCGGTGGATATrACCGGGGCAAGCTGGT'C AGCATCGCCTCGTICATCAAGCCGAACATCCAGAGCrr -116- WO 02136782 WO 0236782PCTIUSOI/46227

GAAGTGCCAAAGACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGGGTACCGTGAGCAAAAAGCGGGCAGTACG

C'ITATCCGCCATGCCGAAGAGC'ITC1TCGGAAAAAGGG

GGCAGACCTC'ITATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGI3CTTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGACTGGGCCCCATATFTG

ATGTATAAGAAAITGACATAA

SEQ ED 14_2A5 ATGAIGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 13 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITGCTCGGGAGC

ACGTITCACCTCGGTGGATATrACCGGGGCAAGCTGATC AGCATCGG1=GCTIAATCAAGCCGAACATCCAGAGCT=

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTrGAAGGGTACCGTGAGCAAAAAGCGGGAAGCAC

GCTTATCCGCCATGCCGAAGAGCLTCTICGGAAAAAAG

GCGCGGACCT1TATGGTGCAACGCCAGGACGTCrGCG AGCGGGTACTATAAAAAGCTCGGC1TCAGCGAACAGGG

CGAAGTCTACGACACACCGCCGGTCGGACCTCATATJT

_____________GATGTATAAGAAA2TGACGTAA SEQ DD 14_2C1 ATGATrGAAGTCAAACCAATAAACGCGGAAGATAC3TA NO: 14 TGAGATCAGGGACCGCATTCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCAGGGGT GCG =TrACCTCGGTGGATATrACCGGGGCAAGCTGGTC AGCATCGCCCCATCAAGCCGAACATCCAGAGCTf

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CrrATCCGCCATGCCGAAGAGC~rC'rrCGGAAAAAAGG

CGCOGACCITIATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGC1TCAGCGAACAGGGrC GAAGTCTACGACACACCGCCGAC-rGGGCCCCATAYI=

GATGTATAAGAAATTGACGTAA

SEQ IID 14_2F1 1 ATGA'ITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 15 TGAGATCAGGCACCGCA'1TCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITGCTCAGGGGT

GCGYICACCITGGTGGATATTACCGGGGCAAGCTGGTC

AGCATCGCCTCCTITCATCAAGCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

C1TATCCGCCATGCCGAAGCGCTT=~CGGAAAAAGG GGCAGACCTC1ATGGTCGCAACGCCAGGACATCTGCGA GCGG3GTACTATAAAAAGCTCGGC'1TCAGCGAACAGGGC GAAGTCTACGACACACCGCCGGCCGGACCCCATA'Ir

GATGTATAAGAAATTGACGTAA

SEQ ID CIMERA ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 16 TGAGATCAGGCACCGCATI'CTCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGA M GCTCAGGGGT GCGTICACCTCGGTGGATATrACCGGGGCAAGCTGATC

AGCATCGCTTCCTITCATCAAGCCGAACAITCAGAGCTT

GA-AGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

_____CACTTGAAGGATACCGCGAGCAAAAAGCGGGCAGTACG

117 WO 02/36782 WO 0236782PCTfIJSOI/46227

C'IATCCGCCATGCCGAAGAGCTTG=CGGAAAAAGGG

GGCAGACCT=ATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGTCAGCGAACAGGGC

GAAGTCTACGACACACCGCCGGTCGGACCTCATATYIG

ATGTATAAGAAATrGACGTAA SEQ DD 10_12D7 ATGATPGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 17 TGAGATCAGGCACCG-NA1TCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCOGGGGC

ACGCT'rCACCTCGGTGGATATTACCGGGGCAAGCTGAT

CAGCAT'CGCTFTCCYCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCACJGTGAGAGGGATGGCG

ACACTTGAAGAGTACCGCGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAGC'1CTTCGGAAAAAG

GGOGCAGACCTCTTATGGTGYCAACGCCAGGACATCTGC

GAGCGGGTACTATAAAAAGCTCGGCTGAGCGAACAAG

GCGAAGTCTACGACATACCGCCGACCGGACCCCATA2Fr ________TI7ATGTATAAGAAATTGACGTAA SEQ ID 10_15F4 ATGATrGAAGTCAAACCAATAAACGGGGAAGATACGTA NO: 18 TGAGATCAGGCACCGCA2FICTCCGGCCGAATCAGCCGC

'ITGAAGCATGTATGTATGAAACCGATFIGCTCAGOGGT

ACGTTCACCTCGGTGGGTATTACCGGGGCAAGGTGGTC

AGCATCGCTCCT=rATCAAGCCGAACATCCAGAGCTr

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGAGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC'ITCTTCGGAAAAAOG

GGGCAGACCITIATGGTGCAAC GCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTICGGCITGFAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATATT

______GATGTATAAGAAATTGACGTAA

SEQ ED 10_17DI ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 19 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGOGGOC

ACG=ICACCTCGGTGGATATTACCGGGOCAAGGTGATC

AGCATCGC'1CC1TCATCAAGCCGAACATCCAGAOCTr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAG'IGAAGGGTACCGCGAGCAAAAAGCGGOCAGTACG

CTTATCCGCCATGCCGAAGAGCTTCTTGGGAAAAAGGG

CGCAGACCTITATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAAGGC

GAAGTCTACGACACACCGCCGQTCGGACCTCATAT1TT

ATGTATAAGAAAITGACGTAA

SEQ ID 10_17F6 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGiTA

TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGA1TGCTCGGGGGC ACGT1TCACCTCGGTGGATATTACCGGGGCAAGCTGGTC AGCATCGCTTCCFICATCAAGCCGAACATrCAGAGCT'r

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC1TGAAGAGTACCGCGAGTCAAAAAGCGGGAAGCAC GCTTATCCGCCATGCCGAAGAGCTc=CGGAAAAAGG _____________GCGCAGACCI=ATGG5TGCAACGCCAG3GACATCrGCG 118 WO 02136782 PTUO/62 PCTIIJSOI/46227

AGCGGGTACTATAAAAAGCTCGGCFPCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGGTCGGACCTCATA'1I

GATGTATAAGAAATTGACGTAA

;Z SEQ IOD 10_18G9 ATGAITGAAGTCAAACCAATAAACGCGGA-AGATACGTA NO:21 TGAGATCAGGCACCGCA'ITCTCCGGCCGAATCAciCCGC

TGGAAGCATGCAAGTATGAAACTGATTTGCTCGGT-GC

ACGTTTCACCTCGGTGGATA'1TACCGGGGCAAGCTGGTC AGCATCGCTTCCTICATCAAGCCGAACA1TCAGAGCTr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGGGA

CACTTGAAGAGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTTrCGGAAAAAGG

GGGCAGACCTCTTATGGTGCAACGCCAGGACATCTGCG

C~1 AGCGGGTAGTATAAAAAGCTCGGCTTCAGCGAACAAGG CGGGGTCTACGACATACCGCCGGTCGGACCTCATATITr GATGTATAAGAAATTC3ACGTAA C~1 SEQ ID 10_1113 ATGATL'GAAGTCAAACCAATAAACGCGGAGOATACGTA NO:22 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCGGGGG*T ACG1T=ACCTCGGTGGATATrATCGGTGGCAAGCTGGTC

AGCATCGCTTCCI=CATCAAGCCGAACATCCAGAGCFP

GAAGGCCGAAAACAGTATCAGCTGAGAGGGATGGCGA

CACFTGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTTCTCGGAAAAAAGG

CGCGGACC I IITIATGGTGCAACGYCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCYPCAGCGAACAGGOC

GAAGTCTACGACATACCGCCGACCGGACCCCATAT1

GATGTATAAGAAAFGACATAA

SEQ ID 10_20D10 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:23 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATIGCTCGGGGGC

ACGC1TCACCTCGGTGGATA2FrACCGGGGCAAGCTGAT

CAGCATCGCCTCCTTCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACACTTGAAGAGTACCGCGAGCAAAAAGCGGGCAGTAC

GC~rATCCGCCATGCCG3AAGAGCTTCTrCGGAAAAAGG GGGCAGACC=1~ATGGTGCAACGCCAGGACATCrGCG AGCGGCTACTATAAAAAGCTCGGCTrCAGCGAACAAGG CGGGGTCTACGACATACCGCCGGjTCGGACCTCATATT= GATGTATAAGAAA ACGTAA SEQ IID 10_23F2 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:24 TGAGATCAG43CACCGCATACTCCGGCCGAATCAGCCGC TITGAAGCATGTATGTATGAAACCGA2IMGCTCGOGGGC ACG1TCACCTCGGTGGATATACCGGOCAAGCTGGTC AGCATCGCTrC: I ICATCAAGCCGAACACCCAGAGCTT GAAGGCCAAAAACAGTATCAGCTGAGAcJOGATGGCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTrIFGGAAAAAGGG

GGAGACCTCTTATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTPCAGCGAACAGOG

GAAGTCTACGACACACCGCCGGTCGGACCTCATAITIG

-119- WO 02/36782 WO 0236782PCTfUSOI/46227

_____________ATGTATAAGAAAITGACGTAA

SEQ ID 10_2B8 ATGATTGAAGTCAAACGAATAA-ACGCGGAAGATACGTA

TGAGATCAGGCACCGCATFCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGArIGCTCGGGGGT ACGMr~CACCTCGGTGGATATTACCGGGGCAAGCTGATC AGCATCGCCTCCTrrCATCAAGCCGAACATCCAGAG$2T

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGAGTACCGCGAGCA-AAAAGCGCOOCAGTACG CTrATCCGGCATGCCGAAGAGCTTGTCGGAAAAAGGG

GGCAGACCTCTTATGGT)GCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTI7CAGCGAACAGGGC GAAGTCTACGACACACCGCCGGTCGGACCTCATATfIT ______ATGTATAAGAAA1TGACGTAA SEQ ID 10_2C7 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:26 TGAGATCAGGCACCGCA'TTrCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATGCTCAGGGGT

GCGT1-CACCTCGGCGGATATrACCGGGGCAAGCTGAT

CAGCATCGCCTCCTTCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATG-GCG

ACAGTCGAAGGGTACCGTGAGCAAAAAGGGGGAAGCA

CGCTCATCCGCCATGCCGA-AGAC3C1rTC1GGAAAAAA 0GGGCGGACCTEIATGGTGCAACGCCAGGACATCTGC

GAGCGGGTACTATAAAAAMXTCGGCTTCAGCGAACAGG

GCGAAGTCTACGACACACCGCCGGTCGI3ACCTCATAYJ'

TGATGTATAAGAAATGACGTAA

SEQ ID 10_3G5 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:27 TGAGATCAGGCACCGCAITCTCCGOCCGAATCAGCCGC TGrGAAGCATGCAAGTATGAAACCGAT1TGCTCGGXiOGC

ACGTITCACCTCGGTGGATATIACCOOGGGCAAGGTGGTC

AGCATCGCTTCCTICAT)CAAGCCGAACATCCAGAGCTFr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCL-rATCCGCCATGCCGAAGAGCTTGTrCGGAAAAAGG

GGGAGACCTTIATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGjCT7CAGCGAA.CAGGG CGAAGTCTACGACATACCGCCGACCGGACCCCATA=1]

______GATGTATAAGAAATFTGACGTAA

SEQ ID 10_4117 ATGKITGAAGTCAAACCGATAAACGCGGA-AGATACGTA NO:28 TGAGATCAGGCACCGCATI7CJCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGAT'ITGCI'CGGGGGC

ACG'1TCACCTCGGTGGATA1rACCGGGGCAAGCrGGTC AGCATCGCICC'TrTCATrCAAGCCGAACATCCAGAGC=

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGGGTACCGTGAGCAAAA-AGCGGGCAGTACG CYTATCCGCCATGCCGAAGAGCTrCTrCGGAAAAAGGG

GGCAGACCTITATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGC'ITCAGCGAACAGGGC

GAAGTCrACGACATACCGCCGACCGGACCCCATAITI 0TGTTAAGAAATTGACGTAA SE 10ID1 ATGATrGAAc3TCAAACCAATAAACGCGGAAGATACGTA 120 WO 02136782 WO 0236782PCT[US01/46227 NO:29TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGiCCGC

TGGAAGCATGCAAGTATGAAACCGAFIGGTCGGGGC

ACGCTTCACCTCGGTGGATATTACCGGGOCAAGCrGGT CAGCATCGCTTCUc I ICATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACGCTTGAAGGGTACCGTGAGGAAAAAGGGGGCAGTAC

GCTTATCCGCCATGCCGAAGAGCTITCGGAAAAAGG

GOGCAGACCTTIATGGTGCA-ACGCCAGGACATCTGCG

AOGfGGGTACTATAAAAAGCTCGGC'1CAGCGAACAGGG CGAAGTCTACGACATACCGCCGGTCGGACCTCATA1I

______GATGTATAAGAAATTGACGTAA

SEQ ID) 10_8C6 ATGAYPGAAGTCAAACCAATAAACGCGGAAGATACGTA NO :30 TGAGATCAGGCACCGCA1TCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT~rGCTC3GOOGT GCGrrrCACCTCGGTGGATATTACCGOOGCAAGCTGATC AGCATCGCCTCCT1CATCAAGCCGAACATCCAGAGCTI?

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGCGAGCAkAAAAGCGOGAAGTAC

GCTTATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAAG

GCGCGGACCTYTATrGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGCTCAGCGAACAAGG

CGGOGTCTACGACATACCGCCGGTCGGACCTCATATEI

GATGTATAAGAAA'TTGACGTAA

SEQ DD 1 10 ATGATITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 31 TGAGATCAGGCACCGCAPT=CCG*JCCGAATCAGCCGC TrGAAGCATGCAAGTATGAAACCGA'I=GCTCGGGGGT

ACGTITCACCTCGGTGGATAITACCAGGGCAAGCTGATC

AGCATCGCCTG'TCATCAAGCCGAACATTCAGAGCFI?

GAAGGCCAAAAACAGTATCAGC--TGAGAGGGATGGCGA

CGC1GAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC GC1TATCCGCCATGCCGAAGAGC2FICTTCGGAAAAAGG GGGCAGACC=11ATGGTGCAACGCCAGGACATCrGTG

AGCGGGTACTATAAAAAGCTGGC'ITCAGCGAACAAGG

CGGGGTCTAGGATATACCGCCGATCGGACCTCATAITX

_____________GATGTATAAGAAA'ITGACATAA

SEQ ID 1 1G3 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:32 TGAGATCAGGCACCGCATI'CCCGGCCGAATCAGCCGC 2FrGAAGCGTGTATGTATGAAACCGATrGCTCGGGGGC ACGTrTCACCTCGGCGGATETrACCAGGGCAAGCTGAT CAGCATCGCTT'CCTrTCATCAAGCCGAACETFCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACGC1TTGAAGGGTACCGCGAGCAkAAAAGCGGGCAGTAC GCTrATCCGCCATGCCGAAGAGCTCTrCGGAAAAAGG

GGGCAGACCTF-ATGGTGCAACGCCAGGACATCTGCG

AGCGGCTAGTATGAAAAGCTCGGCTFCAGCGAACAAOG

CGGGGTCrACGATATACCGCCGATCGGACCTCATATT1

GATGTATAAGAAA'ITGGCATAA

SEQ ID 11113 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:33 TGAGATCAGGCACCGCATACrCCGGCCGAATCAGCCGC

_______TGGAAGCATGCAAGTATGAAACCGATITGCTCAGGGGT

121 WO 02136782 WO 0236782PCTIUSOI/46227 GCGT1TCACCTCGGTGGATATIACCGGGGCAAGCTGATC

AGCATCGCCTCCTTITCATCAAGCCGAACACCCAGAGCIT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'EGAAGGGTACCGTGAGCAAAAAGCGGGGAGTACG

CTTATCCGCCATGCCGAAGCGCIT'ICGGAAAAAAGG

CGGGACC=TATGGTGCAACGCCAGGACATGTGCGA

GCGGGTACTATAAAAAGCTCGGCICAGCGAACAGGGC

GAAGTCTACGACATACCGCCAACTGGGCCCATAT1TG ATGTATAAGAAATrGACGTAA SEQ IID 12_1F9 ATGAITGAAGTCAAACCAATAAAGGCGGAAGATACGTA NO:34 TGAGATCAGGCACCGCATACTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGC

ACGT=TCACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCTICATCAAGCCGAACATCCAGAGCITr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGGGTACCGCGAGCAAAAAGGGGAAGCAC

GCTCATCCGCCATGGCGAAGAGC'ITCTTCGGAAAAAGG

GGGCAGACCTI=ATGGTGCAACGCCAGGACATCTGCG

AGGGGGTACTATAAAAAGCTCGGCI=CAGCGAACAOGGG

CGAAGTCCACGACATACCGCCGACCGGACCCCATATIT

_____TGATGiTATAAGAAATTGACGTAA SEQ ID 12_2(39 ATGATrGAAGTCAAACCAATAAACGCGOAAGATACGTA NO: 35 TGAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATTJGCCGGGGGT

ACGTrrCACCTCGGCGGATATTACCGGGGCAAGCrGGT CAGCATCGC1TCCITCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACAC=GAAGGATACCGTGAOGAAAAAGCGGGCAGTAC

GCITATCCc3CCATGCCGAAGAGCrC=CGGAAAAAGG GGGCAGACCTCITATGGTrGCAACGCCAGGACATCTGCG

AGCGOGTACTATAAAAAGCTCGOCICAGCGAACAGGG

CGAAGTCTACGACACACCGCCGGTCGGACCTCATAII

_____GATGTATAAGAAATI'GACGTAA

SEQ ID 12_3F1 ATGiAFIGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:36 TGAGATCAGGCACCGCATTCICCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGA'rTPCTGOGOC ACGT1CACCTCGGTGGATATrACCGGGGCAAGGTGATC AGCATCGC2FJITC ATCAAGCCGAACATCCAGAGC11?

GAAGGCCAAAAACAGTATCAGCTGAGAGOGATGOCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGAAGTAC

GCTCATCCGCCATGCCGAAGAGCTC7CGGAAAAAGG

GGGCAGACCTIIATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCG(JCTrCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATA=H

GATGTATAAGAAATrGACGTAA SEQ ID 12_5C10 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:37 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATICTCGGGGGC

ACG1TrrCACCTCGGTGGATATTATrCGG*3GCAAGCTGATC

______AGCATCGCCICATCAAGCCGAACATCCAGAGCYI'

-122- WO 02136782 PTUO/62 PCT[US01/46227

GAAGGCCAAAAACAGTATCAGGTGAGAGGGATGGCGA

CACITGAAGAGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTTCTrCGGAAAAAGG

GGOCAGACQTLTATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCTCAGCGAACAGGG

CGAAGTCTACGACGCACCGCCGACCGGACCTCATAFTI

GATGTATAAGAAATrGACGTAA SEQ ID 12_6A10 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 38 TGAGATCAGGCACCGCAICTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGC

ACG'I=CACCTCQOCGGATATTACCCTGGCAAGCrGGT

CAGCATCGCCTCCITCATCAAGCCGAACATCCAGAGGT

TGAAGOCCAAAAACAGTATCACICTGAGAGGGATGGCG

ACAC'=TGAAGGATACCGTGAGCAAAAAGCGGGCAGTAC

GCTrATGCGCCATGCCGAAGAGCTCJTTCGGAAAAAGG

GOOCAGACCTITITATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGC1TAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATATJT

_____________GATC3TATAAGAAA'TGACGTAA SEQ ID 12_6D1 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 39 TGAGAT)CAGGCACCGCATTCT'CCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGAT'ITGCTCGGGGGC

ACG'FTACCrCGGTGGATAMTACCGGGGCAAGCTGATC AGCATCGC2FTCCTCATCAAGCCGAACATCCAGAGCTT7

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTGAAGAGTACCGCGAGCAAAAAGCGOQAAGCAC

GCTCATCCGCCATGCCGAAGAGCTI'C1TCGGAAAAAGG GGGCAGACCT1TATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGC'ITCAGCGAACAAGG

CGGGGTCTACGACATACCGCCTGTCGGACCTCATATIT

________GATGTATAAGAAATrGACGTAA SEQ ID 12_6F9 ATGA'rrGAAGTCAAACCAATAAACGCGGAAGATACGTA TGAGATCAGGCACCGCA'1TCTCCGGCCGAATCAGCCGC 2TGAAGCATGTAAGTATGAAACCGAT1GCTCGGGGGT

ACGTI=CACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCTITATCAAOCCGAACATCCAGAGCTI

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC1TCTTCGGAAAAAGG GGGCAGACGTITATGiGTGCAACGCCAGGACATCTGCG AGCGGCTAGTATAAAAAGCTCGGCITrCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGACCGGACCCCATATII

GATGTATAAGAAATFGACGTAA

SEQ ID 12_6H6 ATGAT17GAAGTCAAACCAATAAACGCGGAAGATACGTA NO:41 TGAGATCAGGCACCGCATACTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGA2TGCTCGGGGGC ACGTICACCTCGGTGGATATrACCGGGGCAAGCTGGTC

AGCATCGCCTCCTITCACCAAGCCGAACATCCAGACIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

_____________CACTTGAAGGGTACCGTGAGCAAAAAGCGGGCAGTACG

123 WO 02/36782 WO 0236782PCTIUS01/46227 CTrATCCGCCATGCCGAAGCCGCTr=rCGGAAAAAAGG CGCGGACCT1TATGO3TGCAACGCCAGGACATCTGCGA GCGGGTAGTATAAAAAGCTCGjGCTTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGACCGGACCCCATAFFI

GATGTATAAGAAATI'GACATAA

SEQ ID 12_7D6 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:42 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACTGAT1GCTCGGGGGC

ACGTITCACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGC'ITCCEITATCAAGCCGAACATCCAGAGCI1'

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG

c'IATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAGGG

GGCAGACCTT=ATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTI'CAGCGAACAAGGC

GGGGTCTACGACATACCGCCGACCGGACCCCATATT

_____________GATGTATAAGAAATTGACGTAA

SEQ ID 12_7011 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:43 TGAGATCAGGCACCGCAT=CCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGC

ACGT1CACCTCGGTGGATATI'ACCGGGGCAAGCGATC AGCATCGCCTCCTICATCAACGCCGAACA2fTCAGAGCTI'

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACITGAAGGATACCGCGAGCAkAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCITCTTCGGAAAAAGGG

GGCAGACCTTIATGGTGCAACGCCAGGACATCTGCGA

GCGGTACTATAAAAAGCTCCGCAGCGAACAGGGC

GAAGTCTACGACACACCGCCGG'TCGGACCTCATATP1G ________ATGTATAAGAAAITrGACGTAA SEQ ID 12F5 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGiTA NO:44 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGA'1TrCTCGGGciGT

ACGTICACCTCGGTGGATATITACCAGGGICAAGCTGATC

AGCATCGCTTCCTTCATAAAGCCGAACATTCAGAGCFT

GAGGGCCAAAAACAGTATCAGC-TGAGAGGGATGGCGA

CAC'IGAAGOGTACCGCGAGCAAAAAGCGGOCAGTACG

CTrATCCGCCATGCCGAAGAGC1fTCFTCGGAAAAAGGG GGCAGACC=flATGGTGCAATGCCAGGACATCTGTGA GCGGGTACTATAAAAAGCTCGGC1=CAGCGAACAAGGC GGGATCTACGACATACCGCCGATCGGACCT)CATAT1TG

ATGTATAAGAAATTGACGTAA.

SEQ ID 12G7 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA

T)GAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC

ITGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGT

AGCATCGCTrCCTTCATAAAGCCGAACA~rCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGC'TGAAGGATACCGTGAGCAAkAAAGCGGGAAGCAC ACTCATCCGCCATGCCGAAGAGCTCTrCGGAAAAAAG _____GCGCAGACC=mATGGTGCAACGCCAGGACATCTGTG 124 WO 02136782 WO 0236782PCTIUSO1/46227

AOCGGGTACTATAAAAAGCTCGGCTCAGCGAACAGGG

CGAAGTCTACGACATACCGCGATCGGACCTCATA=II

GATGTATAAGAAATrGACGTAA SEQ ID 1_2H6 ATGA'TrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:46 TGAGATCAGOCACCGCATTCITCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGA =TGGTCGGGOGT GCGT1CACCTCGGTGGATATTACCGGGGCAAGGTGATG AGCATCGCCTCCTITCATCAAGCCGAACAITPCAGAGCTr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTPQAAGOGTACCGCGAGCAAAAAGCGGGAAGTAC

GCTrATCCGCCATiGCCGAAGAGCT17CICGGAAAAAGG GGGCAGACCT1TATGGTGCAACGCCAGGACATCTGCG AGCGGGTACTATAAAAAGCTCGGC1TTCAGCGAACAAGG CGGGGTCTACGACATACCGCCGATCGGACCTCATA1T=

_____________GATGTATAAGAAATTGACGTAA

SEQ IID 13_12G12 ATGA1TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:47 TGAGATCAGGCACCGCATTCTCCGGGCGAATCAGCCGC '1TAAGCATGTATGTATGAAACCGAT1fTGCTCGGGGGT

ACGTITCACCTCGGTGGATAITACCGGGGCAAGCTGATC

AGCATCGCTTCCT=TAATCAAGCCGAACATCCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CA=TGAAGAGTACCGCGAGCAAAAAGCGGGAAGTAC

GC2FFATCCGCCATGCCGAAGAGCTrC'ICGGAAAAAAG GCGCGGACCI=rATGGTGCAACGCCAGGACATCTGCG AGCGGGTACTATAAAAAGCTGGGC~rCAGCGAACAGGG CGAAGTCTACGACATACCGGCCCiGTCGGACCTCATA=FT

_____________GATGGATAAGAAATT'GACGTAA

SEQ ID 13_6D10 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:48 TGAGATCAGGCACCGCATTCTCCGOCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGATrCGCrCGGAGGC ACGTICACCTCGGTGGATATrACCGOOGCAAGCTGATC AGCATCGCTTCC2FETAATCAAGCCGAACATCCAGAGCET

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGGTACCGTGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC2FrCTTCGGAAAAAGG

GGGCAGACCTC=ATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCTTCAGCGiAACAGGG

CGAAGTCTACGACACACCGCCGGTCGGACCTCATAITT

______GATGTATAAGAAATTIGACGTAA

SEQ ID 13_7A7 ATGATCGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:49 TGAGATCAGJCACCGCATTCCGGCCGAAT)CAGCCGC TTGAAGCATGTATGTATGAAACCGA GCTCAGGAGT

GCGTICACCTCGGCGGATAITACCGOGCAAGCTGAT

CAGCATCGCCTCCTITCACCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGGGGGATGGCG

ACACIGAAGAGTACCGCGAGCAAAAAGCGGGAAGTA

CGCITATCCGCCAT)GCCGAAGAGCTPCTCGGAAAAAG

GGGGCAGACCT1TATGGTGCAACGCCAOGACATICTGC

GAGCGGGTACTATAAAAAGCTCGCTCAGCGAACAGG

_____GGAAGTCTACGACACACCGCCGGTCGGACCTCATATT

125 WO 02136782 WO 0236782PCT[US01/46227

_____________TTGATGTATAAGAAATTGACGTAA

SEQ ED 13_7B12 ATGA2FrGAAGTGAAACCAATAAACGCGGAAGATACGTA NO: 50 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGAGC

ACGMflCACCTCGGTGGATATTACCGGGGCAAGCTGATC AGCATCGCCTCCTTTCATCAAGCCGAACATCCAGAGC1T

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGCGAGCAAAAkAGCGGGCAGTAC GCTTATCCGCCATGCCGAAGAQCTrCTTGGGAAAAAAG GCGCGGACC ITI rGTGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAOCTCGCTT=CAGCGAAGAGOG

CGAAGTCTACGACATACCGCCGACTGGGCCCCATA1T= CATGTATAAGAAG2FTGACGTAA SEQ ED 13_7C1 ATGA'ITGAAGTCAAACCAATAAATGCGGAAGATACGTA NO: 51 TGAGATCAGGGACCGCATACTCCGGCCGAATCAGCCGC 2FrGAAGCATGCAAGTATGAAACCGAT=TGCTCAOGGGT GCGTITACCTCGGTGGATATfACCGGGCAAGCTGATC AGCATCGCCTCJ1T~CAT'CAAGCCGAACATCCAGAACIT GAAGGCCAAkAAACAGTATCAGCTGAGAGGGATGG'CGA CACTrGAAGGATACCGTGAGCAAAAAGGGGTAGTACG c'IATCCGCCATGCCGA.AGAGCrTr=CGGAAAAAAGG

CGCGGACCTTTGTGGTGCAACGCCAGGAGATCTGCGA

GAGGGTACTATAAAAAGCTCGQGC'TCAGCGAACAAGGC

GAAGTCTACGACATACCGCCGACTGGGCCCCATKFEIG

ATC3TATAAGAAATTIGACGTAA SEQ IOD 13_8(6 ATGA~rGAAGTCAA.ACCAATAAACGCGGAAGATACGTA NO: 52 TGAGATCAGGCACCGCKFJ'CTCCGQCCGAATCAGGCGC TGGAAGCATGCAAGTATGAAACCGA =CGCTCGGOGC ACGT1CACCTCGGCGGATATTACCGGGGCAAGCrGAT CAGCATCGC1TCC'=AATCAAGCCGAACATCCAGAGCT

TGAAGGTCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC=TGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CrATCCGCCATGcCCAAGAGC~r=CGGAAAAAAGG

CGCGGACCTPIATGGTGCAACGCCAGGACGTCTGCGA

GCGOGTACTATAAAAAGCTCGGCTTCAGCGAACAAGGC

GGGGTCTACGACATACCGCCGGTCGGACCTCATArjT-G ATGTATAAGAAATrGACGTAA SEQ IID 13_9F6 ATGATI'GAAGTCAAACCAATAAACGOGGAAGATACGTA NO:53 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGATCTGjCGGGOOOG ACG=hCACCTAGGTGGATATTACCGMJOCAAGCTGAT

CAGCATCGCCTCCTICATCAAGCCGAACATCGAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGQGATGGG

ACACTGAAGAGTACCGCGAGCAAAAAGCGGGAAGTA

CGC=ATCCGCCATGCCGAAGAG=CICGGcAAAAAG

GGGCAGACCTEIATGGTGCAACGCCAGGACATCTGC

GA GCGGGTACTATAAAAAGCTCGGC=CAGCGAACA GG

GCGAAGTCTACGACATACCGCCGGTCGGACCTCATAFI

______TGATGTATAAGAAATTGACGTAA

SEQ ID 14-10C9 ATGATrGAAGTcAAAccAATAAAcGCGGAAGATACGTA 126 WO 02/36782 WO 0236782PCTUSOI/46227 NO:54 TGAGATCAGGGACCGCATACTCCGGCCGAATCAGGCGC

TAGAAOCATGCAAGTATGAAACCGATIGCTCAGGGGT

GCGTICACCTCGGTGGATAITACCGGGGCAAGCTGATC

AGCATCGCTCCTCATCAAGCTGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CAC2FGAAGAGTACCGCGAGCAAAAAGCGGGAAGTAC GCTCATCCGCCATGCCGAAGAGCTTCflCGGAAAAAGG GGGCAGACC7fTATGGTGCAACGCCAGGACGTCTGCG

AGCGGGTACTATAAAAAGCTCGGC=CAGCGAACAGGG

CGAAGTCTACGACACACCGCGGTCGGACCTCATAFI=

GATGTATAAGAAGTrGACGTAA SEQ ID 14_10H13 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA 5 TGAGATCAGGCACCGCKTCTCGGCCGAATCAGCCGC TGGAAGCATGCA.AGTATGAAACCGA2FLGCTCAGGOGT

GCGTITCACCTCGGCGGATATT~ACCGGGGCAAGCTGGT

CAGCATCGGCTCGTICATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGiCTGAGAGGGATGGCG

ACACTGAAGAGTACCGGGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAA

GGCGCAGACCmTATGGTGCAACGCCAGGACATCTGC GAGCGGGTACTATAAAAAGCTCGGCT-7CAGCGAACAGG GCGAAGTCTACGACACACCGCCGGTCGGACCTCATATr

YGATGTATAAGAAGTTGACGTAA

SEQ ID 14_10119 ATGAIrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:56 TGAGATCAGGCACCGCATACTCCGGCCGAAT)CAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCAGGGGT GCGTICACCTCGGTGcIATATIACCGGGGCAAGCTGGTC

AGCATCGCCTCCTITATCAAGCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'1GAAGGATACCGTGAGCAAAAAGCGGGCAGTACG CFTATCCGCCATGCCGAAG3AGCTTC1CGGAAAAAAGG CGCGGACC'TITGTGGTGCAACG7CCAGGACATCTGCGA

GCGOGTACTATAAAAAGCTCGGCTTCAGCGAACAGGGC

GAAGTCTACGACACACCGCCGGTCGGACCTCATATVIT

________ATGTATAAGAAATTGAGATAA

SEQ ID 14_11C2 ATGATTGAAGTCAAACCAATAAAkCGCGGAAGATACGTA NO: 57 TGAGATCAGGCACCGCATT'CTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGAGC

ACGTITCACCTCGGCGGATATTACCGGGGCAAGCTGGT

CAGCATCGCTrCCThPCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGOCG

ACAC'TGAAGAGTACCGCGAGCAAAAAGCGGGCAGTAC

G ATCCGCCATGCCGAAGCGCTMC= CGGAAAAAGG GGGCAGACCT1TATGGTGCAACGCCAGGACATCTGCG AGCGGGTACTATAAAAAGCTCGGC2FTCAGCGAACAGG CGAAGTCrACGACACACCGCCGACCGGACCCCATATI-

TGATGTATAAGAAA'TGACGTAA

SEQ ID 14_121)8 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 58 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC FGAAGCATGTAAGTATGAAACCGATIGCJ7GGGGGT 127 WO 02136782 WO 0236782PCT[USOI/46227

ACGTITCACCTCGGCGGATATTACCGGOGCAAGCTGGT

CAGCATCGCCTCC2FITCATCAAGCCGAACATCCAGAGCT

TGAAOGCCAAAA-ACAGTATCAGCTGAGAGGGATGGCG

ACAC1TGAAGGATACCGTGAGCAAAAAGCTGGCAGTAC GCITATCCGCCATGCCGAAGCGCTrC1ITCGGAAAAAAG

GCGCGGACCITGTGGTGCAACGCCAGGACATCTGCG

AGCGGCTACTATAAAAAGCTCGGCTTCAGOGAACAAGG

CGGGGTCTACGACATACCGCCTGTCGGACCTCATA'II

GATGTATAAGAAATTGACGTAA

SEQID

NO:59 14_12H6 SEQ ID 142B6

ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATCCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGIJOGT

GCGTICACCTCGGTGGATMTACCGGGGCAAGCTGATC

ACGCATCGCCTCCI=rATCAAGCCGAACATCCAGAGCITr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CACTTGAAGAGTACCGCGAGCAAAAAGGGOCAGTACG

CTTATCCGCCATGCCGAAGAGC1CTTCGGAAAAAAGG

CGCGGACCTTLGTGGTGCAACGCCAGGACGTCTCGCGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGACTGGGCCCCATAT1TG

ATGTATAAGAAATTGACGTAA

ATGA1TGAAGTCAAACCAATAAATGCGGAAGATAC3TA

TGAGATCAGGCACCGCATTCTCCGGCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGT

ACGTICACCrCGGTGGATATTACCGOGGCAAGCTGATC AGCATCGCTCCTLTrAATCAAGCCGAACATCCAGAG=T

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CFATCCGCCATGCCGAAGAGC'ITC1CGGAAAAAAGG CGCGGACCI=fATGGTGCAACGCCAGGACGTCTGCGA GGGGTACTATAAAAAGCTCGGCTIrCAGCGAACAAGGC GGGGTCTACGACATACCGCCGGTCGcIACCTCATATI=G

ATGTATAAGAAATI'GACGTAA

ATGATTGAAGTCAAACCAATAAATGCGGAAGATAGGTA

TGAGATCAGGCACCGCAFIXTCCGGCCGAATCAOCCGC

TGGAAGCATGCAAGTATGAAACCGAT1GCTCAGGGGT

GCGTITCACCTCGGTGGATATTACCGOQGCAAGCTGGTC

AGCATCGCCC1TCATCAAGCCGAACATCCAGAG=~

GAAGGCCAAAAACAGTATGAGCTGAGAGOGATGGCGA

CACTCGAAGGGTACCGTGAGCAAAAAGCGGGCAGTACG

C1ATCCGCCATGCCGAAGAGCTTCGGAAAAAAGG CGCGGACC1TIGTGGTGCAACGCCAGGACATCTGCGA GTGGGTAGTATAAAAAGCTCGG(TrCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGACTGGGCCCCATATITG

ATGTATAAGAAATTGACGTAA

SEQ BD NO:61 14_2G11 SEQ ID) 14_3B2 ATGA2FPGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 62 TGAGATCAGGCACCGCATTCI2CAGGCCGAATCAGCCGC TGGAAGCATIGCAAGTATGAAACCGAT1GCTCAGGGGT GCGfTrCACCTCGGTGGATATrACCGGGGCAAGCTGGT)C _____AGCATCGCCTCC2FICATCAGGCCGAACATCCAGAG=T 128 WO 02/36782 WO 0236782PCTfUSOI/46227

GAAGTGCCAAAAACAGTATCAGCTGAGAGGGATGGGGA

CAC1TGAAGGATACCGTGAGCAAAAAGCGGGAAGCAC Gc'IATCCCGCCATGCCGAAGCGCTTCTCGGAAAAAAG GCGCGGACJITTT1'ATGGTGCAACGCCAGGACATCI'GCG

AGCGGTACTATAAAAAGCTCGOCTCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGCCGGACCTCATAYII

GATGTATAAGAAATrGACGTA SEQ ID 14_4H8 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:63 TGAGATCAGGCACCGCATTCTCCGOCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGA. GCTCGGGAGC

