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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

• the present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
• a major determinant of hybrid seed corn performance in northern climates is seedling vigor in cold soil or in the cold weather conditions of spring. Cold soils and weather also affect the performance of other crops such as wheat, soybeans, rice, barley, oats, sunflower, and rye. Improving seedling vigor in cold conditions will increase the seedling stand count, extend the effective growing season for a given cold climatic zone, and increase yield in general. Moreover, it will create new opportunities for the use of higher yielding and genetically modified varieties in those regions.
• an inhibition of normal cellular respiration can result in an accumulation of free radicals, reactive oxygen species such as hydrogen peroxide, superoxide, or hydroxyl radicals.
• reactive oxygen species such as hydrogen peroxide, superoxide, or hydroxyl radicals.
• These radicals can cause cellular damage by their high chemical reactivity. They can also dramatically change gene expression, including activation of defense systems, which may be stressful or toxic to the plant.
• thermogenic plants that evolve heat during flowering to volatilized odoriferous compounds that attract insect pollinators.
• thermogenic plants include Sauromautum guttatum, Symplocarpus foetidus, and Arum m ⁇ cul ⁇ tum.
• alternative oxidase mRNA and protein which is regulated by salicylic acid.
• Pathogen infected plant tissue has also been observed to have elevated alternative oxidase levels and increased organ temperature.
• ROS reactive oxygen species
• SHAM salicylhydroxamic acid
• SHAM salivalhydroxamic acid
• induced and systemic acquired pathogen resistance as to tobacco mosaic virus in tobacco. It is less clear what role it may play in resistance to bacteria and fungi (Chivasa, et al., 1997). Salicylate does not appear to be the determinant of non-induced steady-state levels of alternative oxidase, but rather it determines the induced expression levels (Lennon, et al., 1997). Other factors, such as ROS, may also play a role.
• alternative oxidase expression is contributing to pathogen resistance by some unknown mechanism.
• hybrid seed corn production often involves manual or mechanical emasculation of the parent serving as the female and the donation of pollen to it from the other parent.
• Male sterile lines are desired because they would greatly simplify hybrid seed production by eliminating the need for the physical emasculation.
• Various male sterile parents have been proposed and a few implemented, with varied success. The need for more diverse and more effective approaches for producing male sterility is clear.
• Genetic engineering of crop plants has been limited in both the variety and volume of genetic engineering events possible by limitations of selectable markers during transformation. There are relatively few selectable markers employed by plant transformation protocols. This causes several problems. One such problem is that some of the markers are not cleanly selectable. Another is that with so few markers, stacking of multiple genetic engineering traits becomes problematic because the plant to be transformed with a new gene may already possess a transgene construct with the same selectable marker.
• alternative oxidase could be used as a selectable marker.
• tobacco cells treated with inhibitors for the cytochrome respiration pathway such as potassium cyanide
• Such cyanide-treated tobacco cells grew faster when transformed with the alternative oxidase gene under the direction of the 35S promoter which causes a high (higher than normal) expression of the alternative oxidase gene (Vanlerberghe et al., 1997a), presumably because of elevated respiration carried on by the alternative pathway.
• Protein targeting to the mitochondria is another area in which further advances are needed in the art.
• the alternative oxidase genes are nuclear-encoded, but the protein is localized to the mitochondria.
• the import of the protein into the mitochondria is dependent upon transit peptides located at the N-termini of the primary peptide transcripts. These transit peptides are subsequently cleaved off upon entry into the mitochondria.
• Direct N-terminal sequencing of the Sauromatum guttatum mature AOX peptide indicates the start site of the mature peptide (Rhoads and Mclntosh, 1993). This splice-site region is highly conserved in other plant alternative oxidases (Whelan, et al., 1995).
• the mature peptide usually starts with "XST", and the transit peptide has an arginine residue (R) at the minus 2 amino acid position, which is necessary for import (mutagenesis of this arginine inhibits import).
• the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and, (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b).
• the isolated nucleic acid can be DNA.
• the present invention relates to: 1) recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter, 2) a host cell into which has been introduced the recombinant expression cassette, and 3) a transgenic plant comprising the recombinant expression cassette.
• the host cell and plant are optionally a maize cell or maize plant, respectively.
• nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
• Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range.
• Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
• Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
• software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5 th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
• amplified is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template.
• Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
• antisense orientation includes reference to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed.
• the antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
• nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).
• the information by which a protein is encoded is specified by the use of codons.
• amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
• variants of the universal code such as are present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.
• nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)).
• the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize.
• full-length sequence in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of, a native (non- synthetic), endogenous, biologically active form of the specified protein. Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extension, S 1 protection, and ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer- Verlag, Berlin (1997).
• consensus sequences typically present at the 5' and 3' untranslated regions of mRNA aid in the identification of a polynucleotide as full-length.
• the consensus sequence ANNNNAUGG where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5 ' end; consensus sequences at the 3 ' end, such as polyadenylation sequences, aid in determining whether it has a complete 3' end.
• gene activity refers to one or more steps involved in gene expression, including transcription, translation, and the functioning of the protein encoded by the gene.
