MXPA02010012A

MXPA02010012A - Maize cellulose synthases and uses thereof.

Info

Publication number: MXPA02010012A
Application number: MXPA02010012A
Authority: MX
Inventors: Timothy G Helentjaris
Original assignee: Pioneer Hi Bred Int
Priority date: 2000-04-14
Filing date: 2001-04-12
Publication date: 2003-04-25
Also published as: AU2001253413A1; EP1272650A2; WO2001079516A3; CA2406381C; CA2406381A1; HUP0301659A3; WO2001079516A2; HUP0301659A2

Abstract

The invention provides isolated cellulose synthase nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering cellulose synthase levels in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions.

Description

CELLULOSE MAIZE SYNTHETICS AND USES OF THEM TECHNICAL FIELD The present invention is generally related to the molecular biology of plants. More specifically, it relates to nucleic acids and methods for modulating their expression in plants. BACKGROUND OF THE INVENTION Polysaccharides constitute the volume of cell walls of the plant and have traditionally been classified into three categories: cellulose, hemicellulose, and pectin. Fry, S.C. (1988), The growing plant cell wall: Chemical and metabolic analysis, New York,: Longman Scientific & Technical Considering that cellulose is made in the plasma membrane and directly extended in the cell wall, hemicellulosic and pectic polymers are first made in the Golgi apparatus and then exported to the cell wall by exocytosis. Ray, P. M:; et al. (1976) Ber. Deutsch Bot. Ges, Bd. 89, 121-146. The variety of chemical bonds in the hemicellulosic and pectic polysaccharides indicate that there must be dozens of synthases of the polysaccharide in the Golgi apparatus. Darvill et al. , (1980) The primary cells walls of flowering plants. In The Plant Cell (N. E. Tolbert, ed.), Vol. 1 in the Series: The biochemistry of plants: A comprehensive treatise, eds. P. K. Stumpf and E.E. Conn (New York: Academic Press), pp. 91-162.

It has been found that the stem folding power of normal and mutant mutants of barley stem brittle is directly correlated with cellulose concentration in the cell wall Kokubo, et al. , (1989), Plant Physiology 91, 876-882; Kokubo, et al., (1991) Plant Physiology 97, 509-514. As the composition of the stem contributes to numerous important quality factors in the breeding of maize, what is needed in the art are products and methods for the manipulation of the cellulose concentration in the cell wall and the calijdad of the stem of the plant and therefore altering the quality of the stem of the plant to provide, for example, an increase in the ability to stand erect or improving the conservation of forage in the silo. The present invention provides these and other advantages. SUMMARY OF THE INVENTION Generally, it is the object of the present invention to provide nucleic acids and proteins that are related to cellulose synthetases. It is an object of the present invention to provide transgenic plants comprising nucleic acids of the present invention, and methods for the modulation, in a transgenic plant, of the expression of the nucleic acids of the present invention. Accordingly, in one aspect invention related to the isolation of a nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a sequence identity specific to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide that is complementary to the pclinucleotide of (a); and, (c) a polynucleotide comprising a specified number of contiguous nucleotides of a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA. In other aspects the present invention relates to: 1) recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a prcmotor, 2) a host cell into which the recombinant expression cassette has been introduced, and 3) a transgenic plant comprising the recombinant expression cassette. The host cell and plant are optionally corn, wheat, rice, or soybean. DETAILED DESCRIPTION OF THE INVENTION Comprehensive Assessment A. Nucleic Acids and Protein of the Present Invention Unless otherwise stated, the polypeptide polynucleotide sequences identified in Table 1 represent polynucleotides and polypeptides of the present invention. Table 1 is a cross-reference of these polynucleotides and polypeptides for your gene name and the internal database identification number. A nucleic acid of the present invention comprises a polynucleotide of the present invention. A protein of the present invention comprises a polypeptide of the present invention. Table 2 provides a percent identity / similarity calculation of the referenced polynucleotide / polypeptide sequences to identify homologues using methods such as that found in Example 4. TABLE 1 Database, Polypeptide Polynucleotide Name of gene ID NO: SEQ ID NO: SEQ ID NO: Cellulose synthase Cdpgs 5 (cesA-3 Cellulose synthase Cqrael9 (cesA-9, B. Exemplary Utility of the Present Invention The present invention provides utility in such exemplary applications as improving the quality of the stem to improve the position or silage (forage conserved in silo). Further, the present invention maintains an increased concentration of cellulose in the pericarp, while hardening the grain and thus improving its handling ability.

Definitions Units, prefixes, and symbols can be denoted in their accepted SI form. Unless otherwise indicated, the nucleic acids are written from left to right in orientation 5 'to 3'; the amino acid sequences are written from left to right in amino to carboxy orientation, respectively. Numerical ranges recited within the specification are inclusive of the numbers that define the range and include each integer within the defined range. The amino acids can be referred to herein by each of their three normally known letter symbols or by the letter-one symbols recommended by the IUPAC-IUBMB Nucleotide Nomenclature Commission, likewise, they can be referred by their commonly accepted codes of the simple letter. Unless otherwise provided, software, electrical, and electronic terms are used here as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5h edition, 1993). The terms defined below are more fully defined together by reference to the specification. Sections of the section provided throughout the specification are not limitations on the various objects and embodiments of the present invention. By "amplified" is meant the construction of multiple copies of a sequence of a nucleic acid or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Two amplification systems include polymerase chain reaction (PCR), chain ligase reaction (LGR), nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasa systems, of transcription-based amplification (TAS), and rope displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, D. H. the Persing et al. , Ed., American Society for Microbiology, Washington, D.C. (1993). The | Amplification product is called an amplicon. As used herein, "antisense orientation" includes reference to a sequence of the double polynucleotide that is operably linked to a promoter in an orientation where the sense string is transcribed. The string meaningless is sufficiently complementary to a product of endogenous transcription that such translation of the product of endogenous transcription is often inhibited. By "coding" or "coding", with respect to a specified nucleic acid, it means comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise the non-translated sequences (e.g., introns) within the translated regions of the nucleic acid, or such intervening non-translated sequences may be missing (e.g., as in the cDNA). The information why 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, as it is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliate Macronucleus can be used when the nucleic acid is expressed in it When the nucleic acid is prepared or synthetically altered, the advantage can be advantageously taken advantage of known codons of the intentional host where the nucleic acid will be expressed. nucleic acids of the present invention in dicotyledonous monocotyledonous species plants, sequences can be modified to respond to specific codon preferences and GC content preferences of monocots or dicots as these preferences have been shown to differ (Murray et al., Nucí Acids Res. 17: 477-498 (1989)). Thus, the preferred codon of corn for a particular amino acid can be derived from the gene sequences known from corn. The use of corn codon for 28 genes from maize plants is listed in table 4 of Murray et al. , supra. As used herein, "Whole-body sequence" in reference to a specified polynucleotide or its encoded protein means that it has the entire sequence of the native amino acid (non-synthetic), endogenous, biologically (eg, structurally or catalytically). ) active form of the specified protein. Methods for determining whether a sequence is whole body are well known in the art, including such exemplary techniques as northern and western spots, first extension, Sl protection, and ribonuclease protection. For example, see Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). The comparison for knowing whole body homologous sequences (orthologs and / or parologists) can also be used to identify whole body sequences of the present invention. Additionally, general agreement consensus sequences typically present in the 5 'and 3' untranslated regions of the mRNA aid in the identification of a whole-body polynucleotide. For example, the ANNNNAUGG consensus sequence where the underlined codon represents the N-terminal methionine, aids in the determination whether the polynucleotide has a complete 5 'terminus. The consensus sequences at the 3 'end, such as the polyadenylation sequences, aid in the determination whether the polynucleotide has a complete 3' ending. As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if part of the same species, is modified substantially from its native form in composition and / or genomic site by human intervention For example, a promoter operably linked to a heterologous structural gene is of a different species from which the structural gene was derived, or, if part of the same species, one or both are substantially modified from their original form.A heterologous protein can originate from foreign species or, if part of them species, is substantially modified from its original form by human intervention. "Host cell" means a cell that contains a vector and supports the to replication (repetition) and / or expression of the vector. The cells of the host can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Preferably, the host cells are cells of monocotyledonous or dicotyledonous plants. A particularly preferred monocot host cell is a maize host cell.

The term "introduced" includes reference to the incorporation of a nucleic acid into a eukaryotic cell or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (eg, chromosome, plasmid, piastide or mitochondrial DNA), converted into a autonomous, or temporarily expressed, replicon (e.g., transfected mRNA). The term includes such introduction of the nucleic acid as "transfection", "transformation" and "transduction". The term "isolated" refers to material, such as a nucleic acid or a protein, which are: (1) substantially or essentially free of components that normally accompany or interact with it as they are found in their natural environment. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically altered or synthetically produced by deliberate human intervention and / or placed in a different situation within the cell. The synthetic alteration or creation of the material can be done, on the material inside or apart starting from its natural state. For example, a naturally-occurring nucleic acid becomes an isolated nucleic acid if it is altered or produced by non-natural, synthetic methods, or if it is transcribed from DNA that has been altered produced by non-natural, synthetic methods. The isolated nucleic acid can also be produced by the synthetic rearrangement ("shuffling") of a part or parts of one or more allelic forms of the gene of interest. Likewise, a naturally-found nucleic acid (eg, a promoter) is isolated if it is introduced to a different site of the genome. Nucleic acids that are "isolated", as defined herein, are also referred to as "heterologous" nucleic acids. For example, see Compounds and Methods for Site Directed Mutagenesis in Eukariotic Cells, Kiec, U. S. Patent No. 5,565,350; In Vivo Sequence Targeting in Eukaryotic Cells, Zarling et al., WO 93/22443 (PCT / US93 / 03868). As used herein, the "nucleic acid" includes reference to a ribonucleotide polymer deoxyribonucleotide, or chimeras thereof, in any single or double-stranded form, and unless otherwise limited, encompasses known analogs having the essential nature of natural nucleotides in which they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (eg, peptides of nucleic acids). "Nucleic acid library" means a collection of isolated DNA or molecules of nucleic acids. RNA comprising and substantially representing the entire transcribed fragment of a genome of a specified organism, tissue, or a cell type from that organism. Construction of exemplary nucleic acid libraries, such as the cDNA and genomics libraries, is taught in molecular biology references stadar as Berger and Kimmel, Guide to Molecular Cloning Teknology, Methods in Enzymology, Vol. 152, Academia Press, Inc. ., San Diego, CA (Berger); Sambrook et al. , Molecular Cloning-A Laboratory Manual, 2 ed. , Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al. , Eds., Current Protocols, to betwen joint venture Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994). As used herein "operably linked" includes reference to a functional binding between a promoter and a second sequence, wherein the promoter sequence begins and mediates the transcription of the DNA sequence corresponding to the second sequence. Generally, the operably linked means that the nucleic acid sequences to be joined are contiguous and, where necessary, to join two encoded protein regions, contiguous and in the same reading frame. As used herein, the term "plant" includes reference to whole plants, parts of the plant or organs (eg, tiojas, stems, roots, etc.), plant cells, seeds and progeny of the plant. same.

