AU780662B2 - Orthologues of bacterial RuvB:cDNAs and uses thereof - Google Patents

Description

Orthologues of Bacterial RuvB: cDNAs and Uses Thereof BACKGROUND OF THE INVENTION Homologous recombination is a very important mechanism by which all living -igenisms exchange genetic information, thereby generating genetic diversity. Moreover, this mechanism also allows organisms to maintain genetic stability by protecting their genome against physico-chemical or environmental insults. Homologous recombination is a very complex process involving several well-defined biochemical steps, which are catalyzed by the concerted action of many cellular proteins. For example, in E.coli, dozens of genes and their cognate protein products have been implicated in homologous 0o recombination (Kowalczykowski, Dixon, Eggleston, Lauder, S. and Rehrauer, Microb. Rev. 58: 401-465, 1994). The picture is even more complex in eukaryotic organisms.

Despite the complexity of the process, recent genetic, biochemical, and molecular studies have revealed many mechanistic similarities in homologous recombination of prokaryotic and eukaryotic organisms (Camerini-Otero, D. and Hsieh, Ann. Rev.

Biocem. 29: 509-552, 1995). For the sake of understanding, this pathway can be divided into four discrete biochemical steps: 1) initiation 2) homologous pairing and DNA strand exchange 3) branch migration and 4) resolution. In bacteria, the action of various proteins S: such as those encoded by the genes recBCD, recE, recQ and recJ, initiate the S 20 recombination process, step 1, by generating single stranded DNA. The bacterial RecA protein, the product of the recA gene, catalyzes the next step of homology search, pairing, and strand exchange, whereas in eukaryotes, members of the RAD51 gene family are Simplicated in this process. The final step, resolution of the Holliday junction, is attributed to the actions of bacterial RuvC.

o 25 The third step in the process is branch migration, also known as heteroduplex extension. A complex of two proteins, RuvA and RuvB catalyzes this reaction. Extensive characterization of these proteins and their role of in this reaction has been perfoed in "-characterization of these proteins and their role of in this reaction has been performed in WO 01/05975 PCT/US00/16271 -2- E.coli (West Ann. Rev. Gen. 31: 213-244, 1997). The product of the ruvA gene, the 22kDa RuvA protein, exists as a stable tetramer in solution and specifically recognizes and binds to the Holliday junction with very high affinity. This binding is structure specific, independent of the DNA sequence, and Mg2+ dependent. Binding of RuvA to the Holliday junction by itself is not sufficient to cause branch migration either in vitro or in vivo.

Presence of the second component, RuvB, is required.

RuvB is the product of the bacterial gene ruvB. This protein has two NTP binding motifs, known as Walker A and Walker B boxes, as well as other structural motifs common to DNA helicases (West Ann. Rev. Gen. 31: 213-244, 1997). In the absence of metal ions, RuvB exists as a monomer or dimer in solution. However, the functional form of this enzyme is thought to be a hexamer. Two hcxameric RuvB units bind to DNA in a symmetrical manner to form a ring, similar to the hexameric ring formed by other DNA helicases. Binding of ATP or DNA to RuvB is Mg 2 dependent, such that at low concentration ofMg 2 RuvB has very low affinity for DNA and does not hydrolyze ATP.

Raising Mg 2 concentration >20mM increases RuvB's affinity for DNA as well as it's ATPase activity. Circular duplex DNA also stimulates the ATPase activity. RuvA also increases the affinity of RuvB for DNA. RuvB alone is sufficient to promote branch migration in vitro. However, a RuvAB complex functions much more robustly, and requires less Mg 2 Thus, RuvB participates in one of the rate limiting steps in homologous recombination in all living organisms.

Recently, eukaryotic homologues of bacterial RuvB have been cloned and characterized. For example, Kanemaki et al. isolated a 49kDa TBP interacting protein (Tip49) from rat liver. The full-length cDNA of Tip49 revealed its strong homology to bacterial RuvB protein (Kanemaki, Makino, Yoshida, Kishimoto, Koga, A., Yamamoto, Moncollin, Egly, Muramatsu, M. and Tamura, Arch.

Biochem. Biophys., 235: 64-68, 1997). The same scientists also reported the cloning of human Tip49 (Makino, Mimori, Koike, Kanemaki, Kurokawa Inoue, S., Kishimoto, and Tamura, Biochem. Biophys. Res. Commun.. 245: 819-823, 1998).

Similarly, cDNA for a human protein called RuvBL1 has been isolated employing the small (14kDa) subunit of human Replication Protein A, a hetero-trimeric protein involved in DNA replication, recombination and repair, as bait in the yeast two-hybrid assays. Two yeast homologues, scRuvBLl and ScRuvBL2, have also been identified in the yeast genome database. Gene knockout experiments in yeast indicate that scRuvBLl is essential for growth (Qiu, Lin, Thome, Pian, Schlegel, Weremowicz, Parvin, J. and Dutta, J. Biol. Chem. 273: 27786-27793,1998).

Due to the involvement of RuvB in homologous recombination, modulating RuvB expression levels provides the means to modulate the efficiency with which nucleic acids of interest are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides this and other advantages.

Summary of the Invention Generally, it is the object of the present invention to provide nucleic acids and to proteins relating to maize RuvB. It is an object of the present invention to provide: 1) antigenic fragments of the proteins of the present invention; 2) transgenic plants comprising the nucleic acids of the present invention; 3) methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.

According to a first embodiment of the invention, there is provided an isolated nucleic acid encoding a polypeptide having RuvB activity comprising a member selected from the group consisting of: a polynucleotide having at least 85% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9, wherein the sequence identity is based on the entire coding regions and is calculated by the GAP algorithm 20 under default parameters; a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, and a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9; and 25 a polynucleotide which is fully complementary to a polynucleotide of or According to a second embodiment of the invention, there is provided the nucleic acid in accordance with the first embodiment of the present invention, wherein the polynucleotide has at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9, wherein the sequence identity is based on the entire coding regions and is calculated by the GAP algorithm under default parameters.

According to a third embodiment of the invention, there is provided the nucleic acid in accordance with the first embodiment of the present invention, wherein the [R:\LIBFF]I O23Ospec.doc:gcc polynucleotide has at least 95% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9, wherein the sequence identity is based on the entire coding regions and is calculated by the GAP algorithm under default parameters.

According to a fourth embodiment of the invention, there is provided an isolated nucleic acid encoding a polypeptide having RuvB activity comprising a member selected from the group consisting of: a polynucleotide which encodes a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8 and 10, wherein the percent identity is determined over the entire sequence using the GAP algorithm under default parameters; and a polynucleotide which is fully complementary to a polynucleotide of According to a fifth embodiment of the invention, there is provided a method of modulating the level of RuvB in a plant cell, comprising: introducing into a plant cell a recombinant expression cassette comprising a RuvB polynucleotide in accordance with any one of the first to fourth embodiments of the c*o: present invention, operably linked to a promoter; S* culturing the plant cell under plant cell growing conditions; and o expressing said RuvB polynucleotide for a time sufficient to modulate the 20 level of RuvB in said plant cell.

According to a sixth embodiment of the invention, there is provided a method of modulating the level of RuvB in a plant, comprising: introducing into a plant cell a recombinant expression cassette comprising a RuvB polynucleotide in accordance with any one of the first to fourth embodiments of the 25 present invention, operably linked to a promoter; culturing the plant cell under plant cell growing conditions; regenerating a whole plant which possesses the transformed genotype; and expressing said RuvB polynucleotide for a time sufficient to modulate the level of RuvB in said plant.

According to a seventh embodiment of the invention, there is provided an isolated protein having RuvB activity comprising a member selected from the group consisting of: a polypeptide of at least 50 contiguous amino acids from a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and [R:\LIBFF O23Ospec.doc:gcc 4a a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 10, wherein the percent sequence identity is determined over the entire sequence using the GAP algorithm under default parameters; and at least one polypeptide encoded by a member in accordance with the first embodiment of the present invention.

Therefore, the disclosure herein relates to an isolated nucleic acid comprising a member selected from the group consisting of a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of and, a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of(a) or The isolated nucleic acid can be DNA.

In another aspect, the disclosure herein relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.

In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.

In a further aspect, the disclosure herein relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by an isolated .nucleic acid of the present invention.

o S" 20 In another aspect, the disclosure herein relates to an isolated nucleic acid comprising a polynucleotide of specified length which selectively hybridizes under stringent conditions to a polynucleotide of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.

In another aspect, the disclosure relates to a recombinant expression cassette 25 comprising a nucleic acid amplified from a library as referred to supra, wherein the :.**nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to a host cell transfected with this recombinant expression cassette. In some embodiments, the present invention relates to a protein of the present invention that is produced from this host cell.

In yet another aspect, the disclosure relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of [R:\LIBFF]I O23Ospec.doc:gcc 4b the isolated nucleic acids of the present invention. The present invention also provides transgenic seed from the transgenic plant.

Definitions Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.

By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D. C. (1993). The product of amplification is termed an amplicon.

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[R:\LIBFF I O23Ospec.doc:gcc WO 01/05975 PCT/US00/16271 The term "antibody" includes reference to antigen binding forms of antibodies Fab, F(ab) 2 The term "antibody" frequently refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). However, while various antibody fragments can be defined in terms of the digestion of an intact antibody, one of skill will appreciate that such 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 such as single chain Fv, chimeric antibodies comprising constant and variable regions from different species), humanized antibodies comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies bispecific antibodies).

The term "antigen" includes reference to a substance to which an antibody can be generated and/or to which the antibody is specifically immunoreactive. The specific immunoreactive sites within the antigen arc known as cpitopes or antigenic determinants.

These epitopes can be a linear array of monomers in a polymeric composition such as amino acids in a protein or consist of or comprise a more complex secondary or tertiary structure. Those of skill will recognize that all immunogens substances capable of eliciting an immune response) are antigens; however some antigens, such as haptens, are not immunogens but may be made immunogenic by coupling to a carrier molecule. An antibody immunologically reactive with a particular antigen can be generated in vivo or by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al., Science 246: 1275-1281 (1989); and Ward, et al., Nature 341: 544-546 (1989); and Vaughan et al., Nature Biotech. 14: 309-314 (1996).

As used herein, "antisense orientation" includes reference to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.

As used herein, "chromosomal region" includes reference to a length of a chromosome that may be measured by reference to the linear segment of DNA that it comprises. The chromosomal region can be defined by reference to two unique DNA sequences, markers.

WO 01/05975 PCT/US00/16271 -6- The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid.

One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made.

Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another: WO 01/05975 PCT/US00/16271 -7- 1) Alanine Serine Threonine 2) Aspartic acid Glutamic acid 3) Asparagine Glutamine 4) Arginine Lysine 5) Isoleucine Leucine Methionine Valine and 6) Phenylalanine Tyrosine Tryptophan See also, Creighton (1984) Proteins W.H. Freeman and Company.

By "encoding" or "encoded", with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as are present in some plant, animal, and fungal mitochondria, the bacterium Mvcoplasma capricolum, or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.

When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed.

For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)). Thus, the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants are listed in Table 4 of Murray et al., supra.

As used herein "full-length sequence" in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of, a native (nonsynthetic), endogenous, biologically active form of the specified protein. Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extension, S protection, and ribonuclease protection. See, Plant Molecular Biology.: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous WO 01/05975 PCT/US00/16271 -8- (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention. Additionally, consensus sequences typically present at the 5' and 3' untranslated regions of mRNA aid in the identification of a polynucleotide as full-length. For example, the consensus sequence ANNNNAUGG, where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5' end. Consensus sequences at the 3' end, such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3' end.

As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

By "host cell" is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells. A particularly preferred monocotyledonous host cell is a maize host cell.

The term "hybridization complex" includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.

By "immunologically reactive conditions" or "immunoreactive conditions" is meant conditions which allow an antibody, reactive to a particular epitope, to bind to that epitope to a detectably greater degree at least 2-fold over background) than the antibody binds to substantially any other epitopes in a reaction mixture comprising the particular epitope. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols. See Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions.

WO 01/05975 PCT/US00/16271 -9- The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfcction" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed transfected mRNA).

The terms "isolated" refers to material, such as a nucleic acid or a protein, which is: substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment; or if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell genome or subcellular organelle) not native to a material found in that environment. The alteration to yield the synthetic material can be performed on the material within or removed from its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. See, Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Patent No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurring nucleic acid a promoter) becomes isolated if it is introduced by nonnaturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids which are "isolated" as defined herein, are also referred to as "heterologous" nucleic acids.

Unless otherwise stated, the term "maize RuvB nucleic acid" is a nucleic acid of the present invention and means a nucleic acid comprising a polynucleotide of the present invention (a "maize RuvB polynucleotide") encoding a maize RuvB polypeptide. A "maize RuvB gene" is a gene of the present invention and refers to a heterologous genomic form of a full-length maize RuvB polynucleotide.

As used herein, "localized within the chromosomal region defined by and including" with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers.

WO 01/05975 PCT/US00/16271 As used herein, "marker" includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A "polymorphic marker" includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes of that pair to be followed. A genotype may be defined by use of one or a plurality of markers.

As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides peptide nucleic acids).

By "nucleic acid library" is meant a collection of isolated DNA or RNA molecules which comprise and substantially represent the entire transcribed fraction ofa genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques. Methods in Enzymology. Vol. 152, Academic Press, hnc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning A Laboratory Manual. 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wilcy Sons, Inc. (1994).

As used herein "operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

As used herein, the term "plant" includes reference to whole plants, plant organs leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, WO 01/05975 PCT/US00/16271 II including both monocotyledonous and dicotyledonous plants. A particularly preferred plant is Zea mays.

As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.

The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result ofposttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, WO 01/05975 PCITUS00/16271 12 branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Further, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.

As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells whether nor not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as "tissue preferred". Promoters which initiate transcription only in certain tissue are referred to as "tissue specific". A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "repressible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which is active under most environmental conditions.

The term "maize RuvB polypeptide" is a polypeptide of the present invention and refers to one or more amino acid sequences, in glycosylated or non-glycosylated form. The term is also inclusive of fragments, variants, homologs, alleles or precursors preproproteins or proproteins) thereof. A "maize RuvB protein" is a protein of the present invention and comprises a maize RuvB polypeptide.

As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term "recombinant" as used herein does not encompass the alteration of the cell or vector by naturally occurring events WO 01/05975 PCT/US00/16271 -13spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

As used herein, a "recombinant expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.

The term "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity complementary) with each other.

The term "specifically reactive", includes reference to a binding reaction between an antibody and a protein having an epitope recognized by the antigen binding site of the antibody. This binding reaction is determinative of the presence of a protein having the recognized epitope amongst the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to an analyte having the recognized epitope to a substantially greater degree at least 2-fold over background) than to substantially all analytes lacking the epitope which are present in the sample.

Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the polypeptides of the present invention can be selected from to obtain antibodies specifically WO 01/05975 PCT/US00/16271 14reactive with polypeptides of the present invention. The proteins used as immunogens can be in native conformation or denatured so as to provide a linear epitope.

A variety of immunoassay formats may be used to select antibodies specifically reactive with a particular protein (or other analyte). For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine selective reactivity.

The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences at least 2-fold over background).

Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (hetcrologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes to 50 nucleotides) and at least about 60°C for long probes greater than nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 37 0 C, and a wash in IX to 2X SSC (20X SSC 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55 0 C. Exemplary moderate stringency conditions include hybridization in to 45% formamide, 1 M NaCI, 1% SDS at 37 0 C, and a wash in 0.5X to 1X SSC at 55 to Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 370C, and a wash in 0.1 X SSC at 60 to 65 0

C.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal.

WO 01/05975 PCTUS00/16271 Biochem., 138:267-284 (1984): Tm 81.5 OC 16.6 (log M) 0.41 0.61 form) 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1 °C for each 1% of mismatching; thus, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with identity are sought, the can be decreased 10 Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 OC lower than the thermal melting point moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 "C lower than the thermal melting point low stringency conditions can utilize a hybridization and/or wash at 1, 12, 13, 14, 15, or 20 °C lower than the thermal melting point Using the equation, hybridization and wash compositions, and desired Tn,, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32 °C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laborator, 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 (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

As used herein, "transgenic plant" includes reference to a plant which comprises within its genome a hetcrologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics WO 01/05975 PCTIUSOO/16271 16initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgcnic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, nonrecombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

As used herein, "vector" includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons.

Expression vectors permit transcription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and (e) "substantial identity".

As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

As used herein, "comparison window" includes reference to a contiguous and specified segment of a polynucleotide/polypeptide sequence, wherein the polynucleotide/polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide/polypeptide sequence in the comparison window may comprise additions or deletions gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

Generally, the comparison window is at least 20 contiguous nuclcotides/amino acids residues in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in the art.

Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 WO 01/05975 PCT/US00/16271 17- (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al.. Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994).

The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Lnterscience, New York (1995).

Software for performing BLAST analyses is publicly available, through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always 0) and N (penalty score for mismatching residues; always For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) WO 01/05975 PCT/US00/16271 18uses as defaults a wordlength of 11, an expectation of 10, a cutoff of 100, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation of 10, and the BLOSUM62 scoring matrix (see Henikoff& Henikoff(1989) Proc. Nail. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see. Karlin Altschul, Proc. Nat Acad. Sci. USA 90:5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.

However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.

GAP can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence. GAP uses the algorithm of Needleman and Wunsch Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group WO 01/05975 PCT/US00/16271 19of integers consisting of from 0 to 200. Thus, for examplc, the gap creation and gap extension penalties can each independently be: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff& Henikoff(1989) Proc. Natl. Acad. Sci. USA 89:10915).

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Altschul et J. Mol. Bio. 215: 403-410, 1990) or to the value obtained using the GAP program using default parameters (see the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA).

As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino WO 01/05975 PCT/US00/16271 acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) as implemented in the program PC/GENE (Intelligenetics, Mountain View. California, USA).

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, 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 may occur, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypcptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

WO 01/05975 PCT/US00/16271 -21 (ii) The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Optionally, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.

DETAILED DESCRIPTION OF THE INVENTION Overview The present invention provides, among other things, compositions and methods for modulating increasing or decreasing) the level of polynucleotides and polypeptides of the present invention in plants. In particular, the polynucleotides and polypeptides of the present invention can be expressed temporally or spatially, at developmental stages, in tissues, and/or in quantities, which are uncharacteristic ofnon-recombinantly engineered plants. Thus, the present invention provides utility in such exemplary applications as in the control of homologous recombination efficiency or transformation efficiency in plants.

The present invention also provides isolated nucleic acid comprising polynucleotides of sufficient length and complementarity to a gene of the present invention to use as probes or amplification primers in the detection, quantitation, or isolation of gene transcripts. For example, isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for desired transgenic plants, for detecting mutations in the gene substitutions, deletions, or additions), for monitoring upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allelic variants (polymorphisms), orthologs, or paralugs of the gene, or for site directed mutagenesis in eukaryotic cells (see, U.S.

Patent No. 5,565,350). The isolated nucleic acids of the present invention can also be used WO 01/05975 PCT/USO0/16271 -22for recombinant expression of their encoded polypeptides, or for use as immunogens in the preparation and/or screening of antibodies. The isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more genes of the present invention in a host cell, tissue, or plant. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation.

The present invention also provides isolated proteins comprising a polypeptide of the present invention preprocnzyme, procnzyme, or enzymes). The present invention also provides proteins comprising at least one epitope from a polypeptide of the present invention. The proteins of the present invention can be employed in assays for enzyme agonists or antagonists of enzyme function, or for use as immunogens or antigens to obtain antibodies specifically immunoreactive with a protein of the present invention. Such antibodies can be used in assays for expression levels, for identifying and/or isolating nucleic acids of the present invention from expression libraries, for identification of homologous polypeptides from other species, or for purification of polypeptides of the present invention.

The isolated nucleic acids and polypeptides of the present invention can be used over a broad range of plant types, particularly monocots such as the species of the family Gramineae including Hordeum, Secale, Triticum. Sorghum S. bicolor) and Zea Z. mays). The isolated nucleic acid and proteins of the present invention can also be used in species from the genera: Cucurbita, Rosa, Vitis. Juglans. Fragaria, Lotus. Medicago.

Onobrychis, Trifolium, Trigonella. Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus. Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum. Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus. Asparagus. Antirrhinum, Heterocallis. Nemesis, Pelargonium. Panieum, Penniselum, Ranunculus, Senecio, Salpiglossis, Cucumis. Browaalia. Glycine, Pisum, Phaseolus, Lolium, Oryza, and A vena.

Nucleic Acids The present invention provides, among other things, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising a polynucleotide of the present invention.

A polynucleotide of the present invention is inclusive of: WO 01/05975 PCT/US00/16271 23 a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10 and conservatively modified and polymorphic variants thereof, including exemplary polynucleotides of SEQ ID NOS: 1, 3, 5, 7, 9; polynucleotide sequences of the invention also include the maize R2 polynucleotide sequences as contained in plasmids deposited with Amcrican Type Culture Collection (ATCC) and assigned Accession Number 207193.

a polynucleotide which is the product of amplification from a Zea mays nucleic acid library using primer pairs which selectively hybridize under stringent conditions to loci within a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, or the scqucnccs as contained in the ATCC deposit assigned Accession Number 207193; wherein the polynucleotide has substantial sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9; or the sequences as contained in the ATCC deposit assigned Accession Number 207193.

a polynucleotide which selectively hybridizes to a polynucleotide of or a polynucleotide having a specified sequence identity with polynucleotides of or a polynucleotide encoding a protein having a specified number of contiguous amino acids from a prototype polypeptide, wherein the protein is specifically recognized by antisera elicited by presentation of the protein and wherein the protein does not dctcctably immunoreact to antisera which has been fully immunosorbed with the protein; complementary sequences of polynucleotides of(a), or and a polynucleotide comprising at least a specific number of contiguous nucleotides from a polynucleotide of or The polynucleotides of SEQ ID NOS: 1, 3, 5, 7, 9 are contained in plasmids deposited with American Type Culture Collection (ATCC) on April 6,1999 and assigned Accession Number 207193. American Type Culture Collection is located at 10801 University Blvd., Manassas, VA 20110-2209.

The ATCC deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The deposit is provided as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. Section 112.

A. Polynucleotides Encoding A Polypeptide of the Present Invention or Conservatively Modified or Polymorphic Variants Thereof WO 01/05975 PCT/US00/16271 -24- As indicated in above, the present invention provides isolated nucleic acids comprising a polynucleotide of the present invention, wherein the polynucleotide encodes a polypeptide of the present invention, or conservatively modified or polymorphic variants thereof. Accordingly, the present invention includes polynucleotides of SEQ ID NOS: 1, 3, 5, 7, 9, and the sequences as contained in the ATCC deposit assigned Accession Number 207193, and silent variations of polynucleotides encoding a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10. The present invention further provides isolated nucleic acids comprising polynucleotides encoding conservatively modified variants of a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10. Conservatively modified variants can be used to generate or select antibodies immunoreactive to the non-variant polypeptide. Additionally, the present invention further provides isolated nucleic acids comprising polynucleotides encoding one or more allelic (polymorphic) variants ofpolypeptides/polynuclcotidcs. Polymorphic variants are frequently used to follow segregation of chromosomal regions in, for example, marker assisted selection methods for crop improvement.

B. Polynucleotides Amplified from a Zea mays Nucleic Acid Library As indicated in above, the present invention provides an isolated nucleic acid comprising a polynucleotide of the present invention, wherein the polynucleotides are amplified from a Zea mays nucleic acid library. Zea mays lines B73, PHREI, A632, BMS- P2#10, W23, and Mo 17 are known and publicly available. Other publicly known and available maize lines can be obtained from the Maize Genetics Cooperation (Urbana, IL).

The nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing. cDNA libraries can be normalized to increase the representation of relatively rare cDNAs. In optional embodiments, the cDNA library is constructed using a full-length cDNA synthesis method.

Examples of such methods include Oligo-Capping (Maruyama, K. and Sugano, S. Gene 138: 171-174, 1994), Biotinylated CAP Trapper (Carninci, Kvan, et al. Genomics 37: 327-336, 1996), and CAP Retention Procedure (Edery, Chu, et al. Molecular and Cellular Biology 15: 3363-3371, 1995). cDNA synthesis is often catalyzed at 55 0 C to prevent formation of RNA secondary structure. Examples of reverse transcriptases that are relatively stable at these temperatures are SUPERSCRIPT II Reverse Transcriptase (Life Technologies, Inc.), AMV Reverse Transcriptase (Boehringer WO 01/05975 PCT/US00n6271 Mannheim) and RETROAMP Reverse Transcriptase (Epicentre). Rapidly growing tissues, or rapidly dividing cells are preferably used as mRNA sources.

The present invention also provides subsequences of the polynucleotides of the present invention. A variety of subsequences can be obtained using primers which selectively hybridize under stringent conditions to at least two sites within a polynucleotide of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it. Primers are chosen to selectively hybridize, under stringent hybridization conditions, to a polynucleotide of the present invention. Generally, the primers are complementary to a subsequence of the target nucleic acid which they amplify. As those skilled in the art will appreciate, the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired amplification conditions.

In optional embodiments, the primers will be constructed so that they selectively hybridize under stringent conditions to a sequence (or its complement) within the target nucleic acid which comprises the codon encoding the carboxy or amino terminal amino acid residue the 3' terminal coding region and 5' terminal coding region, respectively) of the polynucleotides of the present invention. Optionally within these embodiments, the primers will be constructed to selectively hybridize entirely within the coding region of the target polynucleotide of the present invention such that the product of amplification of a cDNA target will consist of the coding region of that cDNA. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to Thus, the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length. Those of skill will recognize that a lengthened primer sequence can be employed to increase specificity of binding annealing) to a target sequence. A non-annealing sequence at the 5'end of a primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.

The amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra. The resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of WO 01/05975 PCT/USOO/16271 -26the present invention. Methods for protein synthesis from PCR derived templates are known in the art and available commercially. See, Amersham Life Sciences, Inc, Catalog '97, p.

3 54 Methods for obtaining 5' and/or 3' ends of a vector insert are well known in the art.

See, RACE (Rapid Amplification of Complementary Ends) as described in Frohman, M. in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H.

Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc., San Diego), pp. 28-38 (1990)); see also, U.S. Pat. No. 5,470,722, and Current Protocols in Molecular Biology, Unit 15.6, Ausubel, et al., Eds, Greene Publishing and Wiley-Interscience, New York (1995); Frohman and Martin, Techniques 1:165 (1989).

C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of(A) or (B) As indicated in above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide of sections or as discussed above. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising the polynucleotides of or For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated or otherwise complementary to a cDNA from a dicot or monocot nucleic acid library.

Exemplary species ofmonocots and dicots include, but are not limited to: corn, canola, soybean, cotton, wheat, sorghum, sunflower, oats, sugar cane, millet, barley, and rice.

Optionally, the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA libraries can be normalized to increase the representation of rare sequences. Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.

WO 01/05975 PCT/US00/16271 -27- D. Polynucleotides Having a Specific Sequence Identity with the Polynucleotides of(A).

or (C) As indicated in above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides have a specified identity at the nucleotide level to a polynucleotide as disclosed above in sections or above. The percentage of identity to a reference sequence is at least and, rounded upwards to the nearest integer, can be expressed as an integer selected from the group of integers consisting of from 60 to 99. Thus, for example, the percentage of identity to a reference sequence can be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Optionally, the polynucleotides of this embodiment will encode a polypeptide that will share an epitope with a polypeptide encoded by the polynucleotides of sections or Thus, these polynucleotides encode a first polypeptide which elicits production ofantisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide of(A), or However, the first polypeptide does not bind to antisera raised against itself when the antisera has been fully immunosorbed with the first polypeptide. Hence, the polynucleotides of this embodiment can be used to generate antibodies for use in, for example, the screening of expression libraries for nucleic acids comprising polynucleotides of(A), or or for purification of, or in immunoassays for, polypeptides encoded by the polynucleotides of(A), or The polynucleotides of this embodiment embrace nucleic acid sequences which can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.

Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries. This method involves the screening of large collections ofpeptides for individual members having the desired function or structure.

Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 100 amino acids long, and often from about 8 to 15 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide WO 01/05975 PCTfUS00/16271 28 sequence encoding the particular displayed peptide sequence. Such methods are described in PCT patent publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent publication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, CA).

E. Polynucleotides Encoding a Protein Having a Subsequence from a Prototype Polypeptide and is Cross-Reactive to the Prototype Polypeptide As indicated in above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides encode a protein having a subsequence of contiguous amino acids from a prototype polypeptide of the present invention such as are provided in above. The length of contiguous amino acids from the prototype polypeptide is selected from the group of integers consisting of from at least 10 to the number of amino acids within the prototype sequence. Thus, for example, the polynucleotide can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100, contiguous amino acids from the prototype polypeptide. Further, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any integer ofnucleotides from 1 to the number ofnucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

The proteins encoded by polynucleotides of this embodiment, when presented as an immunogen, elicit the production ofpolyclonal antibodies which specifically bind to a prototype polypeptide such as but not limited to, a polypeptide encoded by the polynucleotide of(a) or above. Generally, however, a protein encoded by a polynucleotide of this embodiment does not bind to antisera raised against the prototype polypeptide when the antisera has been fully immunosorbed with the prototype polypeptide. Methods of making and assaying for antibody binding specificity/affinity are well known in the art. Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays and the like.

WO 01/05975 PCT/US00/16271 -29- In a preferred assay method, fully immunosorbed and pooled antisera which is elicited to the prototype polypeptide can be used in a competitive binding assay to test the protein. The concentration of the prototype polypeptide required to inhibit 50% of the binding of the antisera to the prototype polypeptide is determined. If the amount of the protein required to inhibit binding is less than twice the amount of the prototype protein, then the protein is said to specifically bind to the antisera elicited to the immunogen.

Accordingly, the proteins of the present invention embrace allelic variants, conservatively modified variants, and minor recombinant modifications to a prototype polypeptide.

A polynucleotide of the present invention optionally encodes a protein having a molecular weight as the non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptides of the present invention. Molecular weight can be readily determined by SDS-PAGE under reducing conditions. Optionally, the molecular weight is within 15% of a full length polypeptide of the present invention, more preferably within 10% or and most preferably within or 1% of a full length polypeptide of the present invention.

