AU771438B2 - Maize Rad50 orthologue and uses thereof - Google Patents

Description

WO 00/68404 PCT/US00/11086 6 .1- Maize Rad50 Orthologue and Uses Thereof TECHNICAL FIELD The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.

BACKGROUND OF THE INVENTION The RAD50 gene of Saccharomyces cerevisiae plays a crucial role in meiotic recombination as well as DNA repair during vegetative growth (Kupiec, M. and Simchen, Mol. Gen. Genet. 193: 525-531, 1984). The yeast RAD50 gene encodes a 153 kDa protein (Rad50) that contains an ATP- binding site (Walker -B box or P-loop) in the Nterminal region and exhibits ATP-dependent DNA binding in vitro (Raymond, W.E. and Kleckner, Nucleic Acid Res. 16: 3851-3856, 1993). The Rad50 protein also exhibits two, 250 amino acid segments of heptad-repeat sequence, which form alpha helical coiled coil structures (Alani et al., Genetics 122: 47-57, 1989). In yeast, RAD50 deletion mutants show a mitotic hyper-recombinational phenotype. The same mutant exhibits reduced meiotic double strand break formation and recombination (reviewed in Malkova et al., Genetics 143: 741-754, 1996 and Jeggo, Radiation Res. 150: S80-S91, 1998).

Interestingly, similar phenotypes were observed in the deletion mutants for two other yeast genes MRE 11 and XRS2, suggesting an involvement of these genes in doublestrand break repair and homologous recombination (Malkova et al., Genetics 143: 741- 754, 1996; Jeggo, Radiation Res. 150: S80-S91, 1998). Subsequently, Jozhuka and Ogawa demonstrated the interaction of yeast Rad50 and Mrel 1 proteins (Johzuka, K. and Ogawa, Genetics 139: 1521-1532, 1995). Tsukamoto et al., showed the involvement of yeast RAD50, MER 11 and XRS2 as well as HDF1 (yeast homologue of Ku70) in illegitimate or non-homologous end-joining (Tsukamoto, Y. et al.. Mol. Gen. Genet. 255: 543-547, 1997).

Recently, mammalian homologues of yeast RAD50 have been cloned and characterized extensively (Kim, K, et al., J. Biol. Chem., 271: 29255-29264, 1996; Dolganov et al., Mol. Cell. Biol. 16: 4832-4841, 1996; Carney J.P. et al., Cell 93: 477-486, 1998; Trujillo, K.M. et al., J. Biol. Chem. 273: 21447-21450, 1998). Similarly, the Arabidopsis thaliana chromosome II BACF22D22 region (Accession No. AC006223) has been found to contain an open reading frame which encodes a protein with homology to yeast RAD50 (GI: 4263721).

Control of homologous recombination or non-homologous end joining by modulating Rad50 provides the means to modulate the efficiency with which heterologous nucleic acids 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 The present invention describes the maize Rad50 protein, which clearly possesses features characteristic of other Rad50 proteins, and has a calculated molecular weight of -152.5 kDa. The maize Rad50 protein is characterised by the presence of an ATP binding site in the N-terminal region, a second nucleotide binding site in the C-terminal region, putative nuclear localisation signals, and heptad-repeats. The presence of extensive leucine zipper structures appears to be another striking feature of the Rad50 proteins.

These are also found in the maize Rad50 protein and are indicated in bold in Figure 1.

The present invention also describes a maize Rad50 polynucleotide sequence. The maize orthologue of the present invention was used as a probe to map the maize gene to the short arm of chromosome 4.

Generally, it is the object of the present invention to provide nucleic acids and proteins relating to maize Rad50. 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 Stransgenic plant, the expression of the nucleic acids of the present invention.

According to a first aspect of the invention, there is provided an isolated polynucleotide comprising a member selected from the group consisting of: polynucleotide having at least 80% sequence identity to the polynucleotide of SEQ ID NO:1, wherein the sequence identity is based on the entire coding region for each reference sequence and is calculated by the GAP algorithm under default e 30 parameters; and a polynucleotide fully complementary to the polynucleotide of According to a second aspect of the invention, there is provided an isolated polynucleotide comprising a member selected from the group consisting of: a polynucleotide encoding the polypeptide of SEQ ID NO:2; and [R:\ibffJ9923.doc:SAK a polynucleotide fully complementary to the polynucleotide of According to a third aspect of the invention, there is provided an isolated polynucleotide amplified from a Zea mays nucleic acid library using primers which selectively hybridise, under stringent hybridisation conditions, to the polynucleotide of SEQ ID NO:1.

According to a fourth aspect of the invention, there is provided an isolated polynucleotide which selectively hybridises, under stringent hybridisation conditions and a wash in 0.1X SSC at 60 0 C, to the polynucleotide of SEQ ID NO:1.

According to a fifth aspect of the invention, there is provided an isolated polynucleotide comprising a member selected from the group consisting of: the polynucleotide of SEQ ID NO:1; and a polynucleotide comprising at least 30 contiguous nucleotides from the polynucleotide of SEQ ID NO:1; and a polynucleotide fully complementary to the polynucleotide of or According to a sixth aspect of the invention, there is provided a recombinant expression cassette, comprising the polynucleotide of any one of claims 1-8 operably linked to a promoter.

According to a seventh aspect of the invention, there is provided a host cell comprising the recombinant expression cassette of claim 2 or the polynucleotide of any one of claims 1-8.

According to a eighth aspect of the invention, there is provided a transgenic plant comprising a recombinant expression cassette of claim 2 or the polynucleotide of any one of claims 1-8.

o According to a ninth aspect of the invention, there is provided a transgenic seed 25 from the transgenic plant of claim 11.

According to a tenth aspect of the invention, there is provided a method of modulating the level of Rad50 in a plant, comprising: introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of any one of claims 1-8 operably linked to a promoter; S 30 culturing the plant cell under plant cell growing conditions; regenerating a whole plant which possesses the transformed genotype; and expressing of said polynucleotide for a time sufficient to modulate the level of in said plant.

According to a eleventh aspect of the invention, there is provided an isolated protein comprising a member selected from the group consisting of: [R:Aibffl9923.doc:SAK a polypeptide of at least 20 contiguous amino acids from the polypeptide of SEQ ID NO:2; the polypeptide of SEQ ID NO:2; a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO:2, wherein said sequence identity is determined using the GAP program under default parameters; and at least one polypeptide encoded by a member of claim 1.

Therefore, in one aspect, the present invention 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; a polynucleotide which is complementary to the polynucleotide of and, a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of or The isolated nucleic acid can be DNA.

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

ft* f •g f t o fto ot o o t f f* o• f *f* ft*t f f *t*t**f ft**t te ft ftf ft* ft ft ft* ft ft ft ft* [R:\ibff]9923.doc:SAK WO 00/68404 PCT/US00/I 1086 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 present invention 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.

In another aspect, the present invention 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 present invention relates to a recombinant expression cassette 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 present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of 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-1UB 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 5 th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.

WO 00/68404 PCT/US00/11086 -4- 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.

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 are known as epitopes 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).

WO 00/68404 PCTUS00/I 1086 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.

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.

WO 00/68404 PCT/US00/I11086 -6- 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: 1) Alanine Serine Threonine 2) Aspartic acid Glutamic acid 3) Asparagine Glutamine 4) Arginine Lysine 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 Mycoplasma 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.