The cell of the plant, as used herein, further includes, without limitation, cells obtained from or found in: seeds, suspension or culture media, embryos, meristematic regions, lime tissue, leaves, roots, shoots, gametophytes , sporophytes, pollen and microspores. The cells of plants can also be understood to include modified cells, such as protoplasts, obtained from the mentioned tissues. The class of plants that can be used in the methods of the invention is generally as broad as the class of plants most amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. A particularly preferred plant is Zea mayz. As used herein, "polynucleotide" includes reference to a deoxyribopolinucleotide, ribopolynucleotide, or chimeras or analogs thereof having the essential nature of a natural deoxy- or ribonucleotide in which they hybridize, under the conditions of severe hybridization, for substantially the same nucleotide sequence as the naturally occurring nucleotide and / or allows translation into the same amino acid (s) as the naturally occurring nucleotide (s). A polynucleotide can be whole body or a subsequence of a native structural gene or heterol oggoo or regulator. Unless otherwise indicated, the term includes the protein, that the protein is specifically reactive to antibodies raised 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 binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosilation. Furthermore, this invention contemplates the use of both terminal amine variants of the protein, containing methionine and non-methionine, of the invention. As used herein the term "promoter" includes reference to a region of DNA in counterflow from the beginning of transcription and involved in the recognition and binding of RNA polymerase and other proteins to begin transcription A "promoter plant" is a promoter capable of starting transcription in plant cells whether or not its origin is a plant cell. Exemplary promoter plants include, but are not limited to, those obtained from plants, plant viruses, and bacteria comprising genes expressed in plant cells such as Agrobacterium or Rhizobium. Examples of promoters under development 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 that initiate transcription only in certain tissues are referred to as" specific tissue. "A specific promoter-type" cell "primarily handles expression in certain cell types in one or more organs, for example, vascular cells in roots or A "inducible" or "repressible" promoter is a promoter that is under environmental control. Examples of environmental conditions that can be transcribed by the inducible promoters include anaerobic conditions or light preserence. The promoters specific tissue, preferred tissue, specific cell type and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter that is active at most environmental conditions. As used herein, the term "recombinantee" includes the reference to a cell? vector that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a modified cell. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of cellular or expressed native genes that are otherwise abnormally expressed, expressed-low or not at all expressed as result of human intervention. The term "recombinantee" as used here does not cover alteration of the cell or vector by naturally occurring events (eg, spontaneous mutation, transformation / transduction / natural transposition) such as those encountered without human intervention, as used herein a "recombinant expression cassette" is a nucleic acid constructed, recombinantly generated synthetically, with a series of specified nucleic acid elements that allow the transcription of a particular nucleic acid in a host cell. The recombinant expression cassette may be incorporated into a chromosome plasmid, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the portion of the recombinant expression cassette of an expression vector includes, among other sequences, a nucleic acid. to be transcribed, and a promoter. The term "residua" or "amino acid residue" or "amino acid" are used interchangeably here to refer to u? amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid can be a naturally occurring amino acid and, unless otherwise limited, can encompass non-natural analogs of natural amino acids that can function in a similar way as amino acids naturally Found The term "selectively hybridize" includes reference to hybridization, under the conditions of severe hybridization, of a nucleic acid sequence to a specific nucleic acid sequence to a significantly greater degree (eg, at least the 2-fold over of background) than its hybridization to non-designated nucleic acid sequences and to the substantial exclusion of non-designated nucleic acids. Selectively hybridized sequences typically have at least 80% sequence identity, preferably 90% sequence identity, and more preferably 100% sequence identity (i.e., co-consistent) with each other. The term "severe conditions" or "severe hybridization conditions" includes reference to conditions under which the screening will be selectively hybridized to its designated sequence for a greater degree of perceptibility than for other sequences (eg, at least 2- fold over the antecedent). Severe conditions are sequence-dependent and will be different in different circumstances. By controlling the narrowness of the hybridization and / or by washing the conditions, the designated sequences can be identified which are 100% complementary to the probe (probing homolog). Alternatively, the conditions of The specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For the DNA-DNA hybrids, the Tm can approximate the equation of Meinkoth and Wahl, Anal. Biochem. , 138: 267-284 (1984): Tm = 81.5SC + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L; where M is the molarity of the monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under the defined ionic strength and pH) at which 50% of a complementary designated sequence hybridizes to a perfectly balanced probe. The Tm is reduced by approximately C for each 1% of imbalance; thus, Tm, hybridization and / or washing conditions can be adjusted to hybridize to the desired identity sequences. For example, if you search for sequences with > 90% identity, the Tm can be decreased 10aC. Generally, severe conditions are selected to be approximately 5aC lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, very severe conditions can use a hybridization and / or wash in 1, 2, 3, or 4aC lower than the thermal fusion pin (Tm); slightly harsh conditions can use a Hybridization and / or washing at 6, 7, 8, 9, or 10aC lower than the thermal melting point (Tm) the low narrowing conditions can use a hybridization and / or wash at 11, 12, 13, 14, 15, or 20aC lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and Tm desired, those of ordinary skill will understand that variations are inherently described in the narrowness of hybridization and / or wash solutions. If the desired degree of unequalization of results in a Tm of less than 45aC (aqueous solution) or 32aC (formamide solution) it is preferred to increase the concentration of SSC so that a higher temperature can be used. Hybridization and / or wash conditions may be applied for at least 10, 30, 60, 90, 120, or 240 minutes. An extensive guide for nucleic acid hybridization is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization, Nucleic Aicid Probes, Part I, Chapter 2, "Overview of principles of hybridization and strategy of nucleic acid probé assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al. , Eds.,. Greene Publisbing and Wiley-Interscience, New York (1995). As used herein, the term "transgenic plant" includes reference to a plant that comprises within its genome a heterologous polynucleotide. Generally, Heterologous polynucleotides are stably integrated into the genome such that the polinvcleotide is passed on to successive generations. The heterologous polynucleotides can be integrated into the genome alone or as part of a cassette of expression cosebinantee. "Transgenic" is used to include any cell, cell line, callus, tissue, part of the plant or plantai, the genotype of which has been altered by the presence of heterologous nucleic acid that includes those initially altered transgenics as well as those created by crossings sexual or by asexual propagation of the initial transgenics. The term "transgenic" as used herein does not cover genome alteration (chromosomatic or extra-cr?> mosomático) by conventional methods of plant breeding or by naturally occurring events such as randomized cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation. As used herein, the term "vector" includes reference to a nucleic acid used in the introduction of a polynucleotide of the present invention into a host cell. Vectors are often replicons. Expression vectors allow the transcription of a nucleic acid inserted therein. The following terms are used to describe the relationships of the sequence or sequence between a polynucleotide / polypeptide of the present invention with a polynucleotide / polypeptide reference: (a) "sequence or reference sequence", (b) "comparison window", (c) "sequence or sequence of identity ", and (d)" percentage of sequence identity or sequence "(a) As used herein, the" reference sequence "is a defined sequence used as a basis for comparison of the sequence with a polynucleotide / polypeptide of the present invention A reference sequence may be a subset or the integrity of a specified sequence, for example, as a segment of a whole-body cDNA or sequence of the gene, or the entire cDNA or sequence of the gene. As used herein, "comparison window" includes reference to an immediate and specified segment of a polynucleotide / polypeptide sequence, wherein the polynucleotide / polypeptide sequence can be compared to a reference sequence. and where the portion of the polynucleotide / polypeptide sequence in the comparison window may comprise sums or (subtracts) erasures (ie, gaps) compared to the reference sequence. (which does not include sums or subtractions) for the optimal alignment of the two sequences. Generally, the comparison window is so minus 20 nucleotide / amino acid residues in length, and optionally it may be 30, 40.50, 100, or longer. Those of skill in the art understand that to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide / polypeptide sequence, a penalized gap is typically introduced and subtracted from the number of mismatches. Methods of alignment of sequences for comparison are well known in the art. Optimal sequence alignment for comparison can be addressed by the lime homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the alignment homology algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search for the similarity method of Pearson and Lipman, Proc. Nati Acad. Sci. 85: 2444 (1988); for the informatized applications 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 family of BLAST programs that can be used for database similarity searches includes: BLASTN for search of nucleotide sequences or sequences against the database nucleotide sequences; BLASTX for search of nucleotide sequences against the protein sequences of data bae; BLASTP for search of protein sequences against protein database sequences; TBLASTN for search of protein sequences against the nucleotide sequences of the database; and TBLASTX for search of nucleotide sequences against nucleotide database sequences. See, Current Protocols in Molecul [ar Biology, Chapter 19, Ausubel, et al. , Eds., Greene Publishing and Wiley-Interscience, New York (1995); Altschul et a,!., J. Mol. Biol. , 215: 403-410 (1990); and, Altschul et al. , Nucleic Acids Res. 25: 3389-3402 (1997). Software to perform BLAST analyzes is publicly available, for example, through Information from the National Center for Biotechnology (http: //www.ncbi.nlm.nih.gov/). This algorithm first involves the identification of high sequence pairs (HSPs) by identifying short words of length W in the searched sequence, which balance or expectation (E) of 10, a shortcut of 100, M = 5, N = -4, and a comparison of both cordons. For the amino acid sequences, program them. BLASTP uses a long word (W) of 3, an expectation (E) of 10, and a BLOSUM62 matrix account (see Henikoff &Henikoff (1989) Proc. Nati. Acad. Sci. USA. 10915) In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin &P.ltschul, Proc. Nat'l. Acad. Sci USA 90: 5873-5877 (1993)) A measure of similarity provided by the BLAST algorithm is the probability of the smallest sum (P (N)) that gives an indication of the probability by which a balance between two Nucleotides or amino acid sequences would happen by chance. BLAST searches assume that proteins can be planned as random sequences. However, many real proteins comprise regions of non-random sequences that can be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions can be aligned between the unrelated proteins although other regions of the protein are completely dissimilar. Several low-complexity filter programs can be employed to reduce such complexity alignments. For example, SEG (Wooten and Federhen, Comput, Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput, Chem., 17: 191-201 (1993)) can be used for low-complexity filters exclusively (alone) or in combination. Unless otherwise stated, the identity / nucleotide simulation and protein values provided herein are calculated using GAP (GCG Version 10) below the predetermined values. The GAP (Global Alignment Program) can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence. The GAP uses the algorithm of Needleman and Wunsch (JJ Mol. Biol. 443-453, 1970) to find the alignment of two complete sequences that maximize the equilibrium number and minimize the number of gaps. The GAP considers all possible alignments and positions of the gap and creates the alignment with the largest number of paired bases and the smallest number of gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in the units of paired bases. He GAP must make a profit eg hole creation penalized by the equilibrium number for each hole that is inserted.

If a penalized gap extension greater than zero is chosen, the GAP must, in addition, make a profit for each hole inserted from the length of the hole times the gap extension penalty. The default values for the gap creation penalty and the gap extension penalty values in Version 10 of the Wisconsin Genetics software package for the protein sequences are 8 and 2, respectively. For the nucleotide sequences the predefined gap creation penalty is 50 while the predefined gap extension penalty is 3. The penalties for the creation of the gap and the extension of the gap can be expressed as a whole integer selected from the group of integers that They consist of 0 to 100. Thus, for example, the penalties for the creation of the gap and the extension of the gap can each be independently: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50.60 or more The GAP presents a member of the family of the best alignments. There may be many members of this family, but no other member has a good quality. The GAP displays four figures of merit for the alignments: Quality, Proportion, Identity, and Similarity. Quality is the metric maximized to frame the sequences. The ratio is the quality divided by the number of bases in the shortest segment. Percent Jdentity is the percent of the symbols that actually match. The percent Similarity is the percent of the symbols that are similar. Symbols that are in front of the The polynucleotide in the comparison window may comprise additions or subtractions (ie, voids) as compared to the reference sequence (which does not comprise additions or subtractions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions to which the base, nucleic acid, identical or amino acid residue occurs in both sequences to yield the number of matched positions, by dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result per 100 to obtain the percent identity of the sequence. Utilities The present invention provides, among other things, compositions and methods for modulating (ie, increasing or decreasing) the level of polynucleotides and polypeptides of the present invention in plants. In particular, the plinucleotides and polypeptides of the present invention may be expressed temporarily or spatially, for example, in developmental stages, in tissues, and / or in amounts that are atypical of non-recombinantly designed plants. The present invention also provides isolated nucleic acids comprising polynucleotides of sufficient length and complementarity for a polynucleotide of the present invention for use as probes or first amplification in the detection, quantification or isolation of gene transcripts. For example, isolated nucleic acids of the present invention can be used as the probes in the detection of deficiencies at the mRNA level in the selection for the desired transgenic plants, for the detection of mutations in the gene (eg, substitutions, subtracts). : ... is, or additions), for the monitoring of expression regulation or changes in enzyme activity by protecting assays from compounds, for the detection of any number of allelic variants (polymorphisms), orthologs, or gene paralogs, or for the site he directed mutagenesis in eukaryotic cells (see, for example, US Pat. No. 5,565,350). The isolated nucleic acids of the present invention can also be used for the recombinant expression of their encoded polypeptides, or for use as immunogens in the preparation and / or protecting of antibodies. The isolated nucleic acids of the present invention may also be employed for use in sense or counter-sense deletion of one or more genes of the present invention in a host cell, tissue, or plant. The binding of chemical agents that bind, intercalate, stick and / or also conjugate binding for 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 (e.g., prep oenzyme, proenzyme, or enzymes). The present invention also provides proteins comprising an epitope of at least one 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 the expression levels, for identification and / or isolation of nucleic acids of the present invention from * the expression libraries, for the identification of homologous polypeptides of other species, or for polypeptide purification. of the present invention. The isolated nucleic acids and polypeptides of the present invention can be used over a wide range of plant types, particularly monocotyledons such as the species of the Gramineae family, including Hordeum, Sécale, Oryza, Triticum, Sorgum (eg, S. bicolor). ) Y Zea (for example, Z. mays), and dicotyledons such as Glycine. The isolated nucleic acid and the proteins of the present invention can also be used in the genus species: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifo? Ium, Trigonella, Vigna, Ci trus, 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, Nemes is, Pelar gonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Pisum, Phaseolus, Lolium, and Avena. Nucleic Acids The present invention provides, inter alia, nucleic acids isolated from RNA, DNA, and analogs and / or cras thereof, comprising a polynucleotide of the present invention. A polynucleotide of the present invention is inclusive of those in Table 1 and: (a) An isolated polynucleotide encoding a polypeptide of the present invention such as those referenced in Table. ' , including exemplary polynucleotides of the present invention; (b) An isolated polynucleotide which is the amplification product of a plant nucleic acid library using primer pairs which selectively hybridizes under severe conditions to the sites within a polynucleotide of the present invention; (c) An isolated polynucleotide that selectively hybridizes to a polynucleotide of (a) or (b); (d) An isolated polynucleotide having a specified sequence identity with polynucleotides of (a), (b) or (c); (e) An isolated polynucleotide encoding a protein having a specified number of immediate amino acids from a prototype polypeptide, wherein the protein is specifically recognized by elicited antisera taken out by the protein presentation and wherein the protein it is not detectably immunoreactive for ant sera which has been fully immunosorbed with the protein; (f) The complementary polynucleotide sequences of (a), (b), (c), (d) or (e); and (g) An isolated polynucleotide comprising at least a specific number of immediate nucleotides from a polynucleotide of (a), (b), (c), (d), (e) or (f); (h) A polynucleotide isolated from an enriched whole-body cDNA library having the physical-chemical property of selectively hybridizing to a polynucleotide of (a), (b), (c), (d), (e), ( f) or (g); (i) An isolated polynucleotide made by the process of: 1) providing an enriched whole-body nucleic acid library, 2) selectively hybridizing the polynucleotide to a polynucleotide of (a), (b), (c), (d) , (e), (f), (g) or (h), therefore isolating the polynucleotide to invention provides an isolated nucleic acid comprising a polynucleotide of the present invention, wherein the polynucleotides are amplified, under conditions of nucleic acid amplification, from a nucleic acid library of the plant. The conditions of nucleic acid amplification for each of the variety of amplification methods is well known to those of ordinary skill in the art. The nucleic acid library of the plant can be constructed from a monocotyledon such as a cereal crop. Exemplary cereals include corn, sorghum, alfalfa, cañola, wheat, or rice. The nucleic acid library of the plant can also be constructed from a dicotyledon such as soybean. The lines of Zea mayz B73, PHRE1, A632, BMS-P2 # 10, W23, and Mol7 are known and publicly available. Other publicly available corn lines known and available from Maize Genetics Cooperation (Urbana, IL) can be obtained. Wheat lines are available from Wheat Genetics Resource Center (Manhattan, KS). The nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of the intron process. The cDNA libraries can be normalized to increase the representation of relatively rare cDNAs. In optional modalities, and the cDNA library is constructed using an enriched whole-body cDNA synthesis method. Examples of such methods include Oligo-Capping (Maruyama, K. and Sugano, S. Gen 138: 171-174, 1994), Biotinylated CAP Trapper (Carninci, et al., Genomics 37: 327-336, 1996), and CAP Retention Procedure (Edery, E., Chu, LL, et al., Molecular and Cellular Biology: 3363-3371, 1995). Tissues are rapidly developed or rapidly dividing cells are preferred to be used as a source of mRNA for the construction of a cDNA library. The phases of corn growth are described in "How to Corn Plant Develops Special Report No. 48, Iowa State University of Science and Technology Cooperative Service Extension, Ames, Iowa, Reprinted February 1993. A polynucleotide of this modality (or subsequences thereof) can be obtained, for example, by using amplification primers which are selectively hybridized and the former extended, under nucleic acid amplification conditions. , to at least two sites within a polynucleotide of the present invention, or to two sites within the nucleic acid which side and comprises a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid that comprises it. The methods to obtain 5 'endings and / or 3 'of a vector insert are well known in the art. See, for example, RACE (Rapad Amplification of Complementary Ends) as described in Frohman, M.A., in PCR Protocols: A Guide to Methods and ApplJcations, M.A. Innis, D.H. 5 Gelfand, J.J. Sninsky, T.J. White, Eds. (Academia Press, Inc., San Diego), pp. 28-38 (1990)); also see, 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 10 1: 165 (1989). Optionally, the former are complementary to a designated nucleic acid subsequence which they amplify but may have a sequence identity ranging from approximately 85% to 99% relative to the sequence 15 of the polynucleotide to which they are designed to anneal. As those skilled in the art will appreciate, the sites to which the pairs of the first will selectively be hybridized are chosen such that a single immediate nucleic acid can be formed under the conditions 20 of nucleic acid amplification desired. The length of the first in the nucleotides of the group of integers consisting of at least 15 to 50 is selected. Thus, the former may be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length. Those of skill will recognize 25 that a sequence of first elongated osl can be used -.TO* to increase binding specificity (i.e., quenching) to a designated sequence. Unneeded Unei sequence at the 5 'end of a first (a "tail") can be added, for example, to introduce a cloning site at the final ends of the amplicon. The amplification products can be translated using expression systems well known to those skilled in the art. The resulting translation products can. confirmed as polypeptides of the present invention by means of, for example, assaying for the appropriate catalytic activity (eg, specific activity and / or substrate specificity), or verifying the presence of one or more epitopes which are specific to a polypeptide of the present invention. Methods for the synthesis of rotein from templates derived from PCR are known in the art and commercially available. For example, see Amersham Life Sciences, Ine, Catalog '97, p.354. The polynucleotides of the present invention include those amplified using the following primer pairs: SEQ ID NOS: 3 and 4, which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 1; and SEQ ID NOS: 7 and 8, which yield an amplicon comprising a sequence having substantial identity to SEQ ID NO: 5. C. Polynucleotides which selectively hybridize to a polynucleotide of (A) or (E1) As indicated in (c), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein selectively hybridized polynucleotides, under the conditions of selective hybridization, to a polynucleotide of sections (A) or (B) as discussed above. Thus, the polynucleotides of this embodiment can be used for incarnation for isolation detection, and / or quantification of nucleic acids comprising the polynucleotides of (A) or (B). For example, polynucleotides of the present invention can be used to identify, isolate, or amplify the clones partially or whole-body in a deposited library. In some embodiments, the polynucleotides are genomic or sequences isolated from cDNA or otherwise complementary to cDNA from a dicotyledonous or monocotyledonous nucleic acid library. Exemplary monocotyledonous and dicotyledonous species include, but are not limited to: corn, sugarcane, soybean, cotton, wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice. The cDNA library comprises 50% to 95% of whole body sequences (eg, at least 50%, 60%, 70%, 80%, 90%, or 95% of whole body sequences). The cDNA libraries can be normalized to increase the representation of rare sequences. See for example, U.S. Patent No. 5,482,845. Low stringency hybridization conditions are typically, but not exclusively, employed with sequences that have a reduced identity sequence relative to the complementary sequences. Optional moderate and high narrowing conditions may be used for the higher identity sequences. Low low conditions allow selective hybridization of sequences that are approximately 70% to 80% sequence identity and can be used to identify orthologous sequences or paralogical sequences. D. Polynucleotides having a specific sequence identity with the polynucleotides of (A), (B) or (C) As indicated in (d), 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 (A), (B), or (C), above. For example, the identity can be calculated using the algorithms BLAST, CLUSTALW, or GAP under the conditions of predetermined value. The identity percentage for a reference sequence is at least 50% rounded in addition to the nearest whole, can be expressed as an integer selected from the group of integers consisting of 50 to 99. Thus, for example, the percentage of identity to a reference sequence can be at least 60%, 70% , 75%, 80%, 85%, 90%, or 95%. Optionally, polynucleotides of this modality will encode a polypeptide that will share an epitope with a polypeptide encoded by the polynucleotides of sections (A), (B), or (C). Thus, these polynucleotides encode a first polypeptide that elicits antisera production comprising antibodies that are specifically reactive for a second polypeptide encoding a polynucleotide of (A), (B), or (C). However, the first polypeptide does not bind for antisera raised against itself when the antisera has been fully immunosorbed with the first polypeptide. Therefore, the polynucleotides of this embodiment can be used to generate antibodies for use in, for example, the selection of expression libraries for nucleic acids comprising the polynucleotides of (A), (B), or (C), or for the purification of, or immunoassays for, polypeptides encoded by the polynucleotides of (A), (B), or (C). The polynucleotides of this embodiment comprise nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention. Protected polypeptides, for specific binding to antisera, can be conveniently achieved using the peptide display libraries. This method involves the selection of large collections of peptides for individual members that have the desired function or structure. Antibody protected from peptide display libraries is well known in the art. The deployed peptide sequences may be from 3 to 5000 or more amino acids in length, often 5-100 amino acids in length, and often from approximately 8 to 15 amino acids in length. In addition to the direct chemical synthetic methods for the generation of peptide libraries, several recombinant DNA methods have been described. One type involves the deployment of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular unfolding sequence of the peptide. Such methods are described in PCT Patent Publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278.