ACGTITCACCTCGGCGGATATTACCGGGGCAAGCI'GAT

CAGCATCGCCTCCI=CATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAG3GATGGCG

ACACTCGAAGOGTACCGTGAGCAAAAAGCGOGAAGCA

CGCTCATCCGTCCATc3CCGAAGACTCfCGGAAAAAA GGCGCGGACC'1IGTGGTGCAACGCCAGGACGTCT7GC GAGCGGCTACTATAAAAAGCT(.GGC'TrCAGCGAACAGG

GCGAAGTCTACGACACACCGCCGGTCGGACCTCATAFT

_____________TTGATGTATAAGAAATrGACGTAA SEQ ID 14_6A8 ATGA1TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:64 TGAGATCAGGCACCGCATTCT'CCGGCCGAATCAGCCGC 2FrGAkAGCATGTATGTATGAAACCGATITGCTCGOGGGT

ACGTICACCTCGGTGGATATTACCGGGGCAAGCTAGTC

AGCATCGCFTC1TAATCAAGCCGAACATCCAGAGCIT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CA GAAGGATACCGTGAGCAAAAAGCGGGCAGTACG CTrATCCGCCATGCCGAAGAGCTTCTTGGGAAAAAAGG CGCGGACCT1T=TGGTGCAACGCCAGGACATGTGCGA

GCGGGTACTATAAAAAGCTCGGCTFCAGCGAACAGGGC

GAAGTCTACGACACACCGCCGGTCGGACCTCATGTETG

ATGTATAAGAAATTGACGTAA

SEQ ID 146B 10 ATGAITGCAAGTCAAACCAATAAACOCGGAAGATACGTA NO: 65 TGAGATCAGGCACCGCATWCTCCGGCCGAATCAGCCGC TGOAAGCATGCAAGTATGAAACCGA =TGCTCGOGGGT ACG1TCACCTIGGTGGATATTACCGOGGCAAGCTGATC AGCATCGCTTCCT1CATCAAGCCGAACATCCAGAGC'T

GAAGGCCAAAAACAGTATCAGCTGAGAGOGATGGCGA

CACrCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG C'TrATCCGCCATGCCGAAGAGC2FrCTrCGGAAAAAAGG CGCGGACCTTIATG%3TGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTCAGCGAACAAGGC

GGGGTCTACGACATGCCGCCGGTCGGACCTCATAT1TG _____________ATGTATAAGAAGTrGACGTAA SEQ ID 14_6D4 ATGKFPGAAGTCAAACCAATAAACGCGOAAGATACGTA NO: 66 TGAGATCAGGCACCGCATI7CTCGACCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAAC(CGAYIGCTCGGAGGC

ACGTLTPCACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGC1TCC1TAATCAAGCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGGTGAGAGGGATGCGA

______CACITGAGGTACGTAGCAAAAAGCGGGCAGTACG

129 WO 02/36782 WO 0236782PCTIUSOI/46227 CFrATCCGCCATGCCGAAGCGCTTCTrCGGAAAAAGGG

GGCAGACCTCTIATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGC7CAGCGAACAGGGC

GAAGTCTACGACACACCGCCGGTCGGACCTCATKITIG

_____________ATGTATAAGAAATTGACGTAA

SEQ BD 14_7A1 1 ATGATTGAAGTCAAACCAATAAACGGiGAGGATACGTA NO :67 TGAGATCAGGCACCGCAFTrCTCCGGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAFIGCTCAGGGGT

GCG=fCACCCGGTGGATATTACCOGGGOCAAGCITGGTC AGCATCGCCTCCTrTCATCAAGCCGAACATCCAGAGC'TT

GAAGGCGTAAAACAGTATCAGCTGAGAGGGATGGCGAC

ACTCGAAGGGTACCGTGAGCAAAAAGCGGGAAGTACG

CTCATCCGCCATGCCGAAGAGC1TCICGGAAAAAGGG

GGCAGACCTC=ATGGTGCAACGCCAGGACGTCTGCGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGGGC

GAAGTCTACGACACACCGCCGACCGGACCTCATAT1T GATGTATAAGAAA~rGACGTAA SEQ ID 14_7A1 ATGATTGAAGTCAAACCAATAAACGCGGAGQATACGTA NO :68 TGAGATCAGGCACCGCAITCCCGGCCGAATCAGCCGC TG*3AAGCATGCAAGTATGAAACCGAnITGCTCAGGGGT GCG'1TCACCrCGGTGGATATrACCGGGGCAAGCTGGTC AGCATCGCCTCCTIfTCATCAAGCCGAACATCCAGAGCIT

GAAGGTAAAACAGTATCAGCTGAGAGGGATGGCGAC

ACTCGAAGGGTACCGTGAGCAAAAAOGGGGAAGTACG

CTCATCCGCCATGCCGAAGAGCTCICGGAAA.AAGGG

GGCAGACCT=rATGGTGCAACGCCAGGACGTCT)GCGA

GCGOGTACTATAAAAAGCTCGGCTTCAGCGAACAGGGC

GAAGTCTACGAGACACCGCCGACCGGACCTCATATI=

GATGTATAAGAAATTGACGTAA

SEQ IID 14_7A9 ATGATTGAAGTCAAACCAATAAACGCGGAGGATACGTA NO :69 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATTTGCTCGGGOGT

ACG2TPTCACCTCGGCGGATATTACCGGCAAGTGTC AGCATCGCCTC=2TCATCAAGCCAAACATCCAGAGCT

GAAGGCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGGTACCGTGAGCAAAAAGCGGGTAGTACG

=~ATCCGCCATGCCGAAGAGCTrC'rrCGGAAAAAAGG

CGCGGACCTITATGGTGCAACGCCAGGACGTCTGCGA

GCGOGTACTATAAAAAGCTCGGCTPAGCGAACAGGGC

GAAGTCTACGACACACCGCCGGTCGGACC'rCATATFT M

ATGTATAAGAAATTGACGTAA

SEQ ID 14_701 ATGATTGAAGTCAAACCAATAAACGCAGAAGATACGTA

TGAGATCAGGCACCGCATTTCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGA'TrGCTCAGGGGT

GCGTITCACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCTTCCFIAATCAAGCCGAACATCCAGAGGTr

GAAGGCCAAAAACAGTATCAG'ITGAGAGGGATGGCGA

CACTrGAAGAGTACCGTGAGCAAAAAGCGGGAAGTACG CTTATCCGCCATGOCCGAAGCGCTTr=CGGAAAAAQG _____________GGCAGACCTCrrATGGTGCAACGCCAGGACATCTGCGA -130- WO 02136782 WO 0236782PCT[US01/46227 GCGGGTACTATAAAAAGCTCGGCTrCAGCGAACAGGGC

GAAGTCTACGACACACCGCCGGTCGGACCTCATATIG

________ATGTATAAGAAITrGACGTAA SEQ ID) 14_71-19 ATGATrGAAGTCAAACCAATAkACGCGGAAGATACGTA NO:7 1 TGAGATCAGGCACCGCA'1TCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCGGGGGT

ACGTITCACCTCGOCGGATATTACCOOGGCAAGCTGGT

CAGCATCGCCQTrrCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGOATGGCG

ACAcTLGAAGGATACCGTGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAOCTTCITGGAAAAAA

GGCGCGGACC'TT1GTG*3TGCAACGCCAGGACATCTGC GAGCGGGTACTATAAAAAGCTC-GOC1TCAGCGAACAGG

GCGAAGTCTACGACATACCGCCGGTCGGACCTCATA=I

TGATGTATAAGAAAITGACGTAA

SEQ ID 14_SF7 ATGATJ'GAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 72 TGAGATGAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TTGAAGCATGCAAGTATGAAACCGATITGCTCGGGGGT

AC =1TCACCTCGGGGATArrACCGGGGCAAGCTGGT CAGCATCGCCTCC2ICATCAACGCCGAACATCCAGAGCT

TGAAGGCCAAAA.ACAGTATCAGCTGAGAGGGATGGCG

ACACTTGAAGAGTACCGCGAGCAAAAAGCGGOCAGTAC

GCT'rATCCGCCATGCTGAAGCGC1TTTCGGAAAAAAG GCOCGGACCTI GTIGTGCAACGCCAGGACATCTGCA

AGCGGGTACTATAAAAAGCTCGGCTCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGACTGGGCCCCATA71 GATGTATAAGAAA~rGACGTAA SEQ ID 15_10C2 ATGATGAAGTCA-AACCAATAAACGCGGAAGATACGTA NO:73 TGAGATCAGGCACCGCATJ'CTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGATrrGCTCAGGGGT GCGFICACCTCGGTGGATAnFACCGGGGCAAGCTGGTC AGCATCGCCTCCATCAAGCCGAACATCCAGAGCTr GAAGGCCAAAAACAGTATCAGCTGAGAGGGATG*3CGA

CACTGAAGGATACCGTGAGCAAAAAGCGGGAAGTACG

CTCATCCGCCATGCCGAAGAGCITC1TCGGAAAAAGGG

GGCAGACCTCITTATGGTGCAACGCCAGGACAACTGCGA

GCGGGTACTATAAAAAGCTCGGC= CAGCGAACAGGGT GAAGTC'TGACATACCGCCGACCGGACCCCATA1TIG

_____________ATGTATAAGAAATTGACGTAA

SEQ I 15_10D6 ATGAFPGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:74 TGAGATCAGGCACCGCA'TCTCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGA GCTCGGGGGC

ACGFICACCTAGGTGGATA'ITACCGGGGCAAGCTGGT

CAGGATCGCCTCCTITCATCAAGCCGAACATCCAGAGCT

TGAAGGCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACAC=GAAGAGTACCGCGAGCAAAAAGCGGGAAGCA

CGCTCATCCOCCATGCCGAAGAG=CTTCGGAAAAAG

GGGGCAGACCTCTATGGTGCAACGCCAGGACATCTGC

GAGCGGGTACTATAAAAAGGTCGGCITCAGCGAACAGG

A GCAATCTACGACATACCGCCGGTCGQACCTCATA=fl 131 WO 02136782 WO 0236782PCT[USOI/46227

_____________TGATGTATAAGAAAITGACGTAA

SEQ BD 15_11F9 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA TGAGATCAGGCACGCATrCTCCGCCGA.ATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGKFITGCTCAGGGGT

GCGTICACCTrGGTGGATAFTACCGGGGCAAGCTGGTC

AGCATCGCCTCCTITAATCAAGCCGAACATCCAGAGCTI'

GAAGGGCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTTrATCCGCCATGCCGAAGAGCTTC=CGGAGAAAGG

GGCAGACCTIIATGGTQCAACGCCAGGACATCTGCGA

GCGGCTACTATAAAAkAGCTCGCCTI'AGCGAACAGGGC

GA-AGTCTACGACATACCGCCGACCGGACCCCATATIT

GATGTATAAGAAATTGACGTAA

SEQ ID 15_1 1H3 ATGATI7GAAGTCAAACCAATAAACGCGGAAGATACGTA NO:76 TGAGATCAGGCACCGCATACTCCGGGCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCAGGGGT

GCGTITCACCTCGGTGGATATTACCGGOCAAGCTGYATC

AGCATCGCCTC=TICATCAAGCCGAACACCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTrGAGAGGGATGGCGA

CACTTGAAGGGTACCGTGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGCGCrrCTCGGAAAAAAGG

CGCGGACCTI=ATGGTGCAACGCCAGGACATCTGCGA

GCGGTACTATAAAAAGCTCGGC=CAGCGAACAGGGC

GAAGTCrTACGACATACGGCCAACTGGGCCCCATATT=G

_____ATGTATAAGAAATTGACGTAA

SEQ IID 15_12A8 ATGATGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 77 TGAGATCAGGCACCGCA =CTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITGCTCGGGGGT

ACGTTTCACGTCGGTGGATAT]7ACCCGGGGCAAGCTGATC

AGCATCGCCTCC=TATCAAGCCGAACATCCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATG*JCGA

CACTrGAAGGATACCGTGAGCAAAAAGCOGOCAGTACG CTrATCCGCCATGCCGAAGCGCTrCTTCGGAAAAAGGG

GGCAGACCTATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGGrCGGTCAGCGAACAGGGC GAAGTCTACGACATACCGCCGACCGGACCCCATA'1I

_______GATGTATAAGAAMATGACGTAA

SEQ ID 15_12D6 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:78 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATFGCTCAGGGGT

GCG2ITCACCTCGGCGGATATTACCGGCAAGCTGGT CAGCATCGCCTCC1TCAT'CAAGCCGAACATCCAGAGC? TGAAGGCCAAAAACAGTATCAGiCTGAGAGGQATGGCG

ACACITGAAGGGTACCGTGAGCAAAAAGCGGGCAGTAC

GTIATCCGCCATGCCGAAGAGC'TrCTCGGAAAAAAG

GCGCGGACC'IIATGGTGCAACGCCAGGACATCI'GCG

AGCGGGTACTATAAAAAGCTCGGCTI7CAGCGAACAGGG CGAAGTCTACGACACACCGCCGCGTCGGACCTCATA'1I

______GATGTATAAGAAGITGACGTAA

-132 WO 02136782 WO 0236782PCT/USOI/46227 NO:79 TGAGATCAGGCACCGCATACTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAFIGCTCGGGGGT

ACG1TCACCTCGGC3GGATArACCGGOOCAAGCTGGT

;ZCAGCATCGCCTCCTICATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCA-ACTGAGAGGGATGGCG

ACAC'IGAAGGATACCGTGAGCAAAAAGCGGGCAGTAC

GCTTATCCGCCATGCCGAAGACTC1CGGAAAAAAG GCGCGGACC=hIAT)GGTGCAACGCCAGGACGTCTGCG

AGCGGGTACTATAAAAAGCTGGCTTCAGCGAACAGGG

CAAAGTCTACGACATACCGCCGGTCGGACCTCATA'11 GATc3TATAAGAAATGACGTAA SEQ ID 15_12D9 ATGATrGAAGTCAAAGCAATAAACGCGGAGYGATACGTA NO: 80 TGAGATCAGGCACCGCA'1TCTCCGGCCGAATrCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATTITGCTCAGGQGT

ACGYITCACCTCGGGGATA'TA~CCGGGGCAAGCTGGT

C~~1 CAGCATCGCCTCCT1TATCAAGCCGAACATCCAGAGCT

TGAAGGCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACAC=GAAGAGTACCGCGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAGC'TrCGGAAAAAG GGGGCAGACCTCrrATGGTGCAACGCCAGGACATCTGC

GAGCGGGTACTATAAAAAGCTCGGCJCAGCGAACAGG

GCGAAGTCTACGACATACCGCCGGTCGGACCTCATAr TC*ATGTATAAGAAA1TGACATAA SEQ ID 15_3F10 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:8 1 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCAGGGGT

GCGTrTCACCTTGGTGGATATTACCGOGGCAAGCTGATC AGCATCGI=CCrICATCAAGCCGAACATCCAGAGCr GAAGGCCAAAAACAGTATCAGC'TGAGAGGGATGGCG3A CACIGAAG%3GTACCGTGAGCAAAAAGCGGGCAGCACG

CTTATCCGCCATGCCGAAGAGCTCTTCGGAAAAAAGG

CGCGGACC=T-ATGGTGCAACOC3CAGGACA=cGCGA

GAAGTCTACGACACACCGCCGGCGGACCTCATATI

GATGTATACGAAATTGACGTAA

SEQ ID 15_3G1 1 ATGAITGAAG'ITAAACCAATAAACGCGGAAGATACGTA NO :82 TGAGATCAGGCACCGCATACTCCGcJCCGAATCAGCCGC TrGAAGCATGCAAGTATGAAACCGATPGCTCG*JGGGT ACG1TCAC~TCGGCGGATATTACCGGGGCAAGCTGGT CAGCATCGCCTCCTrCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACAC1GAAGAGTACCGCGAGCAAAAAGCGGGCAGTAC

GCTFATCCGCCATGCCGAAGAGCTIQTCGGAAAAAAG

GCGCGGACCTTFGTGGTGCAACGCCAGGACGTC7GCG

AGCGGGOTACTATAAAAAGCTCGGCFCAGCGAACAGOG

CGAAGTCTACGACATACCGCCGGTCGGACCTCATAT1T ATGTATAAGAAAFrGACGTAA SEQ ID 15_4F1 1 ATGAITGAAGTCAAACCAATAAACGCGOAAGATACGTA NO: 83 TAAOATCAGGCACCGCATACTCCGGCCGAATCAGCCGC

'IGAGCATGTATGTATGAAACCGATIGCTCGGGGGC

133 WO 02/36782 WO (1236782PCT/USOI/46227

ACGTITCACCTCGGTGGATATTACCGGGGCAAGCTGGTC

AGCATCGCTTCCTIAATCAAGCCGAACATCCAGAGCTT

GA.AGGCCAAAAACAGTATCAGCTGAGAGTGGATGGCGA

CACITGAAGGGTACCGTGAGCAAAAAGCGGGCAGTACG

C=ATCCGCCATGCCGAAGCGC'TCITCGGAAGAAAGG

CGCGGACCT=1ATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCITCAGCGA.ACAGOGC

GAAGTCTACGACATACCGCCGiACCGGACCCCATAT=T

________GATGTATAAGAAATTGACGTAA

SEQ ID) 15_4H3 ATGATTGAAGTCAAACCAATAAACGCG3AAGATACGTA NO: 84 TGAGATCAGGCACCGCAITGCCGGCCGAATCAGCCGC

TTGAAGCATGCAAGTATGAAACCGATTGCTCGGOOGT

ACG=TCACCTCGGOCGGATATEACCGGGGCAAGCTGGT

CAGCATCGCICCTTCATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGGTGAGAGGGATGGCG

ACA=TGAAGAGTACCGCGAGCAAAAAGCGGGAAGTA

CGCTTATCCGCCATGCCGAAGA&G=FCTTCGGAAAAAA

GGCGCGGACCF1TATGGTGCAACGCCAGGACATCTGC

GAGCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGG

GCGAAGT'CTACGACATACCGCCGACTGOOGCCCATATT

TTGATGTATAAGAAAKIGACGTAA

SEQ ID 15_6D3 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 85 TGAGATCAGOCACCGCATACTCCGOGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATTGCTCGGGQGT

ACGT'ITCACCTCGGTGGATATTACCGGGGCAAGCTGAT)C

AGCATCGCCTCCTTTCATCAAC-FCCGAACACCCAGAGCI7

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGAGTACCGCGAGCAkAAAAGCGGGAAGCAC GCTCATCCGCCATGCCGAAGAGCTrCTrCGGAAAAAGG GGGCAGACCTC1TATGGTGCAACGCCAGGACATCTGCG AGCGGGTACTATAAAAAGCTCGGCTrCAGCGAACAGOG CGAAGTCTACGACATACCGCCGiACCGGACCCCATAT1T GATGTATAAGAAAITrGACGTAAk SEQ ID 15_6(311 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 86 TGAGATCAGGCACCGCATrCT'CCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAT=GCTCAGGO*JT

GCGT=CACCTCGGTGGATATTACCGGGGCAAGCTGGT)C

AGCATCGCCrCCFICATCAAGCCGAACATCCAGAGCTT GAAGGCCAAAAACAGTATCAGCrGAGAGOGATGGCGA CAC1TGAAGAGTACCGGAGCAAAAAGCGGGAAGCAC GCTCATCCGCCATGCCGAAGAGCT'C1TrCGGAAAAAGG GGGCAGACCTTIATGGTGiCAACGCCAGGACATCTGCG AGCGGGTACTATAAAAAGCTrCGGCTrCAGCGAACAGGG CAAAGTCTACGACATACCGCCGGTCGGACCTCATA1TI

______GATGTATAAGAAGITTGACGTAA

SEQ ID 15_9F6 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 87 TGAGATCAGGCACCGCATI'TCCGOGCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGA2FIGCTCGGGGGT ACGT1CACCTCGGCGGATATTAkCCGGGGCAAGCTGAT _____CAGCATCGCCT'CC2FTATCAAGCCGAACATCCAGAGCT -134- WO 02136782 WO 0236782PCTIUS01/46227 TGAAGGCCAAAAACAGTATCAGCTGAGAO7GGATGOCG

ACAGTCGAAGAGTACGCGAGCAAAAAGCGGGCAGTA

CGCTTATCCGCCATGCCGAAGAGC=CTTCGGAGAAAA

GGCGCGGACCTITATGGTGCAACGCCAGGACATCTGC

GAGCGGGTACTATAAAAAGCTCGOCTCAGCGAACAGG

GCGAAGTCTACGACATACCGCCTGTCGGACCTCATATIT

________TGATGTATAAGAAA'GACGTAA.

SEQ lID 15F5 ATGATCGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 88 TGAGATCAGGCACCGCATI7CTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAT~GCTCGGGGGT

ACGT1TACCTCGG'rGGGTACTACCGGGGCAAGCIrGAT CAGCATCGCrrCC'=CATAAAGCCGAACAFPCAGAGCT

TGAGGGCGAAGAACAGTATCAGCTGAGAGOGATGOCG

ACGCTTGAAGGATACCGTGAGCAAAAAGCGOGCAGTAC

GCITATCCGCTATGCCGAAGAGCITG'JTCGAAAAAAAG

GCGCGGACG'TTTATGGTGCAA CGCCAGGACATCTGTG

AGCGOGTACTATAAAAAGCTCGCTC=AGCGAACAGGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATT

GATGTATAAGAAATMGACGTAA

SEQ IID 1 6A1 ATGATPGAAGTCAAACCTATAAACGCGGAAGATACGTA NO: 89 TGAGATCAGGCACCGCATICTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATITGCTCGGGGGT

ACGC1TCACCTCGGTGGATATTACCAGGGCAAGCTGAT CAGCATCGCTrCCTICATAAAGCCGAACAITCAGGGCT

TGAGOGCGAAGAACAGTATCAGGTGAGAGGGATGGCG

ACG7CTCGAAGGGTACCGCGAGCAAAAAGGGGCAGTA CGCTTATCCGCCATGCCGAAGAGCfTC~CGAAAAAAA

GGCGCGGACCTITATGGTGCAATGCCAGGACATCTGT

GAGCGGCTACTATGAAAAGCTCGGCITCAGCGAACAGG

GCGAAGTCTACGACATACCGCCGATCGGACCTCATATI

______TGATGTATAAGAAAITGACGTAA

SEQ IID 16H3 ATGA'1TGACGTCAAACCTATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATTGTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGG*JC

ACGT'ITCACCTCGGCGGATATTACCAGGGCAAGCTGAT

CAGCATCGCCTCCTITCATCAAGCCGAACAT1'CAGAGCT

TGAAGGCCAAAAACAGTATGAGCTGAGAGQOATGGCG

ACAC1TGAAG4JGTACCGCGAGCAAAAAGCGGGAALGTA CGCTCATCCGCCATciCCGAAGAGGTrCTrCGGAAAAAci

GGGGCAGACCTITATGGTGCAATGCCAGGACATCTGT

GAGCGGGTACTATGAAAAGCTCGGCTTCAGCGAACAGG

GCGAAGTCTACGACATACCGCCGATCGGACCTCATA1T _____________TGATGTATAAGAAAFrGACGTAA SEQ ID 1 7C 12 ATGA2TGAAGTCAAACCAATAAGCGCGGAAGATACGTA NO:9 1 TGAGATCAGGCACCGCA'ITCTCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGA M GCTCGGGGGT GCGT1CACCTCGGTGGATA~rACCAGGGCAAGCTGATC AGCATCGCCTCCT1CATCAAGCCGAACATTCAGAGC1T

GAAGGCCAAAAACAGTATCACPGAGAGGGATGGCGA

______CACTTGAAGGGTACCGGAGCAAAAAGCGGGAAGTAC

135 WO 02136782 WO 0236782PCTIUSOI/46227

TATCCGCCATGCCGAAGAGCITCTTCGAAAAAAAG

GCGCGGACCT1TATGGTGCAACGCCAGGACATCTGTG

AGCGGGTACTATGAAAAGCTCGGCTTAGGGAACAGGG

;Z CGAAGTCTACGACATACCGCCGiATCGGACCTCATAT1TI

GATGTATAAGAAATGACGTAA

SEQ ID 1 8D6 ATGA7GAAGTCAAACCAATAAACGCGGAAGATAC3TA NO:92 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATTTGCTCGTGGGOC

ACGTICACCTCGGTGGATATTACCGOGGAAGCTGATC

AGCATCGC3TCC=TCATAAAGCCGAACA TCAGAGCTT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGOCAA

CGC1TGAAGGATACCGTGAGCAAAAAGCGGGAAGTACG

CTATCCGCCATGCCGAAGAGGCITCGGAAAAAGOG

r- GGCAGACCITTATGGTGCAACG-CCAGGACATCTGCGA

GCGGCTACTATGAAAAGCTCGGCITCAGCGAACAGGGC

C~~1 GAAGTGTACGACATACCGCCGATCGGACCTCATAIT1G

_____________ATGTATAAGAAATTGOCATAA

SEQ ID 19C6 ATGA'TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO :93 TGAGATCAGGCACCGCATT'CTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCGGGGGT ACGYPCACCTCGGTGGATATrACCGGGGCAAGCTGAT'C TGCATCGCCTCCTICATCAAGCCGAACATTCAGAGITr

GAAGGGCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTrGAAGGGTACCGCGAGCAAAAAGCCGGGAAGTAC

GCTTATCCGCCATGCCGAAGAGCITJFCGGAAAAAGG

GGGCAGACCTITATc3GTGCAATGCCAGGACATCTGTG

AGAGGOCTACTATGAAAAGCTCGGCITCAGCGAACAAGG

CGGGGTCTACGATATACCGCCGATCGGACCTCATATIT

GATGTATAAGAAKFIX3GCGTAA SEQ ID 1 9D5 ATGATGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:94 TGAGATCAGGCACTGCA'TCTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATTGCTCGGGGGT

ACGTICACCTCGGTGGATATITACCAGGGCAAGCTGATC

AGCATCGC~rCcI=CATAAAGCCGAACA1TCAGAGCFI

GAAGOCCAAAAACAGTATCAGGTGAGAGGGATGGCGA

CGCITGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG

CrCATCCGCCATGCCGAAGAGCTT=TCGGAAAAAGOG

GGCAGACCTITATGGTGCAATGCCAGGACATCTGTGA

GCGGCTACTATAAAAAGCTCGGCTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGATCGGACCTCATAITG

________ATGTATAAGAAATTGACGTAA

SEQ ID 20A 12 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 95 TGAGATCAGGCACCGCMTrCTCCGGCCGAATCAGCCGC

'ITGAAGCATGTATGTATGAAACCGATIGCTCGOGGGT

ACGTCACCrCGGTGGATATTACCAGGGCAAGCTGATC AGCATCGCTT)CCfTCATAATGCCGAACATITCAGAGCI

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGC'IGAAGGGTACCGTGAGGAAAAAGCOGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTFITCCGGAAAAAGG

______GGGTAGACC1TJTATGGTGCAACGCCAGGACATCTGTG -136- WO 02136782 WO 0236782PCTIUSOI/46227 AGCGOGTACTATAAAA.AGCTCGOC1TCAGCGAACAAGG

CGGGATCTACGACATACCGCCGATCGGACGTCATATT

GATGTATAAGAAATTGGCATAA

SEQ ID 20F2 ATGA'1TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:96 TGAGATCAGGCACCGCATCTCCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGAIMGCTCGGGGGT ACG=1CACGTCGGTGGATAYT.ACCGGGGCAAG(2TGATC AGCATCGCTTCCT1CATCAAGCCGAACATTCAGAGCTT GAAGGCCAAAAACAGTATCAGC2TGAGAGGGATGGCGA CAC1TGAAGGGTACCGCGAGCAAAA.AGCGGGAAGTAC GC1TATCCGCCATGCCGAAGAC-rCTTC'ITCGGAAAAAAG GCGCAGACCT1TATGGTGCAACGCCAGGACATCTGTG

AGCGGCTACTATGAAAAGCTCGGCTCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATTT

GATGTATAAGAAMTTGACGTAA

SEQ DD 2.10E+12 ATGATrGAAGTCAAACGAATAAACGCGGAAGATACGTA NO:97 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGT

GCGTICACCI'CGOTGGATATrACCAGGGCAAGCrGATC AGCATCGCTrCC'=CATCAAGCCGAACATrCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCYTGAAGGATACCGTGAGCAAAAAGCGGGAAGCAC

G(2TCATCCGCCATGCCGAAGAGC1TCTTCGGAAAAAAG GCGCAGACCflTATGGTGCAACGCCAGGACATCTGTG

AGCGGGTACTATAAAAA~CTCGGCTTGAGCGAACAGOG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATI

________GATGTATAAGAAATrGACGTAA SEQ ID 23H1 1 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:98 TGAGATCAGGCACCGCA2TCTCGGCCGAATCAGCCGC

TGGAGGCATGTATGTATGAAACCGATLGCTCGGOOGT

ACGYLCACCTCGGTGGATATfACCAGGGCAAGCTGAT'C AGCATCGCTTCC2=CATAAAGCCGAACATTAGAGC'TT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTTGAAGGGTACCGCGAGCAAAAAGCGOOCAGTACG

CTTATCCGCCATGCCGAAGAGC'1TCTCCGAAAAAAAGG CGCGGACCT1TATGGTGCAATGCCAGGACATCTGCGA

GCGGCTACTATGAAAAGCTCGGCTTCAGCGAACAGGC

GAAGTCTACGACATACCACCGATCGGACCTCATAT1TG

ATGTATAAGAAAITGGCATAA

SEQ ID 24(21 ATGATGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:99 TGAGATCAGGCACCGCA'TGCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGATITFGCTCGOGO3GC ACG1TCACCFCGGCGGATATATCGGGACAGGCTGATC AGCATCGCTI'CCTCATCAAGCCGAACATrCAGAGCTT GAAGGCCAAAAACAGTATCAGCI7GAGAGGGATGGCGA

CGCITTAAGGGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC'TC1TCGGAAAAAGG

GGGCAGACGTI=ATGGTGCAACGCCAGGACATCTGTG

AGCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGGG

CA A rCnTACGACATACCGCCGATCGGACCTCATATT 137 WO 02/36782 WO 0236782PCTIUSO1/46227 _______GATGTATAAGAAAGTGACGTA-k SEQ ED 24C6 ATGA'ITGAAGTCAAACCTATAAACGCGGAAGATACGTA NO: 100 TGAGATCAGGCACCGCATI7CTCCGGCCGAATCAGCCGC TrciAAGCATGTATGTATGAAACCGATITGCTCGGGGGT ACGTTrCACCTCGGTGGATATTACCOGGGOCAAGCTGATC AGCATCGC'ITCc'TCATCAAGCCGAACA'rrCAGAGCTT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGC'ITGAAGGGTACCGCGAGCAAAAAGGGGGAAGTAC

GCTrATCCGCCATGCCGAAGAGGCrC2FrCGAAAAAAAG GCGCGGACCT1TATGGTGCAACOCCAGGATAT'CTGT'G AGCGGCTACTATAAAAAGCTCGGC1TCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGATCGGACCTCATA=L

GATGTATAAGAAA'TGGCATAA

SEQ ID 2.40E+08 ATGATrGAAGTCAAACCAATAAACGCGGAA3ATACGTA NO: 101 TGAGATCAGGCACGcCATTCTCCGGCCGAATCAGCCGC

TGGAGOCATGCAAGTATGAAACCGATIGCTCGGGGGC

ACG2FICATCTCGGTGGATATTrACCG%3GGCAAGCTGATC AGCATCGC1TCCTITCATAATGCCGAACATTCAGAGC1T

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGOCG;A

CGCIT7GAAGGATACCGCGAGCAAAAAGCOGGCAGTACG CTTATCCGCCATGCCGAAGAGCTrCITCGGAAAAAGOG GGCAGACCITIATG3TGCAATGCCAGGACATCTGCGA GCGGCTACTATGAAAAGCTCGGCTrCAGCGAACAGGGC GAAGTCrACGACATACCGCCGATCGGACCTCATATF=TG

________ATGTATAAGAAATTGGCATAA

SEQ ID 2_8C3 ATGA'1TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 102 TGAGATCAGGCACCGTATT~rCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGA =TGCTCGGGGGT ACGTITCACCTCGGCGGATA1TATCGGGACAGGCTGATC AGCATCGCCTCCTTCATCAAGCCGAACAMTCAGAGCTIr GAAGO CCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTTGAAGGOTACCGCGAGCAAAAAGOCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTTGITGGAAAAAGGG

GGCAGACCTITATGGTGCAACGCCAGGACATCTGCGA

GCGGCTACTATciAAAAGCTCGGC'ITCAGCGAACAGGGC GAAGTCTACGACATACCGCCGATCGGACCTCATAIT1G

________ATGTATAAGAAATTGACGTAA

SEQ DD 2H3 ATGATTGAAGTCAAACCGATAAACGCGGAAGATACGTA NO: 103 TGAGATCAGGCACCOCATT'CTCCGGCCGAATCAGYCCGC TGGAAGCATGCAAGTATGAAACCGA1TTGCTCGG*3GGT ACGTrrCACCrCGGTGGATATTACCAGGGCAAGCTGATC AGCACCGCTTCCTICATCAAGCCGGACATTCAGAGC'Tr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACrrGAAGGGTACCGCGAGCGAAAAGCGGGAAGTAC GCTCATCCGCCATGCCGAAGAGC2TCTTCGGAAAAAGG

GGGCAGACC'IIATGGTGCAACGCCAGGATATCT)GCG

AGCGGGTACTATAAAAAGCTCGGCFrCAGCGAACAAGG

CGGGGTCTACGATATACCGCCGATCGGACCTCATAT=I

______GATGTATAAGAAATrGACGTAA SEQ IID t3008 IATGATFGAAGTCAAACCAATAAACGCGGAAGATACGTA 138- WO 02/36782 PTUO/62 PCTfUSOI/46227 NO: 104 TGAGATCAGGCACGGCATL'CTGCGGCCGAATCAGCCGC TfrGAkAGCATGTATGTIGAAACCGATTTGCTCGGGGGTG

CGTLCACCTCGGTGGATATTACCAGGGCAAGCTGATCA

GCATCGCTTCC=1CATCAAGCCGAACATTCAGAGCIT

AAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGAC

GCTTGAAGGGTACCGCGAGCAAAAGCGCGGCAGTACGC

TTATCCGCCATGCCGAAGAGCTrC1TCGGAAAAAAGGC GCAGACC1TATGGTGCAACC-TCCAGGACATCTGTGAG CGGGTACTATAAAAAGCTCGGC1TCAGCGAACAG%300G

AAGTCACGACATACCGCCGATCGGACCTCATAITIGA

________TGTATAAGAAJTrGACGTAA SEQ U) 3B_10C4 ATGATTGAAGTCAGACCAATAAACGCGGAAGATACGTA NO: 105 TGAGATCAGOCACCGTATTCTCCGGCCGAATCAGCCGC TTGAAGCATrGTATGTATGAAACCGAYIGCTCGGGGGC ACG'1TCACCTCGGTGGATArACCGOGGCAAGCTGATC AGCATCGCCTCCTITATCAAGCCGAACATTCAGAGCTIr

GAAGGCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CACTrGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTTCTTCGGAAAA.AGOG

GGCAGACCTIATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGC1CAGCGAACAGGGC

GAAGCCTACGACATACCGCCGATCGGACCTCATAITIG

_____________ATGTATAAGAAATGACGTAA

SEQ DD 3B_1007 ATGiATrFiAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 106 TGAGATCAGGCACCGCATI'CTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATIGCTCGOOGGT

ACGT1TCACCTCGGTGGATATrACCGGGGCAAGCTGATC

AGCATCGCCTCCTITATCAAGCCGAACATCAAGCTT

GAAGGCCAAAAACAGTATCAGCTFGAGAGGGATGGCGA

CGC2FGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG CTTATCCGCCATGCCGAAGAGC1TICTTCGGAAAAAAGG CGCGGACCT1TTATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCJCAGCGAACAAGGC

GGGGTCTACGACATACCGCCGATCGGACCCCATAFITG

________ATGTATAAGAAATTGACGTAA

SEQ ID 3B_12B1 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 107 TGAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATIGCTCGGGGGC

ACGFICACCTCGGTGGATA1TACCGGGGCAAGCTGATC

AGCATCGCCTCCTFCATCAAGCCGAACAITCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGQCGA

CACTGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCTTATCCGCCATGCCGAAGAGCTTCTCGGAAAAAGG

GGGCAGACCTI=ATGYGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCTPCAGCGAACAGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATI

GATGTATAAGAAMTTGACGTAA

SEQ IID 33_12D10 ATGA2ITGAAC3TCAAACCAATAAACGCG{3AAGATACGTA NO: 108 TGAGATCAGGCACCGTAITCTCCGGCCGAATGAGCCGC

_____________TGGAAGCATGTATGTACGAAACCGATTIGCTCGGGGGT

-139- WO 02136782 WO 0236782PCTfUSOI/46227 GCGTICACCTCGGTGGATATfACCGGGGCAAGCTGATC

AGCATCGCCTCCYICATCCAGCCGAACATCAGAGCTT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'FrGAAGGATACCGTGAGCAAAAAGCGOCAc3TACG CITATCCGCCATGCCGAAGAGCTrCTrCGGAAAAAAGG