WO 00/68404 PCT/US00/11086 -7- 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, S1 protection, and ribonuclease protection. See, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous (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 WO 00/68404 PCT/USO0/! 1086 -8to 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.

The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" 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 Rad50 nucleic acid" is a nucleic acid of the present invention and means a nucleic acid comprising a polynucleotide of the present invention (a "maize Rad50 polynucleotide") encoding a maize Rad50 polypeptide. A WO 00/68404 PCT/US00/I 1086 "maize Rad50 gene" is a gene of the present invention and refers to a heterologous genomic form of a full-length maize Rad50 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.

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 of a 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 Enzmology, Vol. 152, Academic Press, Inc., 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, ajoint venture between Greene Publishing Associates, Inc. and John Wiley 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 WO 00/68404 PCT/US00/11086 -oused 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, 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 WO 00/68404 PCT/US00/! 1086 -Ii 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, 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 Rad50 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 Rad50 protein" is a protein of the present invention and comprises a maize Rad50 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 WO 00/68404 PCT/US00/I 1086 -12in 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 spontaneous 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 80% 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 WO 00/68404 PCT/US00/11086 13 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 reactive 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 (heterologous 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 0 C for short probes to 50 nucleotides) and at least about 60 0 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 NaCl/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 IX SSC at 55 to WO 00/68404 PCT/US00/1 1086 14- Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37 0 C, and a wash in 0.1X 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.

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 ofmonovalent 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, Tm, 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 Tm 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 11, 12, 13, 14, 15, or 20 °C lower than the thermal melting point Using the equation, hybridization and wash compositions, and desired Tm, 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 OC (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, Laboratory Techniques in Biochemistru 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 heterologous polynucleotide. Generally, the heterologous WO 00/68404 PCT/US00/11 086 15 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 initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" 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 nucleotides/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.

WO 00/68404 PCT/US00/11 1086 -16 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 (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-Interscience, 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 WO 00/68404 PCT/US00/ 1086 17word 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) uses 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. Natl. 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

I

WO 00/68404 PCT/US00/I 1086 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 of integers consisting of from 0 to 200. Thus, for example, 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 al., 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 WO 00/68404 PCT/US00/1 1086 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 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" ofpolynucleotide 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 WO 00/68404 PCT/US00/11086 20 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 polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(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 of non-recombinantly engineered plants. Thus, the present invention provides utility in such exemplary applications as in the control of 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 ofmRNA 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 WO 00/68404 PCTiUSO0/ 11086 -21 compounds, for detection of any number of allelic variants (polymorphisms), orthologs, or paralogs 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 for 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 preproenzyme, proenzyme, 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), Oryza, Avena, 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, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum. Phaseolus, and Lolium.

WO 00/68404 PCT/US00/1 1086 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: a polynucleotide encoding a polypeptide of SEQ ID NO: 2 and conservatively modified and polymorphic variants thereof, including exemplary polynucleotides of SEQ ID NO: 1; the polynucleotide sequence of the invention also includes the maize polynucleotide sequence as contained in a plasmid deposited with American Type Culture Collection (ATCC) and assigned Accession Number 207194.

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 the polynucleotide of SEQ ID NO: 1, or the sequence as contained in ATCC deposit assigned Accession No. 207194, wherein the polynucleotide has substantial sequence identity to the polynucleotide of SEQ ID NO: 1; or the sequence as contained in ATCC deposit assigned Accession No. 207194.

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 detectably immunoreact to antisera which has been fully immunosorbed with the protein; complementary sequences of polynucleotides of or and a polynucleotide comprising at least a specific number of contiguous nucleotides from a polynucleotide of or The polynucleotide of SEQ ID NO: 1 is contained in a plasmid deposited with American Type Culture Collection (ATCC) on April 6,1999 and assigned Accession Number 207194. 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 WO 00/68404 PCT/USOO/11086 23 an admission that a deposit is required under 35 U.S.C. Section 112. The deposited sequence, as well as the polypeptide encoded by the sequence, is incorporated herein by reference and controls in the event of any conflict, such as a sequencing error, with description in this application.

A. Polvnucleotides Encoding A Polypeptide of the Present Invention or Conservatively Modified or Polymorphic Variants Thereof 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 NO: 1, and the sequence as contained in ATCC deposit assigned Accession No. 207194, and silent .variations of polynucleotides encoding a polypeptide of SEQ ID NO: 2. The present invention further provides isolated nucleic acids comprising polynucleotides encoding conservatively modified variants of a polypeptide of SEQ ID NO: 2. Conservatively modified variants can be used to generate or select antibodies immunoreactive to the nonvariant polypeptide. Additionally, the present invention further provides isolated nucleic acids comprising polynucleotides encoding one or more allelic (polymorphic) variants of polypeptides/polynucleotides. 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, PHRE1, A632, BMS- P2#10, W23, and Mol7 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 WO 00/68404 PCT/US00/11086 -24- 138: 171-174. 1994), Biotinylated CAP Trapper (Caminci, 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 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 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.

WO 00/68404 PCTUS00/1 1086 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 the 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 WO 00/68404 PCT/US00/1 1086 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.

D. Polvnucleotides 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 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 of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide of 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 or or for purification of, or in immunoassays for, polypeptides encoded by the polynucleotides of 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 of peptides 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 WO 00/68404 PCT/US00/1 1086 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 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, or 50, 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 such as 2, 3, 4, or 5. The subsequences can be separated by any integer ofnucleotides from 1 to the number of nucleotides 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 WO 00/68404 PCT/US00/1 1086 2S 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.

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 (K i) and/or catalytic activity the microscopic rate constant. kca,) as the native endogenous, full-length protein. Those of skill in the art will recognize that kcat/Kn, 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 kct/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 kcar, K, 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 WO 00/68404 PCT/US00/11 086 -29 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 radioactive procedures. Kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee plot.

F. Polvnucleotides 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 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 polvnucleotide 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, 90, 95, or 100 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 WO 00/68404 PCT/US00/11086 30 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.

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 hexahistidine 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, WO 00/68404 PCT/US00/11 086 -31 for example. Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).

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.

A 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 Laboratoriy 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). 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.

WO 00/68404 PCT/USOO/1 1086 32 A2. Construction of a cDNA Library Construction of a 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 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 are 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 of cDNA 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. See, 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). In 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. See, Edery et al.. Mol. Cell Biol.,15(6):3363-3371 (1995); and, PCT Application WO 96/34981.

WO 00/68404 PCTUS00/1 1086 -33 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 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 Laboratory 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-Interscience, 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 WO 00/68404 PCT/USOO/11086 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 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 of genomic libraries are also commercially available.

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 WO 00/68404 PCT/US00/11086 35 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 the 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 WO 00/68404 PCT/US00/11086 -36polymerization 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 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, environmentallyor developmentally-regulated, 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 WO 00/68404 PCT/US00/11 086 -37 ubiquitin promoter, which can be used to drive expression of the present invention in maize embryos or embryogenic callus.

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 environmental or developmental control. Such promoters are referred to here as "inducible" 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 Adh 1 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-1 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 WO 00/68404 PCT/US00/I 1086 38under 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 enhancers 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 WO 00/68404 PCT/US00/1 1086 -39number 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 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, S1 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.