• heterologous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
• a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
• a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
• host cell is meant a cell which contains a vector and supports the replication and/or expression of the vector.
• Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
• host cells are monocotyledonous or dicotyledonous plant cells.
• a particularly preferred monocotyledonous host cell is a maize host cell.
• transfection or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be inco ⁇ orated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• the nucleic acid may be inco ⁇ orated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• isolated refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with it as found in its 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 at a different location within the cell.
• the synthetic alteration or creation of the material can be performed on the material within or apart 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 which has been altered or produced by non-natural, synthetic methods.
• the isolated nucleic acid may also be produced by the synthetic re-arrangement ("shuffling") of a part or parts of one or more allelic forms of the gene of interest.
• a naturally-occurring nucleic acid e.g., a promoter
• Nucleic acids which are "isolated,” as defined herein, are also referred to as "heterologous" nucleic acids.
• ZmAOXl, ZmAOX2, or ZmAOX3 nucleic acid represents a nucleic acid of the present invention and means a nucleic acid comprising a polynucleotide of the present invention (a "ZmAOXl, ZmAOX2, or ZmAOX3 polynucleotide") encoding a ZmAOXl, ZmAOX2, or ZmAOX3 polypeptide.
• a "ZmAOXl, ZmAOX2, or ZmAOX3 gene” is a gene of the present invention and refers to a full-length ZmAOXl, ZmAOX2, or ZmAOX3 polynucleotide.
• nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
• nucleic acid library is meant a collection of isolated DNA or RNA molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism or of a tissue from that organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al, Molecular Cloning - A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F.M. Ausubel et al, Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
• operably linked includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
• operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
• plant includes reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same.
• Plant cell as used herein, further includes, without limitation, cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues.
• the class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
• polynucleotide includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or chimeras or analogs thereof that have the essential nature of a natural deoxy- or ribo- nucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
• a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are
• polynucleotides as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
• polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
• polypeptide polypeptide
• peptide protein
• proteins are used interchangeably herein to refer to a polymer of amino acid residues.
• the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• the essential nature of such analogues of naturally occurring amino acids is that, when inco ⁇ orated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
• polypeptide polypeptide
• peptide protein
• modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
• this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.
• promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
• a "plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissue are referred to as "tissue specific”.
• a “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
• An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters represent the class of "non-constitutive" promoters.
• a “constitutive” promoter is a promoter which is active under most environmental conditions.
• ZmAOXl, ZmAOX2, or ZmAOX3 polypeptide means a polypeptide of the present invention and refers to one or more amino acid sequences, in glycosylated or non-glycosylated form. The term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof.
• a "ZmAOXl, ZmAOX2, or ZmAOX3 protein” is a protein of the present invention and comprises a ZmAOXl, ZmAOX2, or ZmAOX3 polypeptide.
• recombinant includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified.
• recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention.
• the term "recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
• a "recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell.
• the recombinant expression cassette can be inco ⁇ orated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
• the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.
• amino acid residue or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is inco ⁇ orated into a protein, polypeptide, or peptide (collectively “protein”).
• the amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
• sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
• Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.
• stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence, to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
• stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
• Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
• Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
• Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C.
• T m 81.5 °C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
• the T m is the temperature (under defined ionic strength and pH) at which 50%o of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
• transgenic plant includes reference to a plant which comprises within its genome a heterologous polynucleotide.
• the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
• the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
• Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
• transgenic does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non- recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
• vector includes reference to a nucleic acid used in introduction of a polynucleotide of the present invention into a host cell. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
• sequence relationships between a polynucleotide/polypeptide of the present invention with a reference polynucleotide/polypeptide: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and (d) "percentage of sequence identity”.
• reference sequence is a defined sequence used as a basis for sequence comparison with a polynucleotide/polypeptide of the present invention.
• a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
• comparison window includes reference to a contiguous and specified segment of a polynucleotide/polypeptide sequence, wherein the polynucleotide/polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide/polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the comparison window is at least 20 contiguous nucleotides/amino acids residues in length, and optionally can be 30, 40, 50, 100, or longer.
• Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
• the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
• sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al, J. Mol Biol, 215:403-410 (1990); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analyses is publicly available, e.g., through the
• HSPs high scoring sequence pairs
• Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
• M forward score for a pair of matching residues; always > 0
• N penalty score for mismatching residues; always ⁇ 0.
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• W wordlength
• E expectation
• BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 7. Acad. Sci. USA 90:5873-5877 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P( ⁇ )), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
• a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.
• GAP can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence.
• GAP uses the algorithm of Needleman and Wunsch (J Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
• GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases.
• GAP must make a profit of gap creation penalty number of matches for each gap it inserts.
• gap extension penalty greater than zero
• GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty.
• Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively.
• the default gap creation penalty is 50 while the default gap extension penalty is 3.
• the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100.
• the gap creation and gap extension penalties can each independently be: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.
• GAP presents one member of the family of best alignments.
• GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity.
• the Quality is the metric maximized in order to align the sequences.
• Ratio is the quality divided by the number of bases in the shorter segment.
• Percent Identity is the percent of the symbols that actually match.
• Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
• a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
• the scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
• sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
• sequence identity when percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. WTiere sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
• Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
• percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is - 1 0 - calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• the present invention provides, among other things, compositions and methods for modulating (i.e., increasing or decreasing) the level of polynucleotides and polypeptides of the present invention in plants.
• the polynucleotides and polypeptides of the present invention can be expressed temporally or spatially, e.g., at developmental stages, in tissues, and/or in quantities, which are uncharacteristic of non-recombinantly engineered plants.
• the present invention provides utility in such exemplary applications as enhancing cold tolerance in plants, enhancing disease resistance in plants, achieving male sterility in plants, developing selectable genetic markers for transgenic plant production, and engineering protein targeting to the mitochondria.
• the present invention can be used to enhance cold tolerance in plants.
• the coding regions for any of the three maize alternative oxidase genes will be expressed in a transgenic plant, such as maize, under the direction of tissue-preferred and developmental promoters that cause high level expression in seedlings.
• Two such promoters are those for the GA-regulated alpha-amylase genes and that of a cysteine protease, which cause high- level expression in the seed (scutellum and aleurone).
• a third promoter, that for the beta- glucosidase (Glul) gene causes high-level expression in the seedling mesocotyl, coleoptile, and young leaves.
• Glul beta- glucosidase
• a modified coding region of any of the alternative oxidase genes to produce an alternative oxidase with higher intrinsic activity can be used in the same process described above. This is accomplished using a directed protein engineering strategy involving the alteration of one or both of the cysteine residues at amino acid positions 120 and 170 in ZmAOXl and 102 and 152 in ZmAOX2 with a non-sulfhydryl- containing amino acid such as serine. These cysteine residues are responsible for dimerization of alternative oxidase. The dimeric form is less active than the monomeric form, and also less susceptible to pyruvate stimulation.
• Another option for modifying either of the alternative oxidase genes to produce an altei ⁇ ative oxidase with higher intrinsic activity is using a directed protein engineering strategy involving sequence shuffling of the maize alternative oxidases along with coding regions or synthetic ohgonucleotides for other plant and non-plant alternative oxidases.
• Increased activity may be first assessed by complementing microbes, such as E. coli or yeast, that are mutated in their own alternative oxidase, with the modified alternative oxidases.
• Those complemented with more active alternative oxidases will be expected to grow more actively in vitro.
• the recovered clones are then reintroduced into the plant.
• the enzyme will be more active, other seedling preferred/specific promoters can be used which need not have such a high level of expression.
• the present invention can also be used to enhance disease resistance in plants. Any of the three maize alternative oxidase sequences can be used to enhance disease resistance in plants to various pathogens.
• An exemplary plant is maize.
• Exemplary pathogens include, but are not limited to, viral pathogens.
• this invention enhances inducible resistance to pathogens, but it can be used in constitutive resistance mechanisms as well. Inducible resistance mechanisms involve enhancing the level and timing of alternative oxidase expression following pathogen attack in order to increase expression and thus resistance.
• the present invention can be used in any number of ways to accomplish this goal.
• the coding regions for any of the maize alternative oxidase genes can be expressed in a transgenic plant under the direction of pathogen-inducible promoters that cause elevated expression (where possible, highly elevated expression) following pathogen attack.
• Maize is a particularly preferred transgenic plant.
• Exemplary inducible promoters include inducible maize promoters as disclosed in U.S. Patent Application No. 09/257,583 entitled “Inducible Maize Promoters", filed Feb. 25, 1999.
• Those of skill will recognize other promoters are possible, including promoters having some tissue or developmental preference to their expression that would focus their inducibility to tissues or stages particularly susceptible to the target pathogen.
• these transgenic plants Upon pathogen attack, these transgenic plants have elevated levels of alternative oxidase and enhanced disease resistance.
• the coding region of any of the alternative oxidase genes can be modified to produce an alternative oxidase with higher intrinsic activity. This is accomplished via a directed protein engineering strategy involving alteration of, for example, one or both of the cysteine residues at amino acid positions 120 and 170 in ZmAOXl and 102 and 152 in ZmAOX2, with a non-sulfhydryl-containing amino acid such as serine. These cysteine residues aid in the dimerization of alternative oxidase.
• the dimeric form is less active than the monomeric form, and also less susceptible to pyruvate stimulation.
• the coding region of either of the alternative oxidase genes can be modified to produce an alternative oxidase with higher intrinsic activity. This is accomplished via a directed protein engineering strategy involving sequence shuffling of these two maize alternative oxidases along with coding regions or synthetic oligonucleotides for other plant and non-plant alternative oxidases. Increased activity can be first assessed by complementing microbes, such as E. coli or yeast, that are mutated in their own alternative oxidase, with the modified maize alternative oxidases. Those complemented with more active alternative oxidases will grow more actively in vitro. The recovered clones are then reintroduced into the plant as discussed previously.
• microbes such as E. coli or yeast
• tissue preferred/specific promoters can be used which need not have as high a level of expression.
• a non-inducible resistance is achieved by driving their expression with a promoter that gives appropriate levels of alternative oxidase expression in the desired tissue and/or developmental stages.