Optionally, the polynucleotides of this embodiment will encode a protein having a specific enzymatic activity at least 50%, 60%, 80%, or 90% of a cellular extract comprising the native, endogenous full-length polypeptide of the present invention.

Further, the proteins encoded by polynucleotides of this embodiment will optionally have a substantially similar affinity constant and/or catalytic activity the microscopic rate constant, keat) as the native endogenous, full-length protein. Those of skill in the art will recognize that kt/Km value determines the specificity for competing substrates and is often referred to as the specificity constant. Proteins of this embodiment can have a kcat/Km value at least 10% of a full-length polypeptide of the present invention as determined using the endogenous substrate of that polypeptide. Optionally, the kca/Km value will be at least 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, or 95% the kca/Km value of the full-length polypeptide of the present invention.

Determination of kcat, Km and kcat/Km can be determined by any number of means well known to those of skill in the art. For example, the initial rates the first 5% or less of the reaction) can be determined using rapid mixing and sampling techniques continuous-flow, stopped-flow, or rapid quenching techniques), flash photolysis, or relaxation methods temperature jumps) in conjunction with such exemplary methods of measuring as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance, or WO 01/05975 PCT/US00/16271 radioactive procedures. Kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of(A)-(E) As indicated in above, the present invention provides isolated nucleic acids comprising polynucleotides complementary to the polynucleotides of paragraphs A-E, above. As those of skill in the art will recognize, complementary sequences base-pair throughout the entirety of their length with the polynucleotides of sections have 100% sequence identity over their entire length). Complementary bases associate through hydrogen bonding in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.

G. Polynucleotides Which are Subsequences of the Polynucleotides of(A)-(F) As indicated in above, the present invention provides isolated nucleic acids comprising polynucleotides which comprise at least 15 contiguous bases from the polynucleotides of sections through as discussed above. The length of the polynucleotide is given as an integer selected from the group consisting of from at least to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of. Thus, for example, polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, or 200 contiguous nucleotides in length from the polynucleotides Optionally, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

The subsequences of the present invention can comprise structural characteristics of the sequence from which it is derived. Alternatively, the subsequences can lack certain structural characteristics of the larger sequence from which it is derived such as a poly (A) tail. Optionally, a subsequence from a polynucleotide encoding a polypeptide having at least one linear epitope in common with a prototype polypeptide sequence as provided in above, may encode an epitope in common with the prototype sequence. Alternatively, WO 01/05975 PCT/US00/16271 31 the subsequence may not encode an epitope in common with the prototype sequence but can be used to isolate the larger sequence by, for example, nucleic acid hybridization with the sequence from which it's derived. Subsequences can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.

Construction of Nucleic Acids The isolated nucleic acids of the present invention can be made using standard recombinant methods, synthetic techniques, or combinations thereof In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a monocot. In preferred embodiments the monocot is Zea mays.

The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hcxahistidine marker sequence provides a convenient means to purify the proteins of the present invention. A polynucleotide of the present invention can be attached to a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb. Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).

WO01/05975 PCT/US00/16271 32- A. Recombinant Methods for Constructing Nucleic Acids The isolated nucleic acid compositions of this invention, such as RNA, cDNA.

genomic DNA, or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. While isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art, the following highlights some of the methods employed.

Al. mRNA Isolation and Purification Total RNA from plant cells comprises such nucleic acids as mitochondrial RNA, chloroplastic RNA, rRNA, tRNA, hnRNA and mRNA. Total RNA preparation typically involves lysis of cells and removal of organelles and proteins, followed by precipitation of nucleic acids. Extraction of total RNA from plant cells can be accomplished by a variety of means. Frequently, extraction buffers include a strong detergent such as SDS and an organic denaturant such as guanidinium isothiocyanate, guanidine hydrochloride or phenol.

Following total RNA isolation, poly(A) mRNA is typically purified from the remainder RNA using oligo(dT) cellulose. Exemplary total RNA and mRNA isolation protocols are described in Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). Total RNA and mRNA isolation kits are commercially available from vendors such as Stratagene (La Jolla, CA), Clonetech (Palo Alto, CA), Pharmacia (Piscataway, NJ), and (Paoli Inc., PA). See also, U.S.

Patent Nos. 5,614,391; and, 5,459,253. The mRNA can be fractionated into populations with size ranges of about 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 kb. The cDNA synthesized for each of these fractions can be size selected to the same size range as its mRNA prior to vector insertion. This method helps eliminate truncated cDNA formed by incompletely reverse transcribed mRNA.

A2. Construction ofa cDNA Library Construction ofa cDNA library generally entails five steps. First, first strand cDNA synthesis is initiated from a poly(A) mRNA template using a poly(dT) primer or WO 01/05975 PCT/US00/16271 -33 random hexanucleotides. Second, the resultant RNA-DNA hybrid is converted into double stranded cDNA, typically by reaction with a combination of RNAse H and DNA polymerase I (or Klenow fragment). Third, the termini of the double stranded cDNA are ligated to adaptors. Ligation of the adaptors can produce cohesive ends for cloning.

Fourth, size selection of the double stranded cDNA eliminates excess adaptors and primer fragments, and eliminates partial cDNA molecules due to degradation of mRNAs or the failure of reverse transcriptase to synthesize complete first strands. Fifth, the cDNAs arc ligated into cloning vectors and packaged. cDNA synthesis protocols are well known to the skilled artisan and are described in such standard references as: Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley- Interscience, New York (1995). cDNA synthesis kits are available from a variety of commercial vendors such as Stratagene or Pharmacia.

A number ofcDNA synthesis protocols have been described which provide substantially pure full-length cDNA libraries. Substantially pure full-length cDNA libraries are constructed to comprise at least 90%, and more preferably at least 93% or full-length inserts amongst clones containing inserts. The length of insert in such libraries can be from 0 to 8, 9, 10, 11, 12, 13, or more kilobase pairs. Vectors to accommodate inserts of these sizes are known in the art and available commercially. Sec, e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12 kb cloning capacity).

An exemplary method of constructing a greater than 95% pure full-length cDNA library is described by Carinci et al., Genomics, 37:327-336 (1996). hi that protocol, the cap-structure of eukaryotic mRNA is chemically labeled with biotin. By using streptavidin-coated magnetic beads, only the full-length first-strand cDNA/mRNA hybrids are selectively recovered after RNase I treatment. The method provides a high yield library with an unbiased representation of the starting mRNA population. Other methods for producing full-length libraries are known in the art. Sec, Edery et al.. Mol. Cell Biol.,15(6):3363-3371 (1995); and, PCT Application WO 96/34981.

A3. Normalized or Subtracted cDNA Libraries A non-normalized cDNA library represents the mRNA population of the tissue it was made from. Since unique clones are out-numbered by clones derived from highly WO 01/05975 PCT/USOO/16271 34 expressed genes their isolation can be laborious. Normalization of a cDNA library is the process of creating a library in which each clone is more equally represented.

A number of approaches to normalize cDNA libraries are known in the art. One approach is based on hybridization to genomic DNA. The frequency of each hybridized cDNA in the resulting normalized library would be proportional to that of each corresponding gene in the genomic DNA. Another approach is based on kinetics. If cDNA reannealing follows second-order kinetics, rarer species anneal less rapidly and the remaining single-stranded fraction of cDNA becomes progressively more normalized during the course of the hybridization. Specific loss of any species of cDNA, regardless of its abundance, does not occur at any Cot value. Construction of normalized libraries is described in Ko, Nucl. Acids. Res., 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl.

Acad. 88:1943-1947 (1991); U.S. Patents 5,482,685, and 5,637,685. In an exemplary method described by Soares et al., normalization resulted in reduction of the abundance of clones from a range of four orders of magnitude to a narrow range of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. In this procedure, cDNA prepared from one pool of mRNA is depleted of sequences present in a second pool of mRNA by hybridization. The cDNA:mRNA hybrids are removed and the remaining un-hybridized cDNA pool is enriched for sequences unique to that pool. See, Foote et al. in, Plant Molecular Biology.

A Laboratoy Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique, 3(2):58-63 (1991); Sive and St. John, Nucl. Acids Res., 16(22):10937 (1988); Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995); and, Swaroop et al., Nucl. Acids Res., 19)8):1954 (1991). cDNA subtraction kits are commercially available. See, PCR-Select (Clontech, Palo Alto, CA).

A4. Construction of a Genomic Library To construct genomic libraries, large segments of genomic DNA are generated by fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate molecular biological techniques and WO 01/05975 PCT/US00/16271 instructions sufficient to direct persons of skill through many construction, cloning, and screening methodologies are found in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997).

Kits for construction ofgenomic libraries are also commercially available.

A5. Nucleic Acid Screening and Isolation Methods The cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein.

Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.

The degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.

The nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting WO 01/05975 PCT/US00/16271 -36the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., U.S. Patent No. 4,683,202 (1987); and, PCR Protocols A Guide to Methods and Applications, Innis et al., Eds., Academic Press Inc., San Diego, CA (1990).

Commercially available kits for genomic PCR amplification are known in the art. See. e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have also been described. Wilfinger et al. describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques, 22(3): 481-486 (1997).

In that method, a primer pair is synthesized with one primer annealing to the 5' end of the sense strand of the desired cDNA and the other primer to the vector. Clones are pooled to allow large-scale screening. By this procedure, the longest possible clone is identified amongst candidate clones. Further, the PCR product is used solely as a diagnostic for the presence of the desired cDNA and does not utilize the PCR product itself. Such methods are particularly effective in combination with a full-length cDNA construction methodology, above.

B. Synthetic Methods for Constructing Nucleic Acids The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth.

Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol.

68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22(20): 1859-1862 (1981), using an automated synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res., 12: 6159-6168 (1984); and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is best employed for sequences of WO 01/05975 PCT/US00/16271 37 about 100 bases or less, longer sequences may be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polypeptide of the present invention, for example a cDNA or a genomic sequence encoding a full length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.

For example, plant expression vectors may include a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region one conferring inducible or constitutive, environmentally- or developmentallyregulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

A plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the or promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other transcription initiation regions from various plant genes known to those of skill. One exemplary promoter is the ubiquitin promoter, which can be used to drive expression of the present invention in embryos or embryogenic callus, particularly in maize.

Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise WO 01/05975 PCT/US00/16271 -38environmental or developmental control. Such promoters are referred to here as "induciblc" promoters. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light.

Examples of inducible promoters are the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light.

Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. Exemplary promoters include the anther specific promoter 5126 Patent Nos.

5,689,049 and 5,689,051), glob-I promoter, and gamma-zein promoter. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.

Both heterologous and non-heterologous endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue. Thus, in some embodiments, the nucleic acid construct will comprise a promoter functional in a plant cell, such as in Zea mays, operably linked to a polynucleotide of the present invention.

Promoters useful in these embodiments include the endogenous promoters driving expression of a polypeptide of the present invention.

In some embodiments, isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a nonheterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Patent 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters can be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. Gene expression can be modulated under conditions suitable for plant growth so as to alter the total concentration and/or alter the composition of the polypeptides of the present invention in plant cell. Thus, the present invention provides compositions, and methods for making, heterologous promoters and/or WO 01/05975 PCT/US00/16271 -39enhancers operably linked to a native, endogenous non-heterologous) form of a polynucleotide of the present invention.

Methods for identifying promoters with a particular expression pattern, in terms of, tissue type, cell type, stage of development, and/or environmental conditions, are well known in the art. See, The Maize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3 rd edition, Chapter 6, Sprague and Dudley, Eds., American Society of Agronomy, Madison, Wisconsin (1988).

A typical step in promoter isolation methods is identification of gene products that are expressed with some degree of specificity in the target tissue. Amongst the range of methodologies are: differential hybridization to cDNA libraries; subtractive hybridization; differential display; differential 2-D protein gel electrophoresis; DNA probe arrays; and isolation of proteins known to be expressed with some specificity in the target tissue. Such methods are well known to those of skill in the art. Commercially available products for identifying promoters are known in the art such as Clontech's (Palo Alto, CA) Universal GenomeWalker Kit.

For the protein-based methods, it is helpful to obtain the amino acid sequence for at least a portion of the identified protein, and then to use the protein sequence as the basis for preparing a nucleic acid that can be used as a probe to identify either genomic DNA directly, or preferably, to identify a cDNA clone from a library prepared from the target tissue. Once such a cDNA clone has been identified, that sequence can be used to identify the sequence at the 5' end of the transcript of the indicated gene. For differential hybridization, subtractive hybridization and differential display, the nucleic acid sequence identified as enriched in the target tissue is used to identify the sequence at the 5' end of the transcript of the indicated gene. Once such sequences are identified, starting either from protein sequences or nucleic acid sequences, any of these sequences identified as being from the gene transcript can be used to screen a genomic library prepared from the target organism. Methods for identifying and confirming the transcriptional start site are well known in the art.

In the process of isolating promoters expressed under particular environmental conditions or stresses, or in specific tissues, or at particular developmental stages, a number of genes are identified that are expressed under the desired circumstances, in the desired tissue, or at the desired stage. Further analysis will reveal expression of each particular gene in one or more other tissues of the plant. One can identify a promoter with WO 01/05975 PCT/US00/16271 activity in the desired tissue or condition but that does not have activity in any other common tissue.

To identify the promoter sequence, the 5' portions of the clones described here are analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10 bp located approximately 20 to 40 base pairs upstream of the transcription start site. Identification of the TATA box is well known in the art. For example, one way to predict the location of this element is to identify the transcription start site using standard RNA-mapping techniques such as primer extension, SI analysis, and/or RNase protection. To confirm the presence of the AT-rich sequence, a structure-function analysis can be performed involving mutagenesis of the putative region and quantification of the mutation's effect on expression of a linked downstream reporter gene. See, The Maize Handbook, Chapter 114, Freeling and Walbot, Eds., Springer, New York, (1994).

In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element the CAAT box) with a series of adenines surrounding the trinucleotide G (or T) N G. J. Messing et al., in Genetic Engineering in Plants, Kosage, Meredith and Hollaender, Eds., pp. 221-227 1983. In maize, there is no well conserved CAAT box but there are several short, conserved protein-binding motifs upstream of the TATA box. These include motifs for the trans-acting transcription factors involved in light regulation, anaerobic induction, hormonal regulation, or anthocyanin biosynthesis, as appropriate for each gene.

Once promoter and/or gene sequences are known, a region of suitable size is selected from the genomic DNA that is 5' to the transcriptional start, or the translational start site, and such sequences are then linked to a coding sequence. If the transcriptional start site is used as the point of fusion, any of a number of possible 5' untranslated regions can be used in between the transcriptional start site and the partial coding sequence. If the translational start site at the 3' end of the specific promoter is used, then it is linked directly to the methionine start codon of a coding sequence.

Ifpolypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added can be derived from, for WO 01/05975 PCT/US00/16271 -41 example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8: 4395- 4405 (1988); Callis et al., Genes Dev. 1: 1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.

Use of maize introns Adhl-S intron 1, 2, and 6, the Bronze-1 intron are known in the art.

See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptlI gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. in Enzymol., 153:253-277 (1987). These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene, 61:1-11 WO 01/05975 PCT/US00/16271 -42- (1987) and Berger et al., Proc. Natl. Acad. Sci. 86:8402-8406 (1989). Another useful vector herein is plasmid pBIl01.2 that is available from Clontech Laboratories, Inc.