If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The WO 00/68404 PCT/US00/11086 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 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 nptll 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 ofAgrobacterium 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 WO 00/68404 PCT/US00/1 1086 -41 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 (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 ofmRNA which encodes the enzyme of interest, see, Sheehy et al., Proc. Nat' 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 Haseloffet 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 WO 00/68404 PCT/US00/11086 -42- (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives ofnucleotides complementary to target sequences. A report of similar 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; Feteritz 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 50 to 99.

WO 00/68404 PCT/USO0/I11086 43 Exemplary sequence identity values include 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. Sequence identity can be determined using, for example, the GAP or BLAST algorithms.

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 (kcat/Km), 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 WO 00/68404 PCT/US00/1 1086 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 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 Prokarvotes 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.

WO 00/68404 PCT/US00/ 1086 The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid 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.

and Salmonella (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 WO 00/68404 PCT/US00/11086 -46 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 or pgk (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 large 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. Embrvol. 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.

WO 00/68404 PCT/US00/11086 -47 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.

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. Natl. 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) EMBOJ. 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 WO 00/68404 PCT/US00/I11086 -48 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.

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 ofProkaryotes, Lower Eukarvotes. and Animal Cells Animal and lower eukaryotic yeast) host cells are competent or rendered competent for transfection by various means. There are 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, 111. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming WO 00/68404 PCT/US00/I11086 -49peptide bonds by activation of a carboxy terminal end by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide)) is known to those of skill.

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 ofPlant Cell Culture, Macmillilan Publishing Company, New York, pp. 124- 176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).

WO 00/68404 PCT/USO0O/11086 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 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 WO 00/68404 PCT/US00/l 1086 -51 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 immunoblot 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 Polypeptide 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 ratio of the polypeptides of the present invention in a plant. The method WO 00/68404 PCT/US00/11 1086 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 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 polynucleotide 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 ofpolypeptides 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%, 600/%, 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 WO 00/68404 PCT/US00/11 086 well known in the art. In preferred embodiments, the polypeptides of the present invention are modulated in monocots, particularly maize.

Molecular Markers The present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention. Preferably, 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- 2 1.

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 Rad50 genes of the present invention as well as chromosomal sequences genetically linked to Rad50 genes 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 Rad50 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 Rad50 polynucleotide. In preferred embodiments, the WO 00/68404 PCT/US00/11 086 54 probes are selected from polynucleotides of the present invention. Typically, these probes are cDNA probes or restriction-enzyme treated Pst I) genomic clones. In the present invention probes can be made from the polynucleotide of SEQ ID NO: 1. 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 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 EcoRl, EcoRv, and Sstl.

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 Rad50 polynucleotide 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 Rad50 polynucleotide 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 WO 00/68404 PCT/US00/11086 55 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.

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 et 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 1 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 WO 00/68404 PCT/US00/11086 5 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 ofpolynucleotides 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 in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation ofpolynucleotides 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 Kcat 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 of Zea mays can be used to generate antibody or nucleic acid WO 00/68404 PCT/US00/11086 57probes 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 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 WO 00/68404 PCT/US00/1 1086 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 pM.

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 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 nd 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 The RNA for SEQ ID NO: 1 was isolated from premeiotic ear shoot tissue from maize line A632. 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 ofpoly(A)- RNA from total RNA was performed using POLYATTRACT system (Promega Corporation. Madison, WI). In brief, biotinylated WO 00/68404 PCT/USOO/1 1086 5i 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, MD). The first stand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by SUPERSCRIPT Reverse Transcriptase II at 0 C. The second strand of cDNA was labeled with alpha-"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 pSPORT1 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 M13 forward primers and M 3 reverse primers, or by plasmid isolation. All the cDNA clones were sequenced using M 13 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°C.

Once sufficient colonies were picked, they were pinned onto 22 x 22 cm 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.

WO 00/68404 PCT/US00/11086 After colonies were recovered on the second day, these filters were placed on filter paper pre-wetted 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 pre-wetted 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 37 0 C 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.

Colony hybridization was conducted as described by Sambrook,J., Fritsch, E.F. and Maniatis. (in Molecular Cloning: A laboratory Manual, 2 nd 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: 3, removes clones containing a poly A tail but no cDNA.

5. 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 WO 00/68404 PCTIUS00/1 1086 61 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 twvo or more clones containing overlapping segments of DNA were used to construct contiguous DNA sequences.

Example 4 This example displays structural motifs of the maize Rad5O protein sequence. The highlighted areas indicate nucleotide binding sites. The N-terminal binding site, the Walker-A motif, is known to bind ATP. The putative nuclear localization signals are identified by italics and the bolding indicates the leucine zipper motifs.

Structural motifs of Maize Rad5O protein seguence (SEO ID NO: 2) 1 MSTVDIO4LIK GIRSFDPDNK NVITFFKPLT LIVGPNGAGK TTIIECLKLS 51 CTGELPPNSR SGHTFXTHDPK VAGETETKGQ IKLRFKTAAG KDVVCIRSFQ 101 LTQKASKM~EF KAIESVLQTI MPHTGEKVCL SYRCADMDRE IPALMGVSKA 151 VLENVIFVHQ DESNWPLQDP STLKKKFDDI FSATRYTKAL EVIKKLHKDQ 201 MQEIKTFRLK LENLQTVKDQ A}{KLRENIAQ DQEKSDASKS QMEQLKEKIC 251 GTEREILQME TSLDELRRLQ GQIDIKATER STLLTQQHEK LAALSEENED 301 TDEELMEWQT KFEERIALLE TKISKLVRDM DDEASYSSVL SKQNSELTHE 351 IGKLQAEADA HLTMKHERDS DI KNICTKHN LGPVPEHPFT NDVAMNLTNP.

401 IKARLSSLEN DLLDKKKSNE DQLDVLWKHIY LKINARYSEV DGQIQSKIES 451 MSGILRRRKD KEKERDAAEV ELSKFNLSRI DERERI{MQIE VERKTLALGE 501 RDYDSIISQK RTEVYSLEQK IKVLLREKDI INRNADERVK LGLKKDALES WO 00/68404 62

RGRNPFEKDM

PCTUSOO/1 1086

VDKEYNELRS

551 SKDKLNEIVN EI-KDKIKKVL 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 KS QEAEQEL K VDMF PKVLQD

AFTPDEEDEF

AYVKLVEETI

LLQPTDTIDR

RTRflTLIVEV

SEEELVLLAE

YHQLAERKRE

LQS CMAKQQR

DIHSLEERLL

S KHKQELK.S

HSMKNEEINK

GDAELEMRGP

NAESLAAALL

KDENQ-S IIF FTQS KVTDAR

ANNKRDEQKR

VKKQRMQNSS

PLAEKNLNQH-

HVHE IQQLVK

DDLRDQHRML

EKEQLIVBKX

FQQELDALGR

ISAELNKSKE

SIGSLSAINA

QYKDIEKRYT

IIKELNQQTY

CSAGQKVLAS

RIMEARKOQE

SQEIFD*

EQLTKLRRDM

LENFANGMRE

TAERSKALAM

LADESQKAQA

EVEDLEYALD

NEDMSSAQVR

LLEESLDPLS

LfNCIKGYLD

LLQGQGQLKE.