CGCGGACC'TTATGGTGGAACGCCAGGATATCTGCGA

GCGGGTACTATGAAAAGCTCGGCTCAGCGA-ACAGGGC

GAAGTCTACGACATACCGCCGATCGGACCCCATATTIG

ATGTATAAGAATGACGTAA

SEQ ID 3B_2E5 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 109 TGAGATCAGGCACCGCA2LTCTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATITGCTCGGGGGC

ACGTIITCACCTCGGTGGATATL'ACCGOOGCAAGCTGATC

AGCATCGCCTCCTLCATCAAGCCGAACA1TCAGAGCT GAAGGCCAAAAACAGTATCAGCT7GAGAGGGATGGCGA CAC=rAAGGATACCGTGAGCAAAAAGCGGGCAGTACG CTrATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAGOG CGCGGACCITIATGGTGCAACtJCCAGGACATCTGCGA

GCGGCTACTATGAAAAGCTCGGCTI'CAGCAAACAGGGC

GAAGTCTACGACATACCGCCGATCGGACCTCATATEIG

________ATGTATAAGAAAYI7GACGTAA SEQ ID 3C_10H3 ATGA'ITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 110 TGAGATGAGGCACCGTATTCTCCGGCCGAATCAGCCGC

FTGAAGCATGTATGTATGAAACCGATMGCTCGGGOOC

ACGT1CACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCTITCATCAAGCGCGAACATTCAGAGCT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGGATACCGTGAGCAAAAAGCGGGAAGTACG

CTrATCCGCCATGCCGAAGAGCTTCTCGGAAAAAGGG

CGCAGACCTTTATOOTGCAACGCCAGGATATCTGCGA

GCGGCTACTATAAAAAGCTCGG CAGCGAACAAGGC GGGGTCTACGACATACCGCCGGTCGGACCTCATA1T=TG

ATGTATAAGAAATTGACGTAA

SEQ ID 3C_121110~ ATGATPGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 111 TGAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATIGCTCGGGGGC

ACG2FICACCTCGGTGGATATrACCGGGGCAAGCTGATG AGCATCGCCTCC'TrATCAAGCCGAACAITCAGAGC1

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTGAAGGGTACCGTGGGCAAAAAGCGGGCAGTACG

C=ATCCGCCATGCCGAAGAGCTTCTCGGAAAAAAGG

CGCGGACCT'TIATGGTGCAACGCCAGGACATCTGCGA

GCGGCTACTATGAAAAGCTCGGCITCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGATCGGACCTCATATI=G

ATGTATAAGAAATrGACGTAA SEQ ID 3C_9H8 ATGATPGAAGTCAAACCAATAAACGCGOAAGATACGTA NO: 112 TGAGATCAGGCACCGTA'TCTCCGGCCGAATCAGCCGC 2FTGAAGCATGTATGTATGAAACCGATIGCTCGGGGCC

ACGFICACCTCGGOCGGATATITATCAGGACAGGCTGATC

AGACTCCTrrCATCAAGCCGAACATTCAGAGcE 140 WO 02136782 WO 0236782PCT[USO1/46227

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCTTATCCGCTATGCCGAAGAGCTTCTTCGGAAAAAAG

GCGCGGACC17=ATGGTGCAACGCCAGGATATCTGCG AGCGGCTACTATGAAAAGCTCG*G1TCAGCGAACAGOG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATT

GATGTATAAGAAATTGACGTAA

SEQ lID 4A-lB 11 ATGATrGAAGTCAAACCTATAAACGCGGAAGATACGTA NO: 113 TGAGATCAGGACCGCATACTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATITGCTCGGGGGT

ACGTICACCTCGGTGGATATrACCGGOGGCAAGCTGATC

AGCATCGCCTCCTITCATCAAGCCGAACATCCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGC'TGAAGGGTACCGCGAGCAAAAAGCGGOCAGTACG

CTrATCCGCCATGCCGAAGAGCITCTTCGGAAAAAGG GGCAGACCTIATGGTGCAACGCCAGGACATCX3CGA

GCGGCTACTATGAAAAGCTCGGCTTCAGCGAACAGGOC

GAAGTCrTACGACATACCGCCGATCGGACCTCATATTIG

_____________ATGTATAAGAAATGACGTAA

SEQ IID 4A_1C2 ATGA~rGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 114 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1TGCTCGGOGC

ACGTTCACCTCGGGGATATTAT)CGGGGCAAGCTGATC

AGCATCGCCTCCYTCATCAAGCCGAACA1TICAGAGCFT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CGGTGAAGAGTACCGGGAGCAAAAAGCGGGCAGTACG

CTIATCCGCCATGCCGAAGAGCTP2ICGGAAAAAAGG CGCAGACCT1TATGGTGCAACGCCAGGACATCTGCGA GCGGGTACTATAAAAAGCTCGGCT7CAGCGAACAGGGC

GAAGTCTACGACATACCGCCGATCGGACCTCATATITG

ATGTATAAGAAMTTGACGTAA

SEQ ID 4B_13E1 ATGATTGAAOTCAAACCTATAAACGCGGAAGATACGTA NO:1 15 TGAGATCAGGCACCGCA1ITCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAFLTGCTCGGGGC

ACGTITCACCTCGGTGGATATTACCGGGGCAAGCTGATG

AGCATCGCTTCGTICATCAAGCCGAACATCCAGAGT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGAGTACCGCGAGCAAAAAGCGGGCAGTACG

CYTATCCGCCATGCCGAAGAGCTTCTI'CGGAAAAAAGG

CGCGGACC2IIGTGGTGCAACGCCAGGATATCTGCGA

GCGGCTACTATGAAAAGCTCGGCTTCAGCGAACAGGGC

GAAGTCTACciACATACCGCCGAITCGGACCTCATATI=G

________ATGTATAAGAAATI'GACGTAA

SEQ ID) 4B_13G10 TITACGTCAATICTrATACATCAAAATATGAGGTCCGAT NO: 116 CGIJCGGTATGTCGTAGAC1TCGCCCTGTTCGCTGAAGCC GAGC1~TIATAGTACCCGCTCGCAGATGTCCTGGCG2Tr GCACCATAAAAGGTCCGCGCCTITITCCGAAGAAGCrC

TIPCGGCATGGCGGATGAGCGTGCLTCCCGCTEEEGCTC

GCGGTACCCITCAAGCGTCGCCATCCCTCTCAGCTGATA

_____CTGTITTGGCCTTCAAGCTCTGAATGTTCGGCTGATG

141 WO 02136782 WO 0236782PCT[USOI/46227

AAAGGAGGGATGCTGATCAGCTTGCCCCGGTAATATC

CACCGAGGTGAAACGTGCCCCCGAGCAAATCAGIMCA

TACTJ'GCATGCjrCCAGCGGCTGATTCGGCCGGAGAATG CGGTGCCTGATCTCATACGTATCTCCGCG'1TTGGT _____________TIrGCTCAATCAT SEQ ID) 4B_16E1 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 117 TGAGATCAGOCACCGCA'ITGTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITGCTCGGGGGT

ACGT'TCACCTCGGCGGGATAITACCGGGGCAAGCTGAT

CAGCATCGCCTCC'-ECATCAAGCCGAACATrCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACAC=GAAGGGTACCGICGAGCAAAAAGCGGGCAGTAC

GC=ATCCGCCATGCCGAAGACCrCGGAAAAAGG

GGGCAGACCTEIATGGTGCAACGCCAGGACATCTGG

AGCCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAAGG

CGGGGTCTACGATATACCGCCGATCGGACCTCATAII

GATGTATAAGAAATrGACGTAA SEQ IID 4B_17A1 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 118 TGAGATCAGGCACCGCA'1TCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGGTCGGGGGC

ACG1TCACCrCGGCGGATAUTACCGGGGCAAG3CrGAT CAGCATCGCTTCC2TCATCAAGCCGAGCATCCAGAGCr TGAAG*3CCAAAAACAGTATCAGCTGAGAGGGATGOCG

ACGCTTGAAGAGTACCGCGAGCAAAAAGCGGGCAGTAC

GCFfATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAGG GGGCAGACCF1TATGGTGCAACGCCAGGACATCTGCG AGCGGCTACTATGAAAAGCTCGGG'rrCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATTI

________GATGTATAAGAAA'1TGACATAA SEQ ID 4B_1 8F1 1 ATGATTGAAGTCAATCCAATAAACGCGGAAGATACGTA NO: 119 TGAGATCAGGCACCGCA1TCTCCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGATIGCJ'CGGGGGC

ACGTCTCACCTCGGCGGATATTACCGGGGCAAGCTGAT

CAGCATCGG'ITCCTTCATAATGCCGAACAFPCAGAGCT

TGATGGCCAAAAACAGTATCAGGTGAGAGGGATGGCGA

CACIGAAGGGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTTCTCGGAAAAAAG

GCGCAGACCTITATGGTGCAACGCCAGGACATCTGTG

AGCGGCTACTATGAAAAGCTCG43CTTCAGCGAACAGGG CGAAGTCTACGACATACCGCCGAT)CGGACCTCATA=h7

GATGTATAAGAAA'TGACGTAA

SEQ ID 4B_19C8 ATGATGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 120 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAITTGCTCGGGciGC

ACGI=CACCTCGGCGGATATTACCGGGGCAAGCTGAT

CAGCATCGGCC=ICATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGG

ACGCITGAAGGGTACCGCGAGCAAAA.AGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGACTC=cTCGGAAAAAG

GGAGACCTTLATGGTGCAACGCCAGGACATCTGC

142 WO 02/36782 WO 0236782PCTIUSO1/46227

GAGCGGGTACTATAAAAAGTCTCGGCITCAGCGAACAAG

GCGGGGTCTACGATATACCGCCGATCGGACCTCATATr _____________TGATGTATAAGAAA1TGGCATAA SEQ ID 43_1G4 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 121 TGAGATCAGGCACCGCA=rTC.CGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATLGCTCGGGOT

GCGYTCACCTCGGCGGATATTACCGGGGCAAGCTGAT

CAGCATCGCCTCCTICATCAATCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGTGAGAGOGATOGCG

ACGCTTGAAGGGTACCGCGAGCrAAAAGCGGGAAGTAC GCTrATCCGCCATGCCGAAGAGCTI'C7IrCGGAAAAAAG

GCGCGGACCTITATGGTGCAACGCCAGGATATCTGCG

AGCGGTACTATAAAAAGCTCGC2F=CAGCGAACAGGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATI

________GATGTATAAGAAATTGACGTAA

SEQ ID 43_21C6 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 122 TGAGATCAGGCACCGCAT=GTCGGCCGAATCAGCCGC 3TGAAGCATGTATGTATGAAACCGATIM'GCTCGGGGGC

ACGTMCACCTCGGTGGATA'ITACCGGGOCAAGCTGATC

AGCATCGC~rCCTICATCA-AGCCGAACATTCAGAGCTT

GAAGGCCAAAAACAGTATCACTGAGAGGGATGGCGA

CACTTGAAGAGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCITCTTCGGAAAAAAG

GCGCGGACCTTTATGGTGCAACGCCAGGATATCTGCG

AGCGGCTACTATAAAAAGCTCGGCTrCAGCGAACAAGG CGGGGTCTACGATATACCGCCGATCGGACCTCATA'1J GATGTATAAGAAAFrGACGTAA SEQ IID 4B_2117 ATGA2FTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 123 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

'TOAAGCATGTATGTATGAAACCGATMTGCTCGGGG*JC

ACGTMCACCTCGGTGGATATrACCGGGGCAAGCTGATC AGCATCGCCTCCTICATCAAGCCGAACAITCAGAGCTr

GAAGGCCAAAAAGAGTACCAGCGAGAGGGATGGCGA

CGC2FrGAAGGOGTACCGCGAGCAAkAAAGCGGGCAGTACG CTrAT)CCGCCATGCCGAAGAGGYCTTCGGAAAAAGOG

GGCAGACCTITATGGTGCAACGCCAGGACATCTGCGA

GCGGCTACTATAAAAAGCTCGGCTTCAGCGAACAAGGC

GGGGTCTACGGCATACCGCCGATCGGACCTCATA1TIG

ATGTATAAGAAATTGACATAA

SEQ IID 4B_2H8 ATGA'IrGAAGCCAAACCAATAAACGCGGAAGATACGTA NO: 124 TGAGATCAGGCACCGCITFCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACTGAFITGCTCGGGOGC

ACGTTTCACCTCGGTGGATATTACCGGGGCiAAGCTGAT)C AGCATCGCCTCCT1TCATCAAGCCGAACATTCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATG-GCGA

CGCTrGAAGGGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGCTTCITCGGAAAAAAG

GCGCGGACCT1TATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCTCAGCGAACAGGG

_____________CGAAGTCTACGACATACCGCCGATCGGACCTCATA=1T 143 WO 02136782 WO 0236782PCT[USOI/46227 _____________GATGTATAAGAAATrGACGTAA SP-Q ID 4B_6D8 ATGATrGAAGTCAAACCAATAAACOCGGAAGATACGTA NO: 125 TGAGATCAGGCACCGCATACTCCGOCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1TGCTCGGGGGC

ACGTICACCTCGGTGGATAFI'ACCGGGGCAAGCTGATC

AGCATCGC1CCTICATCAAGCCGAACA1TCAGAGCTr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTPGAAGGGTACCGCGAGCAAAAAGCGGGTAGTACG

C'1TATCCGCCATGCCGAAGAGCTTCTI7CGGAAAAAGGG GGCAGACCT1TATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACATGGC

GAAGTCTACGACATACCGCCGATCGGACCTCATAT1= G

ATGTATAAGAAATT'GACGTAA

SEQ IID 4B_7E,8 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 126 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TGGAAGCATGCATGTATGAAACCGATrTGCTCcKXX3GC

ACGTFCACCTCGGTGGATATTACCGGGGCAAGCT'GATC

AGCATCGC'ITCC1TCATCAAGCCGAACATTCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CACYI'GAAGGGTACCGCGAGCAAAAAGCGGOCAGTACG

CTTATCCGCCATGCCGAAGAGCTrCT1TCGGAAAAAGGG

GGCAGACCTITITI'ATGGTGCAACGCCAGGACATCTGTGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGGOC

GAAGTCTACGACATACCGCCGATCGGACCTCATA1TIG

______ATGTATAAGAAATI'GACGTAA

SEQ ID 4C_8C9 ATGATTGAAGTCAAACCA.ATAAACGCGGAAGATACGTA NO: 127 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGATIGCTCAGGGGT GCGYITACCTCGGTGGATATrACCG*300CAAGCTGATC

AGCATCGCTTCCTICATCAAGCCGAACATCCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTFGAGAGGGATGGCGA

CACTGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTrATCCGCCATGCCGAAGAGCTTC1CGGAAAAAG*30 GGCAGACCT1TATGGTGCAACGCCAGGACATCT)GCGA

GCGGCTACTATGAAAAGCTCGOCTTCAGCGAACAGGGC

GAAGTGTACGACATACCGCCGATCGGACCMATATJ1G

ATGTATAAGAAATTAACATAA

SEQ ID 4H1 ATGATTGAGGTGAAACCGAYFAACGCAGAGGAGACGTA NO: 128 TGAACTAAGGCATAGGATACTCAGACCACACCAGCCGA TAGAGG1TrTATGTATGAAACCGATTACTGTGGTG CGT1CACTrAGGCGGT1ACAGGGGCAAGCTGATIT CCATAGCTCAflCCACCAGGCCGAGCATCCAGAACTCC

AGOOCCAGAAACAATACCAACTCCGAGGTATGGCTACC

'TrGGAAGGITATCGTGACCAGAAAGCGGGATCGAGCCr AATrAAACACGCTGAACAGATCCTTCGGAAGCcGGGGGG

CGGACATGCTATGGTGCAATGCGCGGACATCCGCCGGT

GGCTACTACAAAAAGTTAGGCTrCAGCGAGCAGGGAGA

GGTAHTGAAACGCCGCCAGTAGGACCTCACATCGTAA

_____________TGTATA.AACGCCTCACATAA

SE I 6-14D 10 IATGATFGAAGTCAAACCAATAAACGCc3OAAGATACGTA -144- WO 02136782 WO 0236782PCTIUS01/46227 NO: 129 TGAGATCAGGCACCGCATICTCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGAT1TGCTCGGOGGC ACG'ITrCACCTCGGTGGATATTACCGAGGCAAGCTGATC AGCATCGCCTCCTTCCATCAAGCCGAACATTCAGAGCTr

GAAGGCCATAAACAGTATCAGCTGAGAGGGATGGCGAC

ACIGAAGAGTACCGCGAGCAAAAAGCGGGAAGCACG

CTCATCCGCCATGCCGAACTAGC-ITCTCGGAAAAAGGG

GGCAGACCT1TATGGTGCAACGCCAGGACATCTGCGA

GCGGCTACTATAAAAAGCTCGGCTTCAGCGAACAAGGC

GGGGTCTACGACATACCGCCGGTCGGAC~rCATA2FTG

_____________ATGTATAAGAAA'ITGACGTAA

SEQ MD 6_15G7 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 130 TGAGATCAGGCACCGCATFCTCCGGCCGAATCAGCCGC

ITGAAGCATGTAAGTATGAAACCGATIGCTCGGGGGC

ACG'T=rACCTCGGCGGATA'ITACCGGGGCAAGCrGAT CAOCATCG=TCCT1TCATCAAGCCGAACATTCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGG

ACACTrGAAGGGTACCGCGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAG=TTCGGAAAAAA

GGCGCGGACGTIATGGTGCAAkCGCCAGGACATCTGC GAGCGGGTACTATAAAAAGCTCGGC1CAGCGAACAGG

GCGAAGTCTACGACATACCGCCGGTCGGACCTCATATIT

TGATGTATAAGAAATTGACGTAAk SEQ ID 6_16A5 ATGA2FrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 131 TGAGATCAGGCACCc3CATI'CTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT1GCTCGGGGGT ACGT1CACCTCGGTGGATATrACCGGGGCAAGCTGATC

AGCATCGCCTCCITJCACCAAGCCGAACATTCAGAGCT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTGAAGGATACCGTGAGCAAAAAGCGGGAAGTACG

=TATCCGCCATGCCGAAGAGCTTCTCGGAAAAAGGG

GGCAGACCITIATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTCAGCGAACAAGGC

GGGGTCTACGACATACCGCCGGTCGGACCTCATAFT=G

_____________ATGTATAAGAAATTGACGTAA

SEQ DD 6_16F5 ATGAITGAAGTCAAACCAATA-AACGCGGAAGATACGTA NO: 132 TGAGATCAGGCACCGCMTTCTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGAT=TGCTCGGGGGC

ACGT1CACCTCGGTGGATATTACCGGGGCAAGCTGAT)C AGCATCGTTCC2F1TCATCAAGCCGTACA1TCAGAG=F

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTrATCCGCCATGCCGAAGAGCTTCTrCGGAAAAAGOG

GGCAGACCTITAT'GGTGCAACGCCAGGACATCTGCGA

GGCTACTATAAAAAGCTCGGCITCAGCGAACAAGGC

GGGGTCTACGACATACCGCCGGTCGGACCTCATA1T=TG

ATGTATAAGAAA'TMACGTAA

SEQ ID 6_17C5 ATGATGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 133 TGAGATCAGGCACCGCA~rCTCCGGCCGAATCAGCCGC

'TGAAGCATGCAAGTATGAAGCCGATJGCTCGGGC

145 WO 02/36782 WO 0236782PCTIUSOI/46227

ACGTITCACCTCGGTGGATATI'ACCOGGGCAAGCTGATC

AGCATCGCTTCCTICATCAAGCCGAGCATCCAGAGITr

GAAGGGCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGGAAACCGTGAGCAAAAAGCGGGCAGTAC GCTTATCCGCCATGCCGAAGAGCT~TfCGGAAAAAOG

GOOCAGACCTITATGGTGCAACGCCAGGACATCTGCG

AGGGATTAAGTG GGAAG

CGAAGTCTACGACGTACCGCCGATCGGACGTCA-TATII

_________GATGTATAAC3AAA~rGACGTAA SEQ ID 6_1 8C7 ATGA3TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 134 TGAGATCAGOCACCGCATTCTCCOGGCCGAATCAGCCGC

TGGAAGCATGCAGGTATGAAACCGAT=TGCTCGGGGGC

ACG=TCACCTCGGTGGATATTATCGGGGCAAGCTGATC

AGCATCGC=CCTICATCAAGCCGAACATCCAGAGG1

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCIGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTI'ATCCGCCATGCCGAAGAGG1TTCGGAAAAAGOG GGCAGACCT1TATGGTGCAACGCCAGGATATCTGCGA

GCGOGTACTATAAAAAGCTCGGCTT'CAGCGAACAGGOC

GAAGTITACGACATACCGCCGGTCGGACCTCATAITIG

ATGTATAAGAAATTGACGTAA

SEQ ID 6_18D7 ATGAITGAAGTCAAACCAATAAACGGGGAAGATACGTA NO: 135 TGAGATCAGGCMCCGCAITCTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGATMGCT)CGGGGGT

ACG=ICACCTCGGTGGATATrACCGGGGCAAGCTGATC

AGCATCOCCTCC'=CATCAAGCCGAACATCCAGAGCTT

GAAGOCCAAAAACAGTATCAGCTGAGAGOGATGGCGA

CACTI'GAAGGGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGG1CTPCGrGAAAAAAG GCGCGGACC'1rATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCITCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATAT1T

(ATGTATAAGAAATFGACGTAA

SEQ ID 6_19A10 ATGATTGAAOCCAAACCAATAAAkCGCGGAAGATACGTA NO: 136 TGAGATCAGGACCGCATrCTCCGGCCGAATCAGCCGC

TI'GAAGCATGTATGTATGAAACCGATIGCTCGGGOOT

ACGTYITCACCTCGGTGGATKFI'ACCGGGOCAAGCTGAT)C

AGCATCGCCTCC'IICATCAAGCCGAACATCCAGAGCT7

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'IGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCTrATCCGCCATGCCGAAGAGCITCFICGGAAAAAGG GGGCAGACCT1TATGGTGCAACGCCAGGACATCTGCG AGCGGGTACTATAAAAAGCTCGGG1TCAGCGAACAGGG CGAAGTCTACGACATACCGCCGACCGGACCCCATAT1T GATGTATAAGAAAflGACGTAA SEQ ID 6_19B6 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 137 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGAT1ITGCTCAGGOGT

GCGTICACCTCGGTGGATATTATCGGGGCAAGCTGATC

_____________AGCATCGCTTCC2FICATCAAGCCGAACATTCAGAGC2Fr -146- WO 02/36782 WO 0236782PCTIUS01/46227 GAAGGGCAAAAkACAGTATCAGCTGAGAGGGATGGCGA

CACTCGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CMATCCGCCATGCCGAAGAGCTrC'TrCGGAAAAAOGGG

CGCAGACCTIJATGGTGCAACGCCAGGACATCTGCGA

GCGGCTACTATAAAAAGCTCGGCTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGGTCGGACCTCATATIIX

______ATGTATAAGAAA7TGACGTAA SEQ ID 6_19C3 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 138 TGAGATGAGOCACCGCATTCTCCGOCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGGGGT

ACGTMCACCTCGGCGGATA2FrACCG*300CAAGCrGAT

CAGCATCGCCTCCTTTATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAc3CTGAGAGGGATGGCG

ACACIGAAGGATACCGTGAGCAAAAAGCGGGCAGTAC

GC2FrATCCGCCATGCCGAAGAGC'TCITCGGAAAAAAG GCGCGGACC'1JATGGTGCAACGCCAGGACATCrGCG AGCGGGTACTATAAAAAGGTCGGCnTCAGCGAACAGGG CGAAGTCTACGACATACCGCCGAkTCGGACCTCATATIT

______GATGTATAAGAAATGACGTAA

SEQ ID 6_19C8 ATGAITGAAGTCAAACCAATAAAkCGCGGAAGATACGTA NO: 139 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGA =TGCTCGGGGGT ACGITACACCTCGGTGGATATrACCGGGGCAAGCTGAT

CAGCATCGCCTCCFICATCAAGCCGAACATCCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACACTI7GAAGGATACCGTGAGCAAAAAGGGCAGTAC GCTTATCCGCCAAGCCGAAGAGCTFJC~rCGGAAAAAGG GGGCAGACC'TrATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGGTTCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTTCGGACCTCATATII

______GATGTATAAGGAA'TMACGTAA

SEQ ID 6_20A7 ATOATFGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 140 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGATEGGTCAGGGGC ACGT1ITCACCTCGGCGGATATTACCGGGGCAAGCTGAT CAGCATCGCTrCC'TCATCAAGCCGAACATTCAGATCT

TGAAGGCCAAAAACAGTATCAGCTGMAGAGGGATGGCG

ACACTT'GAAGAGTACCGCGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAGCTCTICGGAAAAAG

GGGGCAGACC1T1ATGGTGCAACGCCAGGACATCTGC

GAGCGOGTAGTATAAAAAGCTCGGGITCAGCGAACAGG

GCGAAGTCTACGACATACCGCCOGTCGGACCTCATATT

TGATGTATAAGAAATTGACGTAA

SEQ ID 6_20A9 ATGATrGAAGTCAAACCAATAAACGCGGGAGATACGTA NO: 141 J7GAGATCAG*CACCGCA1T=TCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAITIGCTCGQGGGC

ACGTJTCACCTCGGTGGATATrACCGGGGCAAGCTGATC AGCATCGCCTCCTITCATCAAGCCGAACA1TCAGAGCTI'

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

______CACTTGAAGOGTACCGCGAGCAAAAAGCGGGCAGTACG

147 WO 02/36782 WO 0236782PCT/UJSOI/46227 CTrATCCGiCCATGCCGAAGAGC'ICTACGGAAAAAAGCG CGCGGACCTFTIATG*3TGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCITCAGCGAACAAGGC

GGGGTCTACGACATACCGCCGGTCGGACCTCATA=~IG

_____ATGTATAAGAAAFIY3ACGTAA SEQ ED 6_20H5 ATGATIriAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 142 TGAGATCAGGCACCGCA'TCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAc3TATGAAACCGAT1TGCTCGGGGGC ACGTICACCTCGGCGGATA1TACCGGGGCAAGCTGAT CAGCATCGCCTC=1ICATCAACGCCGAACATTCAGAGCT

TGAAGGCCAAAAACAGTATCAGGTGAGAGOGATGGCG

ACAC'1TGAAGGATACCGTGAGCAAAAAGCGGGAAGTAC GCTrATCCGCGATGCCGAAc3AGC'Ir=CGGAAAAAAG

GCGCGGACC=TATGGTGCAACGCCAGGACATCTGCG

AGGGCTACTATAAAAAGCTCGG =CAGCGAACAGOG

CGAAGTCTACGACATACCGCCGATCGGACCI'CATATIT

GATGTATAAGAAATTGACGTAA

SEQ IDD 6_21F4 ATGA'TTGAAGTCAAACCAATAA-ACGCOGAAGATACGTA NO: 143 TGAGATCAGGCACCGCGT1TCTCCGGCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATITGCTCGGGOGT

GCG=~CACCTCGGTGGATATrACCGGGGCAAGCPGATC

AGCATCGCCTCCTICATCAAGCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC=GAAGOGTACCGCGAGCAAAAAGCGc3GCAGTACG CTTATCCGCCATGCCGAAGAGCIT'1CGGAAAAAAGG

CGCGGACCTLT]ATOGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGGGC

GAAGTCTACGACGTACCGCCGGT'CGGACCTCATA=1IG ________ATGTATAAGAAATrGACGTAA SEQ ID 6_22C9 ATGATTrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 144 TGAGATCAGGCACCGCATrCTCCGGCCGAATCGGCCGC TrGAAGCATGTATGTATGAAACCGATrGCTCGGGGGC ACGTICACCTCGGTGGATATIrACCGGGGCAAGCTGATC

AGCATCGCCTCCFICATCAAOCCGAACATCCAGGGCTT

GAAGGCAAAAAACAGTATCAGCTGAGAGOGATGGCGA

CAc=GAAGAGTACCGCGAGCAAAA.AGCGGciAAGCAC GC~rATCCGCCATGCCGAAGAGICTrCTCGGAAAAAAG GCGCGGACCr1TATGGTGCAACGCCAGGACTICCGCG AGCGGGTACTATAAAAAGCTCGGCrTICAGCGAACAAGG

AGGGGTCTACGACATACCGCCGGTCGGACCTCATATIT

_____________GATGTATAAGAAATI'GACC3TAA SEQ ID 6_22D9 ATGAITGAAGTCAAACCAATAAACC3CGGAAGATACGTA NO: 145 TGAGATCAGGCACCGTA'1TCTCCGGCCGAATCAGCCGC

TGGAAGCATGCATGTATGAAACCGATTGCTCGAGO

ACGTITCACCTCGGTGGATAITACCGGGGCAAGCTGATC

AGCATCGCCTCCTCMATCAAGCCGAGCATTCAGAGCTI'

GAAGGCCAAAAACAGTATCAGGTGAGAGGGATGGCGA

CACTrGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTTC'TTCGGAAAAAAGG

_____CGCGGACC1TIATGGTGCAACGCCAGGACATCTGCGA 148 WO 02/36782 WO 0236782PCTfUSOI/46227

GCGGGTACTATAAAAAGCTCGGG'LTCAGCGAACAGGGC

GAAGTCTACGACATACG~CCGGTCGGACCTCATAT1TG

ATGTATAAGAAATTGACGTAA

SEQ ID 6_22H19 ATGA'ITGAAGTCAAACCAATAAACGGGGAAGATACGTA NO: 146 TGAGATCAGGCACCGCITrCTCCGGCCGAATCAGCCGC ITGAAGCATGTATGTATGAAACCGA M GCTCGGGGC ACGTITCACCTCGGTGGATATrAkCCGGGGCAAGCTGATC

AGCATCGCCTCCIMCATCAAGCCGAACATTCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTTGATGAGTACCGCGAGCAAAAAGCGGOCAGTACG

CTTATCCGCCATGCCGAAGAGCYrITCGGAAAAAAGG

CGCAGACCITTJAT)GGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGOC~rCAGCGAAGAGGGC GAAGTCTACGACATACCGCCGATCGGACCCCATAT1IG ATGTATAAGAAA'1TGACGTAA SEQ ID 6_23H3 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 147 TGAGATCAGGCACCGCKITTCTCCGGCCGAATCAGCCGC 2TGAAGCATGTATGTATGGAACTGA M GCTCGGJGG ACGT1CACCTCGGTGGATATI'ACCGGGGCAAGCTGATC AGCATCGC1TCCTJCATCAAGCCGAGCAACCAGAGGT

GAAGGCCAAAAACAGTATCAGCI'GAGAGGGATGGCGA

CA=IGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTFCTTCGGAAAAAGGG

GGCAGACCT1TATGGTGCAACGCCAGGACATCTGCGA GCGGGTACTATAAAAAGCTCGGC1TCAGCGAGCAAG3C

GGGGTCTACGACATACCGCCGGTCGGACCTCATATTTG

________ATGTATAAGAAATTGACGTAA

SEQ ID 6_23H7 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 148 TGAGATCAGGCACCGCA'1TCTCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGAT1'GCTCGGGGGC ACGT1CACCTCGGT'GGATATTACCGOJCAAGCTGATC AGCATCGCTrC=I]CATCAAGCCGAACATI'CAGAGC~r

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCITGAAGGATACCGCGAGCAAAAAGCGGOAAGTAC

GCTrATCCGCCATGCAGAAGAGAMTC1TICGGAAAAAAG GCGCGGACCTC2ETATGGTGCAACGCCAGGACATCTGCG

AGCGGGTAGTATAAAAAGCTCGGTTCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATATTI

______GATGTATAAGiAAATTGACGTAA SEQ ID 6_2111 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 149 TGAGATCAGGCACCGCGTTCrCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGATrrGCTCGGGGGC

ACGTITCACGTCGGTGGATATACCGGGGCAAGCTGAT'C

AGCATCGCCTCCFITATCAAGCCGAACATCCAGAGCfT

GAAGOCCAAAAACCGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGGATACCGCGAGCAAAAAGGGGCAGTACG CTTATCCGCCATGCCGAAGAGCTrT'TCGGAAAAAAGG

CGCGGACCTTIATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAA-AAAGCTCGGCYITCAGCGAACAGGGC

_____GAAATCrACGACATACCGCCGATCGGACCTCATATI=G 149 WO 02/36782 WO 0236782PCTfUSOI/46227 ________ATGTATAAGAAAFrGACGTAA SEQ HD 6_3D6 ATGA2FrGAAATCAAACCAATAAACGCGGAAGATACGTA NO: 150 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC 1TGAAGCATGTATGTATGAAACCGAT'ITGCTCGGGGGT ACGTICACCTCGGTGGATATr~ACCGAGGCAAGCTGATC

AGCATCGCCTCCITCATCAAGCCGAACATCCAGAGCTT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CTCTrGAAGGATACCGTGAGCAAAAAGCGCTGCAGTACG CTrATCCGCCATGCCGAAGAGC1TC1TCGGAAAAAGOG

GGCAGACCTITATGGTGCAACGCCAGGACATCTGCGA

GCGGTACTATAAAAAGCTCGCTCAGCGAACAGGGC

GAGGTCTACGACATACCGCCGGTCGGACCTCATATT1TM _____________ATGTATAAGAAA'1TGACGTAA SEQ ID 6_3G3 ATGATGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 151 TGAGATCAGGCACCGCA=rTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATGCTCGGGGGC

ACG1TCACCTCGGTGGATATrACCGGGGCAAGCTGATC

AGCATCGCCTCJITCATCAAGCCGAACATICAGAGCFT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACITrGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG =TATCCGCCATGCCGAAGAGCTTCTrCGGAAAAAAGG

CGCGGACCTL=ATGGTGCAACGCCAGGAGATCT)GCGA

GCGGCTACTATAAAAAGCTCGGTTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGGTCGGACCTCATAT=G

ATGTATAAGAAA'1=GACGTAA SEQ ID 6_3H2 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 152 TGAGATCAGGCACCGCATTrCCGGGGGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGAIMGCTCGJGGGC

ACGT1TACCTCGGTGGATA'ITA.CCGGGGCAAGCTGATC AGCATCGCCTCCT1TGCATCAAGCCGAACATCCAGAGCTT

GAAGOCCAAAA-ACAGTATCAGCTGAGAGOGATGGCGA

CACTrGAAGAGTACCGCGAGCAAAAAGCGGGAAGCAC GCTCATCCGCCATGCCGAAGAG(-CTrFCGGAAAAAGG

GGCAGACCTCTTATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCT)CGGC'1TCAGCGAACAGGG CGAAGTCTACGACATACCGCCGGiTCGGACCTCATA1TI

_____________GATGTATAAGAAATTGACATAA

SEQ ID. 6_4A10 ATGATTGAAGTCAAACCAATAAAGGCGGAAGATACGTA NO: 153 TGAGATCAGOCACCGCATTCTCCGGCCGAATCAGCCGC FGAAGCATGTATGTATGAAACCGA1TGCTCGGGGGC

ACGFTACCTCGGTGGATAYI'ACCGGGGCAAACTGATC

AGCATCGCCTCCTICATCAAGCCGAACATCCAGAGC'TT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTTGAAGGATACCGTGAGCAAAAAGCGOOAAGTACG

CTrIATCCGCCATGCCGAAGAGCTTC'ICGGAAAAAAGG CGCGGACC2FTIATGGTGCAACGCCAGGACATCTGCGA

GCGGCTACTATAAAAAGCTCGGCITCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGGTCGGACCTCATA=flG ______ATGTATAAGAAATrGACGTAA SEQp 6_41 ATA LGAGTCAAACCAATAAACGCGGAAGATACGTA -150- WO 02136782 WO 0236782PCT[USOI/46227 NO: 154 TGAGATCAGGCACCGCGTACTCCGGCCGAATCAGCCGC ITGAAGCATG3TATGTATGAAACCGAYLGCTCGGGGGC ACGT1CACCTCGGTGGATATrACCGGGGCAAGCTGATC COGCATCGCITCCFTCATCAAOiCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATGAGCTGAGAGGGATGGCGA

CAC1GAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG CTrATCCGCCATGCCGAAGAGCTJrCGGAAAAAcGGG GGCAGACC I'TI ATGGTGCAACGCCAGGACATCTGCGA GCGGCTA~TATGAAAAGCTCGGC1CAGCGGACAGGGC GAAGTCTACGACATACCGCCGATCGGACCTCATA TIG _______ATGTATAAGAAATrPGACATAA SEQ ED 6_5D11 ATGKITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 155 TGAGATCAGGCACCGCAITCTCCGOCCGAATCAGCCGG '1TAAGCATGTATGTATGAAACCGA=rGCTICGGGGGC

ACG'I=CACCTCGGTGGATATFACCGGGGCAAGCTGATG

AGCATCG=ECC71CATCAAGCCGAACATCCAGAGCTr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGAGTACCGCGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTCTCGGAAAAAAGG

CGCGGACCTITATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTrCAGCGAACAGGGC GAAGTCTACGACATACCGCCGATCGGACCTCATATP1G ______ATGTATAAGAAATrGACGTAA SEQ ID 6_SF1 1 ATGATTGAAGTCAAACCAATAA-ACGCGGAAGATACGTA NO: 156 TGAGATCAGGCACCGCA1TCTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATITGCTCGGGGGC

ACGT1ITCACCTCGGTGGATATTACCGGGGCAAGCTAATC AGCATCGCTrCCFICATCAAGCCGAACATCCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTGAAGAGTACCGCGAGCAAAAAGCGGGAAGTAC