Other systems for the generation of peptide libraries have aspects of both methods of synthesis, chemical synthesis in vi tro and recombinant methods. See, PCT patent publication Nos. 92/05258, 92/14843, and 97/20078. Also see, U.S. Patent 5,658,754; and 5,643,768 Libraries of displayed peptides, vectors, and selection kits are commercially available from such suppliers as Invitrogen (Carlsbad, CA). E. Polinucleotides encoding a protein having a subsequence of a prototype and cross-reactive polypeptide for the prototyping polypeptide As indicated in (e), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides encode a protein having an immediate amino acid subsequence of a prototype polypeptide of the present invention as provided in (a), above. The immediate amino acid length of the prototype polypeptide of the group of integers consisting of at least 10 to the number of amino acids within the prototype sequence is selected. 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, immediate amino acids of the prototype polypeptide. In addition, the number of such subsequences encoded by a polynucleotide of the instantaneous modality can be any integer selected from the group consisting of 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any nucleotide integer a 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, long production anticuerpoe which specifically to a polypeptide prototype ligated as but not limited to, a polypeptide encoded by the polynucleotide of (a) or (b ) , previously. Generally, however, a protein encoded by a polynucleotide of this modality does not bind antisera raised against | Prototype polypeptide when the antisera has been totally immunosorbed with the prototype polypeptide. The methods of making and assaying for antibody specificity / affinity binding are well known in the art. Lps formats immunoassay examples include ELISA competitive immunoassays, radioimmunoassays, Western blots Western Blots, indjirecto and similar immunofluorescent assay In a preferred assay method, fully inmunosorbed and pooled antisera which is taken for the polypeptide prototype can be used in a bligatorio assay ( binding) competitive to test the protein. The concentration of the prototype polypeptide required to inhibit 50% of the antisera binding for the prototype polypeptide is determined. If the amount of protein required to inhibit binding is less that twice the amount of prototype protein, then it is said that the protein binds specifically to the antisera taken out for the immunogen. Accordingly, the proteins of the present invention embrace allelic variants, conservatively modified variants, and minor recombinant modifications for lin 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 non-glycosylated whole-body polypeptides of the present invention. The molecular weight can be determined rapidly 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 5%, and most preferably within 3%, 2%, or 1% of a polypeptide full length of the present invention. Optionally, polynucleotides of this embodiment encode a protein having a specific enzyme activity at least 50%, 60%, 80% or 90% of a cellular extract comprising the native, endogenous polypeptide whole body of the present invention. In addition, the proteins encoded by the polynucleotides of this embodiment will optionally have a substantially similar affinity constant (Km T) and / or catalytic activity (ie. say, the microscopic proportion constant, kcat) as the endogenous, whole-body native protein. Those of skill in the art will recognize that this value of kcat / Km determines the specificity to compete for substrates and is often called the constant, of specificity. The proteins of this embodiment can have a kcat / Km value of at least 10% of a whole body polypeptide of the present invention as determined using the endogenous substrate of that polypeptide. Optionally, the value of kcat / m will be at least 20%, 30%, 40%, 50% and more preferably at least 60%, 70%, 80%, 90% or 95% the value of kcat / Km of the whole body polypeptide of the present invention. The determination of kcat, Km, and kca / Km can be determined by any number of means well known to those skilled in the art. For example, the initial ratios (ie, the first 5% or less of the reaction) can be determined using rapid sampling and mixing techniques (eg, continuous-flow, stationary-flow, or fast-off techniques), photolysis of flare, or relaxation methods (eg, temperature jumps) together with such exemplary methods of measurement as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance, or radioactive methods. Kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Ho | fstee plotter.

F. Polynucleotides complementary to the polynucleotides of (A) - (E) As indicated in (f), above, the present invention provides isolated nucleic acids comprising the polynucleotides complementary to the polynucleotides of the preceding paragraphs, A-E. As those of skill in the art will recognize, the complementary pair-base sequences along the length integrity with the polynucleotide sections (A) - (E) (ie, have 100% sequence identity over its entire length). The complementary bases associated through hydrogen bonds in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thiamine; and adenine and uracil. G. Polynucleotides, which are subsequences of the polynucleotides (A) - (F) As indicated in (g), above, the present invention provides isolated nucleic acids comprising polynucleotides which comprise at least 15 immediate bases from 'of the polynucleotides of sections (A) to (F) as discussed above. The length of the polynucleotide is given as an integer selected from the group consisting of at least 15 for the length of the nucleic acid sequence from the which the polynucleotide is a subsequence of. Thus, for example, the polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or 100 immediate nucleotides in length of the polynucleotides of (A) - ( F). Optionally, the number of such subsequences encoded by a polynucleotide of the instantaneous modality can be any integer selected from the group consisting of 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any nucleotide integer from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides. Subsequences can be made by synthetic methods in vi tro, biosintetico in vi tro, or in recombinant methods in vivo. In the optional modalities, the subsequences can be done by nucleic acid amplification. For example, the first nucleic acids will be constructed to selectively hybridize to a sequence (or its complement) within, or co-extensive with, the encoded region. The subsequences of the present invention may comprise structural features of the sequence from which it is derived. Alternatively, the subsequences may lack certain structural features of the larger sequence from which this is derived such as a Pol; i (A) tail. Optionally, a subsequence from uh polynucleotide encoding a polypeptide having at least one epitope in common with a prototype polypeptide sequence as provided in (a), above, may encode an epitope in common with the prototype sequence, Alternatively The subsequences can not encode an epi: ope 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 is derived.

Subsequences can be used to modulate or discover the expression of the gene by introducing compounds within the subsequences that bind, intercalate, stick and / or cross-link nucleic acids. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomicin or chloroethylaminoaryl conjugates H. Polynucleotides from an enriched whole-body cDNA library having the physical-chemical property of selectively hybridizing to a polynucleotide of (A) - (G As indicated in (h), above, the present invention provides a polynucleotide isolated from an enriched whole-body cDNA library having the physico-chemical property of selectively hybridizing to a polynucleotide of paragraphs b (A), (B) , (C), (D), (E), (F) or (G) as discussed above. The methods of constructing the enriched whole-body cDNA libraries are known in the art and discussed briefly below. The cDNA library comprises 50% to 95% of whole body sequences at least (eg, at least 50%, 60%, 70%, 80%, 90%, or 95% of whole body sequences). The cDNA library can be constructed from a variety of tissues of a monocotyledone or dicotyledon in a variety of developmental stages. Exemplary species include corn, wheat, rice, cañola, soy, cotton, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and arrpz. Hybridization methods selectively, under the conditions of selective hybridization, a polynucleotide from a whole-body library enriched for a polynucleotide of the present invention is known to those of ordinary skill in the art. Any number of narrowing conditions can be employed to allow for selective hybridization. In the optional modalities, narrowness allows for selective hybridization of sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity over the length of the hybridized region . Enriched whole-body cDNA libraries can be standardized to increase the representation of rare sequences I. Polynucleotide products made by a process of cDNA isolation As indicated in (I), above, the present invention provides an isolated polynucleotide made by the process of: 1) providing an enriched whole-body nucleic acid library] 2) hybridizing the polynucleotide selectively to a polynucleotide of the paragraphs (A), (B), (C), (D), (E), (F), (G) O (H as discussed above, and thereby isolating the polynucleotide from the nucleic acid library.) Enriched whole-body nucleic acid libraries are They are constructed as discussed in paragraph (G) and below.The selective hybridization conditions are as discussed in paragraph (G) .The nucleic acid purification procedures are well known in the art .The purification can be conveniently accomplished. using solid phase methods, such methods are well known to those skilled in the art and equipment is available from commercial suppliers such as Advanced Biotechnologies (Surrey, UK) For example, a polynucleoty of paragraphs (A) - { H) can be immobilized on a support such as a membrane, bead, or particle. For example, see for example U.S. Patent No. 5,667,976. The polynucleotide product of the present process is selectively hybridized to an immobilized polynucleotide and the solid support is subsequently isolated from the non-hybrid polynucleotides by the included methods, but not limiting, centrifugation, magnetic separation, filtration, electrophoresis, and the like. Construction of Nucleic Acids The isolated nucleic acids of the present invention can be made using (a) the standard recombinant methods, (b) the synthetic techniques, or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed, from a monocotyledone such as corn, rice, or wheat, or a dicotyledone such as soybean. The nucleic acids may comprise the sequences conveniently edemas of a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine tag 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 assembler by duplicating and / or expressing a polynucleotide of the present invention. Additional sequences can be added for such genomic The isolation of RNA and the construction of cDNA and genomic libraries is well known to those of ordinary skill in the art. See, for example, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Ed., Greene Publishing and Wiley-Interscience, New York (1995) Al. Enriched whole-body cDNA libraries Different cDNA synthesis protocols have been described which provide enriched whole-body cDNA libraries. Enriched whole-body cDNA libraries are constructed to comprise at least 600%, and more preferably at least 70%, 80%, 90% or 95% whole-body insertions between clones containing the insertions. The insertion length in such libraries can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to accommodate inserts of these sizes are known in the art and are commercially available. For example, see Stratagene's lambda ZAP Express (cDNA vector cloning with 0 to 12 kb cloning capacity). An exemplary construction method greater than 95% pure whole-body cDNA library is described by Carninci et al. , Genomics, 37: 327-336 (1996). Other methods for the production of whole body libraries are known in the art. For example, see Edery et al. , Mol.

Cell Biol. , 15 (6): 3363-3371 (1995); and, PCT Application WO 96/34981. A2 Normal or subtracted cDNA libraries A non-standardized cDNA library represents the mRNA population of the tissue from which they were made. Since the only clones are out-numbered by copies derived from favorably expressed genes, their isolation can be laborious. The normalization of a cDNA library is the process of creating a library in which each clone is represented more equally. The construction of standardized libraries is described in Ko, Nucí. Acids Res. , 18 (19) 5705-5711 (1990); Patanjali et al. , Proc. Nati Acad. USES. , 88: 1943-1947 (1991); U.S.