DLKRHSQEKE

NQFLQLKTTE

RGQDIDYISI

LI IRLALAET

NFQLIVITHD

KKE INQAFWP

DAKRRFLDSK

MLAPFEHLAR

ESSNAEALFQ

FDDLLGVLAH

SSGRGVKSLE

WHNAREEKVK

KEKESLLQEY

SKENEKLKEL

NIDDNLKYRK

RLNSEFNRWQ

MANKDLDRYY

NSDS EGAGTR

FCLNCGILAL

ERFAIILIGQR

LQSILQISAN

KNH-VC PCC ER QLDKLRTI YD

VQMDRDAVEA

EIQLELNFLQ

ASSILERFQK

NALICQKLDEE

QGRHVLCHSQ

TKADVEQLTR

GTLSVYQSNI

TALDKALMRF

SYSYRVVMQT

DEPTTNLDGP

QLAEKYYRVS

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 reference.

EDITORIAL NOTE APPLICATION NUMBER 49754/00 The following Sequence Listing pages 1 to 9 are part of the description. The claims pages follow on pages "63" to WO 00/68404 WO 0068404PCTIUSOO/I 1086 1 SEQUENCE LISTING <110> Pioneer Hi-Bred International, Inc.

<120> Maize Rad5O Orthologue and Uses Thereof <130> 1116-PCT <150> 60/132,575 <151> 1999-05-05 <160> 3 <170> FastSEQ for Windows version <210> <211> <212> <213> <220> <221> <222> 4492

DNA

Zea mnays

CDS

(292) (4239) <400> 1 aattcggcac gagtggatcc attagcaccc cggtacaaaa ccctaaaaac cctaacgccg ctccactgcc cctccttttc tctttccaat agaagttgat agggagatag catccgcaat tggatcggag tgcaagtcgt agagggaggc a tagccgtac ctgaagactc cgttttgcaa ctaggtttgg acttggggct aaaaccctaa caaaaaaacg tcactacgag ggcaatcgct cgtggggcaa gaaccctaac cgattttctc cgtaatgaat ctggccagac g atg agc Met Ser acc gtt gac Thr Val Asp aag atg ctg atc Lys Met Leu Ile ggg att cgg agc Giy Ile Arg Ser gat ccg gac Asp Pro Asp aat aag Asn Lys aac gtc atc acc Asn Val Ile Thr ttc aag ccg ctc Phe Lys Pro Leu ctc atc gtt ggc Leu Ile Val Gly aac ggt gct ggc Asn Gly Ala Gly ace acg atc atc Thr Thr Ile Ilie tgc ctg aag ctt Cys Leu Lys Leu tgc acc ggc gag ctq ccc ccc aac tcc Cvs Thr Glv Glu Leu Pro Pro Asn Ser tct ggc cac ac Ser Gb;, His Thr ttc gtc Phe Val cac gac ccc His Asp Pro ttg cgg ttt Leu Arg Phe gta act gyc gag Val Al1a Gly Giu gaa aca aaa gga Glu Thr Lvs Giv caa att aag Gin Ile Lys atc cgg tcc Ile Arg Ser aag act gca gca Lys Thr Ala Ala aag gat gtg gzg Lys Asp Val Val ttc cag Phe Gin 100 ctt acc caa sag Leu Thr Gin Lys tca aag atg gag tt-t aag gca att gaa Ser Lvs Met Giu Phe Lvs Ala Ile Giu 110 WO 00/68404 agc gtc ctc cag Ser Val Leu Gin act ata aat cca. cac aca Thr Ile Asn Pro His 115 120 agc Ser gtt Val1 tcc S er gac Asp aaa Lys 195 ctg Leu aa t Asn gag Giu a tg Met atc Ile 275 ctt Leu gaa Giu atc Ile gtt Vali tac Tyr t cg Ser aat Asn atc Ile 180 ctt Leu gag Giu att Ilie caa Gin gaa Giu 260 aag Lys gc t Al a tgg Trp agt Sex ctc Leu 340 aga t Arg C aag Lys tg Trp 165 ttc Phe cac His aac Asn gct Ala c tg Leu 245 aca Thr gca Ala gca *Ala caa Gin aaa -Lys 325 tcc Ser :gt :ys ~cc kla 150 :ca Pro tct Ser aag Lys ctt Leu caa Gin 230 aag Lys agt Ser aca Thr ctt Leu aca Thr 310 ctt Leu aaa Lys gct Ala 135 gta Val1 ttg Leu gcc Aia ga t Asp cag Gin 215 gat Asp gaa Giu ttg Leu gag *Giu tct *Ser 295 aaa Lys gta 1Val caa Gin gac Asp ctg Leu cag Gin aca Thr caa Gin 200 act Thr caa Gin a ag Lys gat Asp aga Arg 280 gag Giu ttt Phe aga Arc aa t Asn atg Met gag Giu gac Asp cgc Arg 185 a tg Met gta Vali gaa Giu atc Ile gaa Giu 265 ag t Ser ga a Giu ga a Giu gat IAsp tct Ser 345 gat Asp aat pAsn ccg Pro 170 tat Tyr caa Gin aaa Lys aag Lys tgt Cys 250 ctg Leu aca Thr aat Asn gaa Giu a tg Met 330 gaa Glu aga Arg gtt Vai 155 tca Ser a cg Thr gag Giu gac Asp tca S er 235 ggt Gly aga Arg tta Leu ga a Giu agg Arg 315 gat Asp *tta *Leu Chr lag 3iu 140 ata Ilie aca Thr aaa Ly s atc Ile caa Gin 220 gat Asp acc Thr aga Arg ctt Leu gatI Asp 300 att Ilc gat As; ac~ Th~ ggg Giy 125 att Ile ttt Phe ctt Leu gc t Ala a ag Lys 205 gca Ala gcc Al a gag *Giu *ctt Leu *acg Thr 285 *acc Thr -gcg -Ala -gaa SGiu a cat His Glu cct Pro gt t Val1 a ag Lys ctt Leu 190 act Thr cat His tca Ser aga Arg cag Gin 270 cag Gin gat Asp tta Leu gca Al a gaa Giu 350 Lys gcc Ala cac His aag Lys 175 gaa Giu ttt Phe aag Lys aaa Lys gaa Giu 255 gga Gly cag Gin gag Giu cta Leu tc t *Ser 335 *att Sle gag aaa gtc Val1 tta Leu caa Gin 160 aag Lys gtc Val1 agg Arg ctg Leu tct Ser 240 atc Ile caa Gin cat His gaa Giu gaa Giu 320 tat Tyr gga Gly PCT1JSOO/1 1086 tgc ctc Cys Leu 130 atg ggt Met Gly 145 gat gaa Asp Giu ttc gat Phe Asp ata aag Ile Lys tta aag Leu Lys 210 cgt gaa Arg Giu 225 cag atg Gin Met ctg caa Leu Gin att gac Ile Asp gaa aag Giu Lys 290 cta atg Leu Met 305 aca aaa Thr Lys agc tcc Ser Ser aag ctc Lys Leu 681 729 777 825 873 921 969 L017 1065 1113 116', 1209 1257 1309- 1353 cag gca gaa gct gat gct cac ctg act atg aag cat gaa cga gac tca Gin Ala Glu Ala Asp Ala His Leu Thr Met Lys His Giu Arg Asp Ser 1401 WO 00/68404 355 gac a AspI cat c His I geg Ala2 aat Asn get Ala 435 atg Met get Ala agg Arg gga Gly tat Tyr 515 ata Ilie ge a Ala aag Lys ga t Asp t ac Tyr 595 ita lce iga k.rg gaa Glu 420 zge Arg tca Ser gca Al a gag Glu gaa Giu 500 ag t Ser aat As n ttg Leu gat Asr atc Met 58( aat Asx aaa a Lys P ttt a Phe 1 eta LeuE 405 gat Asp tac1 Tyr ggc Gly gaa Giu aga Arg 485 aga Arg t tg Leu aga Arg Igaa Giu aaa Lys 565 aag Lys gag a Giu ~at Isf icg rhr 190 :ca ~er cag 31n tcC Ser att Ile gtg Val1 470 cat His gac Asp gaa G lu aat Asn age Se r 550 a t Ile aag Lys tta Leu ata Ile 375 ~aat Asn agt Ser tta Leu gaa Glu tt a Leu 455 gag Glu atg Met tat Tyr eag Gin get Ala 53 5 ac Ser aaa Ly s gag Glu aga Arc 360 tge Cys gat Asp ett Leu gat Asp gt t Val1 440 aga Arg ett Leu e aa Gin gat Asp aaa Lys 520 gat Asp aag Lys aag Lys ate Ile tea Ser 600 act Chr gtt Val1 gag Giu gtt V~al 425 ga t Asp egg Arg tea Ser att Ile tea Ser 505 ata Ile gaa Giu gac Asp gta Val1 aat Asn 585 aaa Lys aaa Lys get Ala aat Asn 410 ttg Leu gg t Gly aga Arg aaa Lys gaa Glu 490 att* Ilie aaa Lys aga Arg aag Lys ett Leu 570 eaa Gin tce Ser cat a tg Met 395 gat Asp tgg Trp e ag Gin aaa Lys ttt Phe 475 gte Val1 ata Ile gtg Val1 gt a Val1 etc Leu 555 agg Arg gee Ala eag Gin aat Asn 380 aac Asn ttg Leu aaa Lys ata Ile gat Asp 460 aa t Asn gag Giu ag t S er ctt Leu aaa Lys 540 aat Asn ggg G ly t tt Phe Igaa Glu 365 ctt Leu ctt Leu o tg Leu eae His caa Gin 445 aa a Lys eta Leu agg Arg eag Gln c tg Leu 525 c tg Leu gag Giu agg Arg tgg Trp gc a Ala 605 ggg Gly aca Thr gat Asp tat Tyr 430 tet Ser gag Giu tee Ser aag Lys aaa Lys 510 egg Arg gg t Gly ata Ile aat Asn eet Pro 590 gag Gi1u c eg Pro aac Asn aag Lys 415 ett Leu aag Lys aaa Lys egt Arg aea Thr 495 ega Arg gag Giu t ty Leu gt t Val oct Pro 575 gtg Val1 ea a Gin gtt Val agg Arg 400 aag Lys aaa Lys att Ile gaa Giu ate Ile 480 ett Leu aea Thr aaa Lys aaq Ly s aat Asn 560 ttt Phe gac Asp gag Glu PCTIUSOO/1 1086 370 eet gaa 1 Pro Giu 385 att aaa I Ile Lys aaa tee Lys Ser ata aat Ile Asn gaa tee Giu Ser 450 ege gat Arg Asp 465 gat gag Asp Giu geg ett Ala Leu gaa gta Giu Val gat ata Asp Ile 530 aag gat Lys Asp 545 gag eat Giu His gag aaa Giu Lys aag gaa Lys Giu ett aaa Leu Lys 610 449 497 .545 .593 .641 .689 L737 1785 1833 1881 1929 1977- 202=5 207 3 2121 WO 00/68404 WO 0068404PCT/US 00/1 1086 ttt act cag agc aaa gta act gat gct Phe Thr Gin Ser Lys Val Thr Asp Ala 615 aga Arg 620 gaa caa ttg aca Glu Gin Leu Thr aaa ctt Lys Leu 625 2169 2217 cga aga gat Arg Arg Asp gat gca aaa aga.