GCITATCCGCCATGCCGAAGAGCTrCTCGGAAAAAAG

GCGCGGACC=TATGGTGCAACGCCAGGACATCTGG

AGCGGGTACTATAAAAAGCTCGGCITAGCGAACAGGG

CGAAGTCCACGACATACCGCCGGjTCGGACCTCATArI=

GATGTATAAGAAATTGACGTAA

SEQ ID 6_509 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 157 TGAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGA2TITGCTCGGGGGC ACGM1CACCTCGGCGGATATrACCGGGGCAAGCTAAT

CAGCATCGCCTCCTTTATCAAGCCGAACATTCAGAGCT

TGAAGGCCAAAAACAGTATCAGCT)GAGAGGGATGGCG

ACGCTI'GAAGAGTACCGTGAGCAAAAAGCGGGCAGTAC

GCTTATCCGCCATGCCGAAGAGCIT=CrCGGAAAAAGG GGGCAGACCI I'rTATGGTGCAACGCCAGGATATCTGCG

AGCGGGTAGTATAAAAAGCT'CGC'TTCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATAT1T

GATGTATAAGAAAITGACGTAA

SEQ ID 6_6D5 ATGAITGAAGTCAAACCAATAAACGCGGAAGATGCGTA NO: 158 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC ________TGGAAGCATGCAAGTATGAAACTGAF= GCTCGOGGOC 151 WO 02/36782 WO 0236782PCTfUS01/46227 ACG1TCACCTCGGCGGATATrACCGGGGCAAGCTGAT CAGCATCGCTrCCTICATCAAGCCGAACArrCAGAGCT TGAAGGCCAAAAACAGTATCAGCTGAGAGGciATGG

ACACTTGAAGGGTACCGCGAGCAAAAAGCGGGCAGTAC

GCITATCCGCCATGCCGAAGACrCTrCGGAAAAAAG

GCGGGGGACCTITGTGGTGCAACGCCAGYGACATCTGCG

AGCGGGTACTATAAAAAGCTCGG=rCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATAFI=

_____GATGTATAAGAAA2FTGACGTAA SEQ ID NO:159 6_7D1

SEQID

NO: 160 6_8H3

ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGAnITGCTCAGGGGT GCGT=rACCTCGGTGGATATTAkCCGGGGCAAGCTGATC AGCATCC'iTCC1CATCAAGCCGAACAT-rCAGAGCrr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'ITGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGAGCTGTTCGGAAAAAGGG

GOCAGACCF1TATGGTGCAACGCCAGGACATCTGCGA GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAAG3C

OOOGGTCTACGACATACCGCCGGTCGGACCTGATATFIG

ATGTATAAGAAATrGACGTAA

ATGM'TGAAGTCAAACCAATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATT)CTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATITGCTCGGOGGC

ACGTICACCTCGGTGGATA1TACCGGGGCAAGCTGATC

AGCATCGCCTCC'I=CATCAAGCCGAACATCCAGAGCTT

GAAGOCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCTTGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCIATCCGCCATGCCGAAGAGCITC'ITTCGGAAAAAAG

GCGCGGACL~T1TIfIATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCITAGCGAACAAGG

CGGGGTCTACGACATACCGCGGTCGGACCTCATAF1T GATGTATAAGAAATrGACGTAA ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA TGAGATCAGGCACCGCAITCTCCGOGCCGAATCAGCCc3C

TGGAAGCATGCAAGTATGAAACCGATITGCTCGGGGGC

ACGCTTCACCTrGGTGGATATTACCGGGGCAAGC-rGAT

CAGCATCGCTTCCTICATCAAGCCGAACATTCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACGC'TrGAAGGGTACCGCGAGCAAAAAciCGGGAAGTA CGCITATCCGCCATGCCGAAGAGCTrCGGAAAAAA GGCGCGGACCTI. ATGGTGCAACGCCAGGACATCTGC GAGCGGGTACTATAAAAAGCTCGG CAGCGAACAAG

GCGAAGTCTACGACATACCGCCGGTCGGACCTCATATIT

TGATGTATAAGAAATITGACGTAA

SEQ ID NO: 161 6-9G11 SEQ Bt) 6F1 ATGA1TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 162 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGAFJGCTCGGGGGT

ACGIMCACCTCGGTGGATATTACCGGGGCAAGCTGGTC

_____TGCATCGICC2FTATAAAGCCGAACATTCAGAGCTT 152- WO 02136782 WO 0236782PCTIUJS01/46227

GAAGGCCAAAAACAGTATCAGCTGAGAGOGATGGCGA

CGCTTGATGGATACCGCGAGCAAAAAGCOGGAAGCACG

CTCATCCGCCATGCCGAAGAGC1TC7CGAAAAAAAGG

CGCGGACCTIATGGTGCAATGCCAGGACATCTGTGA

GCGGCTACTATGAAAAGCTCGGCITCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGGTCGGACCTCATATITG

_______ATGTATAAGAAKITGACGTAA

SEQ ID 7_1C4 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 163 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACCGATIGCTCGGGGGC

ACGTITCACCTCGGTGGATA1TACCGGGGCAAGCTGATC AGCATCGCTrCC1TCATCAAGCCGAGCATCCAGAGCT GAAGGCCAAAAACAGTATCAOCrGAGAGGGATGGCGA

CACTTGAAGAGTACCGCGAGCAAAAAGCGGGCAGTACG

C'1ATCCGCCATGCCGAAGAGCTTCTrCGGAAAAAAGG

CGCGGACCTFTATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGITrCAGCGAACAAGGC GGGGTCTACGATATACCGCCGATCGGACCTCATA'r=G

ATGTATAAGAAA'TGACGTAA

SEQ ID 7_2A10 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 164 TGAGATCAGGCACCGCMTCCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACTGA'1=rGCTCGGGGGC

ACGFICATCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCYICATCAAGCCGAACATCCAGAGC'Tr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGC=GAAGGGTACCGCGAGCAAAAAOCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC1-TCITCGGAAAAAAG GCGCGGACCT1TATGGTGCAACGCCAGGACATCrGCG

AGCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAAGG

CGGGGTCTACGATATACCGCCGATCGGACCTATAITI

________GATGTATAAGAAATTGACGTAA

SEQ ID) 7_2A1 1 ATGA2FIGAACTCAAACCAATAAACGCGGAAGATACGTA NO: 165 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC 1TGAAGCATGTATGTATGAAACCGAITGCTCGOGGGC

ACGT=CACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCG~FCC21CATCAAGCCGAACAFrCAGAGCTT

GAAGGCCAAAAACAGTATCAOCTGAGAGGGATGOCGA

CACTTGAAGGGTACCGCGAGGAAAAAGCGGGAAGTAC

GCTTATCCGCCATGCCGAAGAGCTCITCGGAAAAAGG

GGGCAGACCTI=ATGGTGCAACGCCAGGACATCTGCG

AGCGGGTAC7ATAAAAAGCTCGCTCAGCGAACAAGG CGGGGTCTACGACATACCGCCGGTCGGACCTCATAT1T GATGTATAAGAAATI7GACGTAA SEQ ID 7_2D7 ATGATT'GAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 166 TGAGATCAGGCACCGCATCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGAT'TGCrCGGGGGC ACGT1CACCTCGGTGGATATTACCGGGGCAAGCTGATC AGCATCGGCTCCIT IATCA AGCGAACATCCAGAGCITr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

_____________CG=TGAAGGGTACCGTGAGCAAAAAGCGGGAAGTACG

-153- WO 02136782 WO 0236782PCT[USOI/46227

CTCATCCGCCATGCCGAAGAGCITCTTCGCGAAAAAAGG

CGCGGACCT=IATGGTGCAACGCCAGGACATCTGCGA

GTGGGTACTATAAAAAGCTCGOTfCAGCGAACAGGGC ciAAGTCTACGACATACCGCCGGTTCGGACCTGATA2TIG

________ATGTATAAGAAATTGACGTAA

SEQ ID 7_5C7 ATiATIGAAGTCAAACCAATAAACOGGGAAGATACGTA NO: 167 TGAGATCAGGCACCGCA fCTCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGA1TGCTCGGGGGC

ACGTICACCTCOGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTC=T1CATCAAGCCGAACATCCAGAOC2I

GAAOGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTQGAAGGATACCGTGAGCAAAAAGTGGGAAGCACG

CTCATCCGCCATGCCGAAGAGCITTCGGAAAAAAGG

CGCGGACCTITAT'GGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTrCAGCGAACAAGGC GGGGTCTACGATATACCGCCGGTCGGACCTCATA1TIG

________ATGTATAAGAAATTCIACGTAA

SEQ ID 7_9C9 ATGATTGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 168 TGAAATCAGGCACCGCATrCGTCCGGCCGAATCAGCCGC

TTFGAAGCATGTATGTATGAAACCGAITGCTCGGGGGC

ACGTrTCACCTCGGTGGATATTACCGOYGGCAAGCTGATC AGCATCGCTrCCT1CATCAAGCCGAACATCCAGAGC'r

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CA=IGAAGCTGTACCGCGAGCAAAAAGCGTGGAAGTAC

GCTCATCCGCCATGCCGAAGAGCITCTACGGAAAAAAG

GCGCGGACCTTTTATGGTGCAACGCCAOGACATCTGCG

AGCGOTACTATAAAAAGCTCGGTrCAGCGAACAGGG CGAAGTCTACGACATACCGCCGATCGGACCTCATA1TI GATGTATAAGAAATrGACGTAA SEQ ID 9_13F10 ATGATTGAAGTCAAACCAATA.AACGCGGAGGATACGTA NO: 169 TGAGATCAG*GACCGCATTCTCCGGCCGAATCAGCCGC TGGAAGCATGCAAGTATGAAACCGA M GCTCAGGGGT GCGTrTCACC'1GGTGGATATTACCGGGGCAAGCTGGTC

AGCATCGCCTCCTITCATCAAGCCGAACATTCAGAGCTT

GAAGJCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CAc =GAAGAGTACCGCGA.GCAA.AAAGCGGGAAGCAC GCTCATCCGCCATGCCGAAGAGG1T7TTCGGAAAAAGG

GGGCAGACC=CTATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCITCAGCGAACAGOG

CGAAGTCrACGACATACCGCCGACTGGGCCCCATAIIT' GATGTATAAGAAA'rrGACGTAA SEQ ID 9_13F1 ATGATTGAAGTCAAACCAATAAACGCGGAGGATACGTA NO: 170 TGAGATCAGGCACCGCATCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITGCTCAGGGGT

GC=f~CACCTrGGTGGATATTACCGGGGCAAGCTGGTC AGCATCGCCTCCTITCATCAAGCCGAACATrCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTTGAAGAGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC1fTCITCGGAAAAAGG

_____________GGGCAGACCTCTTATGGTGCAACGCCAGGACATCTGCG

-154- WO 02136782 WO 0236782PCTIUSO1/46227

AGCGGGTACTATAAAAAGCTCGGCITCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGAC~TGGGCCCCATATI=

GATGTATAAGAAATrGACGTAA SEQ ID 9_15D5 ATGATTGAACITCAAACCAATAAACGCGGAAGATACCITA NO: 171 TGAGATCAGOCACCGCATFCTCCGOGCCOAATCAGCCGC

TGGACGCATGCAAGTATGAAACCGATIGCTCGGGGGC

ACGTTCACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCTICATCAAGCCGAACATCCAGAGCTr

GAAGOCCAAAAACAGTATCAGCTGAGAGOGATGGCGA

CAC1TGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG C2FrATCCGCCATGCCGAAGAGCITC2FrCGGAAAAAGGG GGCAGACCTGTFrATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGC'ITCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGGTCGGACCTCATA2TPG _______ATGTATAAGA,4AATGACGTAA SEQ ID 9_15D8 ATGATI=GAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 172 TGAGATCAGGCACCGCATACTCCGOCCGAATCAGCCGC TrGAAGCATGTATGTATGAAACCGATG~rCGGGGGT ACGTrTCACCTCGGCGGGATA7TACCGGGGCAAGCTGGT

CAGCATCGCCTCCTICATCAAGCTGAACATCCAGAGCT

TGAAGGCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACAC'=TAAGOGTACCGTGAGCAAAAAGCGGGCAGTAC

GC1TATCCGCCATGCCGAAGCG-CITCGGAAGAAAG GCGCGGACC1TrATGGTGCAACGCCAGGACATCrGCG AGCGGGTACTATAAAAAGCTCGGCrTCAGCGAACAGGG

CGAAGTCTACGACACACCGCCGGTCGGACCCGATATT

GATGTATAAGAAGITGACGTAA

SEQ ID 9_15H13 ATGITrGAAGTCAAGGCAATAAA4CGCGGAAGATACGTA NO: 173 TGAGATCAGGCACCGCATICTCCGG3CCGAATCAGCCGC

TFGAAGCATGTATGTATGAAACCGATATGCTCAGGGGT

GCGTTTACCTGGGTGGATATI'ACCGGGGCAAGCTGATC

AGCATCGCCTCC'TICATCAAGCCGAACATCCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC7TrGAAGAGTACCACGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC"ITCTCGGAAAAAAG

GCGCGGACCT1T=ATGGTGCAACGCCAGGACATCGCG

AGCGGGTACTATAAAAAGCTCGGCTIAGCGAACAGQG

CGAAGTCTACAACACACCGCCGGjTrGGACCTCATAT1T

GATGTATAAGAAAITGACGTAA

SEQ IID 9_18112 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 174 TGAGATCAGGCACCGCA'TCTCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGA=1G~TGGGGGGC ACGT1TACCTCGGCGGATATTACCGGOGCCAAGGT'GAT CAGCATCGCGTCC1TPATCAAGCCGAACATCCAGAGCT

TGTAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'TTGAAGGATACCc3TGAGCAAAAAGCGGGCAGTACA C1TATCCGCCATGCCGAAGAGC'TC1TCGGAAAAAGOG GGCAGACC I'l IATGGTGCAACGCCAGciACATCTGCGA GCCGGTACTATAAAAAGCTCGOC1TCAGCGAACAGOC

_____________GAAGTCTCGACATACCGCCGGTCGGACCTCATATIG,

-155- WO 02136782 WO 0236782PCT/USOI/46227 _____________ATGTATAAGAAATrGACGTAA SEQ II 9_20F12 ATGATTGAAGTAAAACCAATAAkACGCGGAAGATACGTA NO: 175 TGAGATCAGGCACCGCGTTCTCCGGCCGAATCAGCCGC TGGAAGCATGTATGTATGAAACCGATrTGCTCGGGGGC ACGrITACCTCGGTGGATATTACCGGGGCGAGCTGGTC

AGCATCGC'TCTCATCAAGCCGAACATCCAGAGCFI

GAAGGCCAAAAACAGTATCAGCTGAGAG*3GATGGCGA

CAC'TGAAGGGTACCGTGAGCAAAAAGCGGGCAGTACG

C'TTATCCGCCATGCCGAAGAGC'TCTrCGGAAAAAAGG CGCGGACC II13TGGTGCAACGCCAGGACATCTGCGA GCGGGTACTATAAAAAGGTCGGCTrCAGCGAACAAGGC GGGGTCTACGACATACCGCCGCGTCGGAC~rCATA1T1G

_____________ATGTATAAGAAAITGACGTAA

SEQ ID 9_21GB ATGA1TGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 176 TGAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC

TGGAAGCATGTATGTATGAAACTGAITGCTCGGGGC

ACGTrrCACCTCGGCGGATATrACCGGGOGAAGCrGAT CAGCATCGCCCT1CATCAAGCCGAACATCCAGAGCT

TGAAGGGCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACACTCGAAGGATACCGCGAGCAAAAAGCGGGCAGTA

CGCTA-ATCCGCCATGCCGAAGAGCTrCTTCGGAAAAAG GGGGCAGACCTCTTATGGTGCAAkCGCCAGGACATCTGC

GAGCGGGTACTATAAAAAGCTCGGGTCAGCGATCAGG

GCGAAGTCTACGACATACCGCCGGTCGGACCTCATATIT

TGATGTATAAGAAATTGACGTAA

SEQ ID 9_22B I ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 177 TGAGATAAGGACCGCATCCTCCGGCCGAATCAGCCGC

TG-GAAGCATGCAAGTATGAAACCGAFITGGTCGGOOOC

ACGTICACCrCGGTGGATATrACCGGGGCAAGCrGGTC

AGCATCGCCTCCTTTCATCAAGCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAG'TGAAGGGTACCGTGAGCAAAAAGCGOOCAGTACG

CTTATCCGCCATGCCGAAGAGCTTCTCGGAAAAAGOG

GGCAGACC'1IATGGTGCAACGCCAGGACATCTGCGA

GCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGOGC

GAAGTCTACGACTI7ACCGCCGACCGGACCCCATA1T1G

ATGTATAAGAAATTGACGTAA

SEQ ID 9_23A10 ATGATrGA-AGTCAAACCAATAAACGCGGAAGATACGTA NO: 178 TGAGATCAGGCACCGCA'1CCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGAIMGCTCGGGGGC

ACGCTTCACCTCGGTGGATATTACCGGGGCAAGGTGGT

CAGCA'1TGC'1TCCTICATCAAGCCGAACATCCAGAGCF TGAGGGCCAAAAACAGTATCAGCTGAGAGOG3ATGGCG

ACAG'IGAAGGGTACCGCGGGCAAAAAGCGGGCAGTAC

G ATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAGG GGGCAGACCTFIATGGTGCAATGCCAG3ACATCTGCG

AGCGGGTACTATAAAAAGCTCGGCYPCAGCGAACAAGG

CGGGGTCTACGACATACCGCCGGTCGGACCTCATA=~r

______GATGTATAAGAAATTGACGTAA

SID 9 2F6 IATGATTGAAGTCAAACCAATAAACGCGGiAAGATACGTA -156- WO 02/36782 WO 0236782PCTfUSIII/46227 NO: 179 TGAGATCAGGCACCGCATTCTCAGOCCGAATCAGCCGC

TAGAAGCATGCAAGTATGAAACCGATITGCTCAGGGGT

GCG=hTACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCTICATCAAGCCGAACA'TCAGAGCIT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CAC'1TGAAGGATACCGTGAGCAAAAAGCGGGCAGTACG

CTTATCCGCCATGCCGAAGCGC'ITCTCGGAAAAAAGG

CGCGGACCITIGTGGTGCAACGCCAGGACGTCTGCGA

GCGGGTACTATAAAAAGCTCGGC'ITCAGCGAACAGOGC

GAAGTCTACGACATACCGCCGACCGGACCCCATATIT

GATGTATAAGAAATTGACGTAA

SEQ IT) 9_4H10 ATGAITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 180 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACTGATIGCTAGGOGGT

ACGCITCACGTCGGTGGATATTACCGGGGCAAGCTGAT

CAGCATCGCCTCCT=CATCAAGCCGAACATCCAGAGCT

TGAACOGCCAAAAACAGTATCAGCTGAGAGGGATGOCG

ACACTGAAGGGTACCGTGAGCAAAAAGCGOGCAGTAC

GCIATCCGCCATGCCGAAGAGCTCITCGGAAAAAGG

GCGCGGACCTrATATGGTGCAACGCCAGGACATCTGCG

AGCGGGTACTATAAAAAGCTCGCT-AGCGAACAGGG

CGAAGTCTACGACATACCGCCGGTCGGACCTCATAT1T

GATGTATAAGAAATTGACATAA

SEQ ID 9_4H8 ATGATfGAAGTCAAACCAATAAATGCGGAAGATACGTA NO: 181 TGAGATCAGGCACCGCA'IFCTCCGGCCGAATCAGCCGC TrGAACGCATGTATGTATGAAACCGATIGCTCGGAGGC

ACGTPCACCTAGGTGGATATTACCGGGGCAAGCTGAT

CAGCATCGCITrCGTAATCAAGCCGAACATCCAGAGCT TGAAGGCCAAAAkACAGTATCAGCTGAGAGGGATGGCG

ACACTTGAAGGGTACCGTGAGCAAAAAOCGGOCAGTAC

GCITATCCGCCATGCCGAAGAGcITrcTcGGAAA:AAGG GGGCAGACCI=rATGGTGCAACGCCAGGACATCTGCG AGCGGGTACrATAAAAAGCTCGGCTTCAGCGAACAGGG

CGAAGTCTACGACATACCGCGCGTCGGACCTCATAITI

________GATGTATAAGAAAITGACATAA

SEQ ID 9_8H1 ATGAITGAAGTCAAACCAATAACCGCGOAAGATACGTA NO: 182 TGAGATCAGGCACCGCATTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGA=GTCTCGGJOGT

ACG=1CACCTCGGTGG3ATATTACCGGGGCAAGCTGATC

AGCATCGCCTCCTICATCAAGCCGAACATCCAGAGITT

GAAGOCCAAAAACAGTATCAGGTaGAGAGGGATGGCGA

CACTAGAAGGGTACCGCGAGCAAAAAGCGGGCAGTAC

GCTCATCCGCCATGCCGAAGAGCrCICGGAAAAAGG GGGCAGACCT1TATGGTGCAACGCCAGAACATCrGCG

AGCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAQGGG

CGAAGTCTACGACATACCGCCGACCGGACCCCATAT1T

________GATGTATAAGAAATTGACGTAA

SEQ ID 9_9H7 ATGA2FrGAAiTCAAACCAATAAACGCGGAAGATGCGTA NO: 183 TGAGATCACGGCACCGCAITCTCCGGCCGAATCAGCCGC _____________TGGAAGCATGCAAGTATGAAACCGAT1GCTCGGGAGC 157 WO 02136782 WO 0236782PCTUS01/46227

ACGTICACCTCGGTOGATATI'ACCGOGGCAAGCTGATC

AGCATCGCCCI I ICATCAAGCCGAACATCCAGAGCTT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGOCGA

CACTGAAGAGTACCGCGAGCAAAAAGCGGGAAGTAC

GC=ATCCGCCATGCCGAAGACOCTICTTCGGAAAAAAG

GCGCGGACCr=IATGGTGCAACGCCAGGACATCTGCG

AGCGOGTAGTATAAAAAGCTCGGCTTCAGCGAACAGGG

CGAAGTCTACGACATACCGCCTGTCGGACCTCATATI=

_______GATGTATAAGAAAKIGACGTAA

SEQ ID 906 ATGATTGAAG3TCAAACCAATAAACGCGGAAGATACGTA NO: 184 TGAGATCAGGCACCGCA2FTCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITG=CGGGGGT

ACGT1CACCTCGGTGGATATrACCGGGGCAAGCTGATC TGCATCGCCTCCT-ITCATCAAGCCGAACATTCAGAGG2Fr

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CGCITGAAGGGTACCGCGAGCAAAAAGCGGGAAGTAC

GCTTATCCGCCATGCCGAAGAGC=CTCGGAAAAAGG

GGGCAGACCTI=ATGGTGCAATGCCAGGACATCTGT'G

AGAGGCTACTATGAAAACT)CGGCTTCAGCGAACAAGG

CGGTGTACGATATACCGCCGATCGGACCTCATATr

GATGTATAAGAAATTGGCGTAA

SEQ IID 9H1 1 ATGATTGAAGTCAAACCAATAAACGCG3AAGATACGTA NO: 185 TGAGATCAGGCACCGCICTCCGGCCGA-ATCAGCCGT

TGGAAGCATGCAAGTATGAAACCGA=TGCTCGGGOGT

ACGrICACCCGGCGGATATTACCGGGGCAAGCTGAT CAGCATCGC1TCCFICATAAAGCCGAACATTCAGAGCT TGAGGGCGAAGAACAGTATCAGCTGAGAGGGATG*300

ACGCTTGAAGGATACCGTGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAGCTrTTTCGGAAAAAG GGGGCAGACCT1TATGGTGCAATGCCAGGACAT'CTGT

GAGCGGGTACTATAAAAAGCTCGGCTTCAGCGAACAGG

GCGAAGTCTACGACATACCGCCGATCGGACCTCATATIT

TGATGTATAAGAAATrGACGTAA SEQ IID 0_4B 10 ATGATAGAAGTGAAACCGATTAACGCAGAGGATACCTA NO: 186 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAGCGATITACITCGTGGTG

CATTCAC'TTAGGCGGC'rTACAGGGGCAAACTGATT CCATAGC1TCA'TrCCACCAOOCCGAGCACTCAGACCTCG

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGiAAGGTrATCGTGATCAGAAAGCGGGATCGACTCT AATrAAACACGCTGAAGAAATrCTTCGTAAGAGGOGG

CGGACATGCTIGGTGCAATGCGCGGACAACCGCCTCA

GGCTACTACAAAAAGTTAGGCTTCAGCGAGCAGGOAGA

GATAT'ITGATACGCCGCCAGTAGGACCTCACATCCTGAT

_____________GTATAAAAGTGCTCACATAA

SEQI 0_B 11 ATGATAGAOGTGAAACCGATTAACGCAGAGGATACCTA NO: 187 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAAGCGATrrACTTCGTGGTG CAi=rACTAGGCGGCTPTTACGGGGCAAAGTGATIM

_____________CCATAGCTTCMTCCACCAGOCCGAGCACTCAGACCTCG

-158- WO 02136782 WO 0236782PCTIUSOI/46227

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TI'GGAAGGTTATCGTGATCAGAAAGCGGGATCGACTCT

AATfAAACACGTGAACAACTGTAAGAOGGGG

CGGACATGCTITGGTGCAATGC.GCGGACATCCGCCTCA

GGCTACTACAAAAAGTrAGGCTTCAGCGAGCAGGGAGA

GGTAITGAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAAGATCACA

SEQ ID 0_5B3 ATGCTAGAGGTGAAACCGATfAACGCACIAGGATACCTA NO: 188 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAACCGATITAC1TCGTGGTG CAT1TCACTrAGGCGGCT1TACAGGGGCAAACTGATI

CCATAGCTTCATTCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TrGGAAGGTTATCGTGATCAGAAAGCGGGATCGAGTCT

AAITAAACACGCTGAACAACTTC'CGTAAGAGOGG

CGGACTTGCTIGGTGCAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAAG2rAGGC=CAGCGAGCAGGGAGAG GTA1TGATACGCCGCCAGTAGGACCrCACAT)CCTGATG

________TATAAAAGGATCACA

SEQ ID 0_5B4 ATGCTAGAGGTGAAACTGA2FfAACGCAGAGGATACCTA NO: 189 TGAACTAAGGCATAGAATACTCAGACCAA-ACCAGCCGT TAGAAGCGTGTATGTATGAAACCGATITAC1TCGTG-GTG CA2FV7CA=TAGGCGGTIACAGGGGCAAACrGA=I CCATAGc'ICA~rCCACCAGGCCGAGCACTCAGACCTCG AAGGCCAGAACAGTACCAGCrCCGAGGTATGGCTACC

TTGGAAGGEICGTGATCAGAAAGCGGGATCGAGTCT

AATTAAACACGCTGAAGAAA'TrCTrCGTAAGAGCGGGGG CGAACTrG~TITGGT)GTAATGCGCGGACATCCGCCTCAG GCrACTACAAAAAG'TTAGGCICAGCGAGCAGGGAGAG

GTATTGATACGCCGCCAGTAGGACCTCACATCCTGATG

________TATAAAAGGATCACA

SEQ ID 0_5B8 ATGATAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO: 190 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAGCGATTA=ICGTGGTG

CA2FICACTrAGGCGGCTITTACAGGGGCAAACTGATT

CCATAGCTTCAFI'CCACCAGOCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGQCTACC

TTGGAAGGTTATCGTGATCAGAAAGCG*JGATCGAGTCT

AATTAGACACGCTGAACAAA1TC1CGTAAGAGGGG CGGACT1GG1GGTGCAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAAGTTAGGCTCAGCGAGCAGGGAGAG

ATA1TGATACGCCGCCAGTAGGACCTCACATCCTGATG

______TATAAAAGGCTCACA

SEQ ID 0_5C4 ATGATAGAGGTGAAACCGA'TTA.ACGCAGAGGATACCTA NO: 191 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT

TAGAAGCGTGTATGTATGAAACCGMTI'ACTCGTGGTG

CATMCACTIrAGGCGCIrrACAGGGGCAAACTGA1T CCATAGC1CATTCCACCAGGCCGAGCACTCAGGCCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

____TTYGGAAGGTrATCGTGAGCAGAAAGCGGGATCGAGTAT -159- WO 02136782 WO 0236782PCTJUS01/46227 AATTAAACACGCTGAAGAAATrCTrCGTAAGAAQGG CGGACTI'GC1IGGTGCAATGCGCGGACGTCCGCCTCAG GCTACTACAAAAAGTrAGGCTrCAGCGAGCAGGGAGAG ATA71TGACACGCCGCCAGTAGGACCTCACATCCTGATG

________TATAAAAGGATCACA

SEQ ID 0_5D11 ATGATAGAOTGTGAAACCGATL'AACGCAGAGGATACGTA NO: 192 TGA-ACTAAGGCATAGAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAC-CGATTACTFTCGTGGTG

CA=ICACTAGGCGGC I II IACACGOGGCAAACTGAMT

CCATAGCTTCATTGCACCAGGCCGAGCACTCAGACCI'CC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TrGGAAGGTTATCGTGAGCAGAAAGCGGGATCGACTCT AArrAGACACGCTGAACAACrTTCGTAAGAGOGGGG CGGAC'rrGCT=GGTGCAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAGGTTAGGCTCAGCGAGCAGGGAGAG

GTATIGATACGCCGCCAGTAGGACCTCACATCCTGATG

_____________TATAAAAGOC.TCACA

SEQ ID 0_5D3 ATGCTAGAGGTGAAACCGATrAACGCAGAG(3ATACCTA NO: 193 TGAACTAAGGCATAGAATACTC.AGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAAGCGA1TACTCGTGGTG CATTrCAC'IAGGCGGCTATTACAGGGGAAACTGATFI'

CCATAGCTFPCATTCCACCAGGCCGAGCACTCAGAACTCC

AAGOCCAGAAACAGTACCAGCTCCGAGGTATGOCTACC

TrGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT AATTAAACACGCTGAAGAAAMfTTCGTAAGAGOGG

CGGACYFOCTTFGGTGTAATGCGCOGACATCCGCCTCAG

GCTACTACAAAAAGTrAGGCTCAGCGAGCAGOGAGAG

ATATITGAAACGCCGGCAGTAGGACGTCACATCCTGAT

CTATAAAAGGATCACATAA

SEQ ID 0_5D7 ATGATAGAAc3TGAAACCGATTAACGCAGAGGAGACCTA NO: 194 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAACCGATTA=rrGTGGTG CATICAC1TAGGCGGCTE1ACAGGGGCAAACTGATI

CCATAGCITCATTCCACCAGGCCGAGCACTCAGAAGC

GAAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTAC

CTTGGAAGGTTATCGTGATCAG.AAAGCGGGATCGAGTC

TAATTAGACACGCTGAACAACTTC2TCGTAAGAAGGG

GCGAATATGC'TGGTGTAATGCGCGGACAACCGCCTC

AGGCTACTACAAAAAGrrAGGCTICAGCGAGCAGGGAG AGATATTTGATACGCCGCCAGTAGGACCTCACAT)CCrG

ATGTATAAAAGGATCACA

SEQ ID 0_6B4 ATGCTAGAGGTGAAACCGATrAACGCAGAG3ATACCTA NO: 195 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAGCGATITACLTCGTGGTG

CACITCAG=AGGCGGC1TACAGOG3OCAAACTGA1T CCATACTrCATrCCACCAGGCCGAGCACTCAGACCTCC AAGGCCAGAAACAGTACCAGGTCCGAGGTATGGCrACC TTGGAAGGI=rCGTGATCAGAAAGCGGGATCGAGTCT AATrAGACACGCTGAACAAATrCTTCGTAAGAGGGGGG

_____CGGACTGCTIGGTGCAATGCGCGGACATCCGCCTCAG

160- WO 02/36782 PCTfUS01/46227

GCTACTACAAAAA=TAGGCT[CAGCGAGCGGGAAAG

GTAT=GATACGCCGCCAGTAOYGACCTCACATCCTGATG

TATAAAAGGATCACA

;ZSEQ ID 0_6Dlb ATGCTAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO: 196 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGA A CGTGGTG CAT'ITCACIAGGCGGGYI=ACAGGGGCAAACTGATrT7 CCATAGCITrCATrCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGAAGGTTATCGTGATGAGAAAGCGGGATCGAGTCT

AKFI'AGACACGCTGAACAAATrcTPGTAAGAOOOGGGG CGGACATG=T~GGTGCAATGCGCGGACATCCGCCrCA GGCTACTACAAAAAGTTAGG TAGCGAGCAGGGAGA

GGTATIGAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAGGCTCACA

SEQ DI) 0_-6D 1 ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO: 197 TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATITCTCGGGGGC

ACGC1TCACCrCGGTGGATAITACCGGGGCAAGCTGGT CAGCATCGC~rCC1T=ATCAAGCCGAACATCCAGAGCT TGAAGGCCAAAAACAGTATCACICrTGAGAGGGATGGCG

ACGCTTGAAGGGTACCGTGAGCAAAAAGCGGGCAGTAC

GC'TTATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAOG

GGGCAGACCTTIATGGTGCAACGCCAGGACATCTGCG

AGCGO%TACrATAAAAAGCTCGGCTCAGCGAACAGGG CGAAGTCTACGACATACCGCCGGTCGGACCTCATAT1T

_____________GATGTATAAGAAATTGACGTAA

SEQ ID 0_6F2 ATGATAGAGGTGAAACCGATrAACGCAGAGGATACCTA NO: 198 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAAGCGATFAC1CGTGGTG CAT1ITCACTTAGGCGGCTATTACAGGGGCAAACTGATI CCATAGC~rCATrCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TrGGAAGGT1TCGTGAGCAGAAAGCGGGATCGACTCT AATTAGACACGCTGAACAAATrC1TCGTAAGAGOGOGG

CGGACATGC'TTGGTGCAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTTAGGCTrCAGCGAGCAGGGAGA GATATIGATACGCCGCCAGTAG43ACCTCACATCCTGAT

GTATAAAAGGATCACA

SEQ ID 0_6H19 ATGATAGAGGTGAAACCGATrAACGCAGAGGATACCTA NO:199 TGAAGTAAGGCATAAAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAACCGAT'ITACTTCGTGGTG

CATITCACTTAGGCGGGTF=ACGGGCAAACTGA'lM

CCATAGCTTCATTCCACCAGGCCGAGCACTCAGACCTCG

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TIPGGAAGGTTATCGTGAGCAGAAAGGGGATCGACTCT

AATTAGACACGCTGAAGAAATTC1TrCGTAAGAAGGGGG CGAACTfGCTFTfGGTGCAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAAGTTAGGCICAGCGAGCAGGGAGAG

_____________GTnTGACACGCCGCCAGTAGGACCTCACATCCTGATG 161 WO 02/36782 WO 0236782PCT[US01/46227 _____________TATAAAAC3GCTCACA SEQ ID 10_4C10 ATGATAGAGGTGAAACCGATI'AACGCAGAGGATACCTA NO:200 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGA=fAC'ITCGTGGTG CATTrCACTrAGGCGOCTN'TrACAGGGGCAAACT'GAIT CCATAGCTTrCATrCCACCAGGCCGAGCACTCAGAACTCC

AAGGGCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGAAGGTrATCGTGATCAGAAAGCGGGATCGAGYTCF

AATTAAACACGCTGAACAAATTC'ITCGTAAGAGGOGGGG

CGGACNTGCITGGTGCAATGCGCGGACATrCCGCCTCA

GGCTACTACAAAAAG'TTAGGCTTCAGCGAGCAGOGAGA

GATATTTGATACGCCGCCAGTAGGACCTCACATCCT)GAT

GTATAAAAGGCTCACATAA

SEQ ID 10_4D5 ATGATAGAGGTGAAACCGATI'AACGCAGAGGATACCTA NO:201 TGAACTAAGGCATAGAATACTGAGACCAAACCAGCCGA

TAGAAGTGTGTATGTATGAAACCGATJITACTCGTGGT)G

CATMCACTrAGGGGCTIACAGGGGCAAACTGA1T

CCATAGCTCATTCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCI)CCGAGGTATGOGTACC

T-rGGAAGGTTATCGTGAGCAGAAAGCGGGATCGACTG AATrAGACACGCTGAACAAATTCTTCGTAAGAGGGGGG

CGGACTT'GCTGGTGCAATGCGCGGACATCCGCCTCAG

CGCTACTACAAAAAGYTAGGCTrCAGCGAGCAGGGAGAG

GTA'TGATACGCCGCGAGTAGGACCTGCACATCCTGATG

_____________TATAAAAGGATCACATAA

SEQ ID 10_4F2 ATGCTAGAGGTGAAACCGAT~AACGCAGAGGATACCTA NO:202 TGAACTAAGOCATAGAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATG1TGAAAGCGATIT7ACTCGTGGTG CATICACTrAGGCGGC'IIACAGGGGCAAACTGAMr CCATAGCTrCATTCCACCAGGCCGAGCACTGAGAACrCC

AAGGCCAGAAACAGTACCAGCT'CCGAGGTATGGCTACC

TTGGAAGGITrATCGTGAGCAGAAAGCGGGATCGAGTCT AATrAGACACGCTGAAGAAATTCTTCGTAAGAGGOGGG CGGACATGCFTrGGTGTAATGCGCGGACATCCGCCTCA

GOCTACTACAAAAAGTITAGGC'CAGCGAGCAGGGAGA

GATATITAAACGCCGCCAGTAGYGACCTCACATCCTGA

_____________TGTATAAAAGGCTCACATAA

SEQ IID 10_4F9 ATGATAGAGGTG3AAACCGATTAACGCAGAGGATACCTA NO:203 TGAACTAAGOCATAGAATACTCAGACCAAACCAGCCGA TAGAAGTGTGTATGTATGAAACCGA A CGTGGTG CATICAG'IAGGCGGCYTTACAGG4JOCAAACTGATTr