Patents No. 5,482,685, 5,482, 845, 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 tour orders of magnitude to a narrow range of only 1 order of magnitude Proc. Nati, Ac d. Sci. USES. , 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 a mRNA pool is emptied of sequences present in a second pool of mRNA by hybridization. The cDNA: mRNA hybrids are removed and the cDNA remains unhybridized in the pool is enriched for the unique sequences to that pool. See, Foote et al. , Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Tec nigiie, 3 (2): 58-63 (1991); Sive and St, John, Nucí. Acids Res. , 16 (22): 10937 (1988); Current Protocols in Molecular Biology, Ausubel, et al. , Eds., Greene Publishing and Wiley-Interscience,? Ew York (1995); and, Swaroop et al. , Nucí. Acids Res. , 19) 8): 1954 (1991). The cD? A subtraction equipment is commercially available. For example, yea PCR-Select (Clontech, Palo Alto, CA). To build genomic libraries, large segments of AD? genomic by fragmentation, for example using restriction endonucleases, and are ligated with the AD vector? to form concatemers that can be packaged in the appropriate vector. Methodologies for achieving these terminations, and sequencing methods for verifying the nucleic acid sequence are well known in the art. Examples of appropriate molecular biology techniques and sufficient instructions to direct people of skill through much construction, cloning, and protecting methodologies are found in Sambrook, et al. , Molecular Cloning: A Laboratory Manual, 2 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: Academia Press Ine, (1987), Current Protocols in Molecular Biology, Ausubel, bt al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). The equipment for the construction of genomics libraries are also commercially available. The cDNA or genomic library can be screened using a probe based on the sequence of a polynucleotide of the present invention, such as those discovered here. The sodas can be used to hybridize with the 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 hybridization narrowness may be employed in the assay; and either the hybridization or the washing medium can be severe. Nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques. For example, the polymerase chain reaction (PCR) technology can be used to amplify the polynucleotide sequences of the present invention and related genes directly from the genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, example, to clone nucleic acid sequences encoding the proteins to be expressed, to make nucleic acids to be used as probes to detect the presence of desired mRNA in the samples, for nucleic acid sequencing, or for other purposes. The T4 32 protein gene (Boehringer Mánnheim) can be used to improve the performance of long PCR products. PCR-based selection methods have been described. Wilfinger et al. describes a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be removed from the study. BioTechniques, 22 (3): 481-486 (1997). Such methods are particularly effective: it is in combination with a whole-body cDNA construction methodology, above. B. Synthetic Methods for Building 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 tryptophanidite triester solid phase method described by Beaucage and Caruthers, Tetra. Letts. 22 (20): 1859-1862 (1981), for example, using an automated synthesizer, for example, as described in Needham-VanDevanter et al. , Nucleic Acids Res. , 12: 6159-6168 (1984); and, the method with solid support of U.S. Patent No. 4,458,066 Chemical synthesis generally produces a stranded simple oligonucleotide. This can become a double stranded DNA by hybridization with. a complementary sequence, or by polymerization with DNA pclimerase using the simple strand as a template. A forgiving ability will recognize that while chemical synthesis of DNA is best employed for sequences of about 100 bases or less, longer sequences can be obtained by ligation of shorter sequences, Replenishing Expression Cassettes The present invention also provides expression cassettes recombinants comprising a nucleic acid of the present invention. A nucleic acid sequence encoding 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 that can be introduced in the cell of the desired host. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to initiation transcriptional regulator of the sequences that will direct the transcription of the polynucleotide in the intentional host cell, such as the tissues of a transformed plant. For example, Plant Expression Rectors may include (1) a cloned plant gene under the transcriptional control of the 5 'and 3' regulatory sequences and (2) a dominant selectable label. Such plant expression vectors may also contain, if desired, a region-regulating promoter (eg, an inducible or constitutive, environmentally- or developmentally-regulated, cell-tissue-specific / expression-selective), a site starting from the initiation of transcription, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal. A fragment of plant promoter can be employed which will direct the expression of a polynucleotide of the present invention in all tissues of a regenerated plant. Such promoters are referred to here as promoters "constitutive" and are active under most environmental conditions development states differentiation of the cell. Examples of constitutive promoters include cauliflower mosaic virus (CaMV) 35S transcription initiation region, the promoter 1'- or 2'- derived from T-DNA of Agrobacterium turne faeciens, the ubiquitin 1 promoter, the Smas promoter, the alcohol cinnamyl dehydrogenase promoter (US Patent No. 5,683,439 American), the Nos promoter, the pEmu promoter, the rubisco promoter, and the GRPI-8 promoter Alternatively, the plant promoter may direct expression of a polynucleotide of the present invention in a specific tissue or may otherwise be under more precise environmental or developmental control. Such promoters are referred to herein as the "inducible" promoters. Environmental events that can be transcribed by inducible promoters include pathogenic ataciues, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Achim eme promoter is inducible by hypoxia or cold stress, the Hsp70 promoter that is inducible by heat stress, and the PPDK eme promoter is light-inducible, Examples of promoters under developmental control they include eme promoters only initiate transcription, or preferably, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. Exemplary promoters include the anther-specific promoter 5126 (U.S. Patent Nos. 5,689,049 and 5,689,051), glb-1 promoter, and the gamma-zein promoter. Also see, for example, U.S. Patent 60 / 155,859 and 60 / 163,114. The functioning of a promoter can vary, also depending on its location in the genome. Thus, an inducible promoter can become totally or partially constitutive in certain situations. Heterologous and non-heterologous (ie, 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 handle nucleic acid counter expression to reduce, increase, or alter the concentration and / or composition of the proteins of the present invention in a desired tissue. Thus, in some embodiments, the constructed nucleic acid will comprise a promoter, functional in a plant cell, operably linked to a polynucleotide of the present invention. Promoters useful in these modalities include endogenous promoters that direct the expression of a polypeptide of the present invention. In some embodiments, isolated nucleic acids serving as a promoter or enhancer elements at the appropriate position (generally upstream) of a non-heterologous form of a polynucleotide of the present invention can be introduced to regulate up or down expression of a polynucleotide of the present eukaryotic An intron sequence can be added to the 5 'untranslated region or the encoded sequence of the partial encoded sequence to increase the amount of mature message that accumulates in the cytosol. The inclusion of an intron splicing er. The unit of transcription in both express structures, plant and animal, have been shown to increase the expression of the gene in both levels: mRNA and protein up to 1000-pJegar. Buchman and Berg, Mol. Cell Biol. 8: 4395-4405 (1988); Callis et al. , Genes Dev. 1: 1183-1200 (1987). Such improvement of the intron expression of the gene is typically greatest when placed near the 5 'end of the transcription unit. The use of Adhl-S intron 1, 2, and 6 corn intruders, intron Bronze-1 are known in the art. Generally see, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994). The vector comprises the sequences from a polynucleotide of the present invention will typically comprise a marker gene that confers a selectable phenotype on the cells of the plant. Typical vectors useful for gene expression in higher plants are well known in the art and include vectors derived from the tumor-induction plasmid (Ti) of Agrobacterium tumefaciene described by Rogers et al. , Meth. in Enzymol. , 153: 253-277 (1987) A polynucleotide of the present invention can to express oneself in the sense of orientation or counter-sense as desired. It will be appreciated that the control of expression of the gene in orientation or counter-direction can have a direct impact on the observable characteristics of the plant. Conventional technology can be used conveniently to inhibit the expression of the gene in plants. To accomplish this, a nucleic acid segment of the desired gene is cloned and operably linked to a promoter such that the counter sense of the RNA strand will be transcribed. The structure then transforms into the plants and the cords contrasentido of RNA is produced. In the cells of the plant, it has been shown that the opposite RNA inhibits the expression of the gene preventing the accumulation of mRNA which encodes the enzyme of interest, see, for example, Sheehy et al. , Proc. Nat 'l. Acad. Sci. (USA) 85: 8805-8809 (1988); and Hiatt et al. , U.S. Patent? O. 4,801,340. Another method of suppression is sense suppression (ie, co-suppression). The introduction of nucleic acid configured in sense orientation has been shown to be an effective means by which the transcription of designated genes is blocked. For an example of the use of this method to modulate the expression of endogenous genes see,? Apoli et al. , The Plant Cell 2: 279-289 (1990) and the U.S. Patent? O. 5,034,323. Catalytic RNA molecules can also be used or ribozymes to inhibit expression of plant genes. Is it possible to design ribozymes that specifically pair with virtually any designated RNA and stick the phosphodiester backbone to a specific situation, while functionally inactivating the RA? designated. Carrying out this slit, the ribozyme is not altered by itself, and is thus able to recycle and peel other molecules, making it a true enzyme. The inclusion of ribozyme sequences within the RNAs contrasentido 'confers AR activity? - Sticking in them, that is why the activity of the structures is increased. The design and use of AR-specific ribozymes are described in Haseloff et al. , Nature 334: 585-591 (1988). A variety of crosslinking agents, alkylating agents and radical generating species such as the pendant groups in the polynucleotides of the present invention can be used to ligate, label, detect, and / or anchor the nucleic acids. For example, Vlassov, V.V., et al. , Nucleic Acids Ree. (1986, 14: 4065-4076) describes covalent linkages of a single-strand AD fragment with the alkylated derivatives of complementary nucleotides for the designated sequences.A similar work report by the group member is that by Knorre, DG, et al. al., Biochimie (1985) 67: 785-789.Iverson and Dervan also showed sequence-specific heijdidura of AD? simple-cord mediated by the incorporation of a nucleotide modified on which was able to activate the slit (JJ Am. Chem. Soc. (1987) 109: 1241-1243). Meyer R. B., et al.; JJ Am. Chem.

Soc. (1989) 111: 8517-8519, the effect of covalent cross-linking for a designated nucleotide using an alkylating agent complementary to the single-strand of the designated nucleotide sequence. A photoactivated crosslinking for single-strand oligonucleotides mediated by psoralen was discovered by Lee, B.L., et al. , Biochemistry (1988) 27: 3197-3203. The use of Liposuction in the formation of the triple-helix forming the probes was also discovered by Home, et al. , J. Am. Chem. Soc. (1990) 112: 2435-2437. The use of N4, N4-ethanocytosine as: not an alkylating agent to cross-link the single-strand oligonucleotides has also been described by Webb and Matteue: ci, J. Am. Chem. Soc. (1986) 108: 2764-2765; Nucleic Acids Res. (1986) 14: 7661-7674; Feteritz et al. , J. Am. Chen. Soc. 113: 4000 (1991). Various compounds for ligating, detecting, labeling, and / or pasting nucleic acids are known in the art. For example, see 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 of a polypeptide of the present invention (or conservative variants thereof) such as encoded by any of the polynucleotides of the present invention as discussed more fully above (e.g., Table 1). The proteins of the present invention or variants thereof can comprise any number of immediate amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of 10 to the number of residues in a whole body polypeptide of the present invention. Optionally, eeta subsequence of immediate 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 longitv.d. In addition, the number of such subsequences can be any integer selected from the group consisting of 1 to 20, such as 2, 3, 4 or 5. The present invention provides a protein comprising a polypeptide having a specified sequence identity / similarity with a polypeptide of the present invention. The percent identity / similarity of the sequence is an integer selected from the group consisting of 50 to 99. Exemplary sequence identity / similarity values include 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90% and 95%. The identity of the sequence can be determined using, for example, the GAP, CLUSTALW, or BLATS algorithms. As those of usability will appreciate, the present invention includes, but is not limited to, polypeptides catalytically active agents of the present invention (ie, enzymes). Catalytically active polypeptides have a specific activity of at least 20%, 30% or 40% and preferably at least 50%, 60% or 70%, and the most preferred is at least 80%, 90% or 95% of the polypeptide, native, endogenous (non-synthetic). In addition, the substrate specificity (kcat / Km) is optionally substantially similar to the native (non-synthetic) endogenous polypeptide. Typically, the Km will be at least 30%, 40% or 50%, of the native endogenous (non-synthetic) polypeptide; and more preferably at least 60%, 70%, 80% or 90%. The assay methods and measures of quantification of enzyme activity and substrate specificity (kca / Km) are well known to those skilled in the art. Generally, the proteins of the present invention, when presented as an immunogen, elicit production of an antibody specifically reactive for a polypeptide of the present invention. In addition, the proteins of the present invention will not bind for antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for binding determination are well known to those skilled in the art. A preferred immunoassay is a competitive immunoassay. Thus, the proteins of the present invention can be used as Immunogens for the Construction of immunoreactive antibodies 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 can express a protein of the present invention in a recombinantly designed cell such as bacteria, yeast, insect, mammal, or preferably plant cells. The cells produce the protein in a non-natural condition (eg, in quantity, composition, situation, and / or time), because they have been genetically altered through human intervention to do so. Those skilled in the art are expected to be knowledgeable in the numerous expression systems available for the expression of a nucleic acid encoding a protein of the present invention. No effort to describe in detail several of the known methods for the expression of proteins in prokaryotes or eukaryotes will be made. In brief summary, the expression of amino acid nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by incorporation in an expression vector. The / ectors may be convenient for the repetition and integration in prokaryotes or eukaryote, the typical expression of the vectors contain transcription and translation terminators, initiation sequences, and promoters useful for the regulation of the expression of the DNA encoding a protein of the present invention. To obtain high levels of expression of a cloned gene, it is desirable to construct expression vectors that contain, at a minimum, a strong promoter to direct transcription, a riboeoma binding site for translational initiation, and a transcription / translation terminator. A person skilled in the art will recognize that they can make modifications to a protein of the present invention without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the target molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, a methionine added to the amino terminus to provide an initiation site, or additional amino acids (eg, poly His) placed in any terminal to create the purification of sequence conveniently located. Restriction sites or termination codons may also be introduced. Protein synthesis The proteins of the present invention may be constructed using synthetic non-cellular methods. The Protein synthesis in faeolide less than about 50 amino acids in length can be achieved by attaching the C-terminal amino acid sequence to an insoluble support followed by the sequential addition of the remaining amino acids in the sequence. The techniques for the synthesis of the solid phase are described by Barany and Merrifield, Solid-phaee Pepti of Syntheeis, p. 3-284 in The Peptides: Analysis, Synthesis, Biology Vol. 2: Special Methode in Peptide Synthesis, PartA.; Merrifield, et al. , JJ Am. Chem. Soc. 85: 2149-2156 (1963), and Stewart et al. , Solid Phase Peptide Syntheeie, 2 ed. , Pierce Chem. Co., Rockford, III. (1984). Longer length proteins can be synthesized by condensation of the amino and carboxy termini of more intact fragments. Methods for peptide linkage formation by activation of a final carboxy terminus (for example, by the use of N, N'-dicyclohexylcarbodiimide coupling reagent) are known to those skilled. Purification of Lae protein proteins of the present invention can be purified by standard techniques well known in the art. The recombinantly produced proteins of the present invention can be expressed directly or can be expressed as a fusion protein. The recombinant protein is purified by a combination of cell lysis (eg, eonication, French press) and affinity chromatography. For the fusion products, the subsequent digethion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein. The proteins of this synthetic recombinant invention can be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium eulfate, column chromatography, immunopurification methods, and others. , for example, R. Scopes, Protein Purification,: Principies and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies can be raised to proteins as described here. Purification from E. coli can be achieved following the procedures in U.S. Patent No. 4,511,503. The protein can then be expressed from cells expressing the protein and can also be 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. Introduction of nucleic acids into host cells. The method of introducing a nucleic acid from the present invention in a host cell is not critical to the instant invention. Conveniently, transformation or transfection methods are used. According to, a wide variety of methods have been developed to insert a DNA sequence into the genus of a host cell to obtain the transcription and / or translation of the sequence to effect the changes of the oticic faith in the organism. . Thus, any method that provides an efficient introduction of a nucleic acid can be read, A. Transformation of the plant A nucleic acid comprising a polynucleotide of the present invention is optionally introduced into a plant. Generally, the polynucleotide is first incorporated into a recombinant expression cassette or vector. Nucleic acids isolated from the present invention can be introduced into the plants according to the techniques known in the art. Techniques for the transformation of a wide variety of major plant specimens are well known and described in the technical, scientific, and patent literature. For example, see? / Eieing et al. , Ann. Rev. Genet. 22: 421-477 (1988). For example, the DNA structure can be introduced directly into the genomic DNA of the plant cell using techniques such as electrophoration, polyethylene glycol (PEG) foraging, particle bombardment, silicon fiber delivery, or plant microinjection, protoplastoe cells or embryogenic callus. See, for example, Tomes, et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment. pp.197-213 in Plant Cell ue, and Organ Culture, Fundamental Methods. Eds. 0. L. Gamborg and G.C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995; see, U.S. Patent? O. 5,990,387 the introduction of AD structures? using precipitation with PEG are declined in Paszkoweki et al. , Embo 3: 2717-2722 (1984). Electrophoration techniques are described in Fromm et al. , Proc. Nati Acad. Sci. (USA) 82: 5824 (1985). Ballistic transformation techniques are described in Klein et al. , Na ture 327: 70-73 (1987). Transformation techniques mediated with Agrobacterium turne fac i ene are well described in the scientific literature. See, for example, Horsch et al. , Science 233: 496-498 (1984); Fraley et al. , Proc. Nati Acad. Sci. (USA) 80: 4803 (1983); and, Plant Molecular Biology: A Laboratory Manual, Chapter 8, Clark, Ed., Springer-Verlag, Berlin (1997). The structures of AD? Can they be combined with T-AD? It is convenient to flank the regions and enter a conventional vector of Agrobacteri a turne faciene. The virulence function of the host Agroba cterium tumefaciens that will direct the insertion of the structure and adjacent marker in the plant AD? cell when the cell is infected by the bacteria, See, U.S. Patent? O. 5,591,616 Join Me Agrobacterium is mainly useful in the dicotyledonous, certain monocotyledons can be transformed by Agr oba c t eri um. For example, the transformation of Corn Agrobacterium is described in U.S. Patent No. 5,550,318. Other methods of transfection or transformation include (1) transformation mediated by Agrobacterium rhizogenee (see, for example, Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby, Ed., London, Academic Press, 1987, and Lichtenstein, C. P., and Draper, J., In: DNA Cloning, Vol. II, DM GJober, Ed., Oxford, IRI Prees, 1985), Application PCT / US87 / 02512 (WO 88/02405 published april 7 1988) describes the use of A. rhizogenee cord A4 and its plasmid Ri together with the vectors pARC8 or pARC16 of A. tumefaciene (2) uptake of AD? mediated by liposome (see, for example, Freeman et al., Plant Cell Phyeiol. 25: 1353 1984)), (3) the vortex method (see, for example, Kindle, Proc. Nati Acad. Sci. , (USA) 87: 1228 (1990) DNA can also be introduced into plants by direct transference of DNA into pollen as described by Zhou et al. , Methode in Enzymology, 101: 433 (1983); D.