(Palo Alto, CA).

A polynucleotide of the present invention can be expressed in either sense or antisense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable plant characteristics. Antisense technology can be conveniently used to inhibit gene expression in plants. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed.

The construct is then transformed into plants and the antisense strand of RNA is produced.

In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, Sheehy et al., Proc. Nat 7. Acad. Sci. (USA) 85: 8805-8809 (1988); and Hiatt et al., U.S. Patent No.

4,801,340.

Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2: 279-289 (1990) and U.S. Patent No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNAcleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585- 591 (1988).

A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov, V. et al., Nucleic Acids Res (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of WO 01/05975 PCT/US00/16271 -43similar work by the same group is that by Knorre, D. et al.. Biochimie (1985) 67:785- 789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (JAm Chem Soc (1987) 109:1241-1243). Meyer, R. et al., JAm Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home, et al.. JAm Chem Soc (1990) 112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, JAm Chem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Fetcritz et al., J Am. Chem. Soc. 113:4000 (1991). Various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Patent Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and, 5,681941.

Proteins The isolated proteins of the present invention comprise a polypeptide having at least 10 amino acids encoded by any one of the polynucleotides of the present invention as discussed more fully, above, or polypeptides which are conservatively modified variants thereof. The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the present invention. Optionally, this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or The present invention further provides a protein comprising a polypeptide having a specified sequence identity with a polypeptide of the present invention. The percentage of sequence identity is an integer selected from the group consisting of from 60 to 99.

Exemplary sequence identity values include 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.

WO 01/05975 PCT/US00/16271 -44- As those of skill will appreciate, the present invention includes catalytically active polypeptides of the present invention enzymes). Catalytically active polypeptides have a specific activity of at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or and most preferably at least 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (kca /Km) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide. Typically, the Km will be at least 30%, 40%, or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90%. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.

Generally, the proteins of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention. Further, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those of skill in the art. A preferred immunoassay is a competitive immunoassay as discussed, infra. Thus, the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.

Expression of Proteins in Host Cells Using the nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells. The cells produce the protein in a non-natural condition in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by WO 01/05975 PCT/US00/16271 incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. One of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.

Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids poly His) placed on either terminus to create conveniently located purification sequences. Restriction sites or termination codons can also be introduced.

A. Expression in Prokaryotes Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically ofplasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp.

WO 01/05975 PCT/US00/16271 -46and Sahnonella (Palva, et al., Gene 22: 229-235 (1983); Mosbach, et al., Nature 302: 543- 545 (1983)).

B. Expression in Eukaryotes A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a polynucleotide of the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.

Synthesis of heterologous proteins in yeast is well known. Sherman, et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast. Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers Invitrogen).

Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter the CMV promoter, a HSV tk promoter orpgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites an WO 01/05975 PCTIUSOO/16271 -47large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See, Schneider, J. Embryol. Exp. Morphol. 27: 353-365 (1987).

As with yeast, when higher animal or plant host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J Virol. 45: 773-781 (1983)). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol. II a Practical Approach, D.M. Glover, Ed., IRL Press, Arlington, Virginia pp. 213-238 (1985).

Transfection/Transformation of Cells The method of transformation/transfection is not critical to the instant invention; various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they may be directly applied.

Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for effective transformation/transfection may be employed.

A. Plant Transformation A DNA sequence coding for the desired polypeptide of the present invention, for example a cDNA or a genomic sequence encoding a full length protein, will be used to construct a recombinant expression cassette which can be introduced into the desired plant.

WO 01/05975 PCT/US00/16271 48 Isolated nucleic acids of the present invention can be introduced into plants according to techniques known in the art. Generally, recombinant expression cassettes as described above and suitable for transformation of plant cells are prepared. The isolated nucleic acids of the present invention can then be used for transformation. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained.

Transformation protocols may vary depending on the type of plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al (1986) Proc. Nail. Acad. Sci. USA 83:5602-5606, Agrobacterium mediated transformation (see for example, Zhao et al. U.S. Patent 5,981,840; Hinchee et al. (1988) Biotechnology 6:915-921), direct gene transfer (Paszkowski et al (1984) EMBO J. 3:2717- 2722), and ballistic particle acceleration (see, for example, Sanford et al. U.S. Patent 4,945,050; Tomes et al. "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment" In Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995); and McCabe et al. (1988) Biotechnology 6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet. 22:421- 477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Phisiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al.

(1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes et al. "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment" In Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer-Verlag, Berlin (1995) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize) Fromm et al. (1990) Biotechnology 8:833- 839 (maize); Hooykaas-Van Slogteren Hooykaas (1984) Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.

(1985) In The Experimental Manipulation of Ovule Tissues ed. G.P. Chapman et al. pp.

197-209. Longman, NY (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); LI et al.

(1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

WO 01/05975 PCT/US00/16271 -49- The cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports, 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells Animal and lower eukaryotic yeast) host cells are competent or rendered competent for transfection by various means. There arc several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art. Kuchler, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).

Synthesis of Proteins The proteins of the present invention can be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology.

Vol. 2: Special Methods in Peptide Synthesis, Part Merrifield, et al., J Am. Chem. Soc.

2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, 11. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide)) is known to those of skill.

WO 01/05975 PCT/US00/16271 Purification of Proteins The proteins of the present invention may be purified by standard techniques well known to those of skill in the art. Recombinantly produced proteins of the present invention can be directly expressed or expressed as a fusion protein. The recombinant protein is purified by a combination of cell lysis sonication, French press) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein.

The proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Patent No. 4,511,503. The protein may then be isolated from cells expressing the protein and further purified by standard protein chemistry techniques as described herein. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.

Transgenic Plant Regeneration Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillilan Publishing Company, New York, pp. 124- 176 (1983); and Binding, Regeneration ofPlants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).

The regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch et al., Science, 227:1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species WO 01/05975 PCT/US00/16271 51 being transformed as described by Fraley et al., Proc. Natl. Acad. Sci. 80:4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phvs. 38: 467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3 rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wisconsin (1988). One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants.

Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.

Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard WO 01/05975 PCT/US00/16271 -52immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNApositive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant native, nontransgenic). Back-crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated.

Modulating Polvpeptide Levels and/or Composition The present invention further provides a method for modulating increasing or decreasing) the concentration or ratio of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the the ratio of the polypeptides of the present invention in a plant. The method comprises introducing into a plant cell with a recombinant expression cassette comprising a polynucleotide of the present invention as described above to obtain a transformed plant cell, culturing the transformed plant cell under plant cell growing conditions, and inducing or repressing expression of a polynucleotide of the present invention in the plant for a time WO 01/05975 PCT/US00/16271 53 sufficient to modulate concentration and/or the ratios of the polypeptides in the plant or plant part.

In some embodiments, the concentration and/or ratios of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a gene to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, Kmiec, U.S.

Patent 5,565,350; Zarling et al., PCT/US93/03868. And in some embodiments, an isolated nucleic acid a vector) comprising a promoter sequence is transfected into a plant cell.

Subsequently, a plant cell comprising the promoter operably linked to a polynucleolide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or ratios of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and discussed briefly, supra.

In general, concentration or the ratios of the polypeptides is increased or decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned recombinant expression cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra. Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound. Inducible promoters and inducing compounds which activate expression from these promoters are well known in the art. In preferred embodiments, the polypeptides of the present invention are modulated in monocots, particularly maize.

WO 01/05975 PCT/US00/16271 54 Molecular Markers The present invention provides a method of genotyping a plant compnsing a polynucleotide of the present invention. Optionally, the plant is a monocot, such as maize or sorghum. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance.

See, Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer- Verlag, Berlin (1997). For molecular marker methods, see generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in: Genome Mapping in Plants (ed. Andrew H.

Paterson) by Academic Press/R. G. Landis Company, Austin, Texas, pp.7-21.

The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs). RFLPs are the product of allelic differences between DNA restriction fragments resulting from nucleotide sequence variability. As is well known to those of skill in the art, RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed.

Differences in fragment size among alleles represent an RFLP. Thus, the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a gene of the present invention.

In the present invention, the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention. In preferred embodiments, the probes are selected from polynucleotides of the present invention.

Typically, these probes are cDNA probes or restriction-enzyme treated Pst I) genomic clones. The length of the probes is discussed in greater detail, supra, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases WO 01/05975 PCT/US00/16271 in length. Generally, however, the probes are less than about 1 kilobase in length.

Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement. Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and SstI. As used herein the term "restriction enzyme" includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of digesting genomic DNA of a plant with a restriction enzyme; hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; detecting therefrom a RFLP. Other methods of differentiating polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis and chemical mismatch cleavage (CMC). Thus, the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe. Generally, the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention gene, mRNA). The nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the nucleic acid probe comprises a polynucleotide of the present invention.

UTRs and Codon Preference In general, translational efficiency has been found to be regulated by specific sequence elements in the 5' non-coding or untranslated region UTR) of the RNA.

WO 01/05975 PCT/US00/16271 -56- Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res. 15:8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing el al., Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present invention provides 5' and/or 3' UTR regions for modulation of translation of heterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host such as to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference" available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides that can be used to determine a codon usage frequency can be any integer from I to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.

Sequence Shuffling The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. WO 97/20078. See also, Zhang, et al.

Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or WO 01/05975 PCT/US00/16271 -57 in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotidcs which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be a decreased Km and/or increased Kca, over the wild-type protein as provided herein. In other embodiments, a protein or polynculeotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.

Generic and Consensus Sequences Polynucleotides and polypeptides of the present invention further include those having: a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and, a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention. The generic sequence of the present invention comprises each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide, sequence, respectively. The individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phylums, or kingdoms. For example, a polynucleotide having a consensus sequences from a gene family ofZea mays can be used to generate antibody or nucleic acid probes or primers to other Gramineae species such as wheat, rice, or sorghum.

Alternatively, a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence will be at least 9, 10, 15, 20, 25, 30, or 40 amino acids in length, or 20, 30, 40, 50, 100, or 150 nucleotides in length. As those of skill in the art are aware, a conservative amino acid substitution can be used for amino acids which differ WO 01/05975 PCT/US00/16271 58 amongst aligned sequence but are from the same conservative substitution group as discussed above. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.

Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous, or paralogous sequences as provided herein. Optionally, similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability Various suppliers of sequence-analysis software are listed in chapter 7 of 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.

(Supplement 30). A polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001, and most preferably less than about 0.0001, or 0.00001. Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, WI) PILEUP software, Vector NTI's (North Bethesda, MD) ALIGNX, or Genecode's (Ann Arbor, MI) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.

Assays for Compounds that Modulate Enzymatic Activity or Expression The present invention also provides means for identifying compounds that bind to substrates), and/or increase or decrease modulate) the enzymatic activity of, catalytically active polypeptides of the present invention. The method comprises contacting a polypeptide of the present invention with a compound whose ability to bind to or modulate enzyme activity is to be determined. The polypeptide employed will have at least 20%, preferably at least 30% or 40%, more preferably at least 50% or 60%, and most preferably at least 70% or 80% of the specific activity of the native, full-length polypeptide of the present invention enzyme). Generally, the polypeptide will be present in a range sufficient to determine the effect of the compound, typically about 1 nM to 10 jM.

Likewise, the compound will be present in a concentration of from about 1 nM to 10 .iM.

Those of skill will understand that such factors as enzyme concentration, ligand concentrations substrates, products, inhibitors, activators), pH, ionic strength, and WO 01/05975 PCT/US00/16271 59 temperature will be controlled so as to obtain useful kinetic data and determine the presence of absence of a compound that binds or modulates polypeptide activity. Methods of measuring enzyme kinetics is well known in the art. See, Segel, Biochemical Calculations, 2 n d ed., John Wiley and Sons, New York (1976).

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Example 1 This example describes the construction of the cDNA libraries.

Total RNA Isolation Total RNA was isolated from corn tissues with TRIZOL Reagent (Life Technology Inc. Gaithersburg, MD) using a modification of the guanidine isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi (Chomczynski, and Sacchi, N. Anal.

Biochem. 162, 156 (1987)). In brief, plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIZOL Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.

Poly(A)+ RNA Isolation The selection of poly(A)+ RNA from total RNA was performed using POLYATIRACT system (Promega Corporation. Madison, WI). In brief, biotinylated oligo(dT) primers were used to hybridize to the 3' poly(A) tails on mRNA. The hybrids were captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand. The mRNA was washed at high stringency conditions and eluted by RNase-free deionized water.

cDNA Library Construction cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the SUPERSCRIPT Plasmid System (Life Technology Inc. Gaithersburg, WO 01/05975 PCT/USOO/16271 MD). The first stand ofcDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by SUPERSCRIPT Reverse Transcriptase II at The second strand of cDNA was labeled with alpha- 32 P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA molecules smaller than 500 base pairs and unligated adapters were removed by SEPHACRYL-S400 chromatography. The selected cDNA molecules were ligated into pSPORTI vector in between of Not I and Sal I sites.

Example 2 This example describes cDNA sequencing and library subtraction.

Sequencing Template Preparation Individual colonies were picked and DNA was prepared either by PCR with M 13 forward primers and M 13 reverse primers, or by plasmid isolation. All the cDNA clones were sequenced using M13 reverse primers.

Q-bot Subtraction Procedure cDNA libraries subjected to the subtraction procedure were plated out on 22 x 22 cm 2 agar plate at density of about 3,000 colonies per plate. The plates were incubated in a 37 0 C incubator for 12-24 hours. Colonies were picked into 384-well plates by a robot colony picker, Q-bot (GENETIX Limited). These plates were incubated overnight at 37 0

C.

Once sufficient colonies were picked, they were pinned onto 22 x 22 cm 2 nylon membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864 colonies.

These membranes were placed onto agar plate with appropriate antibiotic. The plates were incubated at 37°C for overnight.

After colonies were recovered on the second day, these filters were placed on filter paper prewetted with denaturing solution for four minutes, then were incubated on top of a boiling water bath for additional four minutes. The filters were then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution was removed by placing the filters on dry filter papers for one minute, the colony side of the filters were place into Proteinase K solution, incubated at 37C for 40-50 minutes. The filters were placed on dry filter papers to dry overnight. DNA was then cross-linked to nylon membrane by UV light treatment.

WO 01/05975 PCT/US00/16271 -61 Colony hybridization was conducted as described by Sambrook,J., Fritsch, E.F. and Maniatis, (in Molecular Cloning: A laboratory Manual, 2 d Edition). The following probes were used in colony hybridization: 1. First strand cDNA from the same tissue as the library was made from to remove the most redundant clones.

2. 48-192 most redundant cDNA clones from the same library based on previous sequencing data.

3. 192 most redundant cDNA clones in the entire corn sequence database.

4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA, listed in SEQ ID NO: 11, removes clones containing a poly A tail but no cDNA.

cDNA clones derived from rRNA.

The image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.

Example 3 This example describes identification of the gene from a computer homology search.

Gene identities were determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. et al., (1990) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm.

The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993)) provided by the NCBI. In some cases, the sequencing data from two or more clones containing overlapping segments of DNA were used to construct contiguous DNA sequences.