• the present invention provides methods for the creation of transgenic male sterile plants for the pu ⁇ ose of creating hybrid seed.
• the coding region for one of the maize alternative oxidase genes described herein is used.
• any of the maize alternative oxidase genes can be mutagenized so as to be nonfunctional.
• One method which can be employed to achieve this is transposon insertional mutagenesis.
• mutagenesis may result in male sterility. This can be useful in creating hybrids; the male parent will donate a functional copy of the gene so that the resulting hybrid plant is fertile.
• any of these maize alternative oxidase genes can be expressed in an antisense configuration under the direction of a tassel-specific promoter. Male sterility results when the alternative oxidase expression in the tassel is sufficiently reduced. This strategy requires a restorer gene in the hybrid plant to counteract the antisense expression suppression.
• Alternative oxidase can be used as a selectable marker.
• tobacco cells treated with inhibitors for the cytochrome respiration pathway such as potassium cyanide
• Such cyanide-treated tobacco cells grew faster when transformed with the alternative oxidase gene under the direction of the 35S promoter which causes a high (higher than normal) expression of the alternative oxidase gene (Vanlerberghe et al., 1997a), because of elevated respiration carried on by the alternative pathway.
• Alternative oxidase genes in particular the maize genes presented herein, can be used as selectable markers.
• one or more of the ZmAOX genes can be cotransformed with the target transformation gene.
• the ZmAOX gene would be under the direction of a highly active promoter. The more rapidly growing cells, tissue, or callus would be subcultured, as they represent the successfully transformed tissue.
• the promoter would be preferentially active during early developmental or culturing stages, and have less impact later in development, or even in succeeding generations.
• SHAM inhibitors of alternative oxidase, among which are SHAM
• the resistant variant under the direction of a fairly strong constitutive promoter, although the native ZmAOX promoter may suffice and be advantageous for later in development, is then reintroduced into the plant during cotransformation with the transforming gene of interest.
• the selection medium would contain the inhibitor (as SHAM or disulfiram), and cells which grow are those transformed with the gene of interest and linked to the alternative oxidase selectable marker construct. This selection is even more effective with the addition of SHAM or disulfuran to inhibit the alternative pathway plus an inhibitor of the cytochrome pathway, such as cyanide or antimycin A. In these conditions, respiration only occurs via the inhibitor-resistant alternative oxidase, and only those cells transformed with it survive.
• the alternative oxidase transformation strategies outlined above are additionally useful for enhancing transformation frequency.
• the alternative oxidase pathway is more active following stresses. Particle bombardment, a commonly used transformation technique, is undoubtedly stressful on the tissue.
• the alternative pathway is more active in bombarded tissues. This increased activity is adaptive in that it contributes to reduction of reactive oxygen species that accumulate following stresses such as bombardment and cause damage to the cells and decrease transformation efficiency.
• Increasing the alternative oxidase pathway following transformation as by one of the methods above, or by transiently elevated expression of the gene's coding region by including it in DNA or RNA form on the transforming particles, thus enhances cell survival rates, and hence increases transformation frequency.
• the present invention provides transit peptides for the alternative oxidase genes.
• the transit peptides direct these and other proteins to the mitochondria.
• the transit peptides are a chief determinant for direction of proteins to the mitochondria
• the transit peptide coding region for any of the genes of the present invention can be used to genetically engineer direction of other proteins to the mitochondria. This is achieved by fusing the transit peptide coding region to the N-terminal end of the protein destined to the mitochondria.
• the chimeric protein Upon translation, the chimeric protein is directed to the mitochondria, and upon entry, the transit peptide is proteo lyrically removed by proteases present in the mitochondria, and the liberated protein then resides and functions in the mitochondria.
• Such engineering of protein targeting to the mitochondria is useful in various areas including, but not limited to, herbicide resistance, transformation selectable markers, metabolite production, male sterility (and restoration of fertility), cell cycle control, and apoptosis.
• selectable markers it should be noted that many antimicrobial/antiprokaryote compounds/drugs are known. Many of these affect prokaryotic translation.
• the plant mitochondria and chloroplasts are relatively prokaryotic-like in their translational apparatus, as they were apparently inco ⁇ orated by a primitive eukaryotic cell via endosymbiosis.
• the mitochondrial transit peptide from the alternative oxidase genes can be used to direct genes encoding resistance to such drugs to the mitochondria. As such, they can be used for selectable markers in plant transformation.
• the present invention also provides isolated nucleic acids comprising polynucleotides of sufficient length and complementarity to a gene of the present invention to use as probes or amplification primers in the detection, quantitation, or isolation of gene transcripts.
• isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for desired transgenic plants, for detecting mutations in the gene (e.g., substitutions, deletions, or additions), for monitoring upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allelic variants (polymo ⁇ hisms), orthologs, or paralogs of the gene, or for site directed mutagenesis in eukaryotic cells (see, e.g., U.S. Patent No. 5,565,350).
• the isolated nucleic acids of the present invention can also be used for recombinant expression of their encoded polypeptides, or for use as immunogens in the preparation and/or screening of antibodies.