Hees, Intern. Rev. Cytol. , 107: 367 (1987); Luo et al.

Plant Mol. Biol. Repórter, 6: 165 (1988). The expression of polypeptide encoding ge:? Is can be obtained by injecting the DNA into the reproductive organs of a plant as described by El Pena et al. , Nature, 325.:274 (1987) DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaue et al. , Theor. Appl. Genet , 75:30 (1987); and Benbrook et al. , in Proceedinge Bio Expo 1986, Butterworth, Stor.eham, Mass., pp. 27-54 (1986). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower moeaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco moeaic virus. JB Tracarfection of prokaryotes, minor eukaryotes and animae cells Animal and eukaryotic host cells menoree (for example, yeast) are competent or competent for transfection by various means. There are several well-known methods for introducing DNA into animal cells These include: precipitation of calcium phosphate, fusion of recipient cells with bacterial protoplasts containing DNA, treatment of recipient cells with liposomes containing DNA, DEAE dextran, electrophoration, biolisticae and micro-injection of DNA directly into the cell. The transfected cells are well cultured by means well known in the art. Kuchler, R.J., Biochemical Methods in Cell Cul ture and Virology, Dowden, Hutchinson and Ross, Inc. (1977) Regeneration of transgenic plant The cells of the plant, which result directly or are derived from nucleic acid introduction techniques, can be cultured to regenerate an entire plant that protects the introduced genotype. Such regeneration techniques often rely on the manipulation of certain phytohormones in a culture medium for growth of: ejido. For example, plant cells can be regenerated, from eimplee cells, callus tissue or leaf discs according to standard techniques for plant tissue culture. It is well known in the art that several cells, tissues, and organs of almost any successful plant can be grown to regenerate an entire plant. The regeneration of a plant from protoplast culture is described in Evans et al. , Protoplaste leolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Co., New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 2L-73 (1985). The regeneration of plants from simple protoplaetoe of the plant or several explants is well known in the art. See, for example, Methode for Plant Molecular Biology, A. Weissbach and H. Weieebach, ede., Academic Prese, 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 seedlings in the soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wieconein (1988). For corn traneformation and regeneration see, G?] * Don-Kamm et al. , The Plant Cell, 2: 603-618 (1990) The regeneration of plantlets containing the polynucleotide of the present invention and introduced by Agrobacterium from the leaves of the seedlings (explants) can be achieved as described by Horsch et al. , Science, 227: 1229-1231 (1985). In this procedure, the transformants are grown in preedence of a selection agent and in a medium that leads to the regeneration of shoots in the species of the plant to be transformed as described by Fraley et al. , Proc. Nati Acad. Sci. (USA), 80: 4803 (1983). This procedure typically produces shoots within two to four weeks and these transformant shoots then move to an appropriate root-induction medium containing the selective agent and an antibiotic to prevent bacterial growth. The transgenic plants of the present invention may be fertile or sterile A person with skill will recognize that after the recombinant expression caseette is stably incorporated in the transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of several normal breeding techniques can be used, depending on the species to be crossed. In vegetatively propagated crops, mature transgenic plants can be propagated by harvesting cuttings or by tissue culture techniques to produce multiple identical plants. The selection of desirable transgenics is made and new varieties are obtained that propagate vegetatively for commercial use. In the seed, the propagated crops, mature transgenic plants can be crossed with it-miemae to produce a homozygote of the produced plant. The innate plant produces seed that contains 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, eemillae, hojae, ramae, fruit, and the like, are included in the invention, provided that eetae parts comprise cells comprising the isolated nucleic acid of the present invention. Offspring and variants, and mutants of regenerated plants are also included within the scope of the invention, provides that those parts comprise the introduced nucleic acid sequences. Transgenic plants expressing a polynucleotide of the present invention can be protected for the transmission of the nucleic acid of the present invention by, for example, standard detection techniques and DNA. Expression at the RNA level can be determined initially to identify and quantify positive expression in plants. Can you use standard techniques for the analysis of AR? and PCR amplification assays can be included by using oligonucleotide primers designed to amplify only the heterologous RNA templates and hybridization assay of the solution using heterologous nucleic acid-specific probes. The RNA-positive plants can then be analyzed for protein expression by Western blot analysis using the specifically reactive antibodies of the present invention. In addition, in-vitro hybridization and immunocytochemistry according to standard protocols can be done using specific polynucleotide nucleic acid probe and antibodies, respectively, to localize sites of expression within the transgenic tissue. Generally, transgene lines for the incorporated nucleic acid are normally protected to identify and select the plant with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant which is the homozygote for added heterologous nucleic acid; that is, a tranegenic plant that contains two aggregated nucleic acid sequences, one gene in the same site on each chromosome of one pair of the chromosome. A homozygous transgenic plant can obtain: by copulation (self-pollination) sexually a heterozygous transgenic plant containing a simple aggregated heterologous nucleic acid, germinating some of the seeds produced and analyzing the resulting plants produced to alter the expression of a polynucleotide of the present invention concerning the control of the plant (ie, native, non-transgenic) Crossing-back to a parent plant and outside-crossing with a non-transgenic plant is also contemplated. Level of Modulation and / or Composition of the Polypeptide The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or proportion of the polypeptides of the present invention in a plant or part thereof. The modulation can be effected by increasing or decreasing the concentration and / or the proportion of the polypeptides of the present invention in a plant. The method comprises the introduction, into a plant cell of a recombinant expiation caseette comprising a polynucleotide of the present invention as described above to obtain a transgenic plant cell, culturing the tranegenic plant cell under cell growth conditions of the transgenic plant, and inducing or repressing the expression of a polynucleotide of the present invention in the transgenic plant for a sufficient time to modulate the concentration and / or the proportions of the polypeptides in the transgenic plant or part of the plant. In some embodiments, the concentration and / or proportions of the two polypeptides of the present invention in a plant can be modulated by monitoring, in vivo or in vitro. tro, the promoter of a gene regulates up-down gene expression of the gene. In some embodiments, the encoded regions of native genes of the present invention can be altered via substitution, addition, insertion, or subtraction to decrease the activity of the encoded enzyme. For example, veei K iec, U.S. Patent 5,565,350; Zarling et al. , PCT / US93 / 038 8. And in some embodiments, an isolated nucleic acid (e.g., a vector) comprising a transfected eis promoter sequence in a plant cell. As a consequence, a cell of the plant comprising the promoter operably connected to a polynucleotide of the present invention is selected for by means known in the art as, but not limited to, eur stain, sequencing of DNA, or PCR analysis using the first specific for the promoter and for the gene and detecting the amplicons produced there. A plant or part of the plant, altered or modified by the above modalities is developed under plant formation conditions for a sufficient time to modulate the concentration and / or proportions of the polypeptides of the present invention in the plant. The conditions of plant formation are well known in the art and are discussed briefly, eupra. In general, the conentration or proportions of polypeptides are increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a native control plant, part of the plant, or cell lacking the aforementioned recombinant expression cassette. The modulation in the present invention can occur during and / or subsequent to the growth of: the plant to the desired phase of de-spinning. Modulation of the expreion nucleic acid temporarily 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 orientation or contradictory as discussed in more detail, eupra The induction of expression of a polynucleotide of the present invention can also be controlled by the exogenous administration of an effective amount of inducing compound. The inducible promoters and the inducing compounds which activate the expression to Starting from these promoters are well known in the art. In preferred embodiments, the polypeptides of the present invention are modulated in monocotyledons, particularly corn. UTRs and codon preference In general, translational efficiency has been found to be regulated by specific sequence elements in the 5 'unencoded or untranslated region (5' UTR) of the RNA. consensus translational initiation sequences (Kozak, Nucleic Acide Res. 15: 8125 (1987)) and structure (cap) 7-methylguanosine (Drumond et al., Nucleic Acide Ree., 13: 7375 (1985)). The negative elements include the 5 'UTR intramolecular stem-turn structure (Muesing et al., Cell 48: 691 (1987)) and AUG sequences or open short reading frames preceded by an appropriate 5' UTR.

(Kozak, supra, Rao et al., Mol. And Cell Biol. 8: 284 (1988)).

In accordance with, the present invention provides 5 'and / or 3' untranslated regions for the modulation of the translation of encoded heterologous sequences. In addition, the polypeptide-encoded segments of the polynucleotides of the present invention can be modified to alter the identity of the codon. The use of the altered codon can be used to alter the translational efficiency and / or improve the coding sequence for the expression in a given host such as to refine the use of the codon in a heterologous sequence for expreation in corn. The codon order in the encoded regions of the polynucleotides of the present invention can be analyzed eetaditically using commercially available software packages such as "Preferred Codon" available from the University of Wisconsin Genetics Computer Group. (see Devereaux et al., Nuclei c Acide Ree. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a frequency codon usage characteristic of the encoded region of at least one of the polynucleotides of the present invention. The number of polynucleotides that can be used to determine a codon count frequency can be any integer of 1 to the polynucleotide number of the present invention as provided herein. Optionally, the polynucleotide will e equence the whole body. An exemplary number of sequences for statistical analysis may be at least 1, 5, 10, 20, 50 or 100. Slow sequence The present invention provides methods for the slow sequence using polynucleotides of the present invention, and compositions obtained therefrom. The slow sequence is described in PCT publication No. WO 97/20078. See also, Zhang, J.- H., et al. Proc. Nati Acad. Sci. USES 94: 4504-4509 (1997) Generally, the slow sequence provides means for the generation of polynucleotide libraries having a desired characteristic that can be selected or protected for. Libraries of recombinant polynucleotides are generated from a population of polynucleotides of the related sequence comprising regions of the sequence having the identity of the substantial sequence and can be homologously homologated or in vivo. The sequence-recombined population of polynucleotides comprises a sub-population of polynucleotides that possesses either ventajoeae or deeeadae characteristics and which can be selected by a convenient selection or protected method. The characteristics can be any property or attribute capable of being selected for or detected in a protected system and can include properties of: a coded protein, a transcriptional element, a sequence that controls transcription, RNA processing, RNA stability, structure of chromatin, translation, or other property of the expression of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any trait conferring a selectable or detectable property. In some embodiments, the selected feature will be a diminished Km and / or Kcat increased over the wild-type protein as provided herein. In other modalities, a protein or polynucleotide generated from the slow sequence will have a higher binding ligand affinity than the tipp-wild type non-slow polynucleotide. The increase in such properties may be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value. Generic Sequences and Consensus Polynucleotides and polypeptides of the present invention include those that have: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and, (b) 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. Individual samples encompassed by a polynucleotide having an amino acid or consecutive nucleic acid sequence can be run to generate antibodies or produce nucleic acid probes or primers to protect for homologs in other species, genders, families, orders, classes, row, or kingdoms . For example, a polynucleotide having a consensus sequence from a gene of a Zea maye family can be used to generate antibody or nucleic acid probes or first to other Gramineae species such as wheat, rice or sorghum.