WO 01/05975 T/JO167 PCT[USOO/16271 -62 Example 4 This example shows the conservation of structural motifs in Maize RuvB orthologues. The deduced amino acid sequences of the Maize RuvB orthologucs arc indicated by their SEQ ID number. The amino acids constituting the ATP binding motifs, also known as Walker Box A and Walker Box B, are indicated (Walker Box A is highlighted while Walker Box B is shown in bold). Regions o f high homology to bacterial RuvB are underlined and the putative heptad repeat region is italicized.

SeqId-No6 Seq ld-No8 SeqId-No2 SeqId-No4 SecId-NolO SeqId-No6 SecLId -No8 Seqld-No2 SeqId-No4 SecLId-NolO SeqId-No6 SeqId No8 SecLId -No2 SeqId-No4 SecId-Nol 0 SecLId-No6 SecLId -No8 Seqi dNo2 SecLId-No4 SeaIdNolO SeqId o6 SecId -No8 SeqI dNo2 SeqId -No4 SeqId-Nol 0 SecLId dNo6 SeqId -NoB SeqId dNo2 SecLIdNo4 SeqId-NoIO SecLId -No6 SeqId No8 SeqId -No2 SecLId-No4 SeqId No 10 1

MRIEEVQSTS

MRIEEVQSTS

MRIEEVQSTS

MRIEEVQSTS

MRIEEVQSTS

KKQR IATHTH

KKQRIATHTH

KKQRIATHTH

KKQRIATHTH

KKQRIATHTH

IKGLGLD.AN

I KGLG3LDQAN IKGLGLD .AN IKGLGLD .AN

IKGLGLD.AN

GMSMPLAAGF

GMSMPLAAGF

GMAIALAAGF

GMAIALAAGF

GMAIALAAGF

51 GLAVDMIRQK KMAGRALLLA GPPATGKTA-L ALGIAQELGS GLAVDMIRQK KMAGRALLLA GPPATGKTAL ALGIAQELGS GLAVDMIRQKKMAGRAVLLVGPPATGKTrAL ALGIAQELGS GLAVDMIRQK KMAGRAVLLA GPPATGKTAL ALGIAQELGS QK KMAGRAVLLA GPPATGKTAL A.GIAQELGS 101

EVYSSEVKKT

E VY SSE VKKT

EVYSSEVKKT

EVYSSEVKKT

EVYSSEVKKT

151 YAKS ISHfVII YAKSISHVI I

YAKSIS-VII

YAKS ISHVI I YAKS ISHVII 201 VKRVGRCDS F VKRVGRCDS F VKRVGRCDS F VKRVGRCDS F VKRVGRCDS F 251

PQGGQDILSL

PQGGQDILSL

PQGGQDILSL

PQGQDILSL

PQQ;GQD.XLSL

EVLMENFRR-A

EVLMENFRR-A

EVLMENFRRA

EVLMENFRRA

EVLMENFRRA

GLKTVKGTKQ

GL KTVKGTKQ

SLKTVKGTKQ

SLKTVXGTKQ

SLKTVKGTKQ

ATEYDLEAEE

ATEYDLEAE

ATEYDL BAE

ATEYDLEAEE

ATE YDLEAEE IGLR IKENKE

IGLRIKENKE

IGLRI KENKE IGLRI KENKE

IGLRIKENKE

LKLDPS IVDA LKtAJPS IYDA LKLDSS IYDA

LKLDSSIYDA

LKLDSSIYDA

YVPIPKGEVH

Y-VPIPKGEVH

YVPIPKGEVH

YVPIPKGEVH

YVPZPKGEV.H

VYEGEVIELS

VYEGEVIELS

VYEGEVTELS

VYEGEVTELS

VYEGEVTELS

LIKEKVAVGD

LI KEKVAVGO LI KEKVAVGD

LIKEKVAVGD

LIKE KVAVGD

KKKEIVQDVT

KKKIEVQDVT

K-KKEIVQDVT

KKKEIVQDVT

KKKEIVQDVT

NKVVNVRYIDE

NKVVNRYIDE

NKVVNRYIDE

NKVVNVRYIDE

NKVVNRYIDE

VGQAAAREAA

VGQAAA-REAA

VGQSAAREAA

VGQAAAREAA

100

KVPFCPMVGS

KVPFCPM4VGS

KVPFCPMVGS

KVPFCPMVGS

KVPFCPMVGS

150

PEEAESTTGG

PEEAESTTGG

PEEAESTTGG

PEEAESTTGG

PEEAESTrGG 200

VIYIEANSGA

VIYIEANSGA

VI YIEANSGA VIY IEANSGA

VIYIEANSGA

250

LI-DLDAAIJAQ

LI-DLDAALVAQ

LHDLDAANAQ

LKDLDAANAQ

LHDLDAANqAQ 300

GIAELVPGVL

GIA.ELVPGVL

GIA.ELVPGVL

GIAELVPGVL

GIAELVPGVL

350

GTDMTSPHGI

GTDMTSPHGI

GTDMTS PHGI

GTDMTSPHGI

GTDMTSPHGI

MGQMMKPRKT EITEKLRQEI MGQMMKPRKT EITEKLRQEI MGQMMKPRKT EITEKLRQEI MGQNMKPRKT EITEKLRQEI MGQMMKPRKT EITEKLRQEI 301 FIDEVHMLD I FIDEVHMLD I

FIDEVHMLDI

PIDKVHMLD I FIDEVHMLD I

ECFSYLNRAL

ECFSYLNRAL

ECFSYLNRAL

ECFS YLNRAL

ECFSYLNRAL

ESPLSPIVIL

ESPLSPIVIL

ESPLSPIVIL

ESPLSPIVIL

ESPLSPIVIL

ATNRG ICNVR ATNRG ICNVR

ATNRGICNVR

ATNRGICNVR

ATNRGI CNVR WO 01/05975 SeqId-No6 SeqId -No8 SeqId No2 SecLId -No4 Seq Id No 10 Sec Id dNo6 Seq -Id-No8 SeqId-No2 SecLId -No4 SecLId-No1o 351

PVDLLDRLVI

PVDLLDRLVI

PLLDRLVI

PVDLLDRLVI

PVDLLDRLVI

PCTUSOO/1 6271 400

LAY-LGEIGQQ

LAYLGEIGQQ

LAYLGEIGQ

LAYLGE IGQQ

LAYLGEIGQQ

I RTETYGPTE

IRTETYGPTE

I RTETYGPTE

IRTETYGPTE

I RTETYGPTE MI101LA IRAQ MIQI LA IRAQ MIQ01LA IRAQ M IQ ILA IRAQ MI101LAIRAO

VEEIDIDEES

VEEIDIDEES

VED IDMDEES

VEEIDMDEES

VEEIDMDEES

401

TSLRHAIQLL

TSLRHAIQLL

TSLRHAIQLI

TSLRHAIQLI

TSLRHAIQLI

S PASVVAKTN S PASVVAKTN S PASVVSKTN S PASVVSKTN

SPASVVSKTN

GRE KMCKADL

GREKMCKADL

GREKICKADL

GREKICKADL

GREKICKADL

EEVSGLYLDA

EEVSGLYLDA

EEVSGLYLDA

EEVSGLYLDA

EEVSGLYLDA

450 KS SAR:LLQEQ

KSSARLLQEQ

KSSARLLQEQ

KS SARLLQEQ

KSSARLLQEQ

451 SeqId No6 QERYIT* SecLId NoB QERYIT* SecLIdNo2 QERYIT* SecLIdNo4 QERYIT* SecLId-NolO QERYIT* The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, patent applications, and computer programs cited herein are hereby incorporated by refere nce.

EDITORIAL NOTE APPLICATION NUMBER 58726/00 The following Sequence Listing pages 1 to 17 are part of the description. The claims pages follow on pages "64" to "68".

WO 01/05975 PCT/USOO/16271

I

SEQUENCE LISTING ':110> Pioneer Hi-Bred International, Inc.

':120> Maize Orthologues of Bacterial IRuvB: cDNAs and Uses Thereof ':130> 1121-PCT <150> US 60/144,112 <151> 1999-07-16 <160> 11 <170> FastSEQ for Windows Version <210> 1 ':211> 1845 ':212> DNA <213> Zea mays <:220> <221> CDS <222> (85) (1449) ':400> 1 gacccacgcg tccgccatcc cccgccccca aattttggca gctccacaga aacagagagc gcataaccgg cgttgttggc ggcg atg agg atc gag gag gtg cag tcg acc ill Met Arg Ile Giu Glu Val Gin Ser Thr 1 tcg aag aag cag cgc atc gcc acc cac acc cac atc aag gga ctc ggc 159 Ser Lys Lys Gin Arg Ile Ala Thr His Thr His Ilie Lys Gly Leu Gly 15 20 ctc gac gcc aat ggg atg gcg att gcg ttg gcg gcg ggg ttc gtg ggc 207 Leu Asp Ala Asn Gly Met Ala Ile Ala Leu Ala Ala Gly Phe Val Gly 35 cag tcg gcg gcg cgc gag gcg gcc ggg ctg gcg gtc gac atg att cgc 255 Gin Ser Ala Ala Arg Giu Ala Ala Gly Leu Ala Val Asp Met Ile Arg so cag aaa aag atg gcc ggc cgc gcg gtg ctc ctt gtg ggt ccg ccc gcc 303 Gin Lys Lys Met Ala Giy Arg Ala Val Leu Leu Val Gly Pro Pro Ala 65 acg ggc aag acg gcg cta gcg cic ggc ata gcc cag gag ctc ggc agc 351 Thr Gly Lys Thr Ala Leu Ala Leu Gly Ile Ala Gin Giu Leu Gly Ser 80 aag gtc cct ttc tgc cct atg gta gga tca gaa gtg tac tcc tcg gag 399 Lys Val Pro Phe Cys Pro Met Val Giy Ser Giu Val Tyr Ser Ser Giu 95 100 105 gtc aag aaa act gag gtg ctg atg gaa aat ttc cgt aga gct ata ggt 447 Val Lys Lys Thr Giu Val Leu Met Giu Asn Phe Arg Arg Ala Ile Gly 110 115 120 WO 01/05975PC/JO/67 PCTfUSOO/16271 t tg Leu ct t Leu at t Ile ctg Leu 170 gca Ala aga Arg gaa Glu ata Ile cca Pro 250 cca Pro gtg Val gt t Val1 tat Tyr gc t Ala 330 ata Ile cca Pro 140 cat His tta Leu ggt Gly ggt Gly tat Tyr 220 cag Gin ggt Gly aag Lys aa t As n ttC Phe 300 aac Asn aat Asn aag Lys 125 gaa Glu gta Val gat Asp gat Asp aga Arg 205 gt t Val1 gat Asp ggc Gly act Thr aga Arg 285 att Ile cgt Arg agg Arg aaa Lys gag Glu agc Ser 160 att Ile tac Tyr tct Se r ccc Pro ct t Leu 240 att.

Ile ac Thr gat Asp gtc Val gag Glu 320 tgt Cys gag Giu ag t Se r 145 tta Leu tat Tyr att Ile tt t Phe aa a Lys 225 cat His ttg Leu gaa Glu gaa Giu cac His 305 agc Ser aa t As n gtt Val1 130 aca Thr aag Lys gat Asp gaa Glu gct Al a 210 ggt Gly gac Asp t cc Ser aaa Lys gga Gly 290 atg Met cca Pro gta Val1 gaa Glu ggt Giy gt t Val ctg Leu 180 aat Asn gaa Glu gtC Val1 gat Asp atg Met 260 cgc Arg gca Al a gat Asp tca Ser gga Gly 340 495 543 591 639 687 735 783 831 879 927 975 1023 1071 1119 1167 cca cat ggt ata cca gtg gat ctt ctagaagtgggatatcg Pro His Gly Ile Pro Val Asp Leu LeuApArLeValileAg gat agg ttg gtg att att cgg Asp Arg Leu Val Ile Ile Arg PCTfUSOOf16271 WO 01/05975 aca gag aca Thr Giu Thr ggc cct act gag Gly Pro Thr Giu ata cag ata ttg gct atc cga Ile Gin Ile Leu Ala Ile Arg 375 gca caa gtg Ala Gin Val 380 gag gac att gat Giu Asp Ilie Asp atg Met 385 gat gaa gaa agt Asp Glu Giu Ser gct tat tta Ala Tyr Leu ggc gag Gly Glu 395 atc gga cag cag aca Ile Gly Gin Gin Thr 400 tct tta aga. cat gct Ser Leu Arg His Ala 405 att caa ttg ata Ile Gin Leu Ile cct gcc agc gtg gtc tca. aag act aat Pro Ala Ser Val Val Ser Lys Thr Asn aga gag aaa ata Arg Glu Lys Ile 1215 1263 1311 1359 1407 1449 1509 1569 1629 1689 1749 1809 1845 aag gct gat ctc Lys Ala Asp Leu gaa gtc agt ggg ctc tat ttg gat gcc Giu Val Ser Gly Leu Tyr Leu Asp Ala aaa tcc Lys Ser 440 tcg gct cgg ctg ctc cag gag caa Ser Ala Arg Leu Leu Gin Glu Gin 445 gaa aga tac atc acc Giu Arg Tyr Ile Thr 455 tagatttgga atctagtgaa tgaattttgc ctgttcacgc ttttatcttg ctatctttgt wttwaaaaaa.