• the isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more genes of the present invention in a host cell, tissue, or plant. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation.
• the present invention also provides isolated proteins comprising a polypeptide of the present invention (e.g., preproenzyme, proenzyme, or enzymes).
• the present invention also provides proteins comprising at least one epitope from a polypeptide of the present invention.
• the proteins of the present invention can be employed in assays for enzyme agonists or antagonists of enzyme function, or for use as immunogens or antigens to obtain antibodies specifically immunoreactive with a protein of the present invention.
• Such antibodies can be used in assays for expression levels, for identifying and/or isolating nucleic acids of the present invention from expression libraries, for identification of homologous polypeptides from other species, or for purification of polypeptides of the present invention.
• the isolated nucleic acids and polypeptides of the present invention can be used over a broad range of plant types, particularly monocots such as the species of the family Gramineae including Hordeum, Secale, Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays).
• monocots such as the species of the family Gramineae including Hordeum, Secale, Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays).
• the isolated nucleic acid and proteins of the present invention can also be used in species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Pisum, Phaseolus, Lolium, Oryza, and Avena.
• the present invention provides, among other things, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising a polynucleotide of the present invention.
• a polynucleotide of the present invention is inclusive of: (a) a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 3, 5, 6, 8 including exemplary polynucleotides of SEQ ID NOS: 1, 4, 7;
• a polynucleotide encoding a protein having a specified number of contiguous amino acids from a prototype polypeptide, wherein the protein is specifically recognized by antisera elicited by presentation of the protein and wherein the protein does not detectably immunoreact to antisera which has been fully immunosorbed with the protein;
• a polynucleotide comprising at least a specific number of contiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e), (f) or (g).
• the present invention provides isolated nucleic acids comprising a polynucleotide of the present invention, wherein the polynucleotide encodes a polypeptide of the present invention.
• Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine; and UGG , which is ordinarily the only codon for tryptophan
• UGG which is ordinarily the only codon for tryptophan
• each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention. Accordingly, the present invention includes polynucleotides of SEQ ID NOS: 1, 4, or 7, and polynucleotides encoding a polypeptide of SEQ ID NOS: 2, 3, 5, 6, or 8.
• the present invention provides an isolated nucleic acid comprising a polynucleotide of the present invention, wherein the polynucleotides are amplified from a Zea mays nucleic acid library.
• Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23, and Mol 7 are known and publicly available. Other publicly known and available maize lines can be obtained from the Maize Genetics Cooperation (Urbana, IL).
• the nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing.
• cDNA libraries can be normalized to increase the representation of relatively rare cDNAs.
• the cDNA library is constructed using a full-length cDNA synthesis method. Examples of such methods include Oligo-Capping (Maruyama, K. and Sugano, S. Gene 138: 171-174, 1994), Biotinylated CAP Trapper (Carninci, P., Kvan, C, et al. Genomics 37: 327-336, 1996), and CAP Retention Procedure (Edery, E., Chu, L.L., et al. Molecular and Cellular Biology 15: 3363-3371, 1995).
• cDNA synthesis is often catalyzed at 50-55°C to prevent formation of RNA secondary structure.
• Rapidly growing tissues, or rapidly dividing cells are preferably used as mRNA sources.
• a preferred tissue source from which one can isolate alternative oxidase mRNA is cold-stressed maize seedlings (e.g., seedlings at V3 stage treated for up to 24 hours at 10°C).
• Another preferred tissue source is pathogen-infected maize leaves (e.g., leaves of V6 plants 48 hours after inoculation with Cochliobolus heterostrophus conidia).
• Another preferred tissue source is maize tassels at the early stages of pollen shed.
• the present invention also provides subsequences of the polynucleotides of the present invention.
• a variety of subsequences can be obtained using primers which selectively hybridize under stringent conditions to at least two sites within a polynucleotide of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it.
• Primers are chosen to selectively hybridize, under stringent hybridization conditions, to a polynucleotide of the present invention.
• the primers are complementary to a subsequence of the target nucleic acid which they amplify but may have a sequence identity ranging from about 85% to 99% relative to the polynucleotide sequence which they are designed to anneal to.
• the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired amplification conditions.
• the primers will be constructed so that they selectively hybridize under stringent conditions to a sequence (or its complement) within the target nucleic acid which comprises the codon encoding the carboxy or amino terminal amino acid residue (i.e., the 3' terminal coding region and 5' terminal coding region, respectively) of the polynucleotides of the present invention.
• the primers will be constructed to selectively hybridize entirely within the coding region of the target polynucleotide of the present invention such that the product of amplification of a cDNA target will consist of the coding region of that cDNA.
• the primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50.
• the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length.
• a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence.
• a non-annealing sequence at the 5 'end of a primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.
• the amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra.
• the resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention.
• Methods for protein synthesis from PCR derived templates are known in the art and available commercially. See, e.g., Amersham Life Sciences, Inc, Catalog '97, p.354. Methods for obtaining 5 ' and/or 3 ' ends of a vector insert are well known in the art.
• the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide of sections (A) or (B) as discussed above.
• the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising the polynucleotides of (A) or (B).
• polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
• the polynucleotides are genomic or cDNA sequences isolated or otherwise complementary to a cDNA from a dicot or monocot nucleic acid library.
• Exemplary species of monocots and dicots include, but are not limited to: maize, canola, soybean, cotton, wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice.
• the cDNA library comprises at least 30% to 95%.
• full-length sequences for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% full-length sequences.
• the cDNA libraries can be normalized to increase the representation of rare sequences.
• Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% to 80% sequence identity and can be employed to identify orthologous or paralogous sequences.
• 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.
• Identity can be calculated using, for example, the BLAST or GAP algorithms under default conditions.
• the percentage of identity to a reference sequence is at least 60% and, rounded upwards to the nearest integer, can be expressed as an integer selected from the group of integers consisting of from 60 to 99.
• the percentage of identity to a reference sequence can be at least 70%., 75%, 80%., 85%, 90%, or 95%.
• the polynucleotides of this embodiment will encode a polypeptide that will share an epitope with a polypeptide encoded by the polynucleotides of sections (A), (B), or (C).
• these polynucleotides encode a first polypeptide which elicits production of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide of (A), (B), or (C).
• the first polypeptide does not bind to antisera raised against itself when the antisera has been fully immunosorbed with the first polypeptide.
• the polynucleotides of this embodiment can be used to generate antibodies for use in, for example, the screening of expression libraries for nucleic acids comprising polynucleotides of (A), (B), or (C), or for purification of, or in immunoassays for, polypeptides encoded by the polynucleotides of (A), (B), or (C).
• the polynucleotides of this embodiment embrace nucleic acid sequences which. can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.
• Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries.
• This method involves the screening of large collections of peptides for individual members having the desired function or structure.
• Antibody screening of peptide display libraries is well known in the art.
• the displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5- 100 amino acids long, and often from about 8 to 15 amino acids long.
• several recombinant DNA methods have been described.
• One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence.
• the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides encode a protein having a subsequence of contiguous amino acids from a prototype polypeptide of the present invention such as are provided in (a), above.
• the length of contiguous amino acids from the prototype polypeptide is selected from the group of integers consisting of from at least 10 to the number of amino acids within the prototype sequence.
• the polynucleotide can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids from the prototype polypeptide.
• the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5.
• the subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5. 10, 15, 25, 50, 100, or 200 nucleotides.
• the proteins encoded by polynucleotides of this embodiment when presented as an immunogen, elicit the production of polyclonal antibodies which specifically bind to a prototype polypeptide such as but not limited to, a polypeptide encoded by the polynucleotide of (a) or (b), above.
• a protein encoded by a polynucleotide of this embodiment does not bind to antisera raised against the prototype polypeptide when the antisera has been fully immunosorbed with the prototype polypeptide.
• Methods of making and assaying for antibody binding specificity/affinity are well known in the art.
• Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays and the like.
• fully immunosorbed and pooled antisera which is elicited to the prototype polypeptide can be used in a competitive binding assay to test the protein.
• concentration of the prototype polypeptide required to inhibit 50% of the binding of the antisera to the prototype polypeptide is determined. If the amount of the protein required to inhibit binding is less than twice the amount of the prototype protein, then the protein is said to specifically bind to the antisera elicited to the immunogen.
• the proteins of the present invention embrace allelic variants, conservatively modified variants, and minor recombinant modifications to a prototype polypeptide.
• a polynucleotide of the present invention optionally encodes a protein having a molecular weight as the non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptides of the present invention.
• Molecular weight can be readily determined by SDS-PAGE under reducing conditions.
• 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 full length polypeptide of the present invention.
• the polynucleotides of this embodiment will encode a protein having a specific enzymatic activity at least 50%, 60%), 80%o, or 90% of a cellular extract comprising the native, endogenous full-length polypeptide of the present invention.
• the proteins encoded by polynucleotides of this embodiment will optionally have a substantially similar affinity constant (K m ) and/or catalytic activity (i.e., the microscopic rate constant, k cat ) as the native endogenous, full-length protein.
• K m affinity constant
• catalytic activity i.e., the microscopic rate constant, k cat
• k cat /K m value determines the specificity for competing substrates and is often referred to as the specificity constant.
• Proteins of this embodiment can have a cat /K m value at least 10% of a full-length polypeptide of the present invention as determined using the endogenous substrate of that polypeptide.
• the k ca t/K m value will be at least 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, 80%, 90%, or 95%) the k cat /K m value of the full-length polypeptide of the present invention. Determination of k cat , K m , and k cat /K m can be determined by any number of means well known to those of skill in the art.
• the initial rates i.e., the first 5% or less of the reaction
• the initial rates can be determined using rapid mixing and sampling techniques (e.g., continuous-flow, stopped-flow, or rapid quenching techniques), flash photolysis, or relaxation methods (e.g., temperature jumps) in conjunction with such exemplary methods of measuring as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance, or radioactive procedures.
• Kinetic values are conveniently obtained using a Lineweaver- Burk or Eadie-Hofstee plot.
• the present invention provides isolated nucleic acids comprising polynucleotides complementary to the polynucleotides of paragraphs A-E, above.