CCATAGC'JCA'ITCCACCAGGCCGAGCACTCAGAACT)CC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACG

TrGGAAGGaTLCGTGAGCAGAAAGCGGGATCGAGTCT

AMTAGACACGCTGAACAAATTCGTAAGAGGGGOG

CGGAC1TGCITGGT)GTAATGCGCGGACATCCGCCI'CAG

GCTACTACAAAAAGTTAGCOCTFCAGCGAGCAGGGAGAG

ATAFIGATACGCCGCCAGTAGGACCT)CACATC-CTGATG

_____________TATAAAAGGCTCACATAA

SEQ ID- 110 4G5 'ATGATAGAGGTGAA.ACCGATr7AACGCAGAGGATACCTA -162- WO 02136782 WO 0236782PCTIUJS01/46227 NO :204 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAcGGTGTATGTJGAAAGCGATJ-ACICGTGGTG CArITCA=rAGGCGGCTATACAGGGGCAAACTGATI

CCATAGC=CAITCCACCGGC.CGAGCACTGCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGAAGGTTACCGCGATCAGAAAGCGGGATCGAGTCT

AATTAGACACGCTGAACAAATMCGTAAGAGGGGGG

CGGAC'IrGCITGGTGTAATGCOCGGACATCCGCCTCAG

GCTACTACAAAAAGTTAGGCTTCAGCGAGCAGGGAGAG

ATAITGATACGCCGCCAGTAGGACCTCACATCTGATG

TATAAAAGGCTCACATAA

SEQ ID 10_4H4 ATCrCAGAGGTGAAACCGATrAACGCAGAGGATACCTA NO:205 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGATIAC1TCGTGGTG CATITCACTrAGGCGGC1TITACAGGGGCAAACTGATIT CCATAGCTrCATTCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TrGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT AATTAAACACOGTQGAAGAAATT2FCGTAAGAGGGGG CGGACITGCFITGGTGCAATGC3CGGACATCCGCCTCAG GCrACTACAAAAAGTrAGGCICAGCGAGCAGGGAGAG GTAT1TGATACGCCGCCAGTAGGACCTCACATCCTGATG

TATAAAAGGATCACATAA

SEQ ID 11_3A11 ATGATAGAAGTGAAACCGATFAACGCAGAGGATACCTA NO:206 TGAACTGAGGCATAAAATACTCAGACCAAACCAGCCGA TAGAAGTGTGTATGTATGAAAGCGATIAC1TGTGGTG CA'1TCAC1AGGOCGCTIrTACAGGGGCAAACTGAnrr

CCATAGCGTCATCCACCAGGCCGAGCACCCAGACCTC

CAAGGCCAGAAACAGTACCAGCTCCGAGGTATGOGTAC

CTTGGAAGG'1TATCGTGATCAGAAAGCGGGATCGAGTC TAATrAAACACGCTGAACAAATTCICGTAAGAGGGGG GCGGACTrGCIMGGTGCAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTTAGGCICAGCGAGCAGGGAGA

GGTAT=GAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAGGCTCACATAA

SEQ IID 11_331 ATGCTAGAGGTGAAACCGArrAACGCAGAGGATACCTA NO:207 TGAACTGAGGCATAGAATACTCAGACCAAACCAOCCGA

TAGAAGCGTGTATGTIGAAACCGATIACITCGTGGTG

CAr1TACTTAGGCGGCIT1ACAGGQG3CAAACrGA=n CCATAGCICATrCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAACTCCGAGGTATGGCTACC

TPGGAAGGYIICGTGAGCAGAAAGCGGGATCGACTCT

AATTAGACACGCTGAAGAAA1TCTI'CGTAAGAGGOGG CGGACIGCT1GGTGCAATGCGCGGACATCCGCCTCAG GCTACTACAAAAGGTAGGC1TGAGCGAGCAGGGAGAG

ATA'IIGACACGCCGCCAGTAGGGCCTCACATCCTGATG

________TATAAAAGGCTCACATAA

SEQ ID 11_3B5 ATGATAGAGGTGAAACCGAITAACC3CAGAGGATACCTA NO:208 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGT=GAAAGCGA=I]ACTTCGTGGTG

163 WO 02136782 WO 0236782PCTfUSOI/46227 CATICAC~rAGGCGGCTATITACAGGGGCAAACTGATI

CCATAGCGTCAITCCACCAGGCCGAGCACTCGGAACTC

CAAGGCCAGAAACAGTACCAGCTCCGAGGTATGGOCTAC

CTrGGAAGGTTATCGTGATCAGAAAGCGGGATCGAGTC TAA1TAGACACCGCTGAACAAAKITrCGTAAGAGGGGG

GCGGACATGCT=TGGTGCAATGCGCGGACATCCGCCTC

AGGCTACTACAAAAAG1TAGGCITCAGCGAGCAGGGAG

AGGTATITGATACGCCGCCAGTAGGACCTCACATCCTG

________ATGTATAAAAGGATCACATAA

SEQ ID 11_3C12 ATGATAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO:209 TGAACTAAGGCATAGAATACI'CAGACCAAACCAGCCGT

TAGAAGTGTGTATGTATGAAACCGAFIACTGGTG

CAT1TCAC1TGGGCGGC=flACGGGGGCAAACTGA~ CCATAGCGTCA2TCCACCAGGCCGAGCACCCAGACCTC

CAAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTAC

CTrGGAAGGTrATCGTGATCAGAAAGCGGGATCGAGTC TAA1TAGACACGCTGAACAACTrCGTAAGAGOGGGG GCGGACTITGCT1TGGTGCAATGCGCGGACATCCGCCTCA GGCTACTACAAAAAGITAGO1TrCAGCGAGCAGGGAGA GATAFrCGAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAGGATCACATAA

SEQ ID) 11_3C3 ATGATAGAAGTGAAACCGATFJAACGCAGAGGATACCTA NO:2 10 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAGCGATIACTTCGTGGTG

CACTTCA AGGCGGCTATTACAGGGGCAAACTGAT1=

CCATAGCGTCATTCCACCAGGCCGAGCACTCAGAACTC

CAAGGGCAGAAACAGTACCAGCTCCGAGGTATGGCTAC

=TGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTC

TAATTAAACACG~rGAAGAAAITCTTCGTAAGAGGOGG GCGGA GC GGTGCAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTTAGGGICAGCGAGCAGGGAGA

GGTAITGACACGCCGCCAGTAGGACCTCACATCCTGAT

(TATAAAAGGATCACATAA

SEQ ID 11_3C6 ATGCTAGAOGTGAAACCGATTAACGCAGAGGATACCTA NO:21 1 TGAAGTAAGGCATAAAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATG=flGAAAGCGAYIACICGTGGTG CATITACTAGGCGGC1ITACGGGGGCAAACTGAMI

CCATAGCICAITCCACCAGGCCGAGCACTCAGACCTCG

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TrGGAAGG1rATCGTGAGCAGAAAGCGGGATCGACTCT AAIfTAGACACGCTGAAGAAATfCICGTAAGAGGGGGG

CGGACFTGCTIGGTGCAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAAGTTAGGT'CAGCGAGCAGGGAGAG

ATATYITGATACGCCGCCAGTAGGACCTCACATCCTGATG

TATAAAAGGATCACATAA

SEQ ID 11_3D6 ATGATAGAGGTGAAACGGAITAACGCAGAGGATACCTA NO:212 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAGTGiTGTATGTATGAAACCGA A CGTGGTG CAYICACGCGGCT1TrACAGGGGCAAACT)GA=I

CATAGGTTCAITCCACCAGGCCGAGCACTCAGACCTCC

-164- WO 02/36782 WO 0236782PCTIUSOI/46227

AAGGCCAGAAACAGTACCAGCTGCGAGGTATGGCTACC

ITGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT

AATTAAACACGCTGAACAAATrCTTCGTAAGAGGGGG CGGAC'ITGCT1TGGTGCAATGCGCGGACATCCGCCTCAG GCTACTACAAAAAGTrAGGC'TFCAGCGAGCAGGGAGAG

GTAT=TGATACGCCGCCAGTAGGACCTCACATCCTGATG

TATAAAAGGCTCACATAA

SEQ ID 1_1G12 ATGCTAGAG(3TGAAACCGATTAACGGAGAGGATACCTA NO:21 3 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAGTGTGTATGTATGAAACCGA TAC1TCGTGGTG CA C=GCG=CGGGAATA

CCATAGCITCA'ITCCACCAGGCCGAGCACTCAGAACTCC

AAGGGCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGAAGGTTATCGTGATCAGAAAGCGGGATCGAGTCr AATTAAACACGCTGAAGAAATFCTrCGTAAGAGGOGG CGGAQTGCT1GGTGTAATGCOOGGACATCCGCCTCAG GCTACTACAAAAAGYTAGGC1CAGCGAGCAGGGAGAG

GTAITGAAACGCCGCCAGTAGGACCTCACATCCTGAT

________GTATAAAAGGCTCACATAA

FSEQ ID 1_1111 ATGATAGAAGTGAAACCTATTAACGCAGAGGAGACTTA NO:2 14 CGAACITCGACACAAGATCCTGCGCCCTAATCAGCCGA

TAGAGGCATGCATGTATGAAACGCGATCTGCTGCGGGGC

TCGITCCA GGGCGGGTTCTATCGTGGCCAATTGATC TCGATTGCGAGTTCCACAAAGGI7GAACACTCAGAACT GCAAGGGCAAAAGCAGTATCAATfACGAGGGATGGCG ACCCrCGAAGGAITCCGTGAGCAciAAGGCTGGCTC'TrC GCITA'rrAGGCACGCCGAGGAGATACTACGGAATAAAG GGGCAGATCTG IGGTGTAATGCACGCACGACAGCC TCCGG TACTATAAAAGGCTrGG1TJAGTGAGCACGGC GAAGTL1rCGAAACCCCGCCGcGrTGGGCCGCACATTCTT

ATGTACAAAAGAATCACT

SEQ ID 1_1112 ATGATAGAAGTGAAACCTATTAACGCAGAGGATACTrA NO:2 15 CGAACTTCGACACAGGATCCTGCGCCCTAATCAGCCGT?

AGAGGCATGCATGTATGAAAGCGATCTGCTGCGGGOCT

CGTrCCATGGGCGGGTTCTATCGTGGCAAATI'GATCT CGATrGCGAGTFITCCACCAAGCrGAACACTCAGAACTG GAAGGGCAAAAGCAGTATCAATrACGAGGGATGGCGA CCCTCGAAGGATrCCGTGAGCAGAAGGCTGGCTCTTCG

CTTMTAGGCACGCCGAGGAGATACTACGGAAAAGAGG

GGCAGATCTCTTGGTGTAATGCACGCACGACAGCCG

CCGGTACTATAAAAAGCTrGGTT=AGTGAGCAGGGC GAAA ITIl ICGACACCCCGCCGGTGGGCCGCACATTCTT

ATGTACAAAAGAATCACT

SEQ ID 1_115 ATGATAGAAGTGAAACCTATTAACGCAGAGGATACTTA NO: 216 CGAAATTCGACACAGGATCCTGCGCCCTAATCAGCCGT

TAGAGGCATGCATGTATGAAAGCGATCTGCTGCGOGGC

TCGTrCCATIGGGCGGGTTCTA.TCGTGGCAAKLTGATC

TCGATTGCGAGTTTCACCAAGCTGAACACTCAGACCTG

GAAGGGCAAAAGCAGTATCAA'ITrACGAGGGATGGCGA

____-CCCTCGAAGGATACCGTGATCAGAAGGCTGGCTCTCG

-165- 117n 1VCTTT4Zfl IAK('Y7 CTrATTAGGCACGCCGAGCAGATACTACGGAAAAGAGG

GGCAGATCTGCTIGGTGCAATGCACGCACGACAGCCG

CCGGTrACTATAAAAGGCTGGTI=AGTGAGCAGGGCC ;ZGAAGT1TCGACACCCCGCCGG'TGGGCCGCACAITCTr 1< _____ATGTACAAAAAACTCACT SEQ ID 1_2A12 ATGATAGAAGTGAAkACCTATTAACGCAGAGGATACITrA NO:217 CGAACITCGACACAGGATCCTGCGCCCTAATCAGCCGA

TAGAGGCATGCATGTATGAAAGGGATCTGCTGCGGGC

TCGTrCCAYIGGGCGGG'TrCTATCGTGGCAAATTGATC TCGATI'GCGAGfTTFCCACCAAGCTGAACAGTCAGAACT r- GGAAGGGCAAAAGCAGTATCAAfTACGAGGGATGGCG in ACCCTCGAAGGATACCGTGATCAGAAGGCTGGCTCTAC GCITATrAAGCACGCCGAGGAGATACTACGGAAAAAAG

GGGCAGATCTGCTI=GGTGCAATGCACGCACGTCAGCC

GCCGGTrACTATAAAAGGCF1'GG IAGTGAGCAGGG CGAAATT1TGACACCCCGCCGGTTGGGCCGCACATTCT

________TATGTACAAAAGACTCACT

SEQ ID 1_2B6 ATGATAGAAGTGAAACCTATTAACGCAGAGGAGACTrA NO:2 18 CGAAC'1CGACACAAGATCCTGCGCCCTAATCAGCCG'1T

AGAGGCATGCATGTATGAAACCGATCTGCTGCGGGGCT

CG1TCCATITGGGCGGGFTCTATCGTGGCAAAITGATCT CGATrGCGAGTFJCCACCAAGCTGAACACTCAGAACTG

GAAGGGCAAAAGCAGTATCAATTACGAGGGATGGCGA

CCCTCGAAGGATCCGTGATCAGAACTCTTGOCTrCGC ITrATTAAGCACGCCGAGGAGATACTACGGAAAAGAGGG

GCAGATCTGCTIGGTGCAATGCACGCACGTCAGCCTICC

GGTrACTATAAAAAGCIGGT1EAGTGAGCAGGGCGA AATT1CGAAACCCCGCCGGTGGCGCACATrCTrAT

_____________GTACAAAAGACTCACT

SEQ ID 1_2C4 ATGCTAGAAGTGAAACCTAfTAACGCAGAGGAGACITA NO:219 CGAACITCGACACAAGATCCTGCGCCCTAATCAGCCGA

TAGAGGCATGCATGTATGAAACCGATCTGCTGCGQOOC

TCG2ITCCATTTGGGCGGGTJ'CTATCGTGGCCAAITGATC

TCGATTGCGAGTITCACCAAGCTGAACACTCAGACCTG

CAAGOOCAAAAGCAGTATCAATTACGAGGGATGGCGAC

CCTCGAAGGATACCGTGAGCAGAAGGCrGGCTCTACGC

TTAT'AAGCACGCCGAGGAGCTAGTACGGAAAAAAGGG

GCAGATCrGCTIGGTGCAATGCACGCACGACAGCCGC CGGTrACTATAAAAAGCTrGG3TIAGTGAGCAGGGCrG AAG2IICGACACCCCGCCGGT1'GGGCCGCACATTC'ITA

TGTACAAAAAAATCACT

SEQ ID 1_2D2 ATGATAGAAGTGAAACCTATTAACGCAGAGGATAC'ITA NO:220 CGAACITCGACACAAGATCCTGCGCCCTAATCAGCCGIT

AGAGGCATGCATGTATGAAAGCGATCTGCTGCGGAGCG

CATrCCATMrGGGCGGG'TCTATCGTGGCAAATTGATCT CGATTGCGAG1TCCACAAAGCTGAACACrCAGAACTG

CAAGGGCAAAAGCAGTATCAATTACGAGGGATGOCGAC

CCrCGAAGGATACCGTGATCAGAAGGCTGGCTC1CGC rrATrAGCACGCCGAGGAGATACTACGGAAAAGAGGG

_____________GCAGATATGCTIGGTGCAATGCACGCACGTCAGCCGC

-166- WO 02/36782 WO 0236782PCTfUSO1/46227 CGGTTACTATAAAAGGCTGG1TIAGTGAGCAGGGG AAGTIJCGACACCCCGCCGGTOGGCCGCACATrC=A

________TGTACAAAAGAATCAC=TAA

SEQ ID 1_2D4 ATGATAGAAGTGAAACCTATrAACGCACIAGGATACTrA NO:22 1 CGAAC1CGACACAGGATCCTGYCGCCCTAATCAGCCGA

TAGAGGCATGCATGTATGAAAGCGATCTGCTGCGGGGC

TCGTrCCATITGGGCGGcflTCTATCGTGGCAAATTGATC TCGATTGCGAGT1TCCACCAAGCTGAACACTCAGACCTG

CAAGGGCAAAAGCAGTATCAA'ITACGAGGGATGOCGAC

CCTCGAAGGATACCGTGAGCAGAAGGCTGGCT=TCGC

1TATTAAGCACGCCGAGCAGCTACTACGGAAAAAAGGG GCAGATATGCTITGGTGTAATGCACGCACGTCAGCC3C CGG2FrACTATAAAAGGCTGG=rIAGTGAGCACGGG AAATT7rGAAACCCCGCCGGTTGGGCCGCACATTCTTA

_____________TGTACAAAAGAATCACT

SEQ ID 1_2F8 ATGCTAGAAGTGAAACCTATTAACGCAGAGGATACTA NO:222 CGAAC1TCGACACAGGATCCTGCGCCCTAATCAGCCGIT

AGAGGCATGCATGTATGAAACCGATCTGCTGCGGGGCT

CGTI'CCAYIGGGCGGGfrCATCGTGGCAAAITQATCT CGA1TGCGAGITCCACCAAGCTGAACATTCAGAACTG GAAGvGGCAAAAGCAGTATCAAITACGAGGGATGGCGA CTCTGAAGGATACCGTGATCAGAAGGCTGGCTCTrCG CTIrATTAGGCACGCCGAGGAGATACTACGGAAAAGAGG

GGCAGATATGCTIGGTGCAATGCACGCACGACAGCCG

CCGGTrACTATAAAAAGCXWGGTI=AGTGAGCAGGGC

GAAATIACGACACCCCGCCGGITGGGCCGCACATTCTT

ATGTACAAAAAACTCACT

SEQ ID 1_2HS ATGATAGAAGTGAAACCTATTAACGCAGAGGAGACIA NO:223 CGAAC'ICGACACAAGATCCTGCGCCCTAATCAGCCGTI'

AGAGGCATGCATGTATGAAACCGATCTGCTGCGGOOCG

CG~rCCAYIGGGCGGG'1FGATCGTGGCAAAITGATCT CGATrGCGAGI=CCACCAAGCTGACCACTCAGAACTG

CAAGGGCAAAAGCAGTATCAATTACGAGGGATGGCGAC

CCTCGAAGGATACCGTGAGCAGAAGGCTGGCTCTACGC

TrATrAGGCACGCCGAGCAGATACTACGGAAAAGAGGG GCAGATCTACrIGGTGCAATGCACGCACGTCAGCCGC

CGGTI'ACTATAAAAAGC'ITGGITIAGTGAGCACGGCG

AAATITGAAACCCCGCCGGTTGGGCCGCACATr=TA TGTACAAAAGACTCACTrAA SEQ ID 1_A2 ATGATAGAAGTGAAACCTATrAACGCAGAGGATACTA NO:224 CGAACT]?CGACACAGGATCCTGCGCCGTAATCAGCCGA TAGAGGCATGCATGTATGAAAGCGATCTGCrGCGGGGC

GCGTTCCATTGGGCGGGITCTATCGTGGCAAAYPGATC

TCGATTGCGA=2~CCACCAAGCTGAACACTCAGACCTG CAAGGGCAAAAGCAGTATCAATrfACGAGGGATGGCGAC CCTCGAAG-GATACCGTGAGCAGAAGGCTGGCrcICGC flA1TAGGCACGCCGAGGAGATACTACGGMAAJLAGGG GCAGATATGCTITrGGTGCAATGCACGCACGACAGCCGC

CGGTTACTATAAAAGGTGG'P=AGTGAGCAGGGCG

_____AAGTJTCGACACCCCGCCGGTGGGCCGCACATrA -167- WO 02136782 WO 0236782PCT/USOI/46227

TGTACAAAAGAATCACT

SEQ ID 1_3D6 ATGATAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO:225 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA

TAGAAGTGTGTATGTATGAAACCGATLTTACT=GTGGTG

CAYICACTTAGGCGGCTIACAGGGGCAAACTGAT1T CCATAGC'TrCAITCCACCAGGCCGAGCACTCAGACCTCC

AAGGCAGAAACAGTACCAGC'FCCGAGGTATGGCTACC

TITGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT

AA'ITAAACACGCTGAACAAAT1'CTrCGTAAGAGGGGGG CGGACTTGC=fGGTGCAATGCGCGGACATCCGCCTCAG GCTACTACAAAAAGTrAGGCFTCAGCGAGCAGGGAGAG GTA1TTGATACGCCGCCAGTAGGACCTCACATCCTGATG

TATAAAAGGCTCACATAA

SEQ ID 1_F3 ATGATAGAAGTGAAACCTATrAACGCAGAGGAGACTTA NO:226 CGAAC2FICGACAGAGGATCCTGCGCCCTAATCAGCCGA

TAGAGGCATGCATGTATGAAACGCGATCTGCTGCGGOGGC

TCGTI'CCAFIGGGCGGG'TrCTATCGTGGCCAATTGATC TCGATrGCGAG1TCCACCAAGCTGAACACTCAGAACT

GCAAGGGCAAAAGCAGTATCAATTACGAGGGATGGCG

ACCCTCGAAGGATACCGTGAGCAGAAGGTGGCTC7AC GC1TATTAAGCACGCCGAGGAGATACTACGGAAAAAAG

GOGCAGATCTGCTITGGTGCAATGCACGCACGTCAGCC

GCCGGTfACTATAAAAGGCUJTGGT1TAGTGAGCACGG CGAAA3T1CGACACCCCGCCGGTTGGGCCGCACAFr~r

________TATGTACAAAAGAATCACT

SEQ ID 1_3H2 ATGATAGAAGTGAAACCTAITAACGCAGAGGATACTrA NO:227 CGAACT=CGACACAGGATCCTGCGCCCTAATCAGCCGA

TAGAGOCATGCATGTATGAAACCGATCTGCTGCGOGC

GCGYFCCATTGGGCGGGTACTATCGTGGCCAATTGATC

TCGATTGCGAG2FTCACAAAGCTGAACACTCAGAACT

GCAAGGGCAAAAGCAGTATCAATTACGAGGGATGGG

ACCCTCGAAGGATACCGTGAGCAGAAGGCTGGCTCTAC

GCTTA2FrAAGCACGCCGAGCAGCTACTACGGGAAAAAG

GGGCAGATATGC'TTGGTGCAATGCACGCACGTCAGCC

GCCGGTrACTATAAAAGGCTrGGTITAGTGAGCAGGG

CGAAGT=ICGAGACCCCGCCGGTTGGGCCGCACATTCT

TATGTACAAAAAACTCACT

SEQ ID 1_4C5 ATGATAGAAGTGAAACCTAFJAACGCAGAGGATACT7A NO:228 CGAACTTCGACACAAGATCCTGCGCCCTAAT)CAGCCGA

TAGAGGCATOCATGTATGAAAGCGATCTGCTGCGGGGC

TCGITCCATITGGGCGGGTTCTATCGTGGCAAATTGATC

TCGA'TGCGAGTITCCACAAAGCTGAACACTCAGACCT

GGAAGGGCAAAACCAGTAT)CAA2FrACGAGGGATGGCG ACCCTCGAAGGATACCGTGAGCAGAAGGCTGGCTCrAC GCTrATTAGGCACGCCGAGGAGATACTACGGAAAAGAG

GGGCAGATATGCTLTGGTGCAATGCACGCACGTCAGCC

TCCGGT'rACTATAAAAGGCTrGGTT1AGTGAGCACGGC

GAAATTICGACACCCCGCCGGTTGGGCCGCACA'TTM

ID_ _1 ATGTACAAAAGACTCAC=TAA 14D6 ATGCTAGAAGTGAAACCTATTAAkCGCAGAGGATACTrA -168- WO 02136782 WO 0236782PCT[US01/46227 NO:229 CGAC]CGACACAGGATCCTG-CGCCCIAATCAGCCGA

TAGAGGCATGCATGTATGAAACCGATCTGCTGCGGGGC

TCGTTCCATTGGCGGG-FCTATCGTGGCCAATrGATC TCGAMTGCGAGT1CCACAAAGCTGAACACTCAGACCT

GGAAGGGCAAAAGCAGTATCAAITACGAGGGATGGCG

ACCCTCGAAGGATACCGTGAGCAGAAGOGTGOCTCTAC

GCTrATTAGGCACGCCGAGCAGATACTACGGAAAAGAG GGOCAGATATGCTCI700TGCAATGCACGCACGTCAGCC

GCCGGTTACTATAAAAGGC=GGTIIAGTGAGCAGGG

CGAAGTITPGAAACCCCGCCGGTrGGGCCGCACA=C _____________TATGTACAAAAGACrCACT SEQ ID 1_4111 ATGATAGAAGTGAAACCrATAACGCAGAGGATAC'rrA NO:230 CGAACTTCGACACAGGATCCTGCGCCCTAATCAGCCGTr AGAGGCATGCATGTATGAAAC('GATCTGCr7GCGGGGCr CGTTCCATF1TGGGCGGGTTCTATCGTGGCAAATrGATJr

CGATTGCGA&TICCACCAAGCTGAACACTCAGACCTG

CAAGGOCAAAAGCAGTATCAAYJACGAGGGATGGCGAC

CCTCGAAGGATACCGTGAGCAGAAcX3CTGGCTCTACGC TrAT7AGGCACGCCGAGCAGCrACTACGGAAAAGAGGG GCAGATCTGG2IGGTGCAATGCACGCACGTCAGCGTCC GGTrACTATAAAAGGCTTGG~rTAGTGAGCACGGCGA AGTITACACCCCGCCGGTGGGCCGCACA'rCITAT

GTACAAAAGACTCACT

SEQ ID 1_5H5 ATGCTAGAAGTGAAACCTA'TTAACGCAGACGGAGACTrA NO:23 1 CGAAC=CGACACAAGATCCTGCGCCCTAATCAGCCGTT

AGAGGCATGCATGTATGAAAGCGATCTGCTGCGGGGCT

CGITCCA1TIGGGCGGGTACfATCGTGGCCAX[TIGATCT CGATTGCGAG1TCCACCAAGCTGAACACTCAGAACTG

GAAGGGCAAAAGCAGTATCAATTACGAGGGATGGCGA

CCCTCGAAGGAMTCCGTGAGCAGAAGGCTGGcTCACG

CTTATTAAGCACGCCGAGCAGATACTACGGAAAAGAGG

GGCAGATATGCTTGGTGCAATGCACGCACGTC-AGCCG

CCGGTI'ACTATAAAAAGC1GGITIAGTGAGCACGGC GAAATITrCGACACCCCGCCGGTTGGGCCGCACATT

ATGTACAAAAAACTCAC=TAA

SEQ ID 1_6F12 ATGATAGAAGTGAAACCT ITAACGCAGAGGAGAG'IA NO:232 CGAAC'TCGACACAGGATCCTGCGCCCTAATCAGCCGA

TAGAGOCATGCATGTATGAAAGCGATCTGCTGCGOGGC

TCGTTCCAFJGGOCGGGTrTCTATCGTGGCAAATrGATC TCciATTGCGAGcrrCCACCAAGCTGAACACTCAGACCTA

GAAGGGCAAAAGCAGTATCAATJACGAGGGATGGCGA

CCCTCGAAGGATACCGTGATCAGAAGGCTGGGTGTACG

CITA'ITAAGCACGCCGAGGAGCTACTACGGAAAAGAGG

GGCAGATATGC'1TGGTGCAATGCACGCACGTCAGCCG

CCGGTTACTATAAAAGGCTTGG=IIAGTGAGCACGGC

GAAATTACGAAACCCCGCCGGTTGGGCCGCACATTC'Tr

ATGTACAAAAAAATCACT

SEQ ID 1_616 ATGATAGAAGTGAAACCTATI'AACGCAGAGGATACITA NO:233 CGAAC'1TCGACACAAGATCCI7GCGCCCTAATCAGCCGA

_____TAGAGGCATGCATGTATGAAAGCGATCTGCTGCGOOGC

-169- WO 02/36782 WO 0236782PCTIUSOI/46227 TCGTTCCATTGGGCGGGTTCTATCGTGGCCAATrGATC TCGATTGCGAG1=FCCACCAAGiCTGAACACTCAGACCTG GAAGCAAAAGCAGTATCAATrACGAGGGATGGCGA

CCCTCGAAGGATACCGTGATCAGAAGGCTGGCTC=JCG

C=ATrAAGCACGCCGAGGAGATACTACGGAAAAG:AGG GGCAGATCTG=T1GGTGCAATGCACGCACGTCAGCCG CCGG'TACTATAAAAGGCIfTGGITIAGTGAGCAGGGC GAAAT1CGACACCCCGCCGGFrGGGCCGCACATTCFT

________ATGTACAAAAAAATCACT

SEQ ED 3_h1AlO ATGCTAGAGGTGAAACCGATrAACGCAGAGGATACCA NO:234 TGAACTAAGGCATAGAATAMTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAGCGATIACITCGTGGTG

CAITCACT'FAGGCGGCTAFrACAGGGGCAAACTGA=T CCATAGC'1CAITCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

2fTGGAAGG2TATCGTGAGCAGAAAGCGGGATCGAGTCT

AGTTAAACACGCTGAAGAAATTTCGTAAGAGGGOG

CGGACTTGC=1GGTGTAATGCGCGGACATCCGCCTCAG GCTACTACAAAAAGTTAGGCT1TCAGCGAGCAGGGAGAG ATA1TGAAACGCCGCCAGTACrGACCTCACATCCTGAT

________GTATAAAAGGATCACATAA

SEQ ID 3_14F6 ATGCTAGAGGTGAAACCGATTAACOCAGAGGATACCTA NO:235 TGAACTAAGGCATAGAATA~rCAGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAAGCGATIfTACITCGTGGTG CATTfTCACITAGGCGGCTJTACAGX3OGCAAACFGA=f

CCATAGCTTCATTCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCrCCGAGGTATGGCTACC TTGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCr AATTAAACACc3CTGAAGAA-A'TCTTCGTAAGAGGGGGG CGGACITrGCI=GGTGTAATGCGCGGACGTCCGCCTCAG GCTACTACAAAAAGTrAGGC'ITCAGCGAGCAGGGAGAG

ATATIGAAACGCCGCCAGTAGGACCTCACATCCTGAT

GTATAAAAGGCTCACATAA

SEQ IID 315B2 ATGCTAGiAGGTGAAACCGATI7AACGCAGAGGATACCTA NO:236 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGAT1ACTCGTGGTG CAT=CACTTAGGCGGCTATTACGOGGGCAAACTGA=f

CCATAGC'TCA'TCCACCAGGCCGAGCACTCAGAACTCC

AAGOCCAGAAACAGTACCAGCTCCGAGGTATGQCTACC

TrGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTGT AATTAAACACGCTGAAGAAATCTrCGTAAGAGGOGG CGGACTrGCTITGGTGTAATGCGCGGACATCCGCCTCAG GCTACTACAAAAAGTrAGGCTI'CAGCGAGCAGGGAGAG

ATATITGAAACGCCGCCAGTAGGACCTCACATCCTGAT

GTATAAAAGGATCACATAA

SEQ ED 3_6A10 ATGATAGAAGTGAAACCGA~rAACC3CAGAGGATACCTA NO:237 TGAACTAAGOCATAGA-ATACTCAGACCAA-ACCAGCCGA

TAGAAGCGT)GTATGTATGAAAGCGATIACTL'CGTGGTG

CATJCA=TIAGGCGGCrATrAC'AGGGTGCAAACTGAT _____________CCATAGTCATrCCACCAGGCCGAGCACTCAGAACTCC -170- WO 02136782 WO (i236782PCTIUSOI/46227

AAGGCCAGAAACAGTACCAGCTCCGAG*JTATGGCTACC

TTGGAAGGTTATCGTGAGCAGAAAGGCGGGATCGAGTCT

AAITAAACACGCTGAAGAAATI'=CGTAAGAGGGGGG

CGGACITGCTIGGTGTAATGCGCGGACATCCGCCr)CAG GCTACTACAAAAAG2FrAGGCTrCAGCGAGCAGGGAGAG ATAFIGAAACGCCGCCAGTACiGACCTCACATCCTGAT

GTATAAAAGGATCACATAA

SEQ ID 3_6B 1 ATGCTAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO:238 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA TAGAAGCGTGTATGTATGAAACGCGATJAC1TCGTGGTG CAT1CACITAGGCGGCTATTACAGGGGCAAACTGATr

CCATAGCTTCATTCCACCAGGCCGAGCACCCAGAACTC

CAAGGCCAGAAACAGTACCA~CT-CCGAGGTATGGCTAC

C'1TGGAAGGTrATCGTGAGCAGAAAGCGGGATCGAGTC TAA'17AAACACGCTGAAGA.AA1TrCTCGTAAGAGGGOO

GCGGACTTGCITGGTGTAATGCGCGGACATCCGCGTCA

GGCTACTACAAAAAGTTACGCTTCACGAGCA%3G~jAGrA

GGTATIGAAACGCCGCCAGTAGGACCTCACATCCTGA

________TGTATAAAAGC3ATCACATAA SEQ ID 3_7F9 ATGCTAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO:239 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA

TAGAAGCGTGTATGTATGAAAGCGATIACTTCGTGGTG

CAITCACTTAGGCGGCTATrACGGGCAAACTGA'lM

CCATAGCTTCATTCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCrCCGAGGTATGGCTACC

'ITGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT

AATTAAACACGCTGAAGAAATTCTTCGTAAGAGOGGG

CGGACTGCTIGGTGTAATGCGCGGACATCCGCCT'CAG

GGTACTACAAAAAGTrAGGC'TrCAGCGAGCAGGGAGAG ATAT1T'GAAACGCCGCCAGTAGGACCTCACATCCTGAT GTATAAAAGiGATCACATAA SEQ ID 3_8G1 1 ATGCAGAGGTGAAACCGATTAACGCAGAGGATACCFA NO:240 TGAACTAAGGCATAGAATACTCAGACCCAACCAGCCGA

TAGAAGTGTGTATGTATGAAAGCGA=IACTTCGTGGTG

CAfTCACTAGCGGCTATTACAGQGGCAAACTGATLT

CCATAGCTTCATCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT

AATTAAACACGCTGAAGAAAITC1TrCGTAAGAGGGX3G

CGGACTTGCTITGGTGTAATG-CGCGGACATCCGCCTGAG

GCTACTACAAAAAG~rAGOG1TCAGCGAGCAGGGA GAG

ATATTTGA-AACGCCGCCAGTAGGACCTCACATGCTGAT

______GTATAAAAGGATCACATAA

SEQ ID 4_-iBlO ATGATAGAAGTGAAACCTATTAACGCAGAGGATACCTA NO:241 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGA

TAGAAGTGTGTATGTATGAAACCGATMTACTPCGTGGTG

CATITACTTAGGCGGG-ITIACGGGOCAAACrGATI-r CGATAGCTCATrCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

_____________TTGGAAGcrTrATCGT)GATCAGAAAGCGGGATCGAGTCT 171 WO 02136782 WO 0236782PCTIUSOI/46227

AATTAGACACGCTGAACAAATITCTTCGTAAGAGGGGGG

CGGACATGCTrTGGTGCAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTI'AGGCICAGCGAGCAGGGAGA

GATA TrGAAACGCCGCCAGTAGGACCTCACATCCTGA

________TGTATAAAAGGATCACATAA

SEQ ID 5_2B3 ATGATAGAAGiTGAAACCTATrrAACGCAGAGGATACCTA NO:242 TGAACTAAGGCATAGAATACTCAGACGAAACCAGCCGT

TAGAAGTGTGTATGTATGAAACCGAITACTTCGTGGTG

CAT1TACTTAGGCGGCT1TACGOGGCWAAACTGATL1? CCATAGC1TCATTCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGOCTACC

TTGGAAGG'TrATCGTGATCAGAAAGCGGGATCGAGTCT AAITAGACACGCTGAACAAAT7CTPGTAAGAGGGGQG CGGACATGCTTrGGTGTAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTTAGGCITCAGCGAGCAGGGAGA

GATATITGAAACGCCGCCAGTAGGACCTCACATCCTGA

_____________TGTATAAAAGGATCACATAA

SEQ ID 5_2D9 ATGCTAGANGiTGAAACCGATrAACGCAGAGGATACCTA NO:243 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGN TAGAAGTGTGTATGTATGAAANCGATITA1TrCGTGGTG CATrTCAC=AGGCGGCI=rACAGGGGCAAACrGAI=

CCATAGCTTCAITCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

ITGGAAGGTrATCGTGATCAGAAAGCGGGATCGAGTCT AATPAAACACGCTGAACAAATrC=CGTGAGAGGGOG CGGACATGC2FLGGTGCAATGCGCGGACATCCGCCTCA GOCTAGTACAAAAAG'fTAGGCTTCAGCGAGCAGQGAGA

GGTATIGACACGCCGCCAGTAGGACCTGACATGCTGAT

GTATAAAAGGCTCACATAA

SEQ ID 5_2F10 ATGCTAGAAGTGAAACCTATTAACGCAGAGGATACCTA NO:244 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGA TAGAAGTGTGTATGTATGAAACCGA1TACT7CGTGGTG CATICACTTAGCOCGGC=1ACGGGGGCAAACTGATIT CCATAGCITrCATrCCACCAGGCCGAGCACTCAGACCTCC AAGGCCAGAAACAGTACCAGCrCCGAGGTATGGCTACC