tcacctgtcg ttgatctgct agtgcctgct actgttcgct ctcatcggtg aaccatggat aaaaaaaaaa tggaagt ct c tcacaggtct tgtgttagtc gat tagattg tccggaatct aatggatagc aaaaaaaaaa gaagagaatg tggagcgagc tccagagaag gtcaccggtg gtgcctccac attcttacag aaaaaa tagttgccag acatttcggg acttggtacc caggaattgc gggttgtatt aatgcaactt ctcgaaagtc ggggacggct ggcatattgc cgtgtgtgtt ggcccgaacc gcatggcttt <210> 2 <211> 455 <212> PRT <213> Zea mays <400> 2 Arg Ile Giu Glu Val Gin Ser Thr Ser Lys Lys Gin Arg Ile Ala Met Ala Thr His Thr Ile Ala Leu Ala Gly Leu Ala Val Leu Ilie Lys Gly Leu Leu Asp Ala Asn Gly Ala Ala Gly Phe Gin Ser Ala Ala Arg Giu Ala Ala Val Asp Arg Gin Lys Ala Gly Arg LeU Val Pro Ala Thr Thr Ala Leu Gly Ile Ala Leu Gly Ser Val Pro Phe Cys Pro Met Val Giy Ser Met Giu Asn Val Tyr Ser Ser Lys Lys Thr Giu Val Leu 110 Giu Asn Lys Arg Arg Ala Leu Arg Ile Glu Val 130 Ser Thr Giu Gly Glu Giu Leu Ser Pro Giu Glu Ala Glu Thr Giy Gly Lys Ser Ile Val Ile Ile 145 Leu Tyr Lys Thr Val Asp Ala Leu 180 Lys 165 Ile Thr Lys Gin Lys Leu Asp Ser Val Gly Asp Val 190 Ser Ile 175 Ile Tyr Lys Giu Lys Val PCTIUSOO/16271 WO 01/05975 PTU0167 Glu Ala Asn Ser 195 Ala Thr Giu Tyr 210 Gly Giu Val His Asp Leu Asp Ala 245 Ser Leu Met Gly 260 Lys Leu Arg Gi 275 Gly Ile Ala Giu 290 Met Leu Asp Ile Pro Leu Ser Pro 325 Val Arg Gly Thr 340 beu Asp Arg Leu 355 Met Ile Gin Ile 370 Asp Giu Giu SerI Leu Arg His Ala 405 Thr Asn Gly Arg 420 Gly Leu Tyr Leu 435 Gin Giu Arg Tyr 450 <210> 3 <211> 1912 <212> DNA <213> Zea mays <220> <221> CDS <222> Al a Leu 215 Lys Asn Met Ile Val1 295 Cys Val1 Met Ile Ala 375 Ala Gin Lys Ala Thr 455 Arg 205 Val1 Asp Giy Thr Arg 285 Ile Arg Arg Ile Tyr 365 Glu Gly Ser Leu Leu 445 1458) <400> 3 acccacgcgt ccgcaaattt gttgcggcgg gagagccgga gaggaggcag ctccacagaa acagagagcg cataaccggc ggcgttggcg geg atg agg atc gag gag gtg cag Met Arg Ile Giu Giu Val Gin 1 teg acc teg aag aag cag cgc atc gce acc cac ace cac ate aag gga Ser Thr Ser Lys Lys Gin Arg Ile Ala Thr His Thr His Ile Lys Gly 15 etc ggc cic gac 9cc aat ggg atg gcg att gcg ttg gcg gcg ggg ttc Leu Gly Leu Asp Ala Asn Gly Met Ala Ile Ala Leu Ala Ala Gly Phe 30 gtg ggc cag gcg gcg gcg cgc gag gcg gcc ggg ctg gcg gte gac atg Val Gly Gin Ala Ala Ala Arg Giu Ala Ala Gly Leu Ala Val Asp Met 45 50 114 162 210 258 WO 01/059'75 PCT/UJSOO/16271 att cgc cag aag aag atg gcc ggc cgc gcg gtg ctc ctt gcg ggt ccg 306 Ile Arg Gin Lys Lys Met Ala Gly Arg Ala Val Leu Leu Ala Gly Pro 65 ccc gcc acg ggc aag acg gcg cta gcg ctc ggc ata. gcc cag gag ctc 354 Pro Ala Thr Gly Lys Thr Ala Leu Ala Leu Gly Ile Ala Gin Giu Leu 80 ggc agc aag gtc cct ttc tgt cct atg gta gga tca. gaa. gtg tac tcc 402 Gly Ser Lys Val Pro Phe Cys Pro Met Val Gly Ser Giu Val Tyr Ser 95 100 tcg gag gtc aag aaa act gag gtg ctg atg gaa aat ttc cgt aga gct 450 Ser Glu Val Lys Lys Thr Glu Val Leu Met Giu Asn Phe Axg Arg Ala 105 110 115 ata ggt ttg cgt ata aag gee aac aaa gag gtt tat gaa gga gag gtt 498 Ile Gly Leu Arg Ile Lys Glu Asn Lys Giu Val Tyr Glu Gly Glu Val 120 125 130 135 act gaa ctt tcc cca. gaa gag gct gag agt aca act ggt gga. tat gca. 546 Thr Giu Leu Ser Pro Glu Giu Ala Glu Ser Thr Thr Gly Gly Tyr Ala 140 145 150 aa agc att agc cat gta atc atc agc tta aag act gtt aaa ggg act 594 Lys Ser Ile Ser His Val Ile Ile Ser Leu Lys Thr Val Lys Gly Thr 155 160 165 aag caa ctg aag tta. gat tct tca att tat gat gct ctg atc aag gaa 642 Lys Gin Leu Lys Leu Asp Ser Ser Ile Tyr Asp Ala Leu Ile Lys Glu 170 175 180 aag gtg gca gtg ggt gat gtt ata. iac atc ga gca aet agt gga. gca 690 Lys Val Ala Vai Gly Asp Val Ile Tyr Ile Giu Ala Asn Ser Gly Ala 185 190 195 gtg aea age gtt ggt aga tgt gat tct ttt gct aca gaa tac gat ctt 738 Vai Lys Arg Vai Gly Arg Cys Asp Ser Phe Ala Thr Glu Tyr Asp Leu 200 205 210 215 gee gct gaa gag tat gtt cct atc ccc ass ggt gas gtc cat sag ass 786 Glu Ala Giu Glu Tyr Val Pro Ile Pro Lys Gly Glu Val His Lys Lys 220 225 230 aea gaa att gtg cag get gtc aca ctt cat gec ctt gat gca gcs aat 834 Lys Giu Ile Val Gin Asp Val Thr Leu His Asp Leu Asp Ala Ala Asn 235 240 245 gct cag cca cee ggt ggc cee get ett ttg tcc ctt atg ggc cag etg 882 Ala Gin Pro Gin Gly Gly Gin Asp Ile Leu Ser Leu Met Giy Gin Met 250 255 260 atg aaa cca cga aag, act gaa atc acc gee aaa. cta cgc caa gas att 930 Met Lys Pro Arg Lye Thr Glu Ile Thr Giu Lye Leu Arg Gin Glu Ile 265 270 275 aat sag gtg gte. aet age tat etc get gsa gga. att gca gag ctt gta 978 Asn Lys Vai Val Asn Axg Tyr Ile Asp Glu Gly Ile Ala Giu Leu Val 280 285 290 295 WO 0 1/05975 PTUO/67 PCTIUSOO/16271 cct ggt gtt ttg Pro Gly Val Leu att gat gag gtc Ile Asp Glu Val atg ttg gat atc gaa tgt Met Leu Asp Ile Giu CIS 310 ttt tct tat Phe Ser Tyr aac cgt gca ttg Asn Arg Ala Leu agc cca tta tca Ser Pro Leu Ser cca atc gtg Pro Ile Val 325 act gat atg Thr Asp Met ata ctt gct aca aat agg gga ata tgt aat gta aga Ile Leu Ala Thr Asn Arg Gly Ile Cys Asn Val Arg aca agt Thr Ser 345 cca cat ggt ata Pro His Gly Ile gtg gat ctt cta Val Asp Leu Leu agg ctg gtg att Arg Leu Val Ile cgg aca gag aca Arg Thr Giu Thr ggc cct act gag Giy Pro Thr Giu ata cag ata ttg Ile Gin Ile Leu atc cga gca caa Ile Arg Ala Gin gag gag att gat atg gat gaa gaa agt Glu Giu Ile Asp Met Asp Giu Giu Ser 385 ctt get Leu Ala 390 1026 1074 1122 1170 1218 1266 1314 1362 1410 1458 1518 1578 1638 1698 1758 1818 1878 1912 tat tta ggc gag atc gga cag cag aca Tyr Leu Gly Giu Ile Giy Gin Gin Thr 395 400 tct ttg aga cat Ser Leu Arg His gct att caa Ala Ile Gin 405 aga gag aaa Arg Glu Lys ttg ata tea Leu Ilie Ser 410 cct gec age gtg Pro Ala Ser Val gte Val1 415 tca aag act aat Ser Lys Thr Asn gga Gly 420 ate tgc aag gct gat etc gag gaa gtc agt ggg ctc tat ttg gat gce Ilie Cys Lys Ala Asp Leu Giu Giu Val Ser Giy Leu Tyr Leu Asp Ala 4 2 C 430 435 tee teg gct egg Ser Ser Ala Arg ctc cag gag eaa Leu Gin Glu Gin gaa aga tac ate Glu Arg Tyr Ile tagatttgga atetagtgca teggggggaa gttceggeat attgccgtgt ttggcccgaa ttgcatggct taaaaaaaaa tctcctgtcg ttgatctgct cggcttgaat attgctcgtt gtgtttttat ccctatcttt ttattattte aaaaaaaaaa tggaagtctc tcaeaggtte tttgcagtgc cacgcactgt ettgctcatc gtaaccatgg taaatgteca aaaaaaaaaa gaagagaatg atagtctact etgettgtgt tcgetgatta ggtgtccgga ataatggata taaagcataa aaaa tagttgccag ggtettggag tagtctccar gat tggteae gtetgcctec ggattettac cgaaatgttt ctcgaaagte cgagcaeatt agaagacttg cggtgcagga acgggt tgta agaatgeaac ctacaacnitw <210> 4 <211> 455 <212> PRT <213> Zea mays <400> 4 Met Arg Ile Giu Giu Val Gin Ser Thr Ser Lys 1 5 10 Thr His Thr His Ile Lys Gly Leu Giy Leu Asp 25 Ile Ala Leu Ala Ala Gly Phe Val Giy Gin Ala 40 Ala Gly Leu Ala Val Asp Met Ile Arg Gin Lys Lys Gin Arg Ile Ala Ala Asn Gly Met Ala Ala Ala Arg Giu Ala Lys met Ala Gly Arg WO 01105975 PTU0167 PCT/USOO/16271 Ala Val Leu Gly Val Gly Met Glu Giu Val 130 Ser Thr 145 Leu Lys Tyr Asp Ile Glu Phe Ala 210 Lys Gly 225 His Asp Leu Ser Giu Lys Glu Gly 290 His Met 305 Ser Pro Asn Val Leu Leu Giu Met 370 Met Asp 385 Ser Leu Lys Thr Ser Gly Gln Gin 450 Leu Ala Glu 100 Phe Giu Gly Val1 Leu 180 As n Giu Val Asp Met 260 Arg Al a Asp Ser Gly 340 Arg Gin Giu His Gly 420 Tyr Arg Al a Gin Val1 Arg Giy Gly Lys 165 Ile Ser Tyr His Al a 245 Gly Gin Giu Ile Pro 325 Thr Ieu Ile Ser Al a 405 Arg Leu Tyr Giy 70 Glu Tyr Arg Giu Tyr 150 Gly Lys Gly Asp Lys 230 Ala Gin Glu Leu Glu 310 I le Asp Val Leu Leu 390 Ile Glu Asp Pro Leu Ser Ala Val1 135 Aia Thr Giu Ala Leu 215 Lys Asn Met Ile Val1 295 Cys Val1 Met Ile Ala 375 Ala Gin Lys Al a Pro Gly Ser Ilie 120 Th r Lys Lysa Lys Val1 200 Giu Lys Al a Met Asn 280 Pro Phe Ile Thr Ile 360 Ile Tyr Leu Ile Ala Thr Ser Lys 90 Glu Val 105 Gly Leu Giu Leu Ser Ile Gin Leu 170 Val Ala 185 Lys Arg Ala Glu Glu Ile Gin Pro 250 Lys Pro 265 Lys Val Gly Val Ser Tyr Lcu Ala 330 Ser Pro 345 Arg Thr Arg Ala Leu Gly Ilie Ser 410 Cys Lys 425 Gly Val Lys Arg Ser Ser 155 Lys Val1 Val1 Glu Val1 235 Gin Arg Val Leu Leu 315 Thr His Giu Gin Gi1u 395 Pro Al a Thr Phe Th r Lys 125 Glu Val1 Asp Asp Arg 205 Val1 Asp Gly Thr Arg 285 Ile Arg Arg Ile Tyr 365 Glu Gly Ser Leu Leu Pro Val1 Asn Al a Ile Ser 175 Ile Asp Ile Thr Asp 255 Ile Ile Glu Leu Ile 335 Val1 Pro Ile Gin Val1 415 Glu Lys Ser Ser Ala Arg Leu Leu Gin Glu 440 445 Ile Thr 455 <210> <211> 1886 <212> DNA <213> Zea mays <220> <221> CDS <222> (82) (1446) <400> WO 01/05975 WO 0105975PCT/USO011627 I tcgacccacg cgtccggcgg cggccggcgg gagagagcag ctctagagag aacagagagc gcataactag cggcggcggc g atg agg ata gag Met Arg Ile Giu gag gtg Giu Val caa tog acc tcg Gin Ser Thr Ser cac ato aag ggc otc ggc ctc His Ile Lys Gly Leu Gly Leu gcg gcg ggg ttc gtg ggc cag Ala Ala Gly Phe Val Gly Gin gog gtc gac atg atc cgo cag Ala Val Asp Met Ile Arg Gin ctt gcg ggc ccg 000 goc acg Leu Ala Gly Pro Pro Ala Thr gcg cag gag cto ggc ago aag Ala Gin Glu Leu Giy Ser Lys gaa gtg tac too tca. gag gtc Glu Vai Tyr Ser Ser Giu Val 100 105 tto ogt aga got ata ggt ttg Phe Arg Arg Ala Ile Gly Leu 120 gaa gga gag gtt att gaa ott Giu Gly Glu Val Ile Giu Leu 135 ggt gga. tat gcg aaa ago att Gly Gly Tyr Ala Lys Ser Ile 150 gtc aaa. ggg act aag caa. ttg Val Lys Gly Thr Lys Gin Leu 165 170 ttg ato aag gaa aag gtg gca.

Leu Ile Lys Giu Lys Val Ala 180 185 aat agt gga goa gtg aaa aga Asn Ser Gly Ala Val Lys Arg 200 gaa tat gat ott gaa got gaa Giu Tyr Asp Leu Giu Ala Glu 215 gto oat aag aaa aag gaa ata Val His Lys Lys Lys Glu Ile 230 gag tat gtt cot ato coo aaa ggt gaa Glu Tyr Val Pro Ilie Pro Lys Gly Giu WO 01/05975 WO 0105975PCT/USOO/16271 cag gat gtc aca Gin Asp Val Thr 9 cat gac ctt His Asp Leu gat gca Asp Ala 245 gca aat gcc cag cca Ala Asn Ala Gin Pro caa gyt ggc caa Gin Gly Gly Gin gat Asp 255 att ttg tee ctt Ilie Leu Ser Leu ggc cag atg atg Gly Gin Met Met aag cca Lys Pro 265 cgg aag act Arg Lys Thr gta aac aga Val Asn Arg 285 atc acc gaa aag Ile Thr Giu Lys cgc caa gaa atc Arg Gin Giu Ile aat aag gtg Asn Lys Val 280 cet ggt gtt Pro Gly Val tat atc gac gaa Tyr Ilie Asp Giu atc gca gag ctt Ile Ala Giu Leu ttg ttc Leu Phe 300 att gat gag gte Ile Asp Giu Val atg ttg gat att Met Leu Asp Ile tgc ttt tet tat Cys Phe Ser Tyr ctt aac cgt gcs ttg Leu Asn Arg Ala Leu 315 agc cca tta tca Ser Pro Leu Ser att gtg ata ctc Ile Val Ile Leu acg aat aga gga Thr Asn Arg Gly ata tgt aat gtg aga gga acc gat atg seq agt cca.

Ile Cys Asn Val Arg Gly Thr Asp Met Thr Ser Pro 335 340 345 cat ggt ata cca gtg gac ctt cta.

His Gly Ile Pro Val Asp Leu Leu 350 agg ttg gtg att Arg Leu Val Ile att egg aca Ile Arg Thr 360 atc cga gca Ile Arg Al1a gsa sea tat Giu Thr Tyr 365 ggc cct act gag Giy Pro Thr Giu ata eag ata etg Ile Gin Ile Leu cas gtg Gin Vai 380 gas gag att gat atc gat gaa gaa agt Giu Giu Ile Asp Ile Asp Giu Giu Ser 385 get tat tta ggc Aia Tyr Leu Gly 1023 1071 1119 1167 1215 1263 1311 1359 1407 1456 1516 1576 1636 1696 1756 1816 atc gga eag cag Ile Giy Gin Gin tet ttg aga cat Ser Leu Arg His att eag ttg eta.