Asp Ala Lys Arg ttc ctg gac tcg Phe Leu Asp Ser aaa ctt caa Lys Leu Gin 640 aaa gtt cta Lys Val Leu tct att tta cag ata tct gct Ser Ile Leu Gin Ile Ser Ala aa t Asn 650 gtt gac atg ttt Vai Asp Met Phe caa gac Gin Asp 660 gcc atg aac aaa Ala Met Asn Lys gat gaa cag aaa Asp Giu Gin Lys tta gag aat ttc Leu Giu Asn Phe aat gga atg cgg Asn Gly Met Arg atg ctt gca cct Met Leu Ala Pro gaa cat ttg gct Glu His Leu Ala aag aat cat gta Lys Asn His Val tgc Cys 695 cca tgc tgt gaa Pro Cys Cys Giu gct ttc aca cct Ala Phe Thr Pro gat gag Asp Giu 705 gag gat gag Giu Asp Giu gag aga tct Giu Arg Ser 725 gtg aag aaa caa Val Lys Lys Gin atg caa aac tca Met Gin Asn Ser agt act gca Ser Thr Ala 720 gaa. gct ctt Giu Ala Leu 2265 2313 2361 2409 2457 2505 2553 2601 2649 2697 aaa gct ctg gca Lys Ala Leu Ala atg Met 730 gaa tca tca aat Giu Ser Ser Asn ttt cay Phe Gin 740 caa ttg gat aaa Gin Leu Asp Lys cgg act atc tat Arg Thr Ile Tyr gct tat gtg aag Ala Tyr Val Lys gta gaa gaa acc Val Giu Giu Thr cct cta gca gag Pro Leu Ala Glu aac ttg aat caa Asn Leu Asn Gin ttg gcg gat gaa Leu Ala Asp Giu agt Ser 775 cag aag gcg cay Gin Lys Ala Gin ttt gat gat ctt Phe Asp Asp Leu ttg gyt Leu Gly 785 gtt ctt gcc cat gtt caa aty gac Val Leu Ala His Val Gin Met Asp 790 gat gca gtg gaa Asp Ala Val Giu gcc tta tta Ala Leu Leu 800 caa cay cta Gin Gin Leu caa ccc act Gin Pro Thr 805 gat act att gac Asp Thr Ilie Asp cat gta cat gaa His Val His Giu 2745 ytc aaa Val Lys 820 gaa gta gaa gat Giu Val Giu Asp ctt Leu 825 gaa tat gca ctt Giu Tyr Ala Leu tct agt ggc cga Ser Ser Gly Arg gtc aag tct ttg Val Lys Ser Leu gaa att caa ctg Giu Ile Gin Leu ctg aac ttt ctg Leu Asn Phe Leu 2793 2841 2889 aga aca agy gac aca ttg att gtc gaa gtg gat gat ctt aga gat caa Ary Thr Arg Asp Thr Leu Ile Val Glu Val Asp Asp Leu Arg Asp Gin WO 00/68404 PCTIUSOO/1 1086 cat aga atg His Arg Met aat gct cgg Asn Ala Arg 885 aat gaa gat atg Asn Glu Asp Met agt gct cag gtg Ser Ala Gin Val aga tgg cac Arg Trp His 880 gaa aga ttc Glu Arg Phe gaa gag aaa gtg Glu Glu Lys Val gct tct agc ata Ala Ser Ser Ile caa aaa tct gaa gag gaa Gin Lys Ser Giu Glu Glu gtg ctt cta gct Val Leu Leu Ala gaa aaa gaa caa Glu Lys Giu Gin att gta gaa aag Ile Val Glu Lys ctt tta gaa gag Leu Len Giu Glu ctt gat cca ttg Leu Asp Pro Leu aaa gag aaa gag Lys Giu Lys Glu ttg ttg caa gag Leu Leu Gin Glu tat Tyr 940 aat gct ttg aag Asn Ala Leu Lys caa aag Gin Lys 945 2937 2985 3033 3081 3129 3177 3225 3273 3321 ctg gat gaa Leu Asp Glu tat cat cag ctt Tyr His Gin Leu gaa aga aaa agg Glu Arg Lys Arg gag ttc cag Glu Phe Gin 960 aaa ggg tac Lys Giy Tyr caa gaa ctt gat gct ctt gga aga ctt aat atg aag ata Gin Giu Len Asp Ala Len Gly Arg Leu Asn Met Lys Ile 965 970 975 ttg gat Leu Asp 980 gtt ctt Vai Len 995 tcc aag aaa aac Ser Lys Lys Asn aag ctt aag gaa Lys Leu Lys Glu cag gga agg cat Gin Gly Arg His tgc cat tct Cys His Ser cag tta Gin Leu 1000 cag agt tgc Gin Ser Cys atg gca Met Ala 1005 aaa cag caa Lys Gin Gin aga Arg 1010 ata tca gct gag tta aac aag agc aaa gaa cta ctg cag Ile Ser Ala Giu Len Asn Lys Ser Lys Glu Len Len Gin ggc cag ggc Gly Gin Gly 1025 1015 1020 cag ttg aaa Gin Len Lys aga aac Arg Asn 1030 att gat gac aat ctc aag tac agg aaa aca aag Ile Asp Asp Asn Leu Lys Tyr Arg Lys Thr Lys 1035 1040 gct gat gtg gaa caa Ala Asp Val Giu Gin 1045 ctt act cgt gat Len Thr Arg Asp 1050 ata gaa tca ctt gaa gaa agg Ile Glu Ser Leu Glu Glu Arg 1055 3369 3417 3465 3513 3561 ctg ctt tca ata ggt agc ttg tct gct ata gaa gct gat ctg aaa cgc Leu Len Ser Ile Gly Ser Len Ser Ala Ile Glu Ala Asp Leu Lys Arg 1060 1065 1070 cat tct His Ser 1075 caa gaa aaa Gin Glu Lys gag agg ctt aat Glu Arg Leu Asn 1080 tca gaa ttt aac agg tgg caa Ser Glu Phe Asn Arg Trp Gin 1085 1090 gga aca ctt tct gtt tat caa agt aat Gly Thr Leu Ser Val Tyr Gin Ser Asn 1095 att Ile 1100 tca aag cac aaa caa gag Ser Lys His Lys Gin Glu 3609 I 1105 WO 00/68404 WO 0068404PCTUSOO/1 1086 ctt aaa ctg tca Leu Lys Leu Ser 1.110 ttt ctc cag ctt Phe Leu Gin Leu 1125 tac aag gat atc gag aag Tyr Lys Asp Ile G2lu Lys 1115 cga tat act aat caa Arg Tyr Thr Asn Gin 1120 aag aca act gaa atg Lys Thr Thr Glu Met 1130 gca aac aag gac ttg gac aga Ala Asn Lys Asp Leu Asp Arg 1135 3657 3705 3753 3801 tat tat act gct tta gac Tyr Tyr Thr Ala Leu Asp 1140 aag gct Lys Ala 1145 ctt atg cgg ttc cac agc atg, aag Leu Met Arg Phe His Ser Met Lys 1150 atg gag gag ata aat Met Glu Glu Ile Asn 11S5 aaa ata atc aag gaa Lys Ile Ile Lys Glu 1160 ctg tgg Leu Trp 1165 caa cag aca tac Gin Gin Thr Tyr 1170 aga ggc cag gat att gat tac ata agc ata aat tct gat tct gag ggt Arg Gly Gin Asp Ilie Asp Tyr Ile Ser Ile Asn Ser Asp Ser Giu Gly 3849 1175 1180 1185 gct ggc act Ala Gly Thr cga tca Arg Ser -1190 tac agc tac Tyr Ser Tyr cgc gtt Arg Val 1195 gtt atg caa Val Met Gin act ggt gat Thr Gly Asp 1200 3897 gct gag ctg Ala Giu Leu 1205 gct tct ctt Ala Ser Leu 1220 gaa atg cga ggg Giu Met Arg Gly cgc tgc Arg Cys 1210 agt gct ggt cag aag gtt ctt Ser Ala Gly Gin Lys Val Leu 1215 3945 3993 ata atc aga Ilie Ilie Arg cta gca ctt gcg gaa act ttc tgc ctg aac Leu Ala Leu Ala Giu Thr Phe Cys Leu Asn 1225 1230 tgc ggt Cys Giy 1235 ata ttg gct.