TTGGAAGGYTATCGTGATCAGAAAGCGGGATCGAGTCT

AATTAGACACGCTGAACAAAF'LCGTAAGAGGGGOG

CGGACATGCTIGGTGCAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTrIAGGCT'TCAGCGAGCAGGGAGA

GATATITGAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAGGCTCACATAA

SEQ ID 6_lAl 1 ATGCTAGAGGTGAAACCGATrAACGCAGAGGATAGCTA NO:245 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGATrTACITCGTGGTG

CA=ICACTAGGCGGCITACAGGGGCAAACTGATLT

CCATAGCGTCArrCCACCAGGCCGAGCACTCAGACCTC

CAAGGCCAGAAACAGTACCAGYCTCCGAGGTATGGCTAC

CTI'GGAAGG'TrATCGTGATCAGAAAGCGGGATCGAGTC TAAITAGACACGCTGAACAAATTC1CGTAAGAGOGG

GCGGACATGCTIGGTGCAATGCGCGGACATCCGCCTC

172 WO 02136782 WO 0236782PCTfUSO1/46227

AGGCTACTACAGAAAGIAGGCTTCAOCGAGCAGGGAG

AGGTATIGAAACGCCGCCAGTAGGACCrCACATCCTG _______ATGTAkTAAAAGGCTCACATAA SEQ ID 6_1D5 ATGATAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO:246 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTciTATGTATGAAACCGATTTACTrCGTGGTG CAT1CACTTAGGCGGCTITACAGGGGCAAACTGATI CCATAGC'TCATCCACCAGGCCGAGCACrCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TrGGAAGcYITATCGTGATCAGAAAGCGGGATCGAGTCT AATfAGACACGCTGAACAAAI'TCFCGTAAGAGGOGG

CGGACATG=TIGGTGCAATGCGCGGACATCCGCCTCA

GGCrACTACAAAAAGTTAGOGTCAGCGAGCAGGGGGA GGTA2FITCAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAGGATCACATAA

SEQ ID 6_IFi11 ATGATAGAGGTGAAACCGATrAACGCAGAGGATACCTA NO:247 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGAT1TACITCGTGGTG

CATITCACTTAGGCGGCTITACAGGGGCAAACTGATI

CCATAGCTCATTCCACCAGc3CCGAGCACTCAGACCTCC

AAGQCCAGAAACAGTACCAGCTCCGAGQTATGGCTACC

TTGGAAGGITATCGTGAGCAGAAAGCGGGATCGAGTCT

AA2FIAGACACGCTGAACAAAITCTTCGTAAGAGGGGGG

CGGACATGCI=GGTGCAATGCGCGGACATCCGCCTCA

GGCTACTACAAAAAGTTAGGCI]TCAGCGAGCAOOGAGA

GGTA'1=1'AAACOCCGCCAGTAGGAC ACATCCTGA

______TGTATAAAAGGCTCACATAA

SEQ ID 6_iFi ATGATAGAGGTGAAACCGATITAACGCAGAGGATACCTA NO:248 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGATITAC1CGTGGTG

CATICACTTAGGCGGCI=ACAGGGGCAAACTGMTI'

CCATAGCTTCATTCCACCAGGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGOTATGOCTACC

TrGGAAGGTrATCGTGAGCAGAAAGCGGGATCGAGTCT

AATTAGACACGCTGAACAAATTCTTCGTAAGAGGGOGG

CGGACATGCTITGGTGCAATGCGCGGACATCCGCCTCA

GGCTAGTACAAAAAGTJAGGCTTCAGCGAGCAGGGAGA

GGTAFIGAAACGCCGCCAGTAGGACCTCACATCCTGA

_____________TGTATAAAAGGCTCACATAA

SEQ ID 6_1H1O ATGTAGAGGTGAAACCGAT7AACGCAGAGGATACCTA NO:249 TGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGA1TTAC =?CTGGTG CATITCAC1TAGGCGGCITJACGGGGGCAAACTGATI1 CCATAGCT-rCATTCCACGAGGCGGAGCACTCGGACCTCC

AAC'GCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

1TGGAAGGITATCGTGATCAGAAAGCGGGATCGAGTCT AArrAGACACGCrcGAAGAAATTCTTCGTAAGAGGGOG

CGGACATGCTIGGTGCAATGCGCGGACAT)CCGCCTCA

GGCTACTACAAAAAGTrAGGCTTCAGCGAGCAGGGAGA _____________GGTATITrGACACGCCGCCAGTAGGACCTCACATCCTGAT 173 WO 02/36782 WO 0236782PCTLJSOI/46227

______GTATAAAAAGATCACATAA

SEQ ID 6_1H4 ATGCTAGAAGTGAAACCGATTAACGCAGAGGATACCTA NO:250 TGAACTAAGiGCATAAAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGA'1TACITCGTGGTG CAT1TCACTrAGGCGGC1T1ACG6GCOGGCAAACTGATI CCATAGCTFrCATrCCACCAGGCCGAGCACTCAGACCTCC

AAGGCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

TTGGAAGGTlATCGTGATCAGAAAGCGGGATCGACTCT AA2FTAAACACGCTGAACAAkATTCTTCGTAAGAGGGGOG CGGACATGCTIG%3TGCAATGCGCGGACATCCGCCTCA GGCTACTACAAAAAGTTAGGC2LCAGCGAGCAGGGAGA

GGTAITGAAACGCCGCCAGTAGGACCTCACATCCTGA

_____________TGTATAAAAOGCTCACATAA

SEQ ID 8_iFS ATGATAGAGGTGAAACCGATTAACGCAGAGGATACCTA 1 TGAACTAAGGCATAGAATACTCAGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGATIAfCGTGGTG

CAYICACTAGGCGGCIT=ACAGGGGCAAACTGAF'IT

CCATAGCTITCATFCCACCAGGCCGAGCACTCAGACCT'CC

AAGGCCAGAAACAGTACCAGC'TCCGAGGTATGGCTACC

'1TGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGTCT AA'ITAAACACGCTGAAGAAA'1TC'1TCGTAAGAGGGGGG CGGACTfGCTIGGTGTAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAAGTTAGGCTTCAGCGAGCAGGGAGAG

ATATITGATACGCCGCCAGTAGGACCTCACATCCT'GATG

_____________TATAAAAC3GATCACATAA SEQ ID 8_1G2 ATGATAGAGGTGAAACCGATTAACGCAGAGGATACCTA NO:252 TGAACTAAG*JCATAGAGTACTCAGACCAAACCAGCCGT

TAGAAGTGTGTATGTATGAAACCGATIACTTFCGTGGTG

CATTTAC=AGGCGGCTATACAGGGAAACTGAYTr CCATAGC1TCATTCCACCAGGGCGAGCAGTAGAACTCC

AAGGCCAGAAACAGTACCAGCT'CCGAGGTATGGCTACC

TTGGAAGGTrATCGTGAGCAGAAAGCGGGATCGAGTCT AATrAAACACGCTGAAGAAATrC1CGTAAGAGGGGGG

CGGACTTGCTIGGTGCAATGCGCGGACAT)CCGCCTCAG

GCTACTACAAAAAGTTAGGCTTCAGCGAGCAGGGAGAG

GTATIGAGACGCCGCCAGTAGGACCTCACATCCTGiAT

________GTATAAAAGGCTCACGTAA

SEQ ID 8_1G3 ATGCTAGAG{3TGAAACCGATTIAACGCAGACGGATACTrA NO:253 CGAACTAAGGCATAAAATACTCAGACCAAACCAGCCGA TAGAAGTGTGTATGTATGAAACCGAT A CGTGGTG

CATFICACTTAGGCGGCTATTACAGGGGCAAACTGATYI

CCATAGCTTCATTCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

1TGGAAGGTTATCGTGAGCAGAAAGCGGGATCGAGT)CT AATrAGACACGCTGAAGAAAITCTCGTAAGAGGGGGG

CGGACTTGCI=GGTGTAATGCGCGGACATCCGCCTCAG

GCTACTACAAAAAGTrAGOC1TrCAGCGAGCAGGGAGAG

ATATITGATACGCCGCCAGTAGGACCTCACATCCTGATG

_____________TATAAAAGGATCACGTAA

SEQID 8 1117 ATGCTAGAGGTGAAACCGA-TAACGCAGAGGATACCTA -174- WO 02/36782 WO 0236782PCTIUS01/46227 NO:254 I TGAACTrAAG7GCATAGAATACTCAGACCAAACCAGGCGA TAGAAGTGTGTATGTATGAAACCGAFJACTrCGTGGTG CAT TCACTrAGGCGGCTLTACAGGCAAACTGiA1T CCATAGC'1TTCCACCAGGCCGAGCACTCAGAACTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

'TrGGAAGGITfATCGTGAGCAGAAAGCGGGATCGAGTCT AATrAAACACGCTGAAGAAATCITCGTAAGAGGGGGG CGGACATGG'rGGTGCAATGCGCGGACAT)CCGCCTCA GGCTACTACAAAAAGTTAGGC1CAGCGAGCAGGGAGA

GATATIGAAACGCCGCCAGTAGGACCTCACATCCTGA

TGTATAAAAGGCTCACATAA

SEQ ID NO:255 8_1H9 L 4

ATGCTAGAGGTGAAACCGATTAACGCAGAGGATACCTA

TGAACTAAGOCATAAAATACTC AGACCAAACCAGCCGT TAGAAGTGTGTATGTATGAAACCGATIACTrCGTGGTG CATrrTCACTTAGGCGciCTATrACAG4JOGCAAACTGA=n

CCATAGGITCMTCCACCAOGCCGAGCACTCAGACCTCC

AAGGCCAGAAACAGTACCAGCTCCGAGGTATGGCTACC

2FrGGAAGG3TTATCGTGAGCAGAAAGCGOGATCGAGTCT AATrAGACACGCTGAAGAAATFCTrCGTAAGAcGGGGGG CGGA=2rCTrGGTGTAAT)GCGCGGACATrCCCTCAG GCTACTACAAAAAG1TAGGCYJ'CAGCGAGCAGGGAGAG GTATITGATACGCCGCCAGTACiGACCTCACATGGTrGATG

TATAAAAGOCTCACATAA

ATGATTGAAGTCAAACCTATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCATrCTCCGGCCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGATIGCTCGGOTCGGC

ACGT1CACCTCGGCGGATATTACCGGGGCAAGCTGAT CAGCATCGCTTCCTICATAATGCCGAACA1TCAGAGCT

TGAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCG

ACGCIGAAGGATACCGTGAGCAAAAAGCGGGAAGCA

CGCTCATCCGCCATGCCGAAGAGCTTC1TCGGAAAAAA

GGCGCGGACCTTITAT'GGTGCAACGCCAGGACATCTGT

GAGCGGGTACTATAAAAAGCTCGGGT='CAGCGAACAGG

GCGAAGTCTACGACATACCGCCGATCGGACCTCATATI'

TGATGTATAAGAAATTGACGTAA

SEQ ID NO:256 GATi_21F 12 SEQ ID NO:257 GATi_24G 3

ATGAT[GAAGTCAAACCAATAAACGCGGAAGATACGTA

TGAGATCAGGCACCGCAITCTCCGGCCGAATCAGCCGC

TTGAAGCATGTATGTATGAAACCGAT=TGCTCGOOOOC

ACGTTCACCTCGGTGGATATTACCGG3GCAAGCTGATC

AGCATCGCCTCC=JCATCAAGCCGAACAITAGAGC'TT

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTI'GAAGGGTACCGCGAGCAAAAAGCGGOCAGTACG

G'ITATCCGCCATGCCGAAGAGC2I7TCGGAAAAAGOG GGCAGACCFI=ATGGTGCAATGCCAGGACAT1GTGA

GCGGTI'ACTATGAAAAGCTCGGTTCAGCGAACAGGGC

GAAGTCTACGACATACCGCCGATCGGACCTATA=flG

ATGTATTAGAAMTTGACATAA

SEQ ID GAT1_29G ATGATrGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:258 1 TGAGATCAGGCACCGCITrCTCCGGCCGAATCAGCCGC _____________TrGAAGCATGTATGTATGAAACCGAT=GCTCGGGGGT- 175 WO 02/36782 WO 0236782PCTIUSOI/46227 ACGTITCACCTCGGTGGATATTACGGGGCAAGC7GATC AGCATCGCTTCC'HCATCAAGCCGAACATrCAGAGCTT GAAGGTCCAAAAACAGTATCAGC TGAGAGGGATGGCGA CAC1TGAAGGGTACCCGAGCAAAAAGCGGGTAGTACG C'1TATCCGCCATGCCGAAGAGCTrQTCGGAAAAAGOG

GGCAGACCTIATGGTGCAACGCCAGGACATCTGTGA

GCGGGTACTATAAAAAGCTCGGCTITCAGCGAACAAGGC

GGGGTCTGCGATATACCGCCGATCGGACCTCATAT1TG

_____ATOTATAAGAAATTGGCATAA

SEQ DD) GATi_320 ATGATh3AAGTGAAAGCAATAAACGCGGAAGATACGTA NO:259 1 TGAGATCAGGCACCGCATACTCCGGCCGAATCAGCCGC TI7GAAGCATGTATGTATGAAACCGAYPTGCTCGOOGGC

ACGTICACCTCGGTGGATATTACCGGGGCAAGCTGATC

AGCATCGC2rCcTICATCAAGCCGAACATCCAGAGCI

GAAGGCCAAAAACAGTATCAGGTGAGAGGGATGGCGA

CAC'1GAAGGGTACCGCGAGCAAAAAGCGGCAGTACG

CTTATCCGCCATGCCGAAGAGGITCMCGGAAAAAAGG

CGGAGACCI-ITATGGTGCAACGCCAGGACATCTGTGA

GCGGCTACTATGAAAAG~rCGGCTTCAGCGAACACGC GAAGTCTACGAGATACCGCCGATCGGACCv"ATATITG ATGTATAAGAAATh'GACATAA SEQ ID GAT2_15G ATGA2ITGAAGTCAAACCAATAAACGCGGAAGATACGTA NO:260 8 TGAGATCAGGCACCGCAT1TCTCCGG3CCGAATCAGCCGC

TGGAAGCATGCAAGTATGAAACCGKFITGCCGGGGGC

ACGT1CACCTCGGTGGATATFIACCGGGGCAAGCTGATC

AGCATCGCITCC=TCATAATGCCGAACAITCAGAGCT

GAAGGCCAAAAACAGTATCAG(TGAGAGGGATGGCGA

CGCITGAAGGGTACCGCGAGCAAAAAGCGGGAAGCAC

GCTCATCCGCCATGCCGAAGAGC'ITC'ITCGGAAAAAAG

GCGCAGACCT1TATGGTGCAACGCCAGGACATCI'GTG

AGCGGGTACTATAAAAAGCTCGGC'ITCAGCGAACAGGG

CGAAGTCTACGACATACCGCCGATCGGACCTCATATIT

GATGTATAAGAAATTGACGTAA

SEQ ID GAT2_1911 ATGATTGAAGTCAAACCAATAA-ACGCGGAAGATACGTA NO:26 1 8 TGAGATCAGGCACCGCATACTCCGGCCGAATCAGCCGC

TITGAAGCATGTATGTATGAAACCGATFM'GCTCGGGGGC

ACGTITACCTCGGTGGATATI'ACCGGGGCAAGGTGATC

AGCATCGCcTCATCAAGCCGAACATCCAGAG=T

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACTrGAAGGGTACCGCGAGCAAAAAGCGGGCAGTACG

C'ITATCCGCCATGCCGAAGAGC'TCTTCGGAAAAAAGG

CGCAGACCTELATGGTGCAACGCCAGGACATCTGTGA

GCGG~rACTATGAAAAGCTCGGC'1TCAGCGAACAGGGC

GAAGTCI)GCGACATACCGCCGATCGGACCTCATATIIG

ATGTATAAGAAAflGACATAA SEQ ID GAT'2_21F ATGATJ'GAAGTCAAACCAATAAACGCGGAAGATACGTA NO:262 1 TGAGATCAGGCACCGCATJ'CTCCGGCCGAATCAGCCGC TTGAAGCATGTATGTATGAAACCGA M GCTCGGGGGC

ACG=ICACCTCGGTGGATATTACCGGGGCAAGCTGATC

_____________AGCATCGCTTCCTMCATCAAGCCGAACA'17CAGAGCTT 176 WO 02/36782 WO 0236782PCT[USO0i/46227

CA

GAAGGCCAAAAACAGTATCAGCTGAGAGGGATGGCGA

CACITGAAGGATACCGTGAGCAAAAAGCGGGCAGTAG

CTTATCCGCCATGCCGAAGAGCTTCTTCGGAAAAAGGG

GGCAGACCI=rATGGTGCAACGCCAGGACATCTGTGA GCGGGTACTATAAAAAGGTCGGCTrCAGCGAACAAGGC GGGGTCTACGATATACCGCCGATCGGACCTCATATI=r

ATGTATAAGAAATTGACGTAA

SEQ ID 13_10F6 MIEVKPINAED I Y E ~RflRPNQPLE-ACKYFrDLLRGTFH NO:263 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEGY REQKAGSThIRHAEEIRKKGADLLWCNARTSASGYYKK

_____________LGFSEQGEVYDIPPVGPHILMYKICLT

SEQ ID 13_12G6 MIEVKPINAEDTIYETRHRlRPNQPIRACKYETDLLRGAFH NO: 264 LGGYYRGKLVSIASFHQALEHPELEGQRQYQLRGMATLEG YREQKAGSThIHAELIRKGADLLWCNARTSASGYYK

KLGFSEQGEVYDIPPTGPHJLMYKKLT

SEQ DD 14..2A5 MIEVKPINAEDTYEIRHRLRPNQPLEACKYETDLLOSTFHL NO:265 GGYYRGKLISLASFNQAEBPELEGQKQYQLRGMATLEGYR

EQKAGSTLRHAEELLRKKGADLLWCNARTSASGYYKKL

(FSEQ EYTPPVGPEEIAIYKKLT SEQ ID 14_2C1 M[EVKCPINAED2FiEIRJRLRPNQPLE-ACKYE-IT)LRGAFH NO:266 LGGYYRGKLVSIASFHQAE]HPELEGQKQYQLRGMATLRG

YREQKAGSTLIAEELLRKKGADLLWCNARTSASGYYK

KLGFSE GEVYDTPPTGPHJLMYKKLT SEQ ID 14_2F1 1 MIEVXPINAEDTYEIRRRLRPNQPLE-ACKYETDLLRGAFH NO-267 LGGYYRGKLVSIASFHQAEHPELEGQKQYQLRGMATLEG YREQKAGSTh~iRAEALLRKKGADL-LWCNARTSASGYYK KLGFS GEVYDTPPAGPHILMYKKLT SEQ ID CHIMERA M]BVKPINAFDTYEIRRLRNQPLEACMYETDLLRGAFH NO:268 LGGYYRGKLISIASFHQAIHSELEGQKQYQLRGMATLEGY REQKAGSThIRHAELLRKKGADLLWCNARTSASGYYXK

LGFSEQGEVYDTPPVGPHLMYCKLT

SEQ ID 10_12D7 M[EVKPINAEDTYEIRHIRPNQPLEACKYETDLLGGTLHI NO:269 LGGYYRGKLISIASFHQAEHPELE-GQKQYQLRGMATLEEY REQKAGSThIRHAEELIRKKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPTGPHILMYKKLT

SEQ ID 10_15F4 MIEVK<PINAEDTYEEIRRILRPNQPLEACMfYETDLLRGTFH NO:270 LGGYYRGKLVSIASFHQAEBPELEGQKQYQLRGMA=hE YREQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYK

KLFSEQGGVYDIPPVGPHMIMYKIKLT

SEQ ID 10_171)1 MIEVKPINAEDTYEIR1{R1LRNQPLE-ACKYEMDLLGGTFH NO:27 1 LGGYYRGKLISIASFHQAFRPELRGQKQYQLRGMATLEFGY REQKAGSTURHAEE1WRKKGADLLWCNARTSASGYYKK _____LGFSEQGEVYDTPPVGPHIAvYKKLT SEQ ID 10_17F6 MMIEPINAEDTIYEERRIRPNQPE-A CKYETDLLGG-TFH NO:272 LGGYYRGIKLVSIASFHQAEHSELEGQKQYQLRGMATLEE

YREQKAGSTLHIIIAEELLRKKGADLLWCNARTSASGYYK

GEVYDIPPVGPHELMYKKLT

SEQ ID 10_18G9 M]EVKPINAEDTYEIRHRILRPNQPLEACKYETDLLGGTFH NO:273 LGGYYRGKLVSIASFHQAEHSELEGQKQYQLRGMAThEE

_____YREQKAGSTLIRRAEELLRKKGADLLWCNARTSASGYYK

177 WO 02/36782 WO 0236782PCTIUS01/46227

CA

_______KLGFSEQGGVYDIPPVGPBULMYKKLT

SEQ ID 10_111I3 NMVKPINAEDTYEIRHRILRPNQPLEACKYETDLLGGTFH NO:274 LGGYYRGKLVSIASFHQAEIIPELEGRKQYQLRGMATLEG YREQKAGSMhTRIAEELRKKGADL.LWCNARTSASGYYK

_____________KLGFSEQGEVYDIPPTGPHULMYKIKLT

SEQ BD 10_20D10 MiEVKPINAEDT'YEIRRIRPNQPLE-AGMYETDLLGGTUI NO:275 LGGYYRGKUSIASFHQAEHPELEGQKQYQLRGM.ATLEEY REQKAGSTLTR1AEELLRKKGADLLWCNARTSASGYYKK LGTFSEQGGVYDEPPVGPHLMvYKKLT SEQ ID 10_23F2 MIEVIKPINAEDT-YEIRHILRPNQPL.E-ACMYEITLLOTGTFH NO:276 LO3GYYRGKL VSIASFHQAEBPELBGQKQYQILRGMAThEG YREQKAGSTLtRHAEELLRKXGADLLWCNARTSASGYYX KLGFSE QGEVYDTPPVGPILMY-KXLT SEQ ID 10_2B8 MEEVKPINAEDTYEIRHRILRPNQPLEACKYETIDLLGGTF-H NO:277 LGGYYRGKUSIASFHQAEHPELEGQKQYQLRGMATLEY

REQKAGSTLIRHAEELLRXKGADLLWCNARTSASGYYKK

LGFSEQGEVYDTPVGPHILMYKKLT

SEQ ID) 10_2C7 MIEVKPINAEDTIYE1UMUPJRPNQPLEACKYETDLLRGAFH NO:278 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMATLEGY

RBQKAGSTLIRHAEELRKXGADILLWCNARTSASGYYKK

LGFSEQGEVYDTPPVGPHILMYKKLT

SEQ ID 10_3G5 MIEVKPINAEDTYEIRHRRPNQPLEACKYETDLJX3GTFH NO:279 LGGYYRGKLVSIASFHQAE]HPELBGQKQYQILRGMATL-EG YREQKAGSThIAEELRKKGADLLWCNARTSASGYYK KLGFSEQGEVYDIPPTGPHMhMYKXLT SEQ ID 10_4117 MIEVKPINARDTIYEIRHRTLRNQPLE.ACMYE=LLGGTTFH NO:280 LGGYYRGKLVSIASFHQAEHPELEBGQKQYQLRGMAThEG

YRBQKAGSTLJRAEELLRKXGADL-LWCNARTSASGYYK

LGFSEQGEVYDIPPTGPI[LMYKKLT

SEQ ID 10_61)11 MIEVKPINAFDTYEIRRILPNQPLEACKYETDILGGTLH NO:28 1 LGGYYRGKLVSIASFHQABPEBGQKQYQILRGMAThEG

YREQKAGSTLIRIAEELLRKKGADLLWCNARTSASGYYK

________KLGFSEQGEVYDIPPVGPHILMY.KKLT

SEQ ID 10_8C6 IEVKPNAEDTYEMHRILRNQPL-ACKYETDLLGGAFH NO:282 LGGYYRGKLISIASFHQAIEBPELEGQKQYQLRGMATLEGY

REQKAGSTLIIHAEELRKKGADLLWCNARTSASGYYKK

______LGFSEQGGVYDIE>PVGPHUALYKKLT

SEQ ID 110C MIE VKPINAEDY.ERPJRPNQPLEACKYETDLLGGTFH NO:283 LGGYYQGKLISIASFHQAE11SEUEOQKQYQLRGMATLEGY REQKAGSTi]RHAEELLRKKGADLLWCNARTSVSGYYKK LGFSEQGGVYDITPPIGPHIaMYEKLT SEQ ID 11 G3 MIEVKPINAEDTYEMkHPJRPNQPLE-ACMYETDLLGGTFH NO: 284 LGGYYQGKLISIASFHQAEI{SELEGQKQYQLRGMAThEGiY

REQKAGSTLIHAELRKKGADLLWCNARTSASGYYEK

LGFSEQGGVYDIPPIGPBILMYKKLA

SEQ ID 11113 MIEVK<PINAEDTYERIRRIRPNQPLE-ACMYETDLL(JGAFH NO:285 LGGYYQGKLISIASFHKAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLAEELRKKGADLLWCNARTSVRGYYEK

LGFSEQGGVYDIPPIGPH~MYKk3LT SEQ ID 112 1F9 M]IEVKPINAEDTYEaRHRLRPNQPLEACKYETT)LLGTFH 178 WO 02/36782 WO 0236782PCTIUS01/46227 NO:Z86 LGGYYRGKLISIASFLIQAEHPELEGQKQYQLRGMATLEGY REQKAGSThIIHAEELLRKIKGADLLWCNARTSASGYYKK

LGFSEQGEVHDIPPTGPBE-MYKKLT

SEQ ID 12_2G9 MIEVIKPINAEDTYETRHRJLRPNQPLEACKYETIDLLGGITH NO:287 LGGYYRGKLVSIASFHQAEHPELEGQKQYQLRGMATLEG YREQKAGSThIRALLRKMKGAkDLLWCNARTSASGYYK

IKLGFSEQGEVYDTPPVGPHILMYKIKLT

SEQ ID 12_3F1 MIEVKPIINAEDTYFJIRRhRNQPL-ACKYETDLLGGTFH NO:288 LGGYYRGKLISIASFRiQAEHPELEGQKQYQLRGMATIEGY

REQKAGSTLIRIAEEUIKGADLLWCNARTSASGYYKK

______LGFSEQGGVYDIPPVGPH11MYKKLT SEQ ID 12_5C10 M]EVIKPLNAEDTYE]RHRIRPNQPLEFACKYETTDLLGTFH NO:289 LGGYYRGKLJIASFHQAEHPELEOYQKQYQLRGMAThEEY

RE-QKAGSTLRHAEELLRKGADILWCNARTSASGYYKK

LGFSEQ EYAPPTGPB[LMYKKILT SEQ ID 12_6A10 MIEVKPIhNAEDTIYEURLRPNQPLEACKY=TLLGGTFH NO:290 LGGYYRGKLVSIASFHQAEBPELEGQKQYQLRGMATLEG YREQKAGSThIRHAEELRKGADLLWCNARTSASGYYK ________KLGFSEQGGVYDIPPVGPHaLMYKKT SEQ ID 12_6D1 MIEVKPINAEDTYEJRHIRPNQPLEACMYETDLLGGTFH NO :291 LGGYYRGKLISIASPHQAEHPELE-GQKQYQLRGMAThEEY

REQKAGSTLEIAEURKKGADIIWCNARTSASGYYKK

_____LGFSEQGGVYDIPPVGPIDIMYKKLT

SEQ ID 12_6F9 MIEVKP]NAEDTYEIRBRLRPNQPLEACKYETDLLGGTFH NO:292 LGGYYRGKLISIASFHQAEMPELEGQKQYQLRGMAThEGY

REQKAGSTLIRHAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQ iVYDIPPTGPBELMYKT SEQ ID 12_6116 MEEVKPINAEDTYEIRIRNQPLEACKYETDLLGGTFH NO :293 LGGYYRGKLVSIASFHQAEHPELEGQKQYQLRGMATLRG YREQKAGSThRHAI~EALLRKKGADLLWCNARTSASGYYK KLGiFSEQGEVYDU>PTGPHILMYEIMT SEQ ID 12_7D6 MIEVKCPENAEDTYEIRPRPNQPLE-ACKYETDLLOGTFH NO:294 LGGYYRGKUSIASFHQAEHPELEGQKQYQLRGMATLEGY REQKAGSThiRHAEELLRKKGADLLWCNARTSASGYYKK ________LGFSEQC3GVYDIPPTGPHILMYKKLT SEQ ID 12_7GH1 MIEVKPTNAEDTYEIRRLRPNQPLEACKYETDLLGGTFH NO:295 LGGYYRGKUISIASFHQAEHSELEGQKQYQLRGMATLRGY

RBQKAGSTLTREAEELLRKKGADLLWCNARTSASGYYKX

________LGFSEQGEVYDTPPVGPHMhMYKXLT SEQ ID 1 2F5 MIEVKPINAEDTYERHR1LRPNQPLEACMYETDLLGGTFH NO:296 LGGYYQGiKLISIASFHXAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYKK

_____________LGFSEQGGIYDIPPIGPHILMYKIKLT

SEQ ID 12G7 MEVKPINAEDTYEIT{PRPNQPLEACKYETDLLGGTFH NO:297 LGGYYQGKLJSIASFHKAEHELEGQKQYQLRGMATLEGY RBQKAGSTLTRIIlRKKGADLLWCNARTSVSGYYKK

QEYIPPIGPBILMYKKCLT

SEQ ID 1_2H6 MIEVKPIAEDTYEIRERILRPNQPLEACMYEIDILGGAFH NO:298 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY n RQA GSCTh1HEEL GADL-LWCNARTSASGYYK 179 WO 02136782 WO 0236782PCTIUSOI/46227 LGFSEQGGVYDIPPIGPH]1I1YKKT SEQ ID 13_12G12 MIEVIKPNAEDT1YEIRRIRPNQPLEACMYEThLLGGTFH NO:299 LGGYYRGKUSIASFNQAIEHPELEGQKQYQL-RGMAThEEY REQKAGSTL1RHAEEU-.RKKGADLLWCNARTSASGYYKK

_____________LGFSEQGEVYDIPPVGPHJMHBKKLT

SEQ ID 13_6D10 MTEVKPNAEDTYEIRRRLRPNQPLE-ACMYETDSLGGTFH NO:300 LGGYYRGKLISIASFNQAEHI'ELEGQKQYQLRGMAThEFGY

REQKAGSTILAEELLRKKGADL.LWCNARTSASGYYKK

LGFSEQGEVYDWPVGPHIMYKKLT

SEQ ID 13_7A7 MTEVKPINAEDTIYEIRHRLRPNQPLEACMYETD1LLRSAFH 1 LGGYYRGKUISIASFHQAEEIPELEGQKQYQLRGMAThEEY REQKAGST~iRAELLRKKGADLLWCNARTSASGYYKK

_____LGFSEQGEVYDTPPVGPEHMMYKKLT

SEQ ID 13_7B 12 MIEVKPINAEDTYEIRRR]LRPNQPLEACKYETDLLGSTFHL NO: 302 GGYYRGKUJSIASFHQAEBPELEGQKQYQLRGMATLEGYR

EQKAGSTLJRHAEELLRKKGADLLWCNARTSASGYYKKL

(iFSEQ GEIPPTGPH[LMYKKLT SEQ ID 13_70 IEVKPNAEDiY IRHRRPNQPLEACKYETDLLRGAFH NO: 303 LGGYYRGKLISIASFHQALEBPELEGQKQYQLRGMAThEGY

REQKAGSTLTRHAEELLRKKGADLLWCNARTSARGYYKK

_______LGFSEQGEVYDIPPTGPEILmyiciaT SEQ ID 13_8(36 MIEVKPINAEDTYEIRHRILRNQPLEACKYETDSLGGTFH NO:304 LGGYYRGKLISIASFNQAEHPELEGQKQYQLRGMATLEGY

REQKAGSTILHAELLRKKGADLLWCNARTSASGYYKK

LGFSEQGGVYDIPPVGPBIIMYKKLT

SEQ ID 13_9F6 MIEVKPINAEDTYE]RRILRPNQPLE-ACKYETDLLGGTFH NO:305 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMATLEEY

REQKAGSTLTRHAELRKKGADLLWCNARTSASGYYKK

________LGFSEQGEVYDIPPVGPH]LMYICKLT

SEQ ID 14_1009 MIEVKPINAEDTYEIRHRILRPNQPLEACKYEITDLLRGAFH NO:306 LCK3YYRGKLISIASFHQAEHPELFGQKQYQILRGMAThEEY SEQ ID 14_10H3 M[EVKPINAEDTYEHIRLRPNQPLEACKYETDLILRGAFH NO:307 LGGYYRGKLVSIASPIIQAERHPELEGQKQYQLRGMAThEE YREfQKAGSn' IR A1I P CTGADI.LWCNARTSASGYYK

_____KLGFS]EQGEVYDTPPVGPHILMYKCLT

SEQ ID 14_10H9 MIEVIKPINAEDTYEIRHRIRNQPLEACKYETDLLRGAFH NO:308 LGGYYRGKLVSIASFHIQAEHPELEGQKQYQL-RGMATLEG

YREQKAGSTLIRHAEELLR.KKGADIILWCNARTSASGYYK

_____KLGFSEQQEVYDTPvGPHILMYKKLT SEQ ID 14_11C2 MIEVKPINAEDTYEmERILRPNQPLEACKYETDLLGSTFHL NO:309 c3GYYRGKLVSIASFHQAEBRPELEGQKQYQLRGMATLEEY

REQKAGSTL]RHEALLRKXGADLLWCNARTSASGYYKK

LGFS QEYTPPTGPBILAIYKKLT SEQ ID 14_12D8 MIEVKPINAEDTYERI{JLRNQPLEACKYETDLLGGTFH NO:3 10 LGGYYRGKLVSIASFHQAB]HPELEGQKQYQLRGMATLEG

YREQKAGSTLIRI{AEALLRKKGADLLWCNARTSASGYYK

_____________KLGFRRQGGVYDIPPVGPBI[MYKI<LT

SE I 14 12116 IMIEVKPINAEDTYEURU~LRPNQRLEACKYTL~GF -180- WO 02/36782 WO 0236782PCTJUSOI/46227 NO:3 11 LGGYYRGKUJSIASFHQAEBPELEGQKQYQLRGMATLEEY

REQKAGSTLTREAEELLRKXGADLLWCNARTSASGYYK

L)GFSEQGEVYDIPPTGPHILMYKK-LT

SEQ ID) 14_2B6 MIEVKPINAEDTIY.ERHRIRPNQPLEACKYEIT)LLGGTFH NO:3 12 LGGYYRGKICJSIASFNQAIEHPaLEOQKQYQLRGMAThEGY

REQKAGSTLLRHAEELLRKKGADLLWCNARTSASGYYKK

________LGFSEQGGVYDIPPVGPHIaMYKYLT SEQ ID 14_2(31 MIEVKPINAEDTYEIRHRTLRPNQPLEACKYETDLLRGAFH NO:3 13 LGGYYRGKLVSIASPHQABPELEGQKQYQLRGMATLEG

YREQKAGSTURHAELLRKKGADLLWCNARTSASGYYK

______KLGFSEQGEVYDIPPTGPHILMYKKLT

SEQ ID 14_3B2 MIEVKPINAED IYE1IaHRIRPNQPLEACKYEPDLL-RGAFH NO:3 14 LGGYYRGKLVSIASFHQAE]HPELEGQKQYQLRGMATLEG YRBQKAGSThZiRAEALLRKKGADLLWCNARTSASGYYK

________KLGFSEQGGVYDIPPAGPH]LMYKKLT

SEQ ID 14_4H8 M[EVIKPINAEDTYELRHIRPNQPLEACKYE'IILLGSTFHL NO:3 15 GGYYRGKLISIASFHQAEBPELEGQKQYQLRGMATL-EGYR EQKAGSThIRI{AEELLRKKGADLLWCNARTSASGYYKKL

GFSEQGEVYDTPVGPHILMYKKLT

SEQ ID 14_6A8 MIEVKPINAEDTYEHRIL-RPNQPL.EACMYEfl)LLG3TFH NO:3 16 LGGYYRGKLVSIASFNQAEBPELEGQKQYQLRGMATLEG YREQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYK

LGFSEQGEVYDTPPVGPIIVLMYI(T

SEQ ID 14_6B 10 MEEVKPINAEDTYEIRE{ILRPNQPLEACKYETDLLGGTFH NO:3 17 LGGYYRGKLISIASFHQAE]PELEQKQYQLRGMATLEGY

RRQKAGSTLIRBAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGGVYDMI'PVGPHIlMYfKXLT SEQ ID 14_6D4 MIEVKPTNAEDTYEIRHRLRPNQPLE.ACKYETDL-LGGTFH NO:3 18 LGGYYRGKLISIASFNQAEHP>ELEGQKQYQILRGMATLEGY

REQKAGSTLIRHAEALLRKKGADILLWCNARTSASGYYKK

LGFSEQGEV-YDTPPVGPH[LI%4YKT SEQ IID 14_7A1 1 MIEVKPINAEDTYEIRHRILRPNQPLE-ACKYErIDLRGAFH NO:3 19 LG*3YYRGKLVSIASFHQAEBPELEGLKQYQLRGMATLEG YREQKAGSThJRALLRKGADLLWCNARTSASGYYK

_____________KLOFSEQGEVYDTPPTGPH[LMYKKLT

SEQ ID 14_7A1 M]EVIKPINAEDTYEIRHRILRPNQPLEACMYETDLLRGTFH NO:320 LGGYYRGKLVSIASFHQABPELEGQKQYQLRGMAThEE

YREQKAGSTLTRHAEELLRKKGADLLWCNARTSASGYYK

ICLGFSEQGEVYDTPAGpHI1MYKKLT SEQ ID 14_7A9 M]EVIKPINAEDTYEIRHRLRPNQPLEACKYETDILLGGTFH NO:321 LGGYYRGKLVSIASFHQAKHPELEGQKQYQLRGMATLEG