Ile Gin Leu Leu cct gee age gtg gte gca aag ace aae Pro Aia Ser Vai Vai Ala Lys Thr Asn 415 ggg Gly 420 aga gsa aag stg Arg Glu Lys Met tgc aag Cys Lys 425 get gae etc gag gsa gte age ggg etc tat ttg gat gee Ala Asp Leu Giu Giu Val Ser Gly Leu Tyr Leu Asp Ala 430 435 aaa tee teg Lys Ser Ser 440 tagacttgca get ege etg etc Ala Arg Leu Leu 445 eag gag aa Gin Giu Gin gas aga tac ate Giu Arg Tyr Ilie tctectgetg gettcagtat ctgtttgaat tgttgettgt cgggtgtgtt tattgaccct tggaaggaaa gtcgtagtet tttgtagtae tesegececg ttcaeeecct agct ttgtaa agcctcgaag aeaagtet tg ctgttttttg ttaacaccga egctaatega ecatgcacat agaaatgcat gagagageac ttagccteea ttaagattgg tgtccagaat gatttctttt tctgeaaagt gt ttctgegg gegaagaett eccagecgtg ctgtgtgcee ttaacgctga gcgtcgstct ggaaggaaaa gatteegges caggaattge acgggtgttg tggatggcac WO 01/05975 PCT/USO0/16271 ctggatctct gtccatggcc atcgtaatcc cactgtctgg agcagggttt catgaaaaaa 1876 aaaaaaaaaa 1886 <210> 6 <211> 455 <212> PRT <213> Zea mays <400> 6 Met Arg Ile Glu Glu Val Gin Ser Thr Ser Lys Lys Gin Arg Ile Ala 1 5 10 Thr His Thr His Ile Lys Gly Leu Gly Leu Asp Ala Asn Gly Met Ser 25 Met Pro Leu Ala Ala Gly Phe Val Gly Gin Ala Ala Ala Arg Glu Ala 40 Ala Gly Leu Ala Val Asp Met Ile Arg Gin Lys Lys Met Ala Gly Arg 55 Ala Leu Leu Leu Ala Gly Pro Pro Ala Thr Gly Lys Thr Ala Leu Ala 70 75 Leu Gly Ile Ala Gin Glu Leu Gly Ser Lys Val Pro Phe Cys Pro Met 90 Val Gly Ser Glu Val Tyr Ser Ser Glu Val Lys Lys Thr Glu Val Leu 100 105 110 Met Glu Asn Phe Arg Arg Ala Ile Gly Leu Arg Ile Lys Glu Asn Lys 115 120 125 Glu Val Tyr Glu Gly Glu Val Ile Glu Leu Ser Pro Glu Glu Ala Glu 130 135 140 Ser Thr Thr Gly Gly Tyr Ala Lys Ser Ile Ser His Val Ile Ile Gly 145 150 155 160 Leu Lys Thr Val Lys Gly Thr Lys Gin Leu Lys Leu Asp Pro Ser Ile 165 170 175 Tyr Asp Ala Leu Ile Lys Glu Lys Val Ala Val Gly Asp Val Ile Tyr 180 185 190 Ile Glu Ala Asn Ser Gly Ala Val Lys Arg Val Gly Arg Cys Asp Ser 195 200 205 Phe Ala Thr Glu Tyr Asp Leu Glu Ala Glu Glu Tyr Val Pro Ile Pro 210 215 220 Lys Gly Glu Val His Lys Lys Lys Glu Ile Val Gin Asp Val Thr Leu 225 230 235 240 His Asp Leu Asp Ala Ala Asn Ala Gin Pro Gin Gly Gly Gin Asp Ile 245 250 255 Leu Ser Leu Met Gly Gin Met Met Lys Pro Arg Lys Thr Glu Ile Thr 260 265 270 Glu Lys Leu Arg Gin Glu Ile Asn Lys Val Val Asn Arg Tyr Ile Asp 275 280 285 Glu Gly Ile Ala Glu Leu Val Pro Gly Val Leu Phe Ile Asp Glu Val 290 295 300 His Met Leu Asp Ile Glu Cys Phe Ser Tyr Leu Asn Arg Ala Leu Glu 305 310 315 320 Ser Pro Leu Ser Pro Ile Val Ile Leu Ala Thr Asn Arg Gly Ile Cys 325 330 335 Asn Val Arg Gly Thr Asp Met Thr Ser Pro His Gly Ile Pro Val Asp 340 345 350 Leu Leu Asp Arg Leu Val Ile Ile Arg Thr Glu Thr Tyr Gly Pro Thr 355 360 365 Glu Met Ile Gin Ile Leu Ala Ile Arg Ala Gin Val Glu Glu Ile Asp 370 375 380 Ile Asp Glu Glu Ser Leu Ala Tyr Leu Gly Glu Ile Gly Gin Gin Thr 385 390 395 400 Ser Leu Arg His Ala Ile Gin Leu Leu Ser Pro Ala Ser Val Val Ala 405 410 415 Lys Thr Asn Gly Arg Glu Lys Met Cys Lys Ala Asp Leu Glu Glu Val WO 01/05975

PCT

420 42S 430 Ser Gly Leu Tyr Leu Asp Ala Lys Ser Ser Ala Arg Leu Leu Gin Giu 435 440 445 Gin Gin Giu Arg Tyr Ile Thr 450 455 <210> 7 <211> 1898 <212> DNA <213> Zea mays <220> <221> CDS <222> (166) (1533) <400> 7 attacgccaa gctctaatac gactcactat agggaaagct ggtacgcctg caggtacc tccggaattc ccgggtcgac ccacgcgtcc gtgccttgag gcggcggccg gcgggagac gcagctctag agagaacaga gagcgcataa ctagcggcgg cggcg atg agg ata g~ 'IUSOO/16271 99 ga ag Met Arg Ile Giu 1 gtg Val1 aag Lys Ala gtc Val gc9 Ala cag Gln gtg Val1 cgt Arg gga Gly gga Gly 150 cgc Arg aat Asn gcg Al a atg Met acg Thr ttc Phe act Thr 110 aag Lys gaa Giu gta Val1 acc cac act cac WO 01105975 PCT/USOO/16271 12 gtc aaa iggg act aag caa ttg aaa tta gac ect tca att tat gat gee 705 Val Lys Gly Thr Lys Gin Leu Lys Leu Asp Pro Ser Ile Tyr Asp Ala 165 170 175 180 ttg ate aag gaa aag gtg gca gtg ggt gat gtt ata tac att gaa gca 753 Leu Ile Lys Giu Lys Vai Ala Val Gly Asp Val Ile Tyr Ile Giu Ala 1185 190 195 aat agt gga gca gtg aaa aga gtt ggt aga tgt gat tct ttt gct aca 801 Asn Ser Gly Ala Val Lys Arg Val Gly Arg Cys Asp Ser Phe Ala Thr 200 205 210 gaa tat gat ctt gaa get gaa gag tat gtt cer. atc ccc aaa ggt gaa 849 Giu Tlyr Asp Leu Glu Ala Giu Glu Tyr Vai Pro Ile Pro Lys Gly Giu 215 220 225 gtc cat aag aaa aag gaa ata gtg eag gat gtc aca etc cat gac ctt 897 Val His Lys Lys Lys Giu Ile Val Gin Asp Vai Thr Leu His Asp Leu 230 235 240 gat gca gca aat gee cag cca caa ggt ggc caa gat att ttg tcc ctt 945 Asp Ala Ala Asn Ala Gin Pro Gin Gly Gly Gin Asp Ile Leu Ser Leu 245 250 255 260 atg ggc eag atg atg aag cca egg aag act gaa ate aec gaa aag eta 993 Met Gly Gin Met Met Lys Pro Arg Lys Thr Ci Ile Thr Giu Lys Leu 265 270 275 cge caa gaa ate aat aag gtg gta aac aga tat ate gac gaa gga ate 1041 Arg Gin Giu Ile Asn Lys Val Val Asn Arg Tyr Ile1 Asp Giu Gly Ile 280 285 290 gca gag ctt gta cet ggt gtt ttg ttc att gat gag gtc cac atg ttg 1089 Ala Giu Leu Val Pro Gly Val Leu Phe Ile Asp Giu Val His Met Leu 295 300 305 gat att gaa tgc ttt tet tat ctt aac cgt gca ttg gag age cca tta 1137 Asp Ile Giu Cys Phe Ser Tyr Leu Asn Arg Ala Leu Giu Ser Pro Leu 310 315 320 tea cca artt gtg ata etc get acg aat aga gga ata tgt aat gtg aga 1185 Ser Pro Ile Val Ile Leu Ala Thr Asn Arg Giy Ile Cys Asn Val Arg 325 330 335 340 gga aec gat atg acg agt cca cat ggt ata eca gtg gac ctt cta gat 1233 Gly Thr Asp Met Thr Ser Pro His Gly Ile Pro Val Asp Leu Leu Asp 345 350 355 agg ttg gtg att att egg aca gaa aca tat ggc cet act gag atg ata 1281 Arg Leu Val Ile Ile Arg Thr Giu Thr Tyr Gly Pro Thr Giu Met Ile 360 365 370 cag ata etg get ate ega gea caa gtg gaa gag att gat ate gat gaa 1329 Gin Ile Leu Ala Ile Arg Ala Gin Val Glu iv Ile Asp Ile Asp Glu 375 380 385 gaa agt ett get tat trta ggc gag ate gga cag eag aea tet ttg aga 1377 Glu Ser Leu Ala Tyr Leu Gly Giu Ile Giy Gin Gin Thr Ser Leu Arg 390 395 400 eat get att eag ttg eta tea cet gee age gtg gte gea aag ace aac 1425 His Ala Ile Gin Leu Leu Ser Pro Ala Ser Val Val Ala Lys Thr Asn WO 01/05975 PCTIUSOO0/16271 13 405 410 415 420 ggg aga gaa aag atg tgc aag gct gac ctc gag gaa gtc agc 999 ctc 1473 Gly Arg Glu Lys Met Cys Lys Ala Asp Leu Glu Giu Val Ser Gly Leu 425 430 435 tat ttg gat gcc aaa tcc tcg gct cgc ctg ctc cag gag caa caa gaa 1521 Tyr Leu Asp Ala Lys Ser Ser Ala Arg Leu Leu Gin Giu Gin Gin Glu 440 445 450 aga tac atc acc tagacttgca tctcctgctg tggaaggaaa agcctcgaag 1573 Arg Tyr Ile Thr 455 agaaatgcat tctgcaaagt gcgtcgatct gcttcagtat gtcgtagtct acaagtcttg 1633 gagagagcac gtttctgcgg ggaaggaaaa ctgtttgaat tttgtagtac ctyttttttg 1693 ttagcctcca gcgaagactt gattccggca tattgcttgt tcacgcaccg ttaacaccga 1753 ttaagattgg cccagccgtg caggaattgc cgggtgtgtt ttcaccccct cgctaatcga 1813 tgtccagaat ctgtgtgccc acgggtgttg tattgaccct agctttgtaa ccatgcacat 1873 gatttcttta aaaaaaaaaa aaaaa 1898 <210> 8 <211> 456 <212> PRT <213> Zea mays <400> 8 Met Arg Ile Giu Giu Val Gin Ser Thr Ser Lys Lys Gin Arg Ile Ala 1 5 10 Thr His Thr His Ile Lys Gly Leu Gly Leu Asp Gin Ala Asn Gly Met 25 Ser Met Pro Leu Ala Ala Gly Phe Val Gly Gin Ala Ala Ala Arg Glu 40 Ala Ala Gly Leu Ala Val Asp Met Ile Arg Gin Lys Lys Met Ala Gly 55 Arg Ala Leu Leu Leu Ala Giy Pro Pro Ala Thr Gay Lys Thr Ala Leu 70 75 Ala Leu Gly Ile Ala Gin Giu Leu Gly Ser Lys Val Pro Phe Cys Pro 90 Met Val Gly Ser Giu Val Tyr Ser Ser Giu Val Lys Lys Thr Giu Val 100 105 110 Leu Met Glu Asn Phe Arg Arg Ala Ile Gly Leu Arg Ile Lys Giu Asn 115 120 125 Lys Giu Val Tyr Giu Gly Giu Val Ile Giu Leu Ser Pro Giu Giu Ala 130 135 140 Glu Ser Thr Thr Gly Gly Tyr Ala Lys Ser Ile Ser His Val Ile Ile 145 150 155 160 Gly Leu Lys Thr Val Lys Gly Thr Lys Gin Leu Lys Leu Asp Pro Ser 165 170 175 Ile Tyr Asp Ala Leu Ile Lys Giu Lys Val Ala Val Gly Asp Val Ile 180 185 190 Tyr Ile Giu Ala Asn Ser Gly Ala Val Lys Arg Val Gly Arg Cys Asp 195 200 205 Ser Phe Ala Thr Giu Tyr Asp Leu Giu Ala Giu Glu Tyr Val Pro Ile 210 215 220 Pro Lys Gly Glu Val His Lys Lys Lys Giu Ile Val Gin Asp Val Thr 225 230 235 240 Leu His Asp Leu Asp Ala Ala Asn Ala Gin Pro Gin Gly Gly Gin Asp 245 250 255 Ile Leu Ser Leu Met Gly Gin Met Met Lys Pro Arg Lys Thr Giu Ile 260 265 270 Thr Giu Lys Leu Arg Gin Giu Ile Asn Lys Val Val Asn Arg Tyr Ile WO 01105975 PTUOI67 PCT/USOO/16271 14 Pro Gly Val Leu 285 Phe Asp Glu 290 Vai His Ile Ala Giu Ile Asp Glu Met Leu Asp Cys Phe Ser Leu Asn Arg Ala 305 Giu Ser Pro Leu Ser Ile Val Ile Leu 330 Ser Thr Asn Arg Gly Ile 335 Cys Asn Val Asp Leu Leu 355 Thr Giu Met Thr Asp Met Pro His Gly Arg Leu Val Arg Thr Glu Ile Pro Val 350 Tyr Gly Pro Giu Giu Ile Ile Gin Ile 370 Asp Ile Ile Arg Ala Gin 380 Asp Giu Giu Ala Tyr Leu Glu Ile Gly Gin Ser Leu Arg His 405 Giy Ile Gin Leu Pro Ala Ser Val Val 41i5 Ala Lys Thr Vai Ser Gly 435 Giu Gin Gin 450 <210> <211> <212> <213> Arg Giu Lys Lys Ala Asp Leu Giu Giu 430 Leu tLeu Gin Tyr Leu Asp Ser Ser Ala Giu Arg Tyr Ile 455 1869