Ile Leu Ala ttg gat Leu Asp 1240 gag eca act acg aat Giu Pro Thr Thr Asn 1245 gcg ctg ttg aga ata Ala Leu Leu Arg Ile 1260 cta gat ggg Leu Asp Gly cca Pro 1250 aat gca gag agt Asn Ala Giu Ser ctt gct gct Leu Ala Ala 1255 atg gaa gcc agg Met Giu Ala Arg 1265 aaa ggg cag gag aac ttc cag ttg att gta atc act cat gat gag aga Lys Gly Gin Giu Asn Phe Gin Leu Ilie Val Ile Thr His Asp Giu Arg 1270 1275 1280 ttt gcc cat ctt atc ggt caa agg cag ctt gct gag aag tac tat cga Phe Ala His Leu Ile Gly Gin Arg Gin Leu Ala Giu Lys Tyr Tyr Arg 1285 1290 1295 gtc tcc aag gat gag aac cag cac agc ata att gaa tcc caa gag ata Val Ser Lys Asp Giu Asn Gin His Ser Ile Ile Giu Ser Gin Giu Ile 1300 1305 1310 ttt gac taagggtgtt ctaggaggct gtagcacgca ctcgtttgct agtcgaatcc Phe Asp 1315 4041 4089 4137 4185 4233 4289 agttaattta tacgccgtta gccagaggat acaatctagc tgccaagtac tggtgccaga gcaatgttaa caagctttag gaggctctgt cgtcagttgc gtgaaaccta tccttcgttg ttgtatactt atttaatctg ggaatgtgtg cactgggtga tggatgtttc acgacatcaa tgaatgtttc atcaaaaaaa aaa 4349 4409 4469 4492 <210> 2 WO 00/68404 PCTIUS00/11086 <211> 1316 <212> PRT <213> Zea mays Met 1 Pro Val Leu Phe Ile Arg Ile Cys Met 145 Asp Phe Ile Leu Arg 225 Gin Leu Ile Glu Leu 305 Thr Ser Lys Asp Pro 385 Ile Lys Ile Glu <400> 2 Ser Thr V Asp Asn L 2 Gly Pro A Ser Cys T Val His A Lys Leu A Ser Phe G 1 Glu Ser V 115 Leu Ser T 130 Gly Val S Glu Ser A Asp Asp I 1 Lys Lys L 195 Lys Leu G 210 Glu Asn I Met Glu G Gin Met G 2 Asp Ile L 275 Lys Leu A 290 Met Glu T Lys Ile S Ser Val L 3 Leu Gin A 355 Ser Asp I 370 Glu His I Lys Ala Ser Asn C 4 Asn Ala 435 Ser Met E al ys 0 sn hr sp rg In 00 al yr er sn le 80 eu lu le ln lu 60 *ys la rp er .eu 140 Lla :le Pro %rg lu [20 Arg ;er Asp 5 Asn Gly Gly Pro Phe Leu Leu Arg Lys Trp 165 Phe His Asn Ala Leu 245 Thr Ala Ala Gin Lys 325 Ser Glu Lys Phe Leu 405 Asp Tyr Gly Lys Val Ala Glu Lys 70 Lys Thr Gin Cys Ala 150 Pro Ser Lys Leu Gin 230 Lys Ser Thr Leu Thr 310 Leu Lys Ala Asn Thr 390 Ser Gin Ser Ile Met Leu Ile Lys Gly Ile Arg Ser Phe Asp 10 Ile Gly Leu 55 Val Thr Gin Thr Ala 135 Val Leu Ala Asp Gin 215 Asp Glu Leu Glu Ser 295 Lys Val Gin Asp Ile 375 Asn Ser Leu Glu Leu Thr Lys 40 Pro Ala Ala Lys Ile 120 Asp Leu Gin Thr Gin 200 Thr Gin Lys Asp Arg 280 Glu Phe Arg Asn Ala 360 Cys Asp Leu Asp Val 440 Arg Phe 25 Thr Pro Gly Ala Ala 105 Asn Met Glu Asp Arg 185 Met Val Glu Ile Glu 265 Ser Glu Glu Asp Ser 345 His Thr Val Glu Val 425 Asp Arg Phe Thr Asn Glu Gly 90 Ser Pro Asp Asn Pro 170 Tyr Gin Lys Lys Cys 250 Leu Thr Asn Glu Met 330 Glu Leu Lys Ala Asn 410 Leu Gly Arg Lys Ile Ser Thr 75 Lys Lys His Arg Val 155 Ser Thr Glu Asp Ser 235 Gly Arg Leu Glu Arg 315 Asp Leu Thr His Met 395 Asp Trp Gin Lys Pro Ile Arg Glu Asp Met Thr Glu 140 Ile Thr Lys Ile Gin 220 Asp Thr Arg Leu Asp 300 Ile Asp Thr Met Asn 380 Asn Leu Lys Ile Asp Leu Glu Ser Thr Val Glu Gly 125 Ile Phe Leu Ala Lys 205 Ala Ala Glu Leu Thr 285 Thr Ala Glu His Lys 365 Leu Leu Leu His Gin 445 Lys Thr Cys Gly Lys Val Phe 110 Glu Pro Val Lys Leu 190 Thr His Ser Arg Gin 270 Gin Asp Leu Ala Glu 350 His Gly Thr Asp Tyr 430 Ser Glu Leu Leu His Gly Cys Lys Lys Ala His Lys 175 Glu Phe Lys Lys Glu 255 Gly Gin Glu Leu Ser 335 Ile Glu Pro Asn Lys 415 Leu Lys Lys Ile Lys Thr Gin Ile Ala Val Leu Gin 160 Lys Val Arg Leu Ser 240 Ile Gin His Glu Glu 320 Tyr Gly Arg Val Arg 400 Lys Lys Ile Glu WO 00/68404 WO 0068404PCTIUSOOII 1086 450 Arg Asp 455 Giu Ala Ala Giu Val Leu Ser Lys Phe Asn Leu Ser Arg Ile 4 c :65 ~sp Giu la Leu ;lu Val ~sp Ile 530 .ys Asp hiu His 3iu Lys ys Giu Aeu Lys 610 Lays Leu 625 Laeu Gin Val Leu Asn Phe Ala Arg 690 A~sp Giu 705 Thr Ala Ala Leu Vai Lys Gin His 770 Leu Giy 785 Leu Leu Gin Leu Gly Arg Leu Gin 850 Asp Gin 865 Trp His Arg Phe Giu Gin Leu Ser 930 N.rg Giy Tyr Ile Al a Lys Asp Tyr 595 Phe Arg Ser Gin Ala 675 Lys Giu Glu Phe Leu 755 Leu Vai Gin Vali Giy 835 Arg His Asn Gin Leu 915 Lys Giu Glu 500 Ser Asn Leu Asp Met 