YRBQKAGSTLIRHAEELLRXKGADLLWCNARTSASGYYK

KLGFSEQGEVYDTPPVGPEJMYvrKKLT SEQ ID 14_7G1 MIEVKPNADTYERILRPNQPLEACKYE'DLRGAFII NO :322 LGGYYRGKLUSIASFNQAFIIFELEGQKQYQILRGMATLEEY REQKAGSThIRBkALALRKXGADLLWCNARTSASGYYKK LGFSEQGEVYDTPPVGPFEiLMYKICLT SEQ ID 14_7H9 M]EVIKPINAEDTYEaRRIRPNQPLEACKYETDLLC3GTFH NO:323 LGGYYRGKLVSIASFHQAEHPELEGQKQYQLRGMATLEG YRQAGfSTLRHiAEELLRKKG.ADLLWCNARTSASGYYK 181 WO 02136782 WO 0236782PCT/USOI/46227

;Z

______KLGFSEQGEVYDIPPVGPH[ILMYKKLT

SEQ ID 14_8F7 MIEVKPINARDTIYEIMR=JRNQPLE-ACKYETDILLGGTFH2 NO:324 LGGYYRGKLVSIASFHQAEBPELEGQKQYQLRGMATLEE

YREQKAGSTLTRIABALLRKKGADLLWCNARTSASGYYX

________KLGFSEQGEVYDEPPTGPHJLMYXKLT

SEQ ID 15_10C2 M]EVIKPINAEDTYEIRHRILRPNQPLEACKYETDLL-RGAFH NO:325 LGGY-YRGKLVSIASFHQAE]HPELEGQKQYQLRGMATLEG YREQKAGSThIRHAEELLRKKGADLLWCNARTfASGYYK

_____________KLGPSEQGEVFDII'PTGPEILMYKXLT

SEQ ID 15_10D6 MEVKPINAEDTYEIRHR[LRPNQPLE-ACMYETDLLGGTFH NO:326 LGGYYRGKLVSIASFHQAEBPELEGQKQYQL.RGMAThEE

YREQKAGSMMHIAEELRXGADUZWCNARTSASGYYK

_____________KLGFSEQGEVYDIPPVGPHILMYKKLT

SEQ IOD 15_1019 M]EVKPINAEDTYEIRHRILRPNQPLEACKYETDLLRGAFH NO:327 LGGYYRGKLVSIASFNQAEHPELEGQKQYQL.RGMATLEG

YREQKAGSTLRILAEEILRRKGADLLWCNARTSASGYYK

KLGFSEQGEVYDIE>PTGPHaLMYKKLT SEQ ID 15_11H3 MIEVKPINAEDTYEIRHRIRPNQPL-EACKYETDLLRGAFH NO:328 LGGYYRGKLISIASFHQAEEPELEGQKQYQLRGMAThEGY REQKAGSThTRHAEALLRKKGADLLWCNARTSASGYYKK

_________LGFSEQGEVYDIPPTGPHILMYKKLT

SEQ ID 15_12A8 MIEVKPINAEDTYEIRHRILRPNQPLE-ACKYETDLLGGTFH NO:329 LGGYYRGKLISIASFHQAEBPELEGQKQYQLRGMAThEFGY

REQKAGSTLIRHAEALLRKXGADLLWCNARTSASGYYXX

________LGFSEQGEVYDIPPTGPHaIMYKKL-T SEQ ID 15_12D6 IVIEVKPINAEDTYERIRIRPNQPLE-ACMYEMhLRGAFH NO:330 LGGYYRGKLVSIASFHQAEHPELEGQKQYQLRGMAThEG YREQKAGSTh]RHAEELLRKKGADLLWCNARTSASGYYK _____________KLGFSEQGiEVYDTPPVGPHILMYKKLT SEQ ID 15_12D)8 M]EVKPINAEDTYEIRERLRPNQPLEACKYFJDLLGGTFH NO:33 1 L)GGYYRGKLVSIASFHQAEBPELEGQKQYQLRGMATLEG

YREQKAGSTLIRBAEELLRKKGADLLWCNARTSASGYYX

KLGFSEQGKVYDIPPVGPHLhMYKCKLT SEQ ID 15_12D9 MIEVKPINAEDTYEIR]RPNQPLEFACKYETDLILRQTFH NQ:332 LGGYYRGKLVSIASFHQAEBPELEGQKQYQLRGMATLEE

YREQKAGSTLIRIAEELLRKKGADLLWCNARTSASGYYK

KLGFSEQGEVYDEPPVGPHTLMYTKKLT

SEQ ID 15_3F10 M[EVKPINAEDTYEJRURIRPNQPLEACKYETDLLRGAFJI NQ:333 LGGYYRGKLISIVSFHQAEHPELEGQKQYQLRGMATLEGY REQKAGSnhIRAEELRKGADLLWCNARTSASGYYKK

LGFSEQGEVYDTPPAGPBE-MYTKLT

SEQ IID 15_3G1 1 MIEVKPINAEDTYELZRILRPNQPLEACKYETDLLGGTFH NO:334 LGGYYRGKLVSIASFHQAEBPELEGQKQYQL.RGMATLEE YREQKAGSThIRHAEEIRKKGADLLWCNARTSASGYYK

________KLGFSEQGEVYDIPPVGPHELMYKKLT

SEQ ID) 15_4F1 1 M]EVKPINAEDTYIRHRLRPNQPLEACMYE'IDLLGGTFH NO;335 LCGGYYRGKLVSIASFNQAEHPELEGQKQYQLRGMATLEG

YREQKAGSTLIRHEALLRKKGADLLWGNARTSASGYYK

I ,KLGFSEQGEVYDIPPTGPHILMYKKLT ISQp 15r 341H3 MIEVKPINAEDTYEaRHRLRPNQP LEACKYETDILGTFH -182- WO 02136782 WO 0236782PCTIUSO1/46227 NO:336 LGGYYRGKLVSIASFHQAEHEPELEGQKQYQILRGMAThEE YREQKAGSTaHI{AF~aLRKKGADLLWCNARTSASGYYK

________KLGFSEQGEVYDIPPTGPHJLMYKIKLT

SEQ ID 15_61)3 MIEVIKPINAEDTYE]RHRIRPNQP1E-ACKYEIhLLGGTFH NO:337 LGGYYRGKLISIASFHQAEHPELEQKQYQLRGMATLEBY REQKAGSThIRIAEELLRKKGADLLWCNARTSASGYYKK

________LGFSEQCYTVYDIPPTGPHIIMYKKLT

SEQ ID 15_6G11 MIEVKPINAEDTYELRHPJRPNQPLE'ACKYETDLLRGAFH NO:33 8 LGGYYRGKLVSIASFHQAEHELEGQKQYQL-RGMATLEE

YRBQKAGSTLRHARELLRKKGADLLWCNARTSASGYYK

KLGFSEQGKVYDIPPVGPBILATYKKLT

SEQ ID 15_9F6 M]EVIKPIhTAEDTYEPJIRILRPNQPLEACKYETT)LLGGTFH NO:339 LGGYYRGKLISIASFHQAE[IPELRGQKQYQLRGMATLEEY

REQKAGSTLIAEELRRKGADLLWCNARTSASGYYKK

LOFSEQGEVYDIPPVGPHULMYKKLT

SEQ ID 15E5 MIEVKPINAEDTYEIRE1LRNQPILEACKYE-IDLLGGTFH NO:340 LGGYYRGKLIASFLIKAEHSELEGEEQYQLRGMATLEGY

REQKAGSTLIRYAEELLRKKGADLLWCNARTSVSGYYKK

LGFSEQGEVYDIPPIGPH]LMYKKLT

SEQ ID 1 6A1 MIEVKPINAEDTYEHRIULRPNQPLEAGMYETDLLGGTLJI NO:34 1 LGGYYQGKLISIASFHKAEHSGLEGEEQYQLRGMATLEGY REQKAGSTL1RHAEELLRKKGADLLWCNARTSVSGYYEK

LGFSEQGEVYD]PPIGPHLMYKCLT

SEQ ID 16H13 MID VKPINAEDTYE]RHRLRPNQPLE-ACKYETDLLGGTFH NO:342 LGGYYQGKLISIASFIIQAEHSELEGQKQYQLROMATLEGY REQKAGSTLTRJALkLRKKGADLLWCNARTSVSGYYEK

______LGFSEQGEVYDIPPIGPEILNIYKKLT

SEQ ID 17C12 MIEVKPISAEDTYEIRIIIURPNQPLE-ACMYETDLLGGAFH NO:343 LGGYYQGKLISIASF'HQAEHSELEGQKQYQILRGMAThEGY RRQKAGSThIRHAEELLRKKGADLLWCNARTSVSGYYEK

LGFSEQGEVYDEPPIGPHILMYKKLT

SEQ ID 1 8D6 MIEVKPINAED)TYE1RILRPNQPL-RACKYETDLLGGTFH NO:344 LGQYYRGKLISIASFBXAEHSELEGQKQYQLRGMATLEGY REQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYEK ________LGFSEQGEVYDIPPIGPHLMvYKKLA SEQ ID 19C6 MIEVKPINAEDTYEaRURPNQPLE-ACK-YETLLGGTFH NO:345 LGGYYRGKLICIASPHQAEHSELEGQKQYQLRGM-ATLEGY REQKAGSThJRHAEELRKKGADLLWCNARTSVRGYYEK

_____________LGFSEQGGVYDIPPIGPH]LMYKKLA

SEQ ID 19D5 MI]EVKP]NAEDTYEIRRCILRPNQPLE'FACMYETDLLGGTFH NO :346 LGGYYQGKLISIASFHKAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLIRHAEELLKKGADL.LWCNARTSVSGYYKK

LGFSEQGEVYDIPPIGPHILMYKKLT

SEQ IID 20A12 NMIEVIKPINAEDTYEIRRLRPNQPLEACMYETDLLGGTFH NO:347 LGGYYQGKLISIASFBNAEHSELEGQKQYQLRGMAThEGY

REQKAGSTLIRRAEELLRKKGVDLLWCNARTSVSGYYKK

______LGPSEQGG1YDIPPIGPHI1MYKLA SEQ ID 20F2 M]EVIKPINAEDTYERIRRhRNQPLR-ACMYEfl)LLGGTFH NO:348 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY _____REQKAGSi .TRAPEPTI RKKGADLLWCNARTSVSGYYEK 183 WO 02136782 WO 0236782PC1ZJUSOI/46227

;Z

LGFSEQOEVYDIPPIGPH]LMYKk-LT SEQ ID 2.10OE+12 MVIEVKPJNAEDTYEIR{LRPNQPLE-ACKYETDLLGGAFH NO:349 LGGYYQGKLISIASFHQAEI{SELEOQXQYQLRGMAThEGY

REQKAGSTURIAEELLRKKGADLLWCNARTSVSGYYKK

LGFSEQC'EVYDIPPIGPHILMYKIMT

SEQ ID 23H1 1 miEvKPINAEDTYEIRHPJLRPNQPLE-ACMYFTDLLGGTFII NO:350 LGGYYQGKLISIASFHKAERSELEGQKQYQLRGMAThEGY REQKAGSMMHIRAEaLRKKGAI)LLWCNARTSASGYYEK

________LGFSEQGEVYDIPPIGPH]LMYKKLA

SEQ ID 24C1 MEVKNAEDTYEIIPJLRPNQPLACKYETDILGGTFHI NO:351 L)GGYYRDRLISIASFHQAEEISELR-GQKQYQLRGMAThEGY

REQKAGSTURHAEULRKKGADLLWCNARTSVSGYYKK

LGFSEQGEVYDIPPIGPH]UvfYKIKLT SEQ ID 24C6 MIEVKPIAEDTYEERIRILRPNQPLEACMfYETDLLGGTFH NQ:352 LGGyyRGKLISIASFHQAEHSEGQKQYQILRGMAThEGY

REQKAGSTLTRHAELLRKKGADLLWCNARISVSGYYKIKL

_____________GFSEQGGVYDEPPIGPHILMYKKLA

SEQ ID 2.40E+08 MIEVKNAEDTYEHRLRPNQPLACKYTDLLGGTFH NQ:3 53 LGGyyRGKLISIASFHNAEHSELEGQKQYQLRGMAThE-GY REQKAGSUhIRAEELRKGADLLWCNARTSASGYYEK _______LGFSEQGEVYDIPPIGPBIlvffKKLA SEQ ID 2_8C3 MIEVKPINAFDTYELZRRLRNQPLEACMYETDIIGGTFH NO:354 LGGyyRDRUISIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLIRHAEELLRKKGAI)LLWCNARTSASGYYEK

LGFSEQGEVYDIPPIGPHIMYKKLT

SEQ ID 2H3 MIEVKPINAEDTYEaMHRLRPNQPLE-FACK-YETDLGGTFH NO:355 LGGYYQGKLISTASFHQAGHSELEGQKQYQLRGMATLEG

YRERK.AGSTTRHA.EELLRKKGADLLWCNARISASGYYKK

LGFSEQGGVYDIPPIGPHTMYKLT

SEQ ID 30G8 MIEVKPINAEDTYE]REURI>NQPLEACMfEfDLLGGAFH NQ:356 LGGYYQGKLISIASFHQAE-HSELEGQKQYQLRGMATLEGY

REQKAGSTLIRHABEELRKKGADLJLWCNARTSVSGYYKK

LGFS QEYIPPIGPHILfYKIKLT SEQ ID 3B_10C4 MIEVRPINAEDTYEIRHRILRPNQPLE-ACMYKLDLLGGTFH NO:357 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLRGY REQKAGSTLaHAELLRKKGADLLWCNARTSASGYYKK

______LGFSEQGEAYDIPPIGPHU-MYKKLT

SEQ ID 3B_1007 MIEVKPIN~AEDTYEIRI{RLRPNQPLE-ACMYETDLLGGTFH S LGGyYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY REQKAGSThIRHAELLKKGADLLWCNARTSASGYYKK

_____LGFSEQGGVYDIPPIGPHMYKKLT

SEQ ID 31B_12Bl1 M]EVKPINARDTYEItRRIRPNQPLEACMYETDLLOQTFH NO:359 LGGYYRGKUISIASFHQAEHSELEGQKQYQLROMATLEGY

REQKAGSTLIRHAEELLRKKGADLLWCNARTSASGYYKK

______LGFSEQGEVYDI]PPIGPIUYXXLT

SEQ ID 3B_12D10 MIEVIKIINABDTYETRR]RPNQPLE-ACMYETDLLGGAFH NO:360 LGGYYRGKLISIASFf-FAEHSELEGQKQYQLRGMATLEGY REQKAGSThTRHAEELLRKKGADLLWCNARISASGYYEKL FSEQGEVYDIPPIGPEDIhMYKKLT SEQ IID 13B 2E5 IMIEVKPINAEDTYEIRR1RNQPLE-ACMYETDLJLGTFH -184- WO 02/36782 WO 0236782PCTIUSOI/46227 NQ:361 LGGYYRGKLJSIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLIRIIAEELLRKKGADLLWCNARTSASGYYEK

LGFSKQGEVYDIPPIGPHILMYKKLT

SEQ ED 3C_10113 MIEVKPIh1AEDTYEIRBR1LRPNQPLFACMYETDULLGGTFH NO:362 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMAThBGY

REQKAGSTLIRIAEELLRKKGADLLWCNARISASGYYKKL

______GFSEQGGVYDIPPVGPHJLMYKKLT

SEQ ED 3C_121110 MIEVKPINAEDTYEIRIRILRPNQPL.EACMYETDLLGGTFH NO:363 LGGYYRGKLISIIASFHQAEHSELEGQKQYQLRGMATLEGY RGQKAGSTLBiRAEELLRKKGADL-LWCNARTSASGYYEK

________LGFSEQGEVYDIPPIGPBILMYKKLT

SEQ ID 3C_9H8 MIEVKPINAEDTYETPJ{R1RPNQPLEACMYETDLLGGTFH NO:364 LGGYYQDRLISIASFHQAEHSELEGQKQYQLRGMATLEGY REQKAGSThTRYAEELLRKKGADLLWCNARISASGYYEKL GFSEQ EYIPPIGPI1M~fYYLT SEQ ED 4A-lB 11 MIEVIKPINAEDTYERIRPLRPNQPLEACMYETDLLGGTFH NQ:365 LGGYYRGISIASFHQAEHPELEGQKQYQLRGMATLEGY

REQKAGSTIRUHAEELLKKKGADL.LWCNARTSASGYYEK

LGFSEQGVYIPPIGPHULMYKKLT

SEQ ID 4A_1C2 NMVPINAEDTYERHRILRPNQPLEACKYFIDLLGGThH NO:366 LGGYYRGKLISIASFIIQAEHSELEGQKQYQLRGMATLEEY

RBQKAGSTLEIRAEELLRXKGADLLWCNARTSASGYYKK

LGFSEQ EYIPPIGPBILMYKICLT SEQ ID 4B_13E1 MIEVNAETYEIEJRPNQPEACKYETDLLOGTFH NO:367 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEEYI REQKAGSTLIRHAkEELLRKKGADLLWCNAPJSASGYYEKL ______GFSEQGE IPPIGPIBJ1MYKKLT SEQ ID 4B_13G10 MIEVKPINAEDTYETRHR]LRPNQPLEACMYETLLGGTFH NO:368 LGGYYRGKLISIASFHQAERSELEGQKQYQLRGMATLEGY

REQKAGSTIMHAEELLRKKGADLLWCNARTSASGYYKK

_____LGFSEQGGVYDIPPIGPYBLMYKKLT

SEQ ID 4B_16E1 MIEVK<PINAEDTYEIRHRILRPNQPLEACKYETDLLGGTFH NO:369 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTULRAEELLRKKGADL.LWCNARTSASGYYKK

_____________LGFSEQGGVYDIPPIGPHELMAYKILT

SEQ ID 4B_17A1 MEVKPNADTYERHR]RPNQPLEACKYETDLLGGTFH NO:370 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEEY REQKAGSMhRHEELRKGADLLWCNARTSASGYYEK

LGFSEQGEVYDIPPIGPHTLMYKKLT

SEQ U) 4B_1 8F1 1 MIEVNPINAEDTYFJRBPJLRPNQPLE-ACMYETDLLGGTSH NO:37 1 LGGYYRG.KLSIASFHNAEHSELDGQKQYQLRGMATLEGY

REQKAGSTLIAEELLRKKGADLLWCNARTSVSGYYEK

LGFSEQGEVYDIPPIGPHESMYKKLT

SEQ 4B_19C8 MIEVKPINAEDT1YEIRRLRNQPLEACKYETDLLGGTFH NO:372 LGGYYRGKLSIASFQAHIPEUEGQKQYQLRGMATLEGY

REQKAGSTLIRHAELTRGADLLWCNARTSASGYYKK

_____________LGFSEQGGVYDIPPIGPHILMYKKLAI

SEQ ID NO:373 4B_104 MIEVKINAEDTYETRUkTLRPNQPLE-ACKYETDLLGGAFH

LGGYYRGKLISIASFHQSEITPELEGQKQYQLRGMATLEGY

RELKAOsTLIRakEELLRKKGADLLWCNARISASGYYKICL 185 WO 02/36782 WO 0236782PCTIUSOI/46227

GFSEQGEVYDIPPIGPIHIMYKKLT

SEQ ID 4B_21 C6 MIEVKPII4AED-1-YEIR-1l PPNQPLEACMYETh)ILGGTFH NO:374 LGGYYRGKLJSIASFHQAEHSELRGQKQYQL-RGMAThEEY

REQKAGSTURHAELLRKKGADLLWCNARISASGYYKXL

______GFSEQGGVYDIPPIGPHI[MYKYLT

SEQ ID 4B_2M1 M]EVKPINAEDTYEJEHIRPNQPLEACMYETDLLGGTFH NO:375 LGGYYRGKLISIASFHQAEHSELBGQKQYQL-RGMAThEGY REQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYKK

________LGFSEQGGVYG]PPIGPHILMYKKLT

SEQ ID 4B_2H8 IVIIAKPINAEDTIYEIERRILRPNQPLE-ACKYEIDLLGGTFH NO:376 LGGYfYRGKL.ISIASFHQAEHSEGQKQYQLRGMATLBGY

REQKAGSTLHJRARELRXKGADLLWCNARTSASGYYK

_____________LGFSEQGEVYDIPPIGPI]Tmyxx-LT SEQ ID 4B_6D8 MIEVKPINAFDTYERHRILRPNQPLEACKYE1IDLLGGTFH NO:377 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLIRHAEELLRKGADL-LWCNARTSASGYYKK

______LGFSEHGEVYDIPPIGPHTLMfYKKLT SEQ DD 4B_7E8 MIEVKPINAEDTYERHRLRNQPLE-ACMYETDLLGGTFH NO:378 LGGYYRGKLISIASFHIQAEHSELE-GQKQ YQLRGMATLEGY

REQKAGSTURHAEELLRKXGADLLWCNARTSVSGYYKK

LGFSEQGEVYDIPPIGPBILMYKXIT

SEQ ID 4C_8C9 MIEVIKPINAEDTYEIRRRLRPNQPLEACMYETDLLRGAFH NO:379 LCTGYYRGKLISIASFHQAFILPELEGQKQYQLRGMATLEGY

REQKAGSTLIRHAEELRKKGADLLWCNARTSASGYYEK

EYIPPIGPI19-AlYKKLT SEQ ID 4111 MIEVKPINAEDTIYBIERULRPNQPLE.ACMYETDILGGAFH NO:3 80 LGGYYQGKLISIASFHQAVHSELEGQKQYQLRGMATLG

YREQKAGSTUPJ{AEELLRKKGADLLWCNARTSVSGYYK

KLGFSEQGGVYDIP>PIGPHJLMYKKLT

SEQ 1ID 6_14D10 UMVKPINAEDTYVE]RHRIL-RPNQPLEACMYErDLLGGTFH NO:3 81 LGGYYRGKLISIASFHQAEHSELEGHXQYQLRGMATLEEY

REQKAGSTL]RHAEELLRKKGADLLWCNARTSASGYYKK

______LGFSEQGGVYDIPPVGPHILMYKKLT

SEQ IID 6_15(37 MIEVKPINAEDTYEJRHRILRPNQPLEACKYETDLLGGTFH NO:3 82 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLTPJAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPVGPHILMYKKLT

SEQ BD 6_16A5 M]EVKPINAEDTYEIRHRLRPNQPLE-ACKYETDLLGGTF-H NO:3 83 LGGYYRGKUISIASFHQAEHSELEGQKQYQL-RGMATLEGY

REQKAGSTURHAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGGVYDIPPvopHiLM2YKILT SEQ ID 6_16F5 MIEVKPINAEDTYERHRR-RPNQPLACMYETDLLGGTFH NO:384 LGGYYRGKLISIASFHQAVHSELEGQKQYQLRGMATLEGY

REQKAGSTLJRHAMELRKKGADLLWCNARTSASGYYKK

________LGFSEQGGVYDIPPVGPHILMYKKLT

SEQ ID 6_17C5 MIEVKPINAEDTYEIRRPNQPLEACKYEADILGGTFH NO:385 LCTGYYRGKLISIASFHQAEBiPELEOQKQYQLRGMATLEGN REQKAGSTaHIRAEELLRKKGADLL-WCNARTSASGYYKK _____________LGFSEQGEVYDVPPIGPHMMvYKKLT SEQ ID 61 8C7 MIEVKPIAEDTEIHRIRPNQPLE-ACRYETTDLLGGTFH -186- WO 02/36782 WO 0236782PCTIUS01/46227

CA

NO:386 LGGYYRGKLISIASFHQAEHPELE-GQKQYQLRGMAT-RGY REQKAGSThUIHAEELLRKKGADILWCNARISASGYYKKL GFSE GEVYDLP>PVGPHILMYKIKLT SEQ ID 6_18D7 MIEVKPINAEDTIYEMX 3R TIiR NQPLEACMYETDUX3GTFH NO:387 LGGYYRGKLISLASFHQAEHPELEGQKQYQLRGMATLEGY REQKAGSTLIHiAEELLRKKGADLLWCNARTSASGYYKK

______LGFSEQGGVYDIPPVGPEILMYKKLT

SEQ ID 6_19A10 MIEAKPINAEDTIYE]RHRLRPNQPLEACMYETDLLGGTFH NO:3 88 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEGY REQKAGSTLIRHAE2EKGADLLWCNARTSASGYYKK LCTFSEQGEVYDIPPTGPHI1MYKIMT SEQ ID 6_19B6 M]BVKPIAEDTIY EaIRPJRPNQPLEACMYEIT)LLRGAFH NO:389 LGiGYYRGKLISLASFHQAEHSELEGQKQYQL-RGMATILEGY REQKAGSThIRHAEELRKKGADLLWCNARTSASGYYKK

________LGFSEQGEVYDIPPVGPHILMYKKLT

SEQ ID 6_19C3 MIEVKPINAEDTYEIRHRI.RPNQPLEAcXYETDLLGGTFH NO:390 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEGY

REQKAGSTLIRI{AEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPIGPHILMYKKLT

SEQ ID 6_19C8 MIEKCPINAEDTYEIRUHIRPNQPLEACKYETDLLGGTLH NO:391 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMATLEGY REQKAGSThIRQAEELLRKKGADLLWCNARTSASGYY-KK _____________LGFSEQGGVYDIPPVGiPHILMYKELT SEQ ID 6_20A7 MIEVKPnIAEDTYEIRJRPNQPLE-ACMYETDLLRGTFH NO:392 LGGYYRGKLISIASFHQAEHSDLEGQKQYQLRGMATLEEY

REQKAGSTLHIAEELLR-KKGADL-LWCNARTSASGYYKK

_____________LGFSEQGEVYDLPPVGPHMIMYKKLT

SEQ ID 6_20A9 MIEVKPINAGDTYEIPJRILRPNQPLEACKYE1T)LGGTFH NO:393

LGGYYRGKUISIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTLIRHAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGGVYDIPPVGPHILMYKKLT

SEQ ID 6_20115 MIEVKPINAEDTYEIRHRILRNQPLEACKYE11)LLGGTFH NO:394 LGGYYRGKUSIASFHQAEHSEI-BGQKQYQLRGMATLEGY REQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQCTEVYDIPPIGPHILMYKKLT

SEQ ID 6_21F4 MIEVKYII4AEDTYEIRkVLRPNQPLEACMYETDLLGGAF NO:395 HLGGYYRGKLISIASHQAEHPELEGQKQYQ'LRGMATLEG YREQKAGSTLk{EELLRXKGADLLWCNARTSASGYYK _____KLGFSEQGEVYDVPPVGPHrLMYKKLT SEQ ID 6_22C9 MTEVKPINAEDTYEIRHRILRPNR-PLEACMYETD)LL)GGTFH NO: 396 LGGYYRGKLISIASFHQAFMPGL.EGKKQYQLRGMATLEY

REQKAGSTLIRHAEELLRXKGADLLWCNARTSASGYYKK

LGFSEQGGVYDIPPVGPHMMNYKKLT

SEQ ID 6_22D9 M]EVKP]NAEDTYEIRHR]LRPNQPLEACMYETLLEGTFH NO:397 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEGY REQKAGSThIHiAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPVGPHILMYKKLT

SEQ ID 6_22H19 MIEVICIADTYERPJLRPNQPLACMYETLLGGTFH NO:398 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMAThDEY

_____REQKAGSTLIRHAEULRKKGADLLWCNARTSASGYYKI(

187- WO 02/36782 WO 0236782PCT[US01/46227 LGFSE GEVYDIPPIGPHILMYKKLT SEQ IOD 6_23113 MIEVIKPINAEDTY ERURPNQPLEACMYGTDLLGGTFH NO :399 LGGYYRGKLISIASFHQAEQPELEGQKQYQL-RGMATLE-GY

REQKAGSTLIRHAEELLRXKGADLLWCNARTSASGYYKK

________LGFSEQGGVYDIPPVGPflILMYE3MT SEQ IOD 6_23117 M]EVIKPINAEDTYEIPJIRURPNQPLEACMYETDLLGGTFH NO:400 LGGYYRGKU.SIASFHQAEHSELEGQKQYQLRGMAThE3Y

REQKAGSTLIRHAEELRKGADILLWCNARTSASGYYKKL

GFSEQGGVYD]PVGPE]LMYKKLT

SEQ ID 6_2H1 MIEVKPINAEDTYEIHRVLRPNQPLE-ACMYETLLGGTF 1 HLGGYYRGKLISIASFHQAEBPELEOGQKPYQLRGMATLBG

YRBQKAGSTLIRHAIEELLRKKGADLLWCNARTSASGYYK

______KLGFSjEQGE1YDIPPIGPEILMYKKLT SEQ ID 6_3D6 MIEIKPINAEDTYE]UREJRPNQPLEAGMYETDLLGGTEHL NO:402 GGYYRGKLISIASFHQAEHFELEGQKQYQL-RGMATLEGYR EQKAGSThIRHAEELLRXKGADLLWCNARTSASGYYXKL- OSE GEVYDIPPVGPHILMYILT I SEQ ID 6_3G3 M]EviKpINAEDTyEIRHRILRPNQPLE-AGMYELT)LLGGTFH NO:403 L)GGYYRGKLISIASFHQAEHSELEGQKQYQLRGMAThEGY REQKAGSTLaHIAEE[LRKKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPVGPHILMYKKLT

SEQ ID 6_3112 MIEVKPINAEDTYETZRRIRPNQPLEACMYB'IDLIGGWFH NO:404 LGGYYRGKLISIASFHQAEHPELRGQKQYQILRGMATLEEY REQKAGSTiLIHAEBLRKKGADLLWCNARTSASGYYKX ______LGFSEQ EYIPPVGPFDLMlYKT SEQ ID 6_4A10 M]EVKiPINAEDTYEnIRRLRPNQPLEACMYETDLLGGTFH NO:405 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMATLEGY REQKAGSThIRHAMELRKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPVGPHILMYKKLT

SEQ ID 6_4B 1 MIEVKPINAEDTYEIRERUVLRPNQPLEACMYETDLLGG1T NO:406 BLGGYYRGKLIGIASFHQAEHPEILEGQKQYQLRGMATLE GYREQKAGSThIRHAEELLRKKGADLLWCNARTSASGYY

_____EKLGFSGQGEVYDIPPIGPH]LMYKKLT

SEQ ID 6_5D11 MIEVKPINAEDTYEIRHR]LRPNQP1R-ACMYEII)LLGGTFH NO:407 LGGYYRGKLIIASFIIQAEHPELEGQKQYQLRGMATLEEY

REQKAGSTLJRHAEELLRKKGADLLWCNARTSASGYYKK

______LGFSEQ EYII'PIGPHTLMAYKKLT SEQ ID 6_5F1 1 MIBVKPINAEDTYEaIREIRPNQPLE-ACMYEI1)LLGGTFH NO:408 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEEY REQKAGSThRHAEELLRKKGADLLWCNARTSASGYYKK

________LGFSEQGEVBDIPPVGPHILMYKKLT

SEQ ID 6_5G9 M[EVKPIAEDTYE]RRRPNQPLEACMYEITLLGGTFH NO:409 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLEEY

RBQKAGSTLIHAEEULRKKGADLLWCNARJSASGYYKIKL

________GFSEQGGVYDIPPVGPHILMYKKLT

SEQ ID 6_6D5 MIEVKPIAEDAYERHR]LNQPLE-ACKYETDLLGGTFH NO:4 10 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMAThEGY RBQKAGSTaHJIAEEflLRKXGADLLWCNARTSASGYYKK ______LGFSEQGGVYDIPPVOiPH]LMYKKLT SEQ ID 16-7D 1 iMIEVIKPINAEDTYEIHRLRPNQPLEACMYPDLRAF 188 WO 02136782 WO 0236782PCTIUSO1/46227 NO:41 1 LGGYYRGKUSLASFHQAEHSELEGQKQYQL.RGMATLEGY

RBQKAGSTURI{AEELLRKKGADLLWCNARTSASGYYKK

________LGFSEQGGYYDIPPVGPHELMYJ(XLT

SEQ ID 6_8H3 MIEVKPINAEDTYEIRRILRPNQPLIRACMYETDLLGGTFH NO:412 LGGYYRGKLISIASFHQAEBPELEGQKQYQLRGMAThEGY

REQKAGSTLIRHAEELLRKKGADILLWCNARTSASGYYKK

LGFSEQGVYDIPPVGiPIlaMYKKLT SEQ ID 6_9G1l 1 EVKP]NAEDTYEIFJ{RLRPNQPLE-ACKYE'T)LLGGThH NO:41 3 LGGYYRGKL1SIASFHQAERSELEGQKQYQLRGMATLEGY REQKAGSTLhIIAEELLRKKGADLLWCNARTSASGYYXK

LGFSEQGEVYDIPPVGPHIMYKIKLT

SEQ ID Ff1 MIEVKPINAEDTIY.LMRU-RPNQPLEACMYETDLJLXGTFH NO:4 14 LGGYYRGKLVCIASFHKAEHSELEGQKQYQI-RGMATLDG YRE-QKAGSThERAELLIKKGADLLWCNARTSVSGYYE

________KLGFSEQGEVYDIPPVGPHLLMYKKLT

SEQ ID 7_1C4 MIEVIKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGTFH NO:41 5 LGGYYRGKLISIASFHQAE1HPELEOQKQYQLRGMATLREY RI3QKAGSTLIRIAFELLRKKGADLILWCNARTSASGYYKK

LGFSEQGGVYDIPIGPHI'MYKKLT

SEQ ID 7_2A10 MIEVKPINAEDTYEIRIRILRPNQPLEFACKYETDILIGGTFH NO:4 16 LGGYYRGKUISIASFHQAEHPELEGQKQYQLRGMATLEGY REQKAGSThIRaELLRKKGADLLWCNARTSASGYYKK

LGFSEQGGVYDIPPIGPMJMYXIMT

SEQ ID 7_2A1 1 MIEVIKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGTFH NO:4 17 LGGYYRGKLISIASFHQAEISELEGQKQYQLRGMATLRGY REQKAGSTLHIAEE[I1RKKGADILLWCNARTSASGYYKK

LGFSEQGGVYDIPPVGPH]LMYKIKLT

SEQ ID 7_2D7 MIEVKPINAEDTYEIRHR]LRPNQPLEACKYE11)LLGGTFH NO:41 S L)GGYYRGKLISIASF'HQAEEPELRGQKQYQLRGMATLEGY

REQKAGSTLHIIAEE[RKKGADLLWCNARTSASGYYKX

LGFSEQGEVYDIPPVGPH]LMYKKLT

SEQ ID 7_5C7 MIEVKPINAEDTYEIRHR]ILRPNQPLEACMYETDLLGG1FH NO:419

LGGYYRGKUISIASFHQAEHFELEGQKQYQLRGMATL-EGY

REQKVGSTLRHAEELLRKKGADILLWCNARTSASGYYKK

LGFSEQGGVYDIPPVGPIEL~fIKLT SEQ ID 7_9C9 MIEVKIINAEDTYEIRERLRPNQPLEACMYETLLGGTFH NO:420

LGGYYRGKISIASFHQAEPELEGQKQYQLRGMATLEGY

REQKAGSThIRIIEELLRKKGADLLWCNARTSASGYYKK

_____________LGFSEQGEVYDIPPIGPELMYKKLT

SEQ ID 9_13F10 MIEVKPINAEDTYEIRJRPNQPLEACKYETDLLRGAFH NO:42 1 LGTGYYRGKLVSIASFHQAEHSELEGQKQYQL.RGMATLEE

YREQKAGSTLTRHLAEELLRKKGADL-LWCNARTSASGYYK

KILGFSEQGEVYDIPPTGPHILMYKXLT

SEQ ID 9_13F1 MIEAIKNAEDTYERRLRPNQPLACMYEDLGGTFH NO :422 LGGYYRGKLVSIASFHQAEIITELEGQKQYQLRGMATLEE

YREQKAGSTLTRHAERLLRKKGADL.LWCNARTSASGYYK

KLGFSEQGEVYDIPPVGPHILMYKELT

SEQ ID 9_15D5 MIEVKPINAEDTYERIR{RPNQP1DACKYEDLLGGTFH NO:423 LGGYYRGKLISIASFHQAEBPELEGQKQYQILRGMATLEGY

______REQKAGSTLIRHAERLLRKKXGADLLWCNARTSASGYYKK

189- WO 02136782 WO 0236782PCTfUSOI/46227 T SEQ ID 9_15D8 MIEVICPINAEDTYETHRTL-RPNQPLEACMYETDLLGGTFH NO1:424 LGGYYRGKLVSIASFHiQAFI{PELEGQKQYQL-RGMATLEG YREQKAGSThIRHAEALLRKKGADU-LWCNARTSASGYYK

_______KLGFSEQGEVYDTPPVC*PBIAYKIKLT

SEQ ID 9_15H3 M]EVKPIh4AEDTYEJRERILRPNQPLEACMYETI)MLRGAFH NO:425 LGGYYRGKLISIASFHQAEHPELEGQKQYQLRGMAThEEY HEQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYKK

_____________LGFSEQGEVYNTPPVGPEILMYKKLT

SEQ ID 9_18H2 MIEVIKPINAEDTYEdiRBRLRNQPLEACME1TLLGGTFH NO:426

LGGYYRGKLISIASFHQAEHELVGQKQYQLRGMATLEGY

REQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYKK

LGFSEQGEVYDIPPVGPHILMYKIKLT

SEQ ID 9_20F12 MIEVKPINAEDTYEIRERVLRPNQPL-ACMYEDUGGTF NO:427

HLGGYYRGELVSIASFHQAEHPELEGQKQYQLRGMATLE

GYRBQKAGSTLRHIAEELLRXXGADL.LWCNARTSASGYY

______KKLGFSEQGGVYDIPPVGPBILMYKKLT

SEQ ID 9_21C8 MIEVIKPTNAEDTYERIRRIRPNQPLEACMYBTLLGGTH NO:428 LGGYYRGKLISIASFHQABBPELEGQKQYQLRGMAThEGY REQKAGSThRHAEELLRKGADLLWCNARTSASGYYKK C3EVYDIIPVGPEILMYKKLT SEQ ID 9_22B 1 MIEVNAEDYIRRl-RPNQPLACKYWIDI.GGTFH NO:429 LGYGYYRGKLVSIASFHQAEBPELEGQKQYQLRGMA-fLEG

YREQKAGSTL]RHAELLRKKGADLLWCNARTSASGYYK

KLGFSEQGEVYDLPI>TGPEILMYKKLT

SEQ ID 9_23A10 M[EiVKPINAEDTYEaRELRPNQPLEACKYETDLLGGMhH NO:430

LGGYYRGKLVSIASFHQAFI{PELBGQKQYQLRGMATLEG

YRGQKAGSTLIRHAEELLRKKGADLLWCNARTSASGYYK

______KLGFSEQGGVYDIIPPVGPHEhMYKKLT SEQ ID 9_24F6 MIEVKPINABDTYEIRHRILRPNQPLEACKYET)LLRGAFH NO:43 1 LGGYYRGiKLISIASFHQAEHSELEGQKQYQLRGMATLEGY

REQKAGSTL]PIAEALLRKKGADLLWCNARTSASGYYKK

LGFEQGVYDIPPTGPHIMYKKLT

SEQ ID 9_41110 MM-VKPINAEDTIYIERHRn1RPNQPLEACKYEIDLLGGTLH1 NO:432 LGCJYYRGKISIASFHQAEHPEU3GQKQYQLRGMATLEGY REQKAGSTiLHEELRKKGADL1WGNARTSASGYYKKL

_____________GFSEQGEVYDIPPVGPHILMYKIKLT

SEQ ID 9_4H8 MIEVKPINAEDTYE]RERILRPNQPLEACMYEPDLL1QGTFH NO:433

L)GGYYRGKUISIASFNQAEHPELEGQKQYQLRGMATLEGY

REQKAGSTLTRHAEELLRKKGADLLWCNARTSASGYYKK

LGPSEQC3EVYDIPPVGPHJLMYKKLT SEQ ID 9_8111 MIEVXPrTAEDTYEBRRRPNQPLEACKYETDLLGGTFHL NO:434

GGYYRGKLISIASFHQAEHPELEGQKQYQLRGMATLEGYR

EQKAGSTaHRIAEELLRKKGADLLWCNARTSASGYYKKI

________GFSEQGEVYDIPPTGPHILMYKKLT

SEQ ID 9_9117 MIEVKPINAFDAYEIIRLRPNQPLEACKYETDLLGSTFH NO:435

LGGYYRGKLISIASFHQAEBPELEGQKQYQLRGMATLEY

REQKAGSThIRHAEELLRKKGADLLWCNARTSASGYYKK

_____________LGFEQGEVYDIPPVGPHILMYKKLT

ED 19C6 IMIEVIKPINAEDTYEIRHRILKe FA -190- WO 02/36782 WO 0236782PCTIUSOI/46227 NO:436 LGGYYQGKLISIASFHNAEHSELEGQKQYQLRGMAThBGY REQKAGSThIRHAEELRKKGADLLWCNARTSVSGYYEK

LGFSEQGEVYDIPPVGPHILMYKXLA

SEQ ID 9H111 MIEVKPINABDTYE]RUR1LRPNQPLEACKYETDLLGGTFH NO:437 LGGYYRGKLISIASFHKAEHSELEGEEQYQL.RGMATh.EGY REQKAGSThUiRAIEELLRKKGADLLWCNARTSVSGYYKK

_____________LGFSEQGEVYDIPPIGPHMUMYKKLT

SEQ ID) 0_4B 10 MIlEVIKPINAEDTYELRIK[RPNQPIEACMYESDLLRGAFH NO:438 LGGFYRGKLISIASFHQAEHSDLEGQKQYQILRGMAThEGY RDQKAGSTL1KHAEEILRKRGADMLWCNARTrASGYYKK

EFTPPVGPILMYKRLT

SEQ ID 0_5B 11 MIEVKPINAEDTYELRHEKILRPNQPIEFACMYESDLLRGAFH NO:439 LGGFYGGKLISIASFHQAEHSDLEGQKQYQLRGMAThEGY RDQKAQSTLiKAIEQLLRKRGADMLWCNARTSASGYYK KLGFSEQGEVFETPPVGPHIaMYKKIT SEQ ID 0_5B3 MLRVKPINAEDTYELRHRILRPNQPIEACMYETDLLRGAFH NO:440 LGGFYRGKLISIASFHQAEHSELQGQKQYQLRGMAThEGY

RDQKAGSSLIAEQLLRKRGADLLWCNARTSASGYYKK

LGFSEQ EFTPVGPHlMavYKRIT SEQ IID 0_5B4 MLEVKLINAEDTYELREPJLRPNQPLEACMYETLLRGAF NO:44 1 HLGGFYRGKUISIASFHQAEHSDLEGQKQYQL.RGMATLEG FRDQKAGSSLWH1F GANLLWCNARTSASGYYKK

_____LGFSEQGEVFDTPPVGPBILMYKRIT

SEQ ID 0_5B8 MIEVKPINAEDTYELRHKLRNQPIEACMYESDLLRGAFH NO:442 LGGFYRGKLISIASFHQAEHSDLQGQKQYQLRGMATLEGcY

RDQKAGSSURHAEQ]LRXRGADLLWCNARTSASGYYKK

LGFSEQGEIFDTPVGPHILMYKRLT

SEQ ID 0_5C4 MIEVKPINAEDTYELRHKILRPNQPLEACMYETDLLRGAF NO:443 HLGGFYRGKLISIASFHQAEHSGLQGQKQYQLRGMATLEG YREQKGSSAPRr P CGADLLWCNARTSASGYYKK

LGFSEQGEIFDTPPVGPHMYKRIT

SEQ ID 0_51)11 MJEVKPINAEDTYELPJILRPNQPIEACMYESDLLRGAEH NO :444 LGGFYRGKLISIASFHQAEHSDLQGQKQYQLRGMATLEGY RBQKAc3STLiRAEQLLRKRGADL.LWCNARTSASGYYKR ______LGFSEQCGEVFDTPPVGPHU2MYKRLT SEQ ID 0_51)3 MLEVKPINAEDTYFLRHRIRPNQPIEACMYFSDLLRGAFH NO:445 LGGYYRGKLISIASFHQAEIISELQGQKQYQLRGMATLEGY REQKAGSSLIKHAEEUhRKRGADILWCNARTSASGYYKKL

_____GFSEQGIIFETPPVGPBILMYKRIT

SEQ ID 0_51)7 MIEVKPINAEETYELRHRILRPNQPIEACMYEThLLRGAFII NO:446 LGGFYRGKLISIASFHQAEHSELEGQKQYQLRGMAThEGY R1)QKAGSSLIRHAEQLLRKKGANMLWCNARTTIASGYYK

________KLGFSEQGEIFDTPPVGPEILMYKRIT

SEQ ID 0_6B4 MLEVKPINAEDTYELRHRILRPNQPI]EACMYESDLLRGALH NO:447 LGGFYRGKUSIASFHQAEHSDLQGQKQYQLRGMATLEGF

RDQKAGSSLIRHAEQILRKRGADLLWCNARTSASGYYIK

_____LQFSEQGKVFDTPPVGPHJLMYKRrr SEQ ID 0_61)10 MEVKPINAEDTYELRIIKILRPNQPLEVCMYETDLLRGAF NO:448 HLGGFYRGKLISIASFHQAEI{SDLQGQKQYQLRGMATLEG _____Y1DQKAGSSL]RIAEQILRKRGADMvLWCNARTSASGYYK 191 WO 02136782 WO 2/3782PCTIU-SO1/46227

______KL)GFSEQGEVFETP)PVGPHILMYKRLT

SEQ ID 0_61)11 MIEVKPINAEDTYELRHRILRPNQP]EACMYESDL.LRGAFH NO:449 LGGYYRGKLISIASFHQAEHSDLQGQKQYQILRGMAThEGF

RDQKAGSSLIAEQ]LRKRGADLLWCNARTSASGYYKK

_____________LGFSEQGEVFETPPVGPBILMYICIZT

SEQ ID 0_6F2 MIEVKPINAEDTYELRHRILRNQPIEACMYESDLLRGAFH NO:450 LGGYYRGKLISJASFHQAEHISELQGQKQYQL-RGMATLEGF REQKAGSTLRHAkEQJLRKRGADMLWCNARTSASGYYKK

________LGFSEQGEIFDTPPVGPHRMYKRIT

SEQ ID 0_6H-9 MvIEVKPNAEDTYELRHKILRNQPIEACMYEIDLLRGAFH 1 LGGFYGGKLISIASFHQAEHSDLEOQKQYQLRGMAThEGY RBQKAGSThIRHAEEILRKGANLLWCNARTSASGYYKKL

________GFSEQGEVFDTPPVGPHILMYKRLT

SEQ ID 10_4C10 MIEVKPINAEDTYELH -R JNQPLEVCMYFIDLLRGAF NO:452 BLGGXYRGKLISIASFHQAEHSELQGQKQYQL-RGMAThEG

YRDQKAGSSUMKHAQRRKRGADXLWCNARTSASGYYK

_______KLGPSEQG=FTPVGPIEIMYERLT

SEQ ID 10_4D5 MIVPNADTYLRHILRNQPIEVCMY=rLLRGAFH NO:453 LOGGFYRGKLISIASFHQAEHSDLQGQKQYQIRGMAThBGY

REQKAGSTLRZHAEQILRKRGADLLWCNARTSASGYYKKL

GPSEQGE TPPVGPBILNIYKRrr SEQ ID 10_4F2 MLEVKPINAEDTYELRHRLRPNQPIEACMFESDLLRGAFH NO:454 LGGFYRGKLISIASFHQABHSELQGQKQYQLRGMATLEGY REQKAGSSLaREABEBIRKRGADMLWCNARTSASGYYKK _____LGFSEQGEIFTP VGPBUIMYKRLT SEQ ID 10_4F9 NIIEVKPINAEDTYELRHRILRNQPEYCMYETDILRGAFH NO:455 LGGFYRGKLISIASFHQAEHSELQGQKQYQLRGMATLEGF

REQKAGSSIIRHAEQLR]KRGADLLWCNARTSASGYYKKL

_____________GFSEQGEIFDTPVGPEILMYKRLT

SEQ ID 10_4G5 MIBV<PINAEDTYELRHRILRPNQPIEACMFE-SDLLRGAFH NO :456 LGGYYRGKLISIASFHQAEH-SDLQGQKQYQLRGMAThEG

YRDQKAGSSLIRHAEQ]LRKRGADLLWCNARTSASGYYK

LGPS GCEIFDTPPVGPHJlvfYKRLT SEQ ID 10_4H4 MLEVIKPINAEDTYELRHKILRNQPLEVCMYETDLLRGAF NO:457 HLGGFYRGKLISIASFHQAEHSELQGQKQYQLRGMATLEG YREQKAGSSLIakHEEILRKRGADLLWCNARTSASGYYKK LOFSE GEVFD'rPPVGPHILMYXPM SEQ ID 11_3A1 1 M]EVKPINAEDTYELRBX1LRPNQPIEVCMYEDLLRGAEH NO :458 LGGFYRGKLISIASFHQAIEHPDLQGQKQYQLRGMATLEGY

RDQKAGSSLIKIIAEQILRKRGADLLWCNARTSASGYYKK

LGFSEQGEVFEIPPVGPHRLMYKRLT

SEQ ID 11_3B 1 NEVKPNAEDTYELRIPJLRPNQPIEACMFBTDL.LRGAFH NO:459 LGGFYRGKLISIASFHQAEHSDLQGQKQYQILRGMATLEGF

REQKAGSTLTRRAFRLRKRGADILLWCNARTSASGYYXRL

_____________GFSEQGEIFDTPPVGPBILMYKRLT

SEQ ID 11_3B5 MIEVKPIh4AEDTYELRBRILRPNQPIEACMFESDLLRGAFH NO:460 LGGYYRGKLISIASFHQAEHSELQOQKQYQLRGMATLEGY

RDQKAGSSLIRHAEQ]LRKRGADMLWCNARTSASGYYKK

_____LGFSEQGEVFDTPPVGPHMLMYKRlT SEQ ID 11 3C12 IMIEVKPINAED1 Y h .TR 11 NQPLEVCMYETDLLRGAFH -192- WO 02136782 WO 0236782PCTJUSOI/46227 NO:46 1 LGGFYGGKLISIASFHQAEHPDLQGQKQYQLRGMAThEGY

RDQKAGSSIJRHAEQLLRKRGADLLWCNARTSASGYYKK

_____________LGFSBQGEIFETPPVGPIIILMYKR1T SEQ ID 11_3C3 MIEVIKPINAEDTYELRHK]LRNQPIEACMYESDLLRGALII NO:462 LGGYYRGKLISIASFHQAEHISELQGQKQYQLRGMAThEGY REQIAGSSUKaAEFIRXRGADLLWCNARTSASGYYKKL

EFTPPVGPEILMYKRIT

SEQ ID 11_3C6 MJ.EVKPINAEDTYELRHKILRNQP]EACMFESDLLRGAFH NO:463 LGGFYGGKLISIASFHiQAEHSDLBGQKQYQLRGMATLEGY REQKAGSTURI{AEEaLRKRGADLLWCNARTSASGYYKKL

______GFSBQGEIFDTPPVGPH]LMYKRIT

SEQ ID 11_3D6 MIEVKPINAEDTYELRHRILRPNQPIEVCMYETDLLRGAFH NO:464 LGGFYRGKLISIASF'HQAEHSDLQGQKQYQLRGMAThEGY

REQICAGSSLTKIIAEQILRKRGADLLWCNARTSASGYYKIKL

______GFSEQPEVFDTPPVGPHI[MYKRLT

SEQ ID 1_1G12 MLEVKCPINAEDTYELRHR1LRPNQPIEVCMYFITLLRGAFH NO:465 LCTGFYGGKLISIASFHQAEHSELQGQKQYQLRGMATLEGY

RDQKAGSSLXHAFRILRKRGADTLLWCNARTSASGYYKKL

GFSEQGEVFErPPVGPH]LMYKRLT SEQ ID 1i-Hi M[EVKPINAEETELMRHKILRPNQPIEACMYESDLLRGSFH NO:466 LGGFYRGQLISIASFHXAEHSELQGQKQYQLRGMATLEGF

REQK-AGSSLIHEEILRNXGADLLWCNARTTASGYYKRL

________GFSEHGEVFEI'PPVGPH]LMYKRrr SEQ ID 1_1112 MIEVKPINAEDTYELRHPJLRPNQPLEACMYESDLLRGSFH NO:467 LGGFYRGKLISIASFHQARHSELEGQKQYQLRGMATLEGF REQKAGSSLTRHAEEahRKRGADLLWCNARTITAAGYYKX

LGFSEQEWTPPVGPBILMYKRIT

SEQ ID 1_1115 MIEVKPINAFDTIYFJIURLRPNQPLRACMYESDLLRGSFH NO:468 LGGFYRGKLUSJASFHQAIEHSDLEGQKQYQLRGMAThEGY RDQKAGSSlIAEQILRKRGADLLWCNARTTAAGYYKR LCTFSEQGiEVFIXFPPVGPHILMYXKLT SEQ ID 1_2A12 MIEVKPINAEDTYELRHPJRPNQPIEACMYE-SDLL-RGSFH NO:469 LGGFYRGKIISIASFHQAEQSELEGQKQYQLRGMATLEGY RDQKAGSTLIXBAEEnhRKKGADLLWCNARTSAAGYYKR _____________LGFSEQGEIFDTPPVGPHaMNYKRLT SEQ ID 1_2B6 MIEVKPINAEETYELRHELRPNQPLEACMYETDLRGSFH NO:470 LGGFYRGKLISIASFHQAEHSELE-GQKQYQLRGMATLEGF

RDQKAGSSLIKHAEE!LRKRGADL-LWCNARTSASGYYKXKL

GFSEQGE]FETPPVGPILMYKRLT

SEQ ID 1_2C4 MLEVKCPINAEELRIKLRNQPIEACMYE'IDLLRGSFHf NO:47 1 LGGFYRGQLISIASEHQAEI{SDLQGQKQYQLRGMATLEGY REQKAGSTLIUcHAEELLRKKGADLLWCNARTrAAGYYKK

LGFSEQGEVFDTPPVGPH]LMYKKIT

SEQ ID 1_2D2 MIEVIKPINAEDTYELRRFOLRPNQPLE-ACMYESDLLRSAFH NO :472 LGGFYRGKLISIASFHKAEHSELQGQKQYQLRGMATLEGY

RDQKAGSSL]RHAEELRKRGADMLWCNARTSAAGYY-KR

_____________LGFSEQGEVFDTPPVGPHILMfYKPJ SEQ ID 1_2D4 MIEVKPIAEDTYELU-RPLRNQPIEACMYESDLL-RGSFH NO :473 LGGFYRGKLTSIASFHQAEHISDLQGQKQYQLRGMAThEGY RQK'TAGSSiKEAQLRKGADMLWCNARTSAAGYYK 193 WO 02/36782 WO 0236782PCT/US01/46227

;Z

RLGFSE GEF=PvGPHIAvYKRIT SEQ ID 1_2F8 MLfEVKYINAEDTYELRBRILRPNQPLEACMYETDLLRGSF NO:474 HLOGFYRGKUSIASFHQAEHSELEGQKQYQLRGMATLEG YRDQKAGSSLIRHABEIRKGADMLWCNARTrAAGYYK _____________KLGFSEQGErYDTPPVGPHILMYKKLT SEQ ID 1_2H8 MEV-KPINAEETYELRHK1LRPNQPLE-ACMYETDLLRGAFH NO:475 LGGFYRGK[ISIASFHQADI{SELQGQKQYQLRGMAThEGY REQKAGSThJRHAEQILRKRGADL-LWCNARTSAAGYYKK LGFSEHGEi, 'PVGPHIMY'KRLT SEQ ID 1_3A2 M]EVIKPINAEDTYELRHRILRPNQPIEACMYESDLLRGAFH NO:476 LGGFYRGKLISIASFHQAE{SDLQGQKQYQI.RGMAThEGY REQKAGSSLLHiAEEIRXKGADMLWCNARTIAAGYYIKR

_____________LGFSEQGEVFDTPPVGPHILMYKRIT

SEQ ID 1_3D6 MJEVIKP]NAEDTYELRdEXILNQPIEACMYESDLLQGSFH NQ:477

LGGFYRGQUSIASFHQAEHSDLQGQKQYQILRGMATLEGF

REQKAGSTLJKHXEEILRKGADLLWCNARTSAAGYYKK

_____________LGFSEHGEIFDTPPAGPHIMYKIKLT

SEQ ID 1_3F3 MIEV PNAEETYELRQRIL-RPNQPIEACMYESDURGSFBL NO:478

GGFYRGQLISIASFHQAEHSELQGQKQYQLRGMATLEGYR

EQKAGSTLRIAEEIRKKGADLLWCNARTSAAGYYKRL

GFSEHGEIFDTPPVGPfl1MlYKRfIT SEQ ID 1_3H2 MIEVIKPINAEDTYELRHRJLRPNQPIEACMYEIDLL-RGAFH NO:479 LGGYYRGQUISLASFHKAE-HSELQGQKQYQL.RGMATLEGY REQKAGSTLI~kHEQLR-EKGADMLWCNARTSA-AGYYK _____RLGFSEQGEVFDTPVGPHEn1MYKKLT SEQ ID 1_4C5 MIEVKPINAEDTYELRHK]LRNQPIBACMYESDLLRGSFH NO :480 LGGFYRGKLISIASFBKAEHSDLEGQNQYQLRGMAThEGY

REQKAGSTURIAEEILRXRGADMLWCNARTSASGYYKR

________LGFSEHGEIFDTPVGPIflMYKRLT SEQ ID 1-4D6 M-EVKPINADTYELRRLRPNQPEACMYETDL1RGSFH NO:48 1 LGGFYRGQLISIASFHKABHSDLEGQKQYQI-RGMATLEGY RBQKAGSTLUiRAEQIRRGADMLWCNARTSAAGYYKR

LGFSEQGEVFETPPVGPHJLMYKPRLT

SEQ ID 1I_4111 MIEVKYPNAEDTYELRHRIhRPNQPLE-ACMYEThLLRGSFH NO:482 LGGFYRGKLISIASFLIQAEHSDLQGQKQYQLRGMAThEGY REQKAGSUhJRHAEQLLRKRGADLLWCNARTSASGYYKR

LGFSEHGEVFDTPPVGPILMYKRLT

SEQ ID 15H5 MILEVKPINAEErYELRHKILRPNQPLE-ACMYESDLLRGSFH NO :483 LGGYYRGQLTSIASFHQAEHSELBGQKQYQLRGMATLEGF REQKAGSThIKHAEQ]LRKRGADMLWCNARTSAAGYYKK _____________LGFSEBGEIFDTPPVGPH~lMYKKLT SEQ ID 1_6F12 M[EVKPINAEETYELRHRIILRPNQPIEACMYESDLLRGSFHL NO:484

GGFYRGKLISIASFHQABHSDLEGQKQYQLRGMAMEGYR

DQKAGSTLEiKHELRKRGADMLWCNARTSAAGYYKR

______LGFSEHGE-IY=PVGPHJLMYKKI

SEQ ID 1_6116 M[EVKPINAEDTYELRHKILRPNQPIE-ACMYESDLI-RGSFH NO:485

LGGFYRGQLISIASFHQAEHSDLEGQKQYQLRGMATLEGY

RDQKAGSSLIKIAEEMhRKRGADLLWCNARTSAAGYYKR

LGFSEQGEIFDIPVGPEELMYK=I

SE ID 3_1lAlO IMLEVKPINAEDTYRLRIIRLNQPIEACMYESDLLRGAFH -194- WO 02136782 WO 0236782PCTJUS01/46227 NO:486 LGGYYRGKLISIASFHQAEE{SELQGQKQYQLRGMAThEGY REQIKAGSSLVKTAPP1T

KRGADLLWCNARTSASGYYKK

LGFSEQC3EII=TPVGPH[LMYKR1 SEQ IID 3_14F6 MLEVIKPINAiEDTYELRHRLRNQPIEACMYESDLTRGAFH NO:487 LGGFYRGKLISIASFHQAEHSELQGQKQYQLRGMAThEGY REQKAGSSLKIAET RKIR GADILLWCNARTSASGYYXKL GFsEQGEiFETPPvGPHILIVyKRLr SEQ IID 3_15B2 MaVKPNAEDTYELRHK]LRPNQPLEVCMYEfDL)LRGAF NO:488 BLG<3YYGGKUJSIASFHQAEHSE[LQGQKQYQLRGMAThE

GYREQK-AGSSL]KHAEERKRGADI±WCNARTSASGYK

_____KLGFS QEFTPGPHILMYKRI1T SEQ ID 3_6A10 MIEVKPNAEDTYELRBRLRPNQPIEACMYESDLLRGAFH NO:489

LLJGYYRGKLISIASFHQAEHSELQGQKQYQLRGMATLGY

REQKAGSSUXHIAEEIRKRADLLWCNARTSASGYYKKL

GFSEQGE=HULP VGPBILMYKRIT SEQ ID 3_6B1 MLEVYWNAEDTYaRRILRPNQPIEACMYESDLRGF NO:490 LC3GYYRGKLISIASFHQAEBPELQGQKQYQLRGMATLEGY

REQKAGSSLEKHAEE]LRKGADLLWCNARTSASGYYKL

GFSEQGEVFEPPPVGPEILMYKRIT

SEQ ID 3_7F9 MLEVKPINAEDTYELRHRILRPNQPIEACMYESDLLRGAFH NO:49 1

LGGYYGGKLISIASFHQAEHSDLQGQKQYQLRGMATLEG

YREQKAGSSL~IKAEEILRKRGADLLWCNARTSASGYYKK

_________LGFSEQGEEFETPPVGP{ITMYKR]T

SEQ lID 3_SG1 1 MLEVKPINABDTYELRIIRILRPNQPIEVCMYE-SDLLRGAFH NO:492 LGGYYRGKLISIASFHQAEHSaLQGQKQYQLRGMAThEGY

REQKAGSSLIHEELKGADLLWCNARTSASGYYKIKL

GFsEoGETE=PPvGpHimiKr~r SEQ ID 4_IBlO MEVKPINAEDTYRERllRPNQPIEVCMYET)LTRGF NO:493

LGGFYGGKLISIASFHQAEHSDLQGQKQYQLRGMATLEGY

RDQKAGSSLIRHABQIRKGADMLWCNARTSASGYYKX

LCIFS QEFTPGPI{ILMYER1T SEQ ID 5_2B3 MIEVNAEDTYELRIRLRPNQPLEVCMYETIDLIRGAFH NO:494 LGGFYGGKLISIASFHQAfl{SDLQGQKQYQLRGMATLEGY

RDQKAGSSLJRHAEQILRKGADMLWCNARTSASGYYKK

LGFSEQGE=FI'PVGPHILMYKRI

SEQ ID 5_2D9 MLXVKYINAEDTYELRHFCJRPNQPXEVCMYEXDLLRGAF NO:495

HLGGFYRCGKLSIASFHQAFMSDL)QGQKQYQLRGMATLEG

YRDQKAGSSLKiHAEQIUREGADMLWCNARTSASGYYK

KLCTFSEQGEVFDTPPVGPH]LMMKLT

SEQ ID 5_2F10 ML-EVKPINAEDTYELRIL9RPNQPEVCMYEn)LLRGAF NO:496 HLGGFYGGKLISIASFHQAEHSDLQGQKQYQILRGMAThEG YRDQKAGSSURHAkEQILRKRGADMLWCNARTSASGYYK

______KLGFSEQGEIF=TPVGPBILMYKRLT

SEQ ID 6_IAl1 MLEVKPINAEDTYER{KLRPNQPLEVCMYETDLL-RGAF NO:497 HLGGFYRGKLISIASFHQAEHSDLQGQKQYQLRGMAThEG

YRDQKAGSSLIRRABQ]LRXRGADMLWCNARTSASGYYR

________KLGFSBQGEVEETPPVGPHTLMYKRLT

SEQ D NO:498 6_iDS MIEVKYPhJAUJI YJtixiLK?"NQ'Lk-,v tbj I-KY,

HLGGFYRGKLISIASFHQAEHSDLQGQKQYQIRGMATLEG

YRD KAGSSLIRJIAEQILRKRGADMLWCNARTSASGYYK -195- WO 02136782 WO 0236782PCT[USOI/46227 ________KLGFSBQGEVFE'rPVGPEILIAYKR SEQ ID 6_IF1 1 MIEVKPIAEDTYELRHKIRPNQPLEVCMYBTDLILRGAF NO-499 HLGGFYRGKLISIASFHQAEHSDLQGQKQYQLRGMAThEG

YREQKAGSSLUIRABQILRYKRGADMLWCNARTSASGYYK

KLGFSE QGEVFETPPVGPH~HMYKRIT SEQ DD 6_iFi MLfEVKPINAEDTYELREKELRPNTQPLEVCMYETDLLRGAF NO:500 BLGGFYRGKISIASFHQAEHSELQC3QKQYQLRGMAThE-G SEQ ID 6_1H1O MELEVIKPINAIEDTYELRHKJLRPNQPLEVCMYETDI.LRGAF NO:501 HLGGFYGGKLISIASFHQAEHSDLQC3QKQYQLRGMATI-3G YRDQKAGSSURA LKGADMLWCNARTSASGYYK

KLGFSEQGEVFDTPVGPHILMYKKIT

SEQ ID 6_1114 ML-EVIPINAEDTYELRBKILRPNQPLEVCMYETIDLILRGAF NO:502 HLGGFYGGKLISIASFHQAEHSDLQGQKQYQLRGMATLBG

YRDQKAGSTLTKIAEQILRKRGADMLWCNARTSASGYYK

______KLGFSEQGEVFETPVGPEILMYKRLT

SEQ ID 8_1F8 MLEVKPINAEDTYELRIHRLR-PNQPLEVCMYEITLLRGAFH NO:503 LGGFYRGKLISIASFHQAEHSDLQGQKQYQILRGMATLhEGY RBQKAGSSUKAEI GRADLLWCNARTSASGYYKKL GFSEQGEIFDTI>PVGPHILMYKRrr SEQ ID 8_1G2 MIEVVKPINAEDTYERHVLRPNQP.EVCME7LLRGAF NO:504

BLGGYYRGKUISIASFHQAEHSELQGQKQYQLRGMATLEG

YREQKAGSSL.TKHAEEIRKRGADLLWCNARTSASGYYKK

LGFSEQGEVF=TPVGPHML1YKRLT SEQ ID 8_1G3 MLEVKPNAEDTYELRBML.RPNQPIEVCMYETDU-RGAF NO:505 HLGGYYRGKUISIASFHQAEHSELQGQKQYQLRGM.AThEGi YPEQKAGSS IRHAEEILKRGADLLWCNARTSASGYYKK LGFS QEFTPVGPH]MYKRIT SEQ ID 8_1117 MEVIKPINAEDTYELRHR]LRPNQPIEVC-MYETDLLRGAFH NO:506 LGiFYRGKLISIASFHQAEHSELQGQKQYQILRGMATLEGY REQKAGSSUKAE

KGADMLWCNARTSASGYYKK

LGFSEQGEEWETPPVGPHELMYKRLT

SEQ IID 8_1119 MLEVIKPINAEDTYELRHKILNQPLEVCMYETDLLRGAF NO:507 HLGGYYRGKLJSIASFHQAEHSDL)QGQKQYQLRG3MATLE

GYRRQKAGSSLTRHAEERKRGADLLWCNARTSASGYYK

_______KWGFSEQGEVFDTPPVTPHMYKRLT

SEQ ID GATi_21F MIEVKPINAEDT 1 mflILR1 ~PNQPLEACKYETDILGGTFH NO:508 12 LGGYYRGKLISIASFHNAEHSEIEGQKQYQLRGMATLhEGY RRQKAGSMhRHLLRKKGADLLWCNARTSVSGYYKK LGFSEQGEVYDIPPIGPHrLMYKKLT SEQ ID GATi_24G miEviKPINAEDTYERHRiLRPNQPLEACMYETDLLGGTFH NO :509 3 LGGYYRGKLISIASFHQAEHSELEGQKQYQLRGMATLhEGY

RBQKAGSTURHAEELLRKKGADLLWCNARTFVSGYYEK

_____LGFSEQGEVYDIPPIGPYIMYEKLT

SEQ ID GATI-29G miEvKPINAEDTYEIRHR]LRPNQPLE-ACMYEThLLGGTFH 10 1 LGGYYRGKLISIASFHQAIEISEIEGQKQYQLRGMATLEGY RBQKAGSTLaHIAEaLRIKKGADLLWCNARTSVSGYYKK LGFS QGVCDIPPIGPHIIUAfYKIKLA 196- WO 02/36782 WO 0236782PCTfUSOI/46227 11 1 LGGYYRGKLISIASFHQAEHI'ELEGQKQYQLRGMAThEGY

REQKAGSTLIHFELKKGADLLWCNARTSVSGYYEK

LGFSBQC3EVYDIPPIGPHILMYKKLT SEQ DI) GAT2_150 MIEVKPINARDT1YE'IRHRILRPNQPLEACKYETDLLGGTFH 2 8 LGGYYRGKLISIASFHNAEHISELBGQKQYQL-RGMATLEGY

REQKAGSTLIRHAEELLRKKGADTLLWCNARTSVSGYYKK

LGFSEQGEVYDIPPIGPHILMYKIKLT

SEQ ID GAT2_19H MIEVKPINAEDTYEIRHRTLRPNQPLEACMYETDLLGGTFH 13 8 LGGYYRGKLJISIASFHQAEHPELEGQKQYQLRGMATLEcY

REQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEK

LGPSEQGEVCDIPPIGPHlvfYXKLT SEQ ID GAT2_21F MIEVKPINAEDTYEIRHRILRPNQPLE-ACMYETDUJ3GTFH 14 1 L)GGYYRGKLISIASFHQAEHiSELEGQKQYQLRGMATLRGY REQKAGSThIAEELLRKKGADLLWCNARTSVSGYYKK LGFSEQG%3VYDIPPIGPEILMYKKLT SEQ ID B. AACTGAAGGAGGAATCTC NQ:515 licheniform is ribosome binding site 197

Claims (25)

1. An isolated or recombinant polynucleotide, such as herein described, comprising a O nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO: 1, S wherein said polynucleotide encodes a polypeptide having glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formamide with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 °C for 15 minutes.

2. The isolated or recombinant polynucleotide of claim 1, wherein the polypeptide catalyzes the acetylation of glyphosate with a kcat/Km of at least 10 mMl 1 min' for glyphosate. (N

3. The isolated or recombinant polynucleotide of claim 1, wherein the polypeptide catalyzes the acetylation of aminomethylphosphonic acid.

4. An isolated or recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide having glyphosate N-acetyltransferase activity, the polypeptide comprising an amino acid sequence comprising at least 20 contiguous amino acids of SEQ ID NO:6. The isolated or recombinant polynucleotide of claim 4, wherein the polypeptide comprises an amino acid sequence comprising at least 50 contiguous amino acids of SEQ ID NO:6.

6. The isolated or recombinant polynucleotide of claim 4, wherein the polypeptide comprises an amino acid sequence comprising at least 100 contiguous amino acids of SEQ ID NO:6.

7. The isolated or recombinant polynucleotide of claim 4, wherein the polypeptide comprises an amino acid sequence comprising at least 125 contiguous amino acids of SEQ ID NO:6.

8. The polynucleotide of claim 1, further comprising a nucleotide sequence encoding an N-terminal chloroplast transit peptide.

9. A nucleic acid construct comprising a polynucleotide of claim 1. The nucleic acid construct of claim 9, wherein said polynucleotide is operably linked to a promoter, where the promoter is heterologous with respect to the polynucleotide and effective to cause sufficient expression of the encoded polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the nucleic acid construct. -198-

11. The nucleic acid construct of claim 10, wherein the polynucleotide sequence of claim 1 functions as a selectable marker.

12. The nucleic acid construct of claim 9, wherein the construct is a vector.

13. The vector of claim 12, comprising a second polynucleotide sequence encoding a second polypeptide that confers a detectable phenotypic trait upon a cell or organism expressing the second polypeptide at an effective level.

14. The vector of claim 13, wherein the detectable phenotypic trait functions as a selectable marker. The vector of claim 14, wherein the detectable phenotypic trait consists of herbicide resistance, pest resistance or a visible marker.

16. The vector of claim 12, wherein the vector comprises a T-DNA sequence.

17. The vector of claim 12, wherein the vector is a plant transformation vector.

18. A composition comprising a nucleotide of claim 1.

19. A cell comprising at least one polynucleotide of claim 1, wherein the polynucleotide is heterologous to the cell. The cell of claim 19, wherein the polynucleotide is operably linked to a regulatory sequence.

21. A cell transduced by the vector of claim 12.

22. The cell of claim 19 or 21, wherein the cell is a transgenic plant cell.

23. An isolated or recombinant polypeptide, such as herein described, comprising an amino acid sequence encoded by a nucleotide sequence which hybridizes under stringent conditions to the complement of SEQ ID NO: 1, wherein said polypeptide has glyphosate N-acetyl transferase activity and said stringent conditions comprise hybridization conditions comprising 50% formamide -199- 00 with 1 mg heparin at 42 °C carried out overnight, and a wash comprising 0.2XSSC at 65 °C for minutes. O 24. The isolated or recombinant polypeptide of claim 23, wherein the polypeptide catalyzes the acetylation of glyphosate with a kcat/Km of at least 10 mlv 1 min-' for glyphosate. The isolated or recombinant polypeptide of claim 24, wherein the polypeptide C catalyzes the acetylation of glyphosate with a kcat/Km of at least 100 mM-'min' for glyphosate.

26. The isolated or recombinant polypeptide of claim 25, wherein the polypeptide

28. The isolated or recombinant polypeptide of claim 27, wherein the polypeptide catalyzomprises the acetylatmino acid sequence omethylprising at least 50 contiguous amino acids of SEQ ID N:6.cid. O 27. An isolated or recombinant polypeptide, such as described 27, wherein, having glyphosate N-acetyltransferase activity, the polypeptide comprising an amino acid sequence comprising at least comprises an amino acid sequence comprising at least 100 contiguous amino acids of SEQ ID NO::6. 28. The isolated or recombinant polypeptide of claim 27, wherein the polypeptide comprises an amino acid sequence comprising at least 50 contiguous amino acids of SEQ ID NO:6.

29. The isolated or recombinant polypeptide of claim 27, rther comprisein the polypeptide comprises an amino acid sequence comprising at least 100 contiguous amino acids of SEQ ID NO:6. The isolated or recombinant polypeptide of claim 27, wherein the polypeptide comprises an amino acid sequence comprising at least 125 contiguous amino acids of SEQ ID NO:6.

31. The isolated or recombinant polypeptide of claim 23, further comprising an N- terminal chloroplast transit peptide.

32. The polypeptide of claim 23, comprising a secretion sequence or a localization sequence.

33. The polypeptide of claim 32, wherein the localization sequence comprises a chloroplast transit sequence. -200-