DNA

Zea mays <220> <221> CDS <222> (64) (1377) <400> 9 cgacccacgc gtccgggcag ctccacagaa acagagagcg cataaccggc ggcgttggcg gcg atg agg atc gag gag gtg cag tcg acc tcg aag aag cag cgc atc Met Arg Ile Glu Giu Val Gin Ser Thr Ser Lys Lys Gin Arg Ile gcc acc cac acc Ala Thr His Thr gcg att gcg ttg Ala Ile Ala Leu cgc gcg gtg ctc Arg Ala Val Leu atc aag gga ctc ggc etc gac gcc Ile Lys Gly Leu Gly Leu Asp Ala aat ggg atg Asn Gly Met atg gcc ggc Met Ala Gly geg gcg ggg ttc Ala Ala Gly Phe ggc cag aag aag Gly Gin Lys Lys ctt gcg ggt Leu Ala Gly ccc gee acg ggc aag acg gcg cta Pro Ala Thr Giy Lys Thr Ala Leu gcg ggc Ala Gly ata gcc cag gag Ile Ala Gin Giu gyc agc aag gtc Gly Ser Lys Val ttc tgt cct atg Phe Cys Pro Met gta Val s0 gga tca gaa gtg tac tee tcg gag gtc Gly Ser Giu Val T'yr Ser Ser Glu Val aaa act gag gtg Lys Thr Giu Val 348 atg gaa aat ttc met Glu Asn Phe aga gct ata ggt ttg cgt ata aag gaa aac aaa Arg Ala Ile Gly Leu Arg Ile Lys Giu Asn Lys WO 01/05975 PCTIUSOO/16271 gag Glu agt Ser tta Leu tat Tyr 160 atc Ile ttt Phe aaa Lys cat His ttg Leu 240 gaa Glu gaa Glu cac His agc Ser aat Asn 320 gtt Val aca Thr aag Lys 145 gat Asp gaa Glu get Ala ggt Gly gac Asp 225 tce Ser aaa Lys gga Gly atg Met cca Pro 305 gta Val act Thr aaa Lys 135 aag Lys aag Lys gtg Va1 gaa Glu aaa Lys 215 get Ala atg Met aat Asn cct Pro ttt Phe 295 ata Ile aca Thr gag gct gag Glu Ala Glu 125 atc ate age Ile Ile Ser tct tea att Ser Ser Ile gtt ata tac Val Ile Tyr 175 tgt gat tct Cys Asp Ser 190 cct ate ccc Pro Ile Pro 205 gte aca ctt Val Thr Leu caa gat att Gin Asp Ile gaa ate acc Glu Ile Thr 255 tat ate gat Tyr Ile Asp 270 gat gag gtc Asp Glu Val 285 gca ttg gag Ala Leu Glu gga ata tgt Gly Ile Cys ccg gtg gat Pro Val Asp 335 ggc cet act Gly Pro Thr 444 492 540 588 636 684 732 780 828 876 924 972 1020 1068 1116 ett eta gat agg ctg gtg att att egg aea gag aca tat Leu Leu Asp Arg Leu Val Ile Ile Arg Thr Giu Thr Tyr WO 01105975 gag atg ata Glu Met Ilie atg gat gaa Met Asp Giu 370 PCTIUSODII 6271 ata ttg gct atc Ile Leu Ala Ile gca caa gtg gag Ala Gin Val Glu gag att gat Glu Ile Asp 365 gaa agt ctt gct Glu Ser Leu Ala tta ggc gag atc gga cag cag aca Leu Gly Giu Ile Gly Gin Gin Thr 380 tct ttg Ser Leu 385 aga cat gct att Arg His Ala Ilie ttg ata tca cct Leu Ile Ser Pro gcc agc gtg gtc tca Ala Ser Val Val Ser 395 gat ctc gag gaa gtt Asp Leu Giu Glu Vai 415 act aat gga aga Thr Asn Gly Arg aaa atc tgc aag Lys Ilie Cys Lys gct Ala 410 1164 1212 1260 1308 1356 1407 1467 1527 1587 1647 1707 1767 1827 1869 agt ggg ctc tat Ser Giy Leu Tyr gat gcc aaa tcc Asp Ala Lys Ser gct cgg ctg ctc cag gag Aia Arg Leu Leu Gin Giu caa caa gaa Gin Gin Giu tac atc acc Tyr Ile Thr tagatttggg tcacctgtcg tggaagtctc gaagagaatg tggagcgagc tccagagaag gtcaccggtg gtgcctccac attcttacag aaatgtttct t taatggg ta <210~ <211, <212' <213.

tagttgccag acatttcggg acttggtacc caggaattgc gggttgtatt aatgcaactt acaacatata catacactac -438

PRT

SZea mays ctcgaaagtc ggggacggct gg cat at tgc cgtgtgtgtt ggcccgaacc gcatggcttt gacctcctgc tttatgtaaa atctagtgaa ttgatctgct tgaattttgc agtgcctgct ctgttcacgc actgttcgct ttttatcttg ctcatcggtg ctatctttgt aaccatggat attatttcta aatgtccata ccaaattaaa attcgtttta aaaaaaaaaa aa tcacaggtct tgtgt tagtc gattagattg tccggaatct aatggatagc aagcttaaca gcttagataa <400> Arg Ile Glu His Thr His Ala Leu Ala Val Gin Ser Thr Ser Lys Lys Gin Arg Ile Ala Lys Gly Leu Gly 25 Gly Asp Ala Asn Gly Met Ala Ala Gly Arg Ala Leu Ala Ile Aia Giy Phe Val Gin Lys Lys Pro Ala Vai Giy Ile Leu Leu Aia Gly Ala Thr Giy Ala Gin Giu Ser Lys Val Cys Pro Met Ser Giu Val Ser Ser Glu Val Lys Lye Thr Glu Val Leu Met Giu Asn Phe Val Tyr Giu 115 Thr Thr Gly 130 Lys Thr Val Ala Ile Gly Arg Ser Gly Giu Val Thr Giu Ile Lys Giu Asn Lye Glu .110 Pro Giu Giu Ala Glu Ser 125 His Val Ile Ile Ser Leu Gly Tyr Ala Lys Ser Ile Ser 135 Lye Gin Leu Lys Giy Thr 150 Glu Lys Val Aia Lye 'Leu 155 Val Gly 170 Ser Ser Ile Asp Ala Leu Ilie Lye 165 Glu Ala Asn Ser Gly Asp Val Ile Tyr Ile 175 Ser Phe Ala Val Lye Arg Val Gly Arg Cys Asp WO 01/05975 PCTUSOOII6271 17 180 185 190 Ala Thr Giu Tyr Asp Lieu Giu Ala Glu Glu Tyr Val Pro Ilie Pro Lys 195 200 205 Gly Glu Val His Lys Lys Lys Glu Ile Val Gin Asp Val Thr Leu His 210 215 220 Asp Leu Asp Ala Ala Asri Ala Gin Pro Gin Gly Gly Gin Asp Ile Leu 225 230 235 240 Ser Leu Met Gly Gin Met Met Lys Pro Arg Lys Thr Giu Ile Thr Glu 245 250 255 Lys Leu Arg Gin Giu Ile Asn Lys Val Val Asn Arg Tyr Ile Asp Giu 260 265 270 Gly Ilie Ala Glu Leu Val Pro Gly Vai Leu Phe Ile Asp Giu Val His 275 280 285 Met Leu Asp Ile Giu Cys Phe Ser Tyr Leu Asn Arg Ala Leu Giu Ser 290 295 300 Pro Leu Ser Pro Ile Val Ile Leu Ala Thr Asn Arg Gly Ile Cys Asn 305 310 315 320 Val Arg Gly Thr Asp Met Thr Ser Pro His Gly Ile Pro Val Asp Leu 325 330 335 Leu Asp Arg Leu Vai Ile Ile Arg Thr Glu Thr Tyr Gly Pro Thr Giu 340 345 350 Met Ile Gin Ile Leu Ala Ilie Arg Ala Gin Val Giu Giu Ile Asp Met 355 360 365 Asp Giu Giu Ser Leu Ala Tyr Leu Gly Giu Ilie Gly Gin Gin Thr Ser 370 375 380 Leu Arg His Ala Ilie Gin Leu Ile Ser Pro Ala Ser Val Val Ser Lys 385 390 395 400 Thr Asn Gly Arg Glu Lys Ile Cys Lys Ala Asp Leu Glu Giu Val Ser 405 410 415 Gly Leu Tyr Leu Asp Ala Lys Ser Ser Ala Arg Leu Leu Gin Glu Gin 420 425 430 Gin Giu Arg Tyr Ile Thr 435 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide based upon an adaptor used for cDNA library construction and poiy(dT) to remove clones which have a poiy(A) tail but no cDNA insert.

<400> 11 tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa 3(

Claims (43)

1. An isolated nucleic acid encoding a polypeptide having RuvB activity comprising a member selected from the group consisting of: a polynucleotide having at least 85% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9, wherein the sequence identity is based on the entire coding regions and is calculated by the GAP algorithm under default parameters; a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, and a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9; and a polynucleotide which is fully complementary to a polynucleotide of or

2. The nucleic acid of claim 1, wherein the polynucleotide has at least sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9, wherein the sequence identity is based on the entire coding regions and is S* calculated by the GAP algorithm under default parameters.

3. The nucleic acid of claim 1, wherein the polynucleotide has at least S° sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 3, and 9, wherein the sequence identity is based on the entire coding regions and is calculated by the GAP algorithm under default parameters.

4. An isolated nucleic acid encoding a polypeptide having RuvB activity comprising a member selected from the group consisting of: a polynucleotide which encodes a polypeptide having at least 25 sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS: 2, *0 0 4, 6, 8 and 10, wherein the percent identity is determined over the entire sequence using the GAP algorithm under default parameters; and a polynucleotide which is fully complementary to a polynucleotide of

5. A recombinant expression cassette comprising a member of any one of claims 1-4, operably linked to a promoter.

6. A non-human host cell comprising the recombinant expression cassette of claim

7. A transgenic plant comprising the polynucleotide of any one of claims 1-4. [R:\LIBFF]J0230spec.doc:gcc

8. The transgenic plant of claim 7, wherein said plant is a monocot.

9. The transgenic plant of claim 7, wherein said plant is a dicot. The transgenic plant of claim 7, wherein said plant is selected from the group consisting of: corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.

11. A transgenic seed from the transgenic plant of any one of claims 7-10, wherein the seed comprises the polynucleotide.

12. A method of modulating the level of RuvB in a plant cell, comprising: introducing into a plant cell a recombinant expression cassette to comprising a RuvB polynucleotide of any one of claims 1-4, operably linked to a promoter; culturing the plant cell under plant cell growing conditions; and expressing said RuvB polynucleotide for a time sufficient to modulate the level of RuvB in said plant cell.

13. A method of modulating the level of RuvB in a plant, comprising: introducing into a plant cell a recombinant expression cassette comprising a RuvB polynucleotide of any one of claims 1-4, operably linked to a promoter; culturing the plant cell under plant cell growing conditions; 20 regenerating a whole plant which possesses the transformed genotype; and expressing said RuvB polynucleotide for a time sufficient to modulate the level of RuvB in said plant.

14. The method of claim 13, wherein said plant is maize. S 25 15. An isolated protein having RuvB activity comprising a member selected from the group consisting of: a polypeptide of at least 50 contiguous amino acids from a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and a polypeptide having at least 90% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 10, wherein the percent sequence identity is determined over the entire sequence using the GAP algorithm under default parameters; and at least one polypeptide encoded by a member of any one of claims 1-4. [R:\LIBFF]10230spec.doc:gcc 66

16. A plant produced by the method of claim 13.

17. The plant of claim 16, wherein the plant is selected from the group consisting of: corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.

18. A transgenic seed produced by the plant of claim 16, wherein the seed comprises the polynucleotide.

19. A method of increasing homologous recombination, comprising: introducing into a plant cell a polynucleotide of interest and the RuvB polynucleotide of any one of claims 1-4 to produce a plant cell which possesses the transformed genotype; culturing the plant cell under plant growing conditions; and expressing the RuvB polynucleotide for a time sufficient to increase homologous recombination of the polynucleotide of interest. The method of claim 19, wherein the plant cell is from a monocot or a dicot.

21. The method of claim 20, wherein the plant cell is maize.

22. The method of claim 19, wherein the RuvB polynucleotide and the polynucleotide of interest are introduced into the plant cell simultaneously.

23. The method of claim 19, wherein the RuvB polynucleotide is introduced into a plant cell prior to the introduction of the polynucleotide of interest. 20 24. A plant cell produced by the method of any one of claims 19-23. The plant cell of claim 24, wherein the plant cell is from a monocot or a dicot.

26. The plant cell of claim 25, wherein the plant cell is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice and barley. 25 27. A plant produced by the method of any one of claims 19-23. S: 28. The plant of claim 27, wherein the plant is a monocot or a dicot.

29. The plant of claim 28, wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice and barley. The plant of claim 27, wherein the RuvB polynucleotide was introduced prior to the introduction of the polynucleotide of interest.

31. A transgenic seed of the plant of any one of claims 27-30, wherein the seed comprises the polynucleotide of interest.

32. A method of increasing transformation efficiency, comprising: [R:\LIBFF]I O23Ospec.doc:gcc 67 introducing into a plant cell a polynucleotide of interest and the RuvB polynucleotide of any one of claims 1-4 to produce a plant cell which possesses the transformed genotype; culturing the plant cell under plant growing conditions; and expressing the RuvB polynucleotide for a time sufficient to increase transformation efficiency of the polynucleotide of interest.

33. The method of claim 32, wherein the RuvB polynucleotide and the polynucleotide of interest are introduced into the plant cell simultaneously.

34. The method of claim 32, wherein the RuvB polynucleotide is introduced into the plant cell prior to the introduction of the polynucleotide of interest. The method of claim 32, wherein the plant cell is from a monocot or a dicot.

36. The method of claim 35, wherein the plant cell is maize.

37. A plant cell produced by the method of claim

38. The plant cell of claim 37, wherein the plant cell is a monocot or a dicot.

39. The plant cell of claim 38, wherein the plant cell is selected from the group consisting of: maize, soybean, sorghum, canola, wheat, alfalfa, cotton, rice and barley.

40. A plant produced by the method of any one of claims 32-35.

41. The plant of claim 40, wherein the plant is a monocot or a dicot.

42. The plant of claim 41, wherein the plant is selected from the group consisting 20 of: maize, soybean, sorghum, canola, wheat, alfalfa, cotton, rice and barley.

43. The plant of claim 40, wherein the RuvB polynucleotide was introduced prior to the introduction of the polynucleotide of interest.

44. A transgenic seed produced by the plant of any one of claims 40-43, wherein the seed comprises the polynucleotide of interest. o* 25 45. The isolated nucleic acid of claim 1, substantially as hereinbefore described with reference to any one of the examples.

46. A recombinant expression cassette comprising the nucleic acid of claim operably linked to a promoter.

47. A non-human host cell comprising the recombinant expression cassette of claim 46.

48. A transgenic plant comprising the polynucleotide of claim

49. Transgenic seed of the plant of claim 48, wherein the seed comprises the polynucleotide. A method of modulating the level of RuvB in a plant cell, substantially as hereinbefore described with reference to any one of the examples. [R:\LIBFF]I O23Ospec.doc:gcc 68

51. A method of modulating the level of RuvB in a plant, substantially as hereinbefore described with reference to any one of the examples.

52. The isolated protein of claim 15, substantially as hereinbefore described with reference to any one of the examples.

53. A method of increasing homologous recombination, substantially as hereinbefore described with reference to any one of the examples.

54. A method of increasing transformation efficiency, substantially as hereinbefore described with reference to any one of the examples. 1o Dated 14 July, 2004 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 9 ow o 0 9 s *f *SOO [R:\LIBFF] I O23Ospec.doc:gcc