580 Asn Thr Arg Ile Asp 660 Asn Asn Asp Arg Gin 740 Vali Al a Leu Pro Lys 820 Val Thr Arg Al a Lys 900 Ile Glu Arg 485 Arg Leu Arg Glu Lys 565 Lys Giu Gin Asp Leu 645 Ala Gly His Glu Ser 725 Gin Glu Asp Al a Thr 805 Giu Lys Arg Met Arg 885 Ser Val1 Lys 470 His Met Asp Tyr Giu Gin Asn Ala 535 Ser Ser 550 Ile Lys Lys Glu Leu Arg Ser Lys 615 Met Asp 630 Gin Ile Met Asn Met Arg Val Cys 695 Phe Val 710 Lys Ala Leu Asp Giu Thr Giu Ser 775 His Val 790 Asp Thr Val Giu Ser Leu Asp Thr 855 Leu Asn 870 Glu Giu Giu Giu Giu Lys Giu Ser Gin Asp Lys 520 Asp Lys Lys Ile Ser 600 Val1 Al a Ser Lys Giu 680 Pro Lys Leu Lys Ile 760 Gin Gin Ile Asp Giu 840 Leu Glu Lys Giu Lys 920 Leu Ile Ser 505 Ile Glu Asp Val1 As n 585 Lys Thr Lys Ala Arg 665 Met Cys Lys Ala Leu 745 Pro Lys Met Asp Leu 825 Giu Ile Asp Val1 Leu 905 Leu Leu Giu 490 Ile Lys Arg Lys Leu 570 Gin Ser Asp Arg Asn 650 Asp Leu Cys Gin Met 730 Arg Leu Al a Asp Arg 810 Giu Ile Val1 Met Lys 890 Val Leu Gin 475 Val1 Ile Val1 Val1 Leu 555 Arg Ala Gin Al a Arg 635 Val1 Glu Al a Glu Arg 715 Giu Thr Al a Gin Arg 795 His Tyr Gi Giu Ser 875 Ala Leu Glu Glu Giu S er Leu Lys 540 Asn Gly Phe Giu Arg 620 Phe Asp Gin Pro Arg 700 Met Ser Ile Giu Ala 780 Asp Val1 Al a Leu Val 860 Ser Se r Leu Giu Tyr Arg Gin Leu 525 Leu Giu Arg Trp Ala 605 Giu Leu Met Lys Phe 685 Al a Gin Ser Tyr Lys 765 Phe Ala His Leu Gi u 845 Asp Al a Ser Al a Ser 925 Asn Lys Lys 510 Arg Gly Ile Asn Pro 590 Glu Gin Asp Phe Arg 670 Glu Phe Asn Asn Asp 750 As n Asp Val Giu Asp 830 Leu Asp Gin Ile Glu 910 Leu Ala 480 Thr Leu 495 Arg Thr Giu Lys Leu Lys Val Asn 560 Pro Phe 575 Val Asp Gin Giu Leu Thr Ser Lys 640 Pro Lys 655 Leu Giu His Leu Thr Pro Ser Ser 720 Ala Glu 735 Ala Tyr Leu Asn Asp Leu Glu Ala 800 Ile Gin 815 Ser Ser Asn Phe Leu Arg Val Arg 880 Leu Giu 895 Glu Lys Asp Pro Leu Lys 935 940 Gin Lys Leu Asp Giu Giu Tyr His Gin Leu Ala Giu Arg Lys Arg Glu WO 00/68404 PCT/US00/11086 9 945 950 955 960 Phe Gin Gin Glu Leu Asp Ala Leu Gly Arg Leu Asn Met Lys Ile Lys 965 970 975 Gly Tyr Leu Asp Ser Lys Lys Asn Glu Lys Leu Lys Glu Leu Gin Gly 980 985 990 Arg His Val Leu Cys His Ser Gin Leu Gin Ser Cys Met Ala Lys Gin 995 1000 1005 Gin Arg Ile Ser Ala Glu Leu Asn Lys Ser Lys Glu Leu Leu Gin Gly 1010 1015 1020 Gin Gly Gin Leu Lys Arg Asn Ile Asp Asp Asn Leu Lys Tyr Arg Lys 1025 1030 1035 1040 Thr Lys Ala Asp Val Glu Gin Leu Thr Arg Asp Ile Glu Ser Leu Glu 1045 1050 1055 Glu Arg Leu Leu Ser Ile Gly Ser Leu Ser Ala Ile Glu Ala Asp Leu 1060 1065 1070 Lys Arg His Ser Gin Glu Lys Glu Arg Leu Asn Ser Glu Phe Asn Arg 1075 1080 1085 Trp Gin Gly Thr Leu Ser Val Tyr Gin Ser Asn Ile Ser Lys His Lys 1090 1095 1100 Gin Glu Leu Lys Leu Ser Gin Tyr Lys Asp Ile Glu Lys Arg Tyr Thr 1105 1110 1115 1120 Asn Gin Phe Leu Gin Leu Lys Thr Thr Glu Met Ala Asn Lys Asp Leu 1125 1130 1135 Asp Arg Tyr Tyr Thr Ala Leu Asp Lys Ala Leu Met Arg Phe His Ser 1140 1145 1150 Met Lys Met Glu Glu Ile Asn Lys Ile Ile Lys Glu Leu Trp Gin Gin 1155 1160 1165 Thr Tyr Arg Gly Gin Asp Ile Asp Tyr Ile Ser Ile Asn Ser Asp Ser 1170 1175 1180 Glu Gly Ala Gly Thr Arg Ser Tyr Ser Tyr Arg Val Val Met Gin Thr 1185 1190 1195 1200 Gly Asp Ala Glu Leu Glu Met Arg Gly Arg Cys Ser Ala Gly Gin Lys 1205 1210 1215 Val Leu Ala Ser Leu Ile Ile Arg Leu Ala Leu Ala Glu Thr Phe Cys 1220 1225 1230 Leu Asn Cys Gly Ile Leu Ala Leu Asp Glu Pro Thr Thr Asn Leu Asp 1235 1240 1245 Gly Pro Asn Ala Glu Ser Leu Ala Ala Ala Leu Leu Arg Ile Met Glu 1250 1255 1260 Ala Arg Lys Gly Gin Glu Asn Phe Gin Leu Ile Val Ile Thr His Asp 1265 1270 1275 1280 Glu Arg Phe Ala His Leu Ile Gly Gin Arg Gin Leu Ala Glu Lys Tyr 1285 1290 1295 Tyr Arg Val Ser Lys Asp Glu Asn Gin His Ser Ile Ile Glu Ser Gin 1300 1305 1310 Glu Ile Phe Asp 1315 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide based upon an adaptor used for cDNA library construction and poly(dT) to remove clones which have a poly(A) tail but no cDNA insert.