• complementary sequences base-pair throughout the entirety of their length with the polynucleotides of sections (A)-(E) (i.e., have 100% sequence identity over their entire length).
• Complementary bases associate through hydrogen bonding in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
• the present invention provides isolated nucleic acids comprising polynucleotides which comprise at least 15 contiguous bases from the polynucleotides of sections (A) through (F) as discussed above.
• the length of the polynucleotide is given as an integer selected from the group consisting of from at least 15 to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of.
• polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous nucleotides in length from the polynucleotides of (A)-(F).
• the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5.
• the subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.
• the subsequences of the present invention can comprise structural characteristics of the sequence from which it is derived. Alternatively, the subsequences can lack certain structural characteristics of the larger sequence from which it is derived such as a poly (A) tail.
• a subsequence from a polynucleotide encoding a polypeptide having at least one linear epitope in common with a prototype polypeptide sequence as provided in (a), above may encode an epitope in common with the prototype sequence.
• the subsequence may not encode an epitope in common with the prototype sequence but can be used to isolate the larger sequence by, for example, nucleic acid hybridization with the sequence from which it's derived.
• Subsequences can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids.
• Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates. Construction of Nucleic Acids
• the isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof.
• the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a monocot.
• the monocot is Zea mays.
• the nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention.
• a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide.
• translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
• a hexa- histidine marker sequence provides a convenient means to purify the proteins of the present invention.
• a polynucleotide of the present invention can be attached to a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention.
• nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb.
• Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).
• RNA, cDNA, genomic DNA, or a hybrid thereof can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art.
• oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. Isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art.
• Substantially pure full-length cDNA libraries are constructed to comprise at least 90%, and more preferably at least 93% or 95% full-length inserts amongst clones containing inserts.
• the length of insert in such libraries can be from 0 to 8, 9, 10, 11, 12, 13, or more kilobase pairs.
• Vectors to accommodate inserts of these sizes are known in the art and available commercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12 kb cloning capacity).
• a non-normalized cDNA library represents the mRNA population of the tissue it was made from. Since unique clones are out-numbered by clones derived from highly expressed genes their isolation can be laborious. Normalization of a cDNA library is the process of creating a library in which each clone is more equally represented. Construction of normalized libraries is described in Ko, Nucl. Acids. Res., 18(19):5705- 5711 (1990); Patanjali et al, Proc. Natl. Acad. U.S.A., 88:1943-1947 (1991); U.S. Patents 5,482,685, and 5,637,685.
• Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species.
• cDNA prepared from one pool of mRNA is depleted of sequences present in a second pool of mRNA by hybridization.
• the cDNA:mRNA hybrids are removed and the remaining un-hybridized cDNA pool is enriched for sequences unique to that pool. See, Foote et al. in, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique, 3(2):58-63 (1991); Sive and St. John, Nucl.
• cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech, Palo Alto, CA).
• genomic libraries large segments of genomic DNA are generated by fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate molecular biological techniques and instructions sufficient to direct persons of skill through many construction, cloning, and screening methodologies are found in Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods in
• the cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent.
• the nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques.
• PCR polymerase chain reaction
• PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other pu ⁇ oses.
• the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.
• PCR-based screening methods have been described. Wilfinger et al. describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques, 22(3): 481-486 (1997). Such methods are particularly effective in combination with a full-length cDNA construction methodology, above.
• 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. Enzymo 68: 90-99 (1979); the phosphodiester method of Brown et al, Meth. Enzymol 68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al, Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caru hers, 7etr ⁇ . Letts.
• the present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention.
• a nucleic acid sequence coding for the desired polypeptide of the present invention for example a cDNA or a genomic sequence encoding a full length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell.
• a recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
• plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker.
• plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
• a plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant.
• Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation.
• constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1 '- or 2'- promoter derived from T-DN A of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other transcription initiation regions from various plant genes known to those of skill.
• CaMV cauliflower mosaic virus
• the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control.
• promoters are referred to here as "inducible" promoters.
• Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light.
• promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
• exemplary promoters include the anther specific promoter 5126 (U.S. Patent Nos. 5,689,049 and 5,689,051), glob-1 promoter, and gamma-zein promoter.
• the operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.
• the nucleic acid construct will comprise a promoter functional in a plant cell, such as in Zea mays, operably linked to a polynucleotide of the present invention.
• Promoters useful in these embodiments include the endogenous promoters driving expression of a polypeptide of the present invention.
• isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non- heterologous form of a polynucleotide of the present invention so as to up- or down- regulate expression of a polynucleotide of the present invention.
• endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Patent 5,565,350; Zarling et al, PCT7US93/03868), or isolated promoters can be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
• Gene expression can be modulated under conditions suitable for plant growth so as to alter the total concentration and/or alter the composition of the polypeptides of the present invention in plant cell.
• the present invention provides compositions, and methods for making, heterologous promoters and/or enhancers operably linked to a native, endogenous (i.e., non- heterologo s) form of a polynucleotide of the present in ention.
• polypeptide expression it is generally desirable to include a polyadenylation region at the 3 '-end of a polynucleotide coding region.