Alternatively, a polynucleotide having a consensus sequence generated from the orthologous genes can be used to identify or allele orthologs from another 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 skilled in the eetán conecientee art, a conservative eubstitution of the amino acid can be used for amino acids that differ between the aligned sequence but are of the same group of conservative eubetitución as discussed above. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acids in length of the consensus sequence. Similar sequences used for the generation of a generic or rabbit sequence include any number and combination of allelic variants of the same gene, ortholog, or paralogical sequences as provided herein. Optionally, the sequences used to generate a generic sequence or consensus are identified using the probability of the smallest sum (P (N)) of the BLAST algorithm. Several software providers of sequence analysis are listed in Chapter 7 of Current Protocols in Molecular Biology, F.M Ausubel et al. ,: 3de. , Current Protocole, a joint venture between Greene Publishing Aeeociates, Inc. and John Wiley & Sons, Inc. (Supplement 30). A sequence of polynucleotide is considered similar to a reference sequence if the probability of the smallest sum 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 preferred less than about 0.0001, or 0.00001. Similar polynucleotides can be aligned and a generic or consensus sequence generated by the multiple sequence alignment software available from several commercial providers such as Genetice Computer Group 'e (Madieon, Wl) PILEUP eoftware, Vector NTI's (North Bethesda, MD) ALIGNX, Genecode 's (Ann Arbor, MI) SEQUENCHER. Conveniently, predefined parameters of such software may be used to generate generic or consecutive sequences. Machined Applications The present invention provides machines, data structure, and processes for planning or analyzing the polynucleotides and polypeptides of the present invention. A. Machines: data, data structure, procedure, and function The present invention provides a machine having a memory comprising: 1) data representing a sequence of a polynucleotide or polypeptide of the present invention, 2) a structure of data that reflects the eubyacente organization and structure of the data and facilitates the access to the program to elements of the data corresponding to components of the logical subordinate of the sequence, 3) the processes to carry out the analysis, analysis, planning of the sequence, and 4) optionally, a function or utility for the polynucleotide or polypeptide . Thus, the present invention provides a memory for storing data that can be accessed by a computer program to implement a process for making use, analysis, or modeling of a sequence of a polynucleotide, with memory comprising data representing the sequence of a polynucleotide of the present invention. The machine of the present invention is typically a digital computer. The term "computer" includes one or more laptops or desktops, computer workstations, servers (including intranet or internet servers), large computer systems, and any integrated system comprising any of the foregoing, regardless of whether the The process, memory, input, or performance of the computer are remote or local, as well as any network management that interconnects the module of the computer. The term "computer" is the computer code of the US Patent and Trademark Office or the European Patent Office when the data representing the polypeptide or polynucleotide sequence of the present invention is used for the purposes of the invention. searches for patentability. The present invention contemplates the proportion of the data as a sequence of a polynucleotide of the present invention including in a computer the readable medium. As those of skill in the art will be aware, the nemoria form of a machine of the present invention, or the particular mode of the readable medium of the computer, is not a critical element of the invention and can take a variety of forms. The memory of such a machine includes, but is limited to, ROM, or RAM, or computer readable media such as, but not limited to, media communication! magnetic fields such as computer disks or hard disk drives, or media such as CD-ROMs, Yls, DVDs, and the like, The present invention further contemplates providing a structure of the data that is also contained in the memory. The structure of the data can be defined by the computer programs that define the processes (see below) or can be defined by the storage programming of the separated data and program recovery of the subprograms, or systems. Thus, the present invention provides a memory for storing a data structure that can be accessed by a computer programmed to implement a process for effecting the use, analysis, or modeling of a sequence of a polynucleotide. The memory comprises data representing a polynucleotide having the sequence of a polynucleotide of the present invention. The data is stored inside the memory. In addition, a data structure, stored within the memory, is associated with the data that reflects the underlying organization and structure of the data to facilitate program access to elements of [data corresponding to the sub-component and logical component of the data. sequence. The structure of the data allows the polynucleotide to be identified and manipulated by such programs. In an extensive embodiment, the present invention provides a data structure containing data representing a sequence of a polynucleotide of the present invention stored within a computer-readable medium. The data of the structure are organized to reflect the logical structuring of the sequence, so that the sequence is analyzed easily by the programs of the software capable of accessing the structure of the data. In particular, the structure of the data of the present invention organizes the reference sequences of the present invention in a way that allows the software tools to perform a wide variety of analysis using the logical elements and sub-elements of each sequence. An example of such a data structure looks like a information and can be structured in this way, such as information related to the entire sequence, for example, if the sequence is a full length the viral gene, a mammal house guardian gene or ur. EST from a clone X, the information related to the downstream regions of non-encoded 3 ', for example, hair pin structure, and information related to the various domains of the encoded region, for example, Zinc finger . This structure of: data is an open and robust structure to accommodate newly generated data and acquired knowledge. Such a structure is also a flexible structure. It can be arranged down to a 1-D string to facilitate mining data and analysis steps, such as grouping, repetitive-masking, and HMM analysis. In the meantime, such a data structure can also extend the associated attributes in the multiple dimensions. Indicators can be established among the attributes evaluated when it is necessary to facilitate the management and processing of the data in a comprehensive genomic recognition base. In addition, such an object-oriented data structure. The polymorphism can be represented by an :: amylia or sequence class of objects each of which has an internal structure as discussed above. The common traits are abstracted and assigned to the father's object, considering that each object of the child repreeer.ta a specific variant of the family or claee. Such data structure allows the data to be recovered efficiently, updated and integrated by the software applications associated with the database of the sequence and / or recognition base. The present invention contemplates providing processes for carrying out analysis and modeling, which are described in the following section. Optionally, the present invention contemplates that the machine of the present invention will in some way include a utility or will function for the polynucleotide or "polypeptide" of the present invention. The function or utility of the polynucleotide or polypeptide may be a function or utility for the sequence data, 15 per ee, of the tangible material. Exemplary function or utilities include the name (by the International Union of Biochemistry and Molecular Biology: rules of nomenclature) or the function of the enzyme or protein repreened by the polynucleotide or polypeptide of the present invention; the The metabolic pathway of the protein represented by the polynucleotide or polypeptide of the present invention; the substrate or structural product or paper of the protein repreezed by the polynucleotide or polypeptide of the present invention; or, the phenotype (for example, a raego 25 agronomic or pharmacological) affected by the modulation of expression or activity of the protein represented by the polynucleotide or polypeptide of the present invention. B. Modeling and computational analysis The present invention provides a process for modeling and analyzing data representative of a polynucleotide or sequence ie polypeptide of the present invention. The process comprises the input of the sequence data of a polynucleotide or polypeptide of the present invention into a machine having a hardware or software system for analogy modeling, by developing the data structure to facilitate access to the data of the invention. the sequence, manipulating the data to model or analyze the structure or activity of the polynucleotide or polypeptide, and displaying the results of the modeling or analysis. Thus, the present invention provides a process for effecting the use, analysis, or modeling of a polynucleotide sequence or sequence of the derived peptide through the use of a computer having a memory. The process comprises 1) placing within the memory the data representing a polynucleotide having the sequence of a polynucleotide of the present invention, developing within the memory a structure ie data associated with the data and reflecting the underlying organization and structure of the data. loe data to facilitate the access of the program to elements of the data that correspond to the sub-components sequential logic, 2) programming the computer with a program that contains sufficient instructions to perform the procedure to effect the use, analysis, or modeling of the polynucleotide sequence or peptide sequence, and, 3) executing the program on the computer while granting program access to the data and for the structure of the data within the memory. A variety of modeling and analysis tools are well known in the art and are commercially available. Included among the modeling / analysis tools are methods for: 1) recognizing the eolapadae sequences (eg, from a sequencing project) with a polynucleotide of the present invention and creating an alignment called "contig"; 2) identifying the sites for the restriction enzyme of a polynucleotide of the present invention; 3) identifying the products of a polynucleotide digestion ribonuclease Tl of the present invention; 4) identify the first PCRs with the same minimum-complementarity; 5) compute the dietary requirements of the sequences in an alignment, phylogenetic reconstruction of trees using distance methods, and calculate the degree of divergence of two encoded protein regions; 6) identify the models as encoded regions, terminations, repeats, and other polynucleotide sequencing models of the present invention; 7) identify the RNA secondary structure; 8) identify the motif of the sequence, isoelectric point, secondary structure, hydrophobicity, and antigenicity in polypeptides of the present invention; 9) translation of polynucleotides of the present invention and subsequent-linking of polypeptides of the present invention; and 10) comparison of doe proteins or nucleic acid sequences and identification of points of similarity or inequality between them. The processes for carrying out analysis and modeling can be independently produced or can be obtained from commercial suppliers. Exemplary analyzes and modeling tools are provided in products such as InforMax (Bethesda, MD) Vector NTI Suite (Version 5.5), Intelligenetics (Mountain View, CA) PC / Gene program, and Genetics Computer Group's (Madieon, Wl) Wisconsin Package (Version 10.0); these tools, and the functions they perform, (as provided and discovered by the programs and the literature that accompanies them) are incorporated herein for reference and are described in greater detail in section C below. Thus, in an extemal mode, the present invention provides a media-reader machine that contains a computer program and data, comprising a program stored in the media containing the instructions sufficient to implement a process for effecting the use, analysis, or modeling of a representation of a polynucleotide or sec: uence of the peptide. The data stored in the media represents a sequence of a polynucleotide having the sequence of a polynucleotide of the present invention. The media also includes a data structure that reflects the underlying organization and structure of the data to facilitate program access to data elements that correspond to the logical sub-components of the sequence, the structure of the data to the be inherent in the program and the way in which the program organizes and accesses the data C. Search for homology As an example of such a comparative analysis, the present invention provides a process of identifying a homologous candidate (ie, a ortholog or paralogo) of a polynucleotide or polypeptide of the present invention. The process comprises entering the sequence data of a polynucleotide or polypeptide of the present invention into a machine having a hardware or software system for sequence analysis, developing data structures to facilitate access to the data of the invention. the sequence, manipulating the data to analyze the structure of the polynucleotide or polypeptide, and displaying the results of the analysis. A homologous candidate has a statistically significant probability of having the same biological function (for example, catalyzes the same reaction, junctions to protein homologs / nucleic acids, has a similar structural role) as the reference sequence to which it compares. Accordingly, the polynucleotides and polypeptides of the present invention have utility in the identification of homologs in animals or other plant species, particularly those in the Gramineae family such as, but not limited to, sorghum, wheat or rice. The process of the present invention comprises obtaining data representing a polynucleotide or polypeptide test sequence. Test sequences can be obtained from a nucleic acid of a plant animal. The test sequences can be obtained directly or indirectly from the sequence of the databases including, but not limited to, those such as: GenBank, EMBL, GenSeq, SWISS-PROT, or those available online through the UK Human Genome Mapping Project (HGMP) Genome Web. In some modalities, the test sequence is obtained from plant species other than corn whose function is uncertain but will be compared to the test sequence to determine the similarity of the sequence or identity of the sequence. The data from the test sequence is entered into a machine, such as a computer, containing: i data representing a reference sequence and, i) a hardware system or sequence comparison software to compare the reference sequence * and the test sequence for similarity or sequence identity. The exemplary sequence comparison sevenmae are provided for in the sequence analysis software such as those provided by Genetics Computer Group (Madison, Wl) or InforMax (Bethesda, MD), or Intelligenetics (Mountain View, CA). Optionally, the comparison of the sequence is established using the BLAST or GAP suite programs. Generalmer.te, a smaller sum probability value (P (N)) of less than 0.1, or alternatively, less than 0.01, 0.001, 0.0001, or 0.00001 using the BLAST 2.0 algorithm collection under the predefined parameters identifies the sequence of test as a homologous candidate (ie, an ally, ortholog, or paralog) of the reference sequence. Those of skill in the art will recognize that a homologous candidate has an increased statistical probability of having the misi or similar function as the gene / protein represented by the test sequence. The reference sequence may be the sequence of a polypeptide or a polynucleotide of the present invention. The reference sequence or the test sequence is optionally at least 25 amino acids or at least 100 nucleotides in length. The length of the reference or test sequences can be the length of the polynucleotide or polypeptide described, respectively, above in the sections entitled "Nucleic Acids" (particularly section (g)), and "Protein" As those of skill in the art eetán conecientes, the greater identity / uniformity of the sequence between a reference sequence of known function and a test sequence, the greater probability that the test sequence will have the same or similar function as the sequence reference. The results of the comparison between the test and reference sequences are made (for example, displayed, printed, recorded) via any of several performance and / or media (eg, computer, monitor, hard-die-hard) , or media reader) Nucleic acid detection The present invention further provides methods for the detection of a polynucleotide of the present invention in a nucleic acid sample suspected of containing a polynucleotide of the present invention, such as a cell lysate of a plan. a, particularly a corn lysate. In some embodiments, a gene cognate with a polynucleotide of the present; The invention or portion of the moiety can be amplified prior to contacting the nucleic acid molecule with a polynucleotide to form a hybridization complex. The polynucleotide hybridized under severe conditions to a gene encoding a polypeptide of The present invention, Hybridization complex formation is used to detect a gene encoding a polynucleotide of the present invention in the nucleic acid sample. Those of skill will appreciate that an isolated nucleic acid comprising a polynucleotide of the present invention will lack cross-hybridization in the sequences in common with non-target genes that would yield a • false positive result, Detection of the hybridization complex can be achieved using any number of well-known methods. For example, the nucleic acid sample, or a portion thereof, may be hybridized by formats including, but not limited to, the dissolution phase, solid phase, mixed fae, or in vitro hybridization eneay. The detectable label suitable for use in the present invention includes any composition detectable by epecpecic, radioisotopic, photochemical, biochemical, irimunochemical means, electrical, optical or chemical means. The useful labels in this The invention includes biotin for labeling with conjugated labeling streptavidin, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Other labels include ligands that bind to the labeled antibodies with the fluorophores, agents 25 of chemiluminescents, and enzymes. Labeling the acids The nucleic acid of the present invention is readily achieved such as by the use of labeled first PCR. Although the present invention has been described in some detail by way of illustration and example for the 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 a cDNA library Total RNA can be isolated from corn tissues with the TRIzole reagent (Life Technology Inc. Gaithersburg, MD) using a modification of the ieothiocyanate / phenol-guanidine acid process as described by Chomczyneki and Sacchi (Chomczynski, P., and Sacchi, N. Anal.