<400> 3 tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa

Claims (21)

1. An isolated polynucleotide comprising a member selected from the group consisting of: polynucleotide having at least 80% sequence identity to the polynucleotide of SEQ ID NO: 1, wherein the sequence identity is based on the entire coding region for each reference sequence and is calculated by the GAP algorithm under default parameters; and a polynucleotide fully complementary to the polynucleotide of

2. An isolated polynucleotide comprising a member selected from the group 0o consisting of: a polynucleotide encoding the polypeptide of SEQ ID NO:2; and a polynucleotide fully complementary to the polynucleotide of

3. An isolated polynucleotide amplified from a Zea mays nucleic acid library using primers which selectively hybridise, under stringent hybridisation conditions, to the polynucleotide of SEQ ID NO:1.

4. An isolated polynucleotide which selectively hybridises, under stringent hybridisation conditions and a wash in 0.1X SSC at 60 0 C, to the polynucleotide of SEQ ID NO:1. An isolated polynucleotide comprising a member selected from the group consisting of: the polynucleotide of SEQ ID NO: 1; and a polynucleotide comprising at least 30 contiguous nucleotides from the polynucleotide of SEQ ID NO: 1; and a polynucleotide fully complementary to the polynucleotide of or 25

6. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises at least 85% identity to SEQ ID NO:1.

7. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises at least 90% identity to SEQ ID NO: 1. 30 8. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises at least 95% identity to SEQ ID NO: 1. A recombinant expression cassette, comprising the polynucleotide of any one of claims 1-8 operably linked to a promoter.

10. A host cell comprising the recombinant expression cassette of claim 9 or the polynucleotide of any one of claims 1-8. [R:\1ibffl9923.doc:SAK 64

11. A transgenic plant comprising a recombinant expression cassette of claim 9 or the polynucleotide of any one of claims 1-8.

12. The transgenic plant of claim 11, wherein said plant is a monocot.

13. The transgenic plant of claim 11, wherein said plant is a dicot.

14. The transgenic plant of claim 11, wherein said plant is selected from the group consisting of maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet. A transgenic seed from the transgenic plant of any one of claims 11-14.

16. A method of modulating the level of Rad50 in a plant, comprising: introducing into a plant cell a recombinant expression cassette comprising a Rad50 polynucleotide of any one of claims 1-8 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 of said polynucleotide for a time sufficient to modulate the level of Rad50 in said plant.

17. The method of claim 13, wherein said plant is selected from the group consisting of maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.

18. An isolated protein comprising a member selected from the group consisting of: a polypeptide of at least 20 contiguous amino acids from the polypeptide of SEQ ID NO:2; 25 the polypeptide of SEQ ID NO:2; a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO:2, wherein said sequence identity is determined using the GAP program under default parameters; and at least one polypeptide encoded by a member of claim 1. o 30 19. The isolated polypeptide of claim 18, wherein the polypeptide comprises at least 85% identity to SEQ ID NO:2.

20. The isolated polypeptide of claim 18, wherein the polypeptide comprises at least 90% identity to SEQ ID NO:2.

21. The isolated polypeptide of claim 18, wherein the polypeptide comprises at least 95% identity to SEQ ID NO:2. [R:\Iibff]9923.doc:SAK 4A

22. A Rad50 polynucleotide, substantially as hereinbefore described with reference to any one of the examples.

23. A recombinant expression cassette comprising the polynucleotide of claim 22, operably limited to a promoter.

24. A host cell comprising the recombinant expression cassette of claim 23 or the polynucleotide of claim 22. A transgenic plant comprising the recombinant expression cassette of claim 23 or the polynucleotide of claim 22.

26. A method of modulating the level of Rad50 in a plant, substantially as hereinbefore described with reference to any one of the examples.

27. An isolated Rad50 protein, substantially as hereinbefore described with reference to any one of the examples. Dated 8 January, 2004 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *s* 4 0* *0 o 0* S *S *S [R:\libff]9923.doc:SAK