• the poiyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
• the 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
• An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8: 4395- 4405 (1988); Callis et al, Genes Dev. 1 : 1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of maize introns Adhl-S intron 1, 2, and 6, the Bronze- 1 intron are known in the art.
• the vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells.
• Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al, Meth. in Enzymol., 153:253-277 (1987).
• a polynucleotide of the present invention can be expressed in either sense or anti- sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable plant characteristics.
• Antisense technology can be conveniently used to inhibit gene expression in plants. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced.
• antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al, Proc. Nat l. Acad. Sci. (USA) 85: 8805-8809 (1988); and Hiatt et al, U.S. Patent No. 4,801,340.
• Another method of suppression is sense suppression.
• Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes.
• this method to modulate expression of endogenous genes see, Napoli et al, The Plant Cell 2: 279-289 ( 1990) and U.S. Patent No. 5,034,323.
• Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA- cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al, Nature 334: 585- 591 (1988).
• cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids.
• Vlassov, V. V., et al, Nucleic Acids Res (1986) 14:4065-4076 describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences.
• a report of similar work by the same group is that by Knorre, D. G., et al, Biochimie (1985) 67:785- 789.
• the isolated proteins of the present invention comprise a polypeptide having at least 10 amino acids encoded by any one of the polynucleotides of the present invention as discussed more fully, above, or polypeptides which are conservatively modified variants thereof.
• the proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in a full length polypeptide of the present invention.
• this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length.
• the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5.
• the present invention further provides a protein comprising a polypeptide having a specified sequence identity with a polypeptide of the present invention.
• the percentage of sequence identity is an integer selected from the group consisting of from 50 to 99. Exemplary sequence identity values include 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Sequence identity can be determined using, for example, the GAP or BLAST algorithms.
• the present invention includes catalytically active polypeptides of the present invention (i.e., enzymes).
• Catalytically active polypeptides have a specific activity of at least 20%, 30%, or 40%, and preferably at least 50%, 60%), or 10%, and most preferably at least 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide.
• the substrate specificity k cat /K m
• the K m will be at least 30%, 40%, or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90%.
• Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.
• the proteins of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention. Further, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those of skill in the art. A preferred immunoassay is a competitive immunoassay as discussed, supra. Thus, the proteins of the present invention can be employed as immunogens for constructing antibodies immuno reactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.
• nucleic acids of the present invention may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells.
• the cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.
• the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by inco ⁇ oration into an expression vector.
• the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
• Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention.
• transformation/transfection is not critical to the instant invention; various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect ph ⁇ notypic changes in the organism. Thus, any method which provides for effective transformation/transfection may be employed.
• a DNA sequence coding for the desired polypeptide of the present invention will be used to construct a recombinant expression cassette which can be introduced into the desired plant.
• the preferred method of plant transformation for the present invention by particle bombardment of immature maize embryos. See, e.g., Tomes, et al, Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment, pp.197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods, eds. O. L. Gamborg and G.C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995, and Songstad, D.D., B.M. Hairston and C.L. Armstrong 1993. Stable Transformation of Maize by Microprojectile Bombardment of Immature Embryos. Agronomy Abstracts p. 183.
• Isolated nucleic acid acids of the present invention can be introduced into plants according to techniques known in the art. Generally, recombinant expression cassettes as described above and suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et al, Ann. Rev. Genet. 22: 421-477 (1988).
• the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, polyethylene glycol (PEG), poration, particle bombardment, silicon fiber delivery, or microinjection of plant cell protoplasts or embryogenic callus.
• DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
• the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the ceil is infected by the bacteria. See, U.S. Patent No. 5,591,616.
• tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al, Plant Cell Physiol 25: 1353 (1984)), (3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Scl, (USA) 87: 1228 (1990).
• DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al, Methods in Enzymology, 101 :433 (1983); D. Hess, Intern Rev. Cytol, 107:367 (1987); Luo et al, Plant Mol. Biol. Reporter, 6:165 (1988).
• polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by 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 Neuhaus et al, Theor. Appl Genet., 75:30 (1987); and Benbrook et al, in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986).
• plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
• Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means.
• eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means.
• methods of introducing DNA into animal cells include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and micro-injection of the DNA directly into the cells.
• the transfected cells are cultured by means well known in the art. Kuchler, R.J., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).
• the proteins of the present invention can be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield, et al, J. Am. Chem. Soc. 85: 2149-2156 (1963), and Stewart et al, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, 111.
• Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide) are known to those of skill.
• the proteins of the present invention may be purified by standard techniques well known to those of skill in the art. Recombinantly produced proteins of the present invention can be directly expressed or expressed as a fusion protein.
• the recombinant protein is purified by a combination of cell lysis (e.g., sonication, French press) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein.
• the proteins of this invention, recombinant or synthetic may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R.
• Transgenic Plant Regeneration Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium. For transformation and regeneration of maize, see Gordon-Kamm et al, The Plant Cell, 2:603-618 (1990). Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant.
• Plant regeneration from cultured protoplasts is described in Evans et al, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
• Transgenic plants of the present invention may be fertile or sterile