Biochem. 162,156 (1987)). In short, the sample of plant tissue is sprayed into the liquid nitrogen before the addition of the TRIzol reagent, and then further homogenized with a mortar and pestle. The addition of chloroform followed by centrifugation ee directs for the separation of an aqueous phase ac and an organic phase. The total RNA is recovered by precipitation with isopropyl alcohol from the aqueous phase. The selection of poly (A) + RNA from total RNA is performed using the PolyATact system from (Promega Corporation.

Madieon, Wl). The first biotinylated oligo (dT) are used to hybridize poly (A) 3 'tails in the mRNA. The hybrids are captured using streptavidin coupled to the paramagnetic particles and a position of the magnetic separation. The mRNA is then washed at high stringency conditions and eluted by deionized water or RNaea-free. You can achieve e-mail from cDNA and unidirectional cDNA library by using Super Script Plaemad Syetem (Life Technology Inc. Gaithereburg, MD). The first cDNA cord is synthesized by priming a first oligo (dT) containing a Not I site. The reaction is catalyzed by Super Script Reverse Tranecriptase II at 45SC. The second cord of cDNA is labeled with alpha-32P-dCTP and a portion of the reaction analyzed by agarose gel electrophoresis to determine the sizes of the cDNA. The cDNA molecules smaller than 500 base pairs and the unbound adapters are removed by chromatography with Sephacryl-S400. The cDNA molecules (selected are ligated into the pSPORTl vector between the Not I and Sal I sites. Alternatively, the cDNA libraries can be prepared by any of many available methods. For example, the cDs can be: plasmid vectors by preparing the cDNA libraries first by preparation of the cAD libraries in Uni-ZAP ™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Syetems, La Jolla, CA). The Uni-ZAP? TMX, R libraries are converted into the plasmid libraries according to the protocol provided by Stratagene. In the conversion, the inertions of the cDNA in the plasmid vector pBluescript will be contained. In addition, cDNAs can be introduced directly into the precut Bluescript II SK (+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturing protocol (GIBCO BRL). Products). Once the cDNA inserts are in the plasmid vectors, the plasmid cDNAs are prepared from randomly chosen bacterial colonies containing recombinant pBluescript plasmids, or the inserted cDNA sequences are amplified via the polymerase chain reaction using the primer eepecific for sequences of the vector flanking the inserted cDNA sequences. The insertion of amplified cDNA or plasmicjloe DNAs are sequenced in the sequencing reactions of first generation partial cDNA sequences (labeled sequence or "ESTs" see Adame et al., (1991) Science 252: 1651-1656). The resulting ESTs are analyzed using a fluorescent sequencer Perkin Elmer Model 377. Example 2 This method describes the construction of an enriched whole cDNA library. A enriched whole-body cDNA library using one of two variations of the method of Caminci et al. , Genomice 37: 327-336, 1996. These variations are based on the chemical introduction of a biotin group into the diol residue of the 5 'cap structure of eukaryotic mRNA to select the first cord of cAD? full body. The selection occurs by entrapping the biotin residue in the capped eardrops with streptavidin-coated magnetic beads followed by RNase I treatment to remove the incompletely synthesized cDNAs. The second cord of cDNA is synthesized using established procedures such as those provided in Life Technologies' (Rockville, MD) "Super Script Plaemad System for cD? Synthesis and Plaemad Cloning" kit. Libraries made by this method have been shown to contain 50% to 70% of whole body cADs. The first methods of cord synthesis are detailed below. An asterisk denotes that the reagent was obtained from Life Technologies, Inc. A. First cord of cDNA ententee method 1 (with trehalose) mRNA (lOug) 25μl * Not I first (5ug! LOμl * 5x ls cord buffe c 43μl * 0.1m DTT 20μl * dNTP mix lOmm lOμl BSA 10ug / μl lμl Trehalose (purchased) 59.2μl RNaea inhibitor (Promega) 1.8μl * Superscript II RT ~ 200u / μl 20μl 100% glycerol 18μl Water 7 μl The mAR? and the first? ot I mix and deenaturalize at 65aC for 10 min. This is then cooled in the ice and other components are added to the tube. The incubation is at 45 aC for 2 min. Twenty microliters of RT (reverse transcriptaea) is added to the reaction and the program is started in the thermocycler (MJ) Reeearch, Waltham, MA): Paeo 1 45aC lOmin Paeo 2 45aC -0.3aC / c: j.clo, 2 seconds / cycle, Paeo 3 go to 2 for 33 cycles Step 4 35aC 5min Step 5 45aC 5min Paeo 6 45aC 0.2aC / cycle, 1 sec / cycle, Paeo 7 go to 7 for 49 cycles Paeo 8 55aC 0.1aC / cislo, 12 sec / cycle, Paeo 9 go to 8 for 49 cycles Step 10 55aC 2min Step 11 60aC 2min Step 12 go to 11 for 9 times stored at -20aC. C. Oxidization of the diRNA group of mRNA for biotin labeling. The first cDNA strand is spun down and washed once with 70% EtOH. The pellets were re-suspended in 23.2μl of DEPC treated with water and placed on ice. Prepare 100 mM fresh NaI04, and then add the following reagents: mRNA: lst cDNA (start n 20μg mRNA) 46.4μl 10OmM NaI04 (freshly prepared) 2.5μl 3M NaOAc pH4.5 l.lμl To make 100mM NaI04, use 21.39μg of NaI04 for lμl of water. Wrap the tube in foil and incubate on the ice for 45min. After incubation, the reaction is then precipitated in: 5M NaCl lOμl 20% SDS 0.5μl isopropanol 61μl Incubate on the ice for at least 30 min, then bring it down at maximum speed at 4aC for 30 min and wash once with ethanol 70% and then 80% ethanol. D. Biotinylation of the diol group of mRNA Resuspend DNA in HOμl of DEPC treated with water, then add the following reagents: 20% SDS 5μl 2M NaOAc pH 6.1 5μl lOmm biotin hydrazide (freshly prepared) 300μl Wrap in aluminum foil and incubate overnight at room temperature. E. Treatment RNaea I Precipitate DNA in: 5M NaCl lOμl 2M NaOAc pH 6.1 75μl mRNA-biotinylated cDNA 420μl 100% EtOH (2.5Vol) 1262.5μl (Perform this precipitation in two tubes and separate 420μl of DNA in 210μl each, add 5μl of 5M NaCl, 37.5μl of 2M NaOAc pH 6.1, and 531.25μl of 100% EtOH). Store at -20aC for at least 30 min. Hile down the AD? at 4aC at maximum speed for 30 min. and wash twice with 80% EtOH, then dissolve the AD? in 70μl of water-free RNase. Group two tubes and finish with 140μl. Add the following reagents: RNase One lOU / μl 40μl lst CDNA: R? A 140μl 10X buffer 20μl Incubate at 37SC for 15min. Add 5μl of 40μg / μl tAR? of yeast to each sample for capture. F. Capture of lst full length cDNA Block the beads with the yeast tRNA: Count the yeast lml tRNA 40μg / μl 5μl Incubate on ice for 30min with mixing, wash 3 vecee with lml of 2M NaCl, 5 OmmEDTA, pH 8.0, Resuspend the beads in 800μl of 2M NaCl, 50mm EDTA, pH 8.0, add RNase I mueetra treated with 200μl, and incubate the reaction for 30min at room temperature. Capture the beads using the magnetic position, save the supernatant, and start the following washes: 2 washes with 2mACl, 50mm EDTA, pH 8.0, 1ml each, 1 wash with 0.4% SDS, 50μg / ml tRNA, 1 wash with lOmm Tris-Cl pH 7.5, 0.2mm EDTA, lOmm NaCl, 20% glycerol, 1 wash with 50μg / ml tRNA, 1 wash with the lst cDNA buffer G. Synthesis of the second cord of cDNA Resuspend the beads in: * SX first buffer 8μl * O. lmM DTT 4μl * 10mm dNTP mix 8μl * 5X 2nd buffer 60μl 100% EtOH 750μl EtOH 100% 600μl glycogen lμg / μl 2μl glycogen lμg / μl 2μl H. Ligation of the adapter J Re-suspend the pellet in 26μl of water and use lμl for the TAE gel. Prepare the reaction as follows 2nd cord where cDNA 25μl * 5X T4 DNA ligase buffer lOμl * Salt I adapters lOμl * T4 DNA ligase 5μl Mix gently, incubate the reaction overnight at 16aC, add 2μl of ligase second day and incubate at room temperature for 2 hrs (optional). Add 50μl of water to the reaction and use lOOμl of phenol to clean the DNA, transfer 90μl of the upper phase into a new tube and precipitate in: Glycogen lμg / μl 2μl Upper phase DNA 90μl NH40ac 7.5M 50μl EtOH 100% 300μl Precipitate all night at -20aC down the hair illa to 4eC and wash in 70% EtOH, dry the pellet. I. Digestion of Not I enough, they are fixed on 22 x nylon membranes 22 c using the Q-bot. Each membrane holds 9.216 or 36.864 colonies. These membranes are placed on an agar plate with an appropriate antibiotic. The plates are incubated overnight at 37 ° C. After the colonies are recovered on the second day, these filters are placed on a paper filter preheated with denaturing dissociation for four minutes, then incubated on top of a boiling water bath for an additional four minutes. The filters are then placed on the pre-moistened filter paper with neutralizing solution for four minutes. After the excess solution is removed by placing. Loe filter in the dry filter paper for one minute, the side of the filter colony is put in solution Proteinase K, incubated at 37 aC for 40-50 minutes. The filters are placed on dry filter papers to dry overnight. The DNA is then cross-linked to the nylon membrane by treatment with UV light. Hybridization of the colony is directed as described by Sambrook, J., Fritsch, E.F. and Maniatis, T., (in Molecular Cloning: A Laboratory Manual, 2nd Edition). The following probes can be used in the hybridization of the colony: 1. The first cDNA cord from the mRNA tissue as the library was made from the removal of the most redundant clones 2. 48-192 of the most redundant cDNA clones from the same library based on the previous sequencing data. 3. The 192 most redundant cDNA clones in the data base of the entire maize sequence. 4. An oligonucleotijdo Sal-A20: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA, removes clones that contain a poly A tail but no cDNA. 5. Loe clonee of cDNAs derived from mRNA. The image of the autoradiograph is examined in the computer and the intensity signal and the direction of the cold colony of each colon: .a is analyzed. Re-formation of cold colonies from 384 plates to 96 plates is conducted using Q-bot. Example 4 This example describes identification of the gene from a computer homology search. Identities of the gene can be determined by BLAST conduction (Baeic Local Alignment Search Tool; Altechul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; also see www.ncbi.nlm.nih. gov / BLAST /) searches under the predefined parameters for the consistency for sequences contained in the "nr" database of BLAST (comprising the non-redundant translations of GenBank CDS, the sequence derived from the structure -dimensional Brookhaven Protein Data Bank, the last major load of the SUISS-PROT protein sequence database, EMBL, and DDBJ data base). The cDNA sequences are analyzed for similarity so that all publicly available DNA sequences contained in the "nr" datum database that match the BLASTN algorithm. The AD sequences are translated into all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database as used by the BLASTX algorithm.

(Gish, W. and States, D. J. Náture Genetice 3: 266-272 (1993)) provided by NCBI. In some cases, sequencing data from two or more clones containing overlapping segments of DNA are used to construct the immediate DNA sequences. Sequence alignments and percent identity calculations can be performed using the MEERGIGN Megalign program from bioinformatics computing euite (DNASTAR Inc., Madison, Wl) Multiple sequence alignment can be performed using the alignment method Cluetal of (Higgins and Sharp (1989) CABIOS 5: 151-153) with the predefined parameters HOLLOW (GAP) PENALTY = 10, LENGTH of the HOLLOW (GAP) PENALTY = 10). The predefined parameters for pairwise alignments using the Clustal method 7i, i? jA Í. . , ATCC 97366. The DNA segment from pML103 contains a 1.05 kb promoter fragment of Sall-Ncol from the 27 kD zein maize gene and a 0.96 kb Smal-Sall fragment from the 3 'terminus of the corn 10 kD zein in the vector pGem9Zf (+) (Promega). The vector and insertion of DNA can be ligated to 15aC overnight, essentially as described by (Maniatis). The ligated DNA can then be used to transform E. coli XLl-blue (Epicurian Coli XLl Blue: Stratagene). Transforming bacteria can be screened by the restriction enzyme digestion of the DNA plastron and the limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase DNA Sequencing Kit, U. S. Biochemical). The resulting plasmid structure comprises a transgene encoded in the 5 'to 3' direction, the 27 kD zein maize promoter, a cDNA fragment encoding the instantaneous polypeptides, and the 10 kD zein 3 'region. The tranegenes described above can be introduced into the maize cell by the following procedure. Immature maize embryos of cariopsae can be dried in de-trailing pathways derived from corn crosses produced by lines H99 and LH132. Embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm in length. The embryos are then placed with the lateral-axis facing down and in contact with N6 agarose-solidified medium (Chu et al. (1975) Sci. Sin. Peking.18: 659-668) l The embryos are placed in the dark at 27 aC. Embryogenic callous friable eme coneiete in undifferentiated cell masses with the somatic and embryoid proembryoids carried on suepensor structures proliferated from the scutellum of these immature embryos. The embryogenic callus isolated from the primary seedlings can be cultured in the N6 medium and eub-cultured in this medium every 2 to 3 weeks The p35S /? C plasmid (Hoechst Ag, Frankfurt, Germany) or the equivalent can be used in the experiments. of traneformation to provide an electable marker. This plasmid contains the Pat gene (see European patent publication 0 242 236) which encodes phosphinotricin acetyl traneferaea (PAT). The PAT enzyme confers the re-entition to the herbicidal inhibitors glutamine eintetaea such as the phosphinotricin 1 gene pate in p35S / Ac is under the control of the 35S promoter from the Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812) and the 3 'region of the nopaline and intetase gene from T-DNA of the plaemido Ti of AgroJbacterium umefaeciene. The particle bombardment method (Klein et al (1987) Nature 327: 70-73) can be used to transfer the genes to callus cell culture. According to this method, the gold particles (lμm in diameter) are . ii.iiMn.i. TO . TO , .. . coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5M solution) and base-free spermidine (20 μL of a l.OM solution) are added to the particles. The suspension is vortex during the addition of these solutions. After 10 minutes, the tubes are centrifuged briefly (5 sec at 15,000 rpm) and the supernatants removed. The particles are re-suspended in 200μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol from the rinse is re-performed and the resuspended particles in a final volume of 30μL of ethanol an aliquot of (5μL) of the DNA-coated gold particles can be placed in the center of a Ka ton flywheel (Bio-Rad) Labs). The particles are then accelerated in the corn tissue with a Biolistic PDS-1000 / He (Bio-Rad Inetruments, Hercules CA), using a helium pressure of 70,306 kg / cm2 (1000 psi), a gap distance of 0.5cm and a flying distance of 1. Ocm. For the bombardment, embryogenic tissue on filter paper on agarose-solidified N6 medium. The tissue is placed as a thin layer and covering a circular area approximately 5cm in diameter. The petri dishes containing the fabric can be placed in the camera of the PDS-1000 / He approximately 8cm from the top screen. He Uu ?? . * - .. air in the chamber then evacuates to a vacuum of 81.12cm (28 inches) of Hg. The macrocarrier is accelerated with a helium shock wave using unites. rupture membrane that explodes when the He pressure in the shock tube reaches 70,306 kg / cm2 (1000 psi). Seven days after the bombardment the tissue can be transferred to an N6 medium containing glufosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly in this medium. After an additional 2 weeks the tissue can be transferred to a fresh N6 medium containing glufoeinate. After 6 weeks, areas of approximately 1 cm in diameter of actively growing calli can be identified in some of the plates containing the glufosinate-supplemented iaryium. These calluses may continue to grow when they are sub-cultivated in the selective medium. Plants can be regenerated from transgenic calluses by first transferring the tissue clusters to the N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to the regeneration medium (Fromm et al., (1990) Bio / Technology 8: 833-839) Example 6 This example describes the expression of transgenes in dicotyledonous cells. An eemilla- expired caeeette faith & encoding instantaneous polypeptides. To induce somatic embryos, cotyledcns, 3-5 mm in length from the sterilized surface, immature seeds of the soybean cultivar A2872, can be cultured in light or dark at 26 aC on an appropriate agar medium for 6-10 weeks. . The somatic embryos which produce secondary embryos are then cut and placed in a convenient liquid medium. After repeated selection for clusters of somatic embryos that were multiplied as early embryos, embryonic in globular eetadium, the pellets remain as described below. The culture embryogenic suspeneion of eoja cultivar can be maintained in 35mL of liquid culture medium in a rotary shaker, at 150 rpm, at 26aC with fluorescent light at a time of 16: 8 hours day / night. The cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue in 35 mL of liquid culture medium. The embryogenic soybean culture can then be transformed by the particle bombardment method (Klein et al., (L987) Nature (London) 327: 70-73, U.S. Patent No. 4,945,050 American). A Du Pont Biolistic PDS 1000 / HE instrument can be used for these transformations. A selectable marker gene that can be used for ü £ jaatul, facilitate the transformation of soybean is a tranegene composed of promoter 35S Virue of the Moeaico of the Cauliflower (Odell et al., (1985) Nature 313: 810-812), the gene hygromicin phosphotransferaea of the plasmid pJR225 (of E. coli; Gritz et al., (1983) Gene 25: 179-188) and the 3 'region of the nopaline synthetase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaeciene. The seed expression cassette comprising the 5 'phaseolin region, the fragment coding for ins: antáneoe polypeptides and the 3' phaseolin region can be added as a restriction fragment. This fragment can then be inserted into a single restriction site of the vector carrying the marker gene. To 50μL of an euspeneión of 60mg / mL with lμm of gold particles are added (in order): 5μL DNA (1 μg / μL), 20μl of spermidine (0.1M), and 50μL of CaCl2 (2.5M). The preparation of the particle is then stirred for three minutes, spun in a microfuge for 10 seconds and the supernatants removed. The DNA-coated particles are washed once in 400μL of 70% ethanol and resuspended in 40μL of anhydrous ethanol. The suspension of DNA / particulate can be eonicated tree times for one second at a time. Five micro-inches of the DNA-coated gold particles are then bled into each disk of the macro carrier. Approximately 30Ó-400mg of a suspension of Two-week-old culture of "age" is placed in an empty 60x15 mm petri dish and the re-fluid liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 tissue plates are normally bombarded. The pressure of metal rupture is set at 77,336 Kg / cm2 (1100 psi) and the chamber is evacuated to a vacuum of 81.12cm (28 inches) of mercury. The tissue is placed approximately 8. 89cm (3.5 inches) off the retention screen and bombed three times. The next bombardment, the tissue can be divided by the MiLtad and can put back in the liquid and cultivated as described above. Five to seven days after the bombardment, liquid culture media can be exchanged with fresh culture media, and eleven to twelve days after bombardment with fresh culture media containing 50 mg / mL hygromycin. This means of selective culture can be refreshed weekly. Seven to eight weeks after the bombardment, the green, transformed tissue can be observed growing from the untransformed, necrotic embryogenic clusters. The isolated green tissue is removed and inoculated into the individual flasks to generate new clonally propagated tissue, embryogenically transformed culture suspensions. Each new line can be treated as an independent transformation event. These euspeneions can then be subcultured and can be maintained as the clusters of immature embryos or can, regenerate in whole plants by maturation and germination of individual somatic embryos. Example 7 This example describes the expreation of a transgene in microbial cells. The cDNAs encoding instantaneous polypeptides can be inserted into the T7 expression vector E. coli pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56: 125-135) which uses the bacteriophage T7 AR? polymerase / T7 promoter system. Plasmid pBT430 was first constructed by destroying EcoR I and Hind III sites in pET-3a at their original positions. An adapter oligonucleotide containing EcoR I and the Hind III sites was inserted into the BamH I site of pTT-3a. Eeta creation pET-3aM with the only eitios for additional cloning for the insertion of genes in the expression vector. Then, the Nde I site in the translation initiation position was converted to a Neo I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430 The DNA plasmid containing a cDNA can be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment can then be purified on a NuSieve GTG 1% low agarose gel melting point (FMC). The buffer and agarose contain 10 μg / ml of etidium bromide for the localization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase (Epicenter Technologies) according to the instructions of the manufacturer, precipitated in ethanol, dried and resuspended in 20μL of water. Appropriate adapter oligonucleotides can be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA). The fragment containing the ligated adaptadoree can be purified from the excess adapters by using low point agaroea as described above. The pBT430 vector is digested, dephosphorylated with alkaline phosphatase (ΔEB) and deeproteinized with phenol / chloroform as described above. The vector pBT430 prepared and the fragment can then be ligated at 16 ° C for 15 hours followed by transformation into electrocompetent DH5 cells (GIBCO BRL). Transformants can be selected on agar plates containing LB culture media and 100 μg / mL ampicillin. Transformants containing the gene are protected by encoding the instant polypeptides then for correct orientation with respect to the T7 promoter by restriction enzyme analysis. For high-level expression, a plasmid clone with the insertion of the cAD? in the correct orientation JÉ * .-. -i relative to the T7 promoter can be transformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol. 189: 113-130). Cultures were grown in LB culture medium containing ampicillin (100mg / L) at 25aC. At an optical density at 600 nm of approximately L, IPTG (inducer, isopropylthio-β-galactoside) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25 a. The cells are then harvested by centrifugation and re-suspended in 50μL of 50mM Tris-HCl at pH 8.0 containing O.l M DTT and 0.2mM of phenyl-methylsulfonyl fluoride. A small amount of lmm glass beads can be added and the mixture sonicated 3 times for approximately 5 seconds each time with a microprobe eo? The mixture is centrifuged and the protein concentration of the supernatant is determined. One microgram of protein of the soluble fragment of the culture can be separated by gel electrophoresis with SDS-polyacrylamide. The gels can be obeyed for protein bands that migrate to the expected molecular weight, Example 8 This example describes a method for identifying plants that contain mutants in the gene of interest and a strategy for identifying the function of those genes. This example is found in the work with the CQRAD17 gene, defined in U.S. Patent Application 09 / 371,383 and covered there as Seq. ID No. 25, which is a member of the same gene family as SEQ ID Nos. 1 and 5 of the present application. A pejrsona of ability in the art could conceive soon the ueo of this procedure with the discovered sequences in the current application. The Trace Utility System for Corn (TUSC) is a method that uses genetic and molecular techniques to facilitate the study of gene function in corn. Studying the function of the gene implies that the sequence of the gene is already known, and the method works on the backward path: from the sequence to the phenotype. This type of application is called "reverse or reverse genetics" which contrasts with "front" methods that are designed to identify and isolate the gene (s) responsible for a particular trait (phenotype). Pioneer H-Bred International Inc., has a proprietary collection of corn genomic DNA from approximately 42,000 individual Fl planks (Reverse genetics for Maite, Meeley, R. and Briggs, S., 1995, Maiz Genet, Coop.? Ewslett 69:67, 82). The genome of each of these individuals contains multiple copies of the transposable element family, Mutator. { Mu) The Mu family is highly mutagenic; in the presence of the active element z-DR, these elements transpose throughout the genome, inserting in the gene regions, and often breaking the function of the gene. Collecting genomic DNA from a large number (42,000) of individuals, Pioneer has assembled a mutagenized corn genome library. The insertion events of Mu are predominantly heterozygous; Given the recessive nature of most ineerional mutation, the Fi plants appear wild-type. Each of the Fi plants is the same to produce F2 seeds, which are collected. Generating the offspring of F2, insertional mutations eegregadae in a Mendelian fashion to be used to investigate the effect of a mutant alíelo on the phenotype. The TUSC system has been used successfully by several laboratories to identify the function of a variety of genes (cloning and characterizing the corn gene Anl, Bensen, RJ, et al., 1995, Plant Cell; 7: 75-84; Diverection of C-function activity in maize fJLower development, Mena, M., et al., 1996 , Science 274: 1537rl540; Analyeie of a chemical plant defenee mechaniem in grasses, Frey, M., et al., 1997, Science 277: 696-699; The control of maize spikelet merietem fate by the APETALA2-like gene indeterminate epikelet 1 , Chuck, G., Meeley, RB, and Hake, S., 1998, Genes &Development 12: 1145-1154; A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplanet gene expression, Roy, LM and Barkan, A., 1998, J. Cell Biol. 141: 1-11) PCR Screening for the Mu inserts in CQRAD17 Combined in the 48 plant plantae each, was subjected to PCR with GSPl or GSP2 and Mu TIR. The pools that were confirmed to be positive by hybridization of the dot-blot were attached to CQRAD17 cDNA as a (probé) probe were subjected to spot-on-gel analysis in order to determine the size of 1-oe amplified fragments. The pools in which the clean fragments were identified underwent extensive analysis to identify the individual plants within the pools that contained the insertion (ee) Mu. Lae eemillae to par de plantae Fi identified in this way were planted in the field. Leaf disks of twenty plants in each F2 row were collected and the genomic DNA was isolated. The same twenty plants were the same and the seed of F3 saved. The pooled DNA (from 20 plantae) of each of the twelve filae ee was subjected to PCR using first GSPl or GSPJ2 and Mu TIR as mentioned above. Three pools identified to contain Mu inserts were subjected to individual plant analysis and homozygous dentified. The Mu site insertion sites with the surrounding signature sequences are identified below: Allocate 1: TCTTCACCA- l-GGTCCTTCG Allelo 2: GTCGAAATT- Wu-TTCTTCAGC Allocate 3: GCTCACGGG- Mu- GAAGTTTAT All three insert ethene within 200 nucleotides of each other which read the outdoor frame, suggesting that this region, ie the gene, could represent a hot spot for Mu inception. One of the ineercionee, alíelo 3, is in the region predicted to code for a transmembrane domain. It is expected that each of these inserts renders the gene inactive. The above examples are provided to illustrate the invention but do not limit its scope. Other variants of the invention will be readily apparent to a person of ordinary skill in the art and will encompass the appended claims. All publications, patents, patent applications, and computer programs cited herein shall be incorporated by reference for reference.

Claims

CLAIMS 1. An isolated nucleic acid comprising a polynucleotide having at least 80% sequence identity, as determined by the GAP algorithm under the predefined parameters, for a polynucleotide of SEQ ID NO: 1. An isolated nucleic acid comprising a polynucleotide having at least 80% sequence identity, as determined by the GAP algorithm under the predefined parameters, for a polynucleotide of SEQ ID NO: 5. 3. A polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2 and 6. 4. A polynucleotide selected from the group consisting of SEQ ID NOS: 1 and 5. 5. A polynucleotide that is complementary to a polynucleotide. of SEQ ID NO. l or SEQ ID NO. 5. A recombinant expression caseette, comprising a member of any of the claims 1 to 5, operably linked, in sense or counter-sense or orientation, to a promoter. 7. A huéeped cell comprising the recombinant expression cassette of claim 6 8. Transgenic plant nail comprising the cassette The recombinant expression of claim 6. 9. The transgenic plant of claim 8, wherein said plant is a monocot. 10. The transgenic plant of claim 8, wherein said plant is a dicotyledonous plant. 11. The transgenic plant of claim 8, wherein said plant is selected from the group consisting of: corn, soybean, sunflower, orgo, cañola, wheat, alfalfa, cotton, rice, barley, millet, peanut, and cocoa. 12. A seed of the transgenic plant of claim 8 13. A method of modulating the cellulose synthetase level in a plant cell, comprising: (a) introducing a recombinant exprease caseette, into a plant cell , of claim 6; (b) cultivating the plant cell under cell cellar development conditions; and (c) induction of expression of said polynucleotide for a sufficient time to modulate the level of cellulose synthetase in said plant cell. The method of claim 13, wherein the cell of the plant is corn, wheat, rice, or soybean. 15. A method of modulating the level of cellulose synthetaea in a plant, comprising: (a) introduction | of a caeeette of recombinant expression, in a plant cell of claim 6; (b) cultivating the plant cell under conditions of cell development of the plant; (c) regeneration of a plant from said plant cell; and (d) induction of the expression of said polynucleotide during an efficient time to modulate the level of celluloea synthetase said plant. 16. The method of claim 15, wherein the plant is corn, wheat, rice, or soybean. 17. An isolated protein comprising a member selected from the group consisting of: (a) a polypeptide selected from the group consisting of SEQ ID NOS: 2 and 6 (b) a polypeptide having at least 80% identity of the sequence, and having at least one epitope in common with, a polypeptide selected from the linking group of SEQ ID NOS: 2 and 6, wherein said identity of the sequence is determined by the GAP algorithm under the predefined parameters.; and, (c) at least one polypeptide encoded by a member of any of claims 1 to 5. 18. A method of modifying the expiry of cellulose synthetaea in a gene in a corn plant comprising: (a) identification, from a population of maize, plants mutagenized with the transposable element Mu, eeae plantae containing one or more insertions of Mu within a polynucleotide of either of claims 1 to 5; (b) select those plants that kill the expreation of the modified celluloea synthetase gene. 19. The method of claim 18, wherein the expulsion of the celluloea gene si: ntetase ee down-regulates. 20. The method of claim 18, wherein the expression of the cellulose synthetase gene is up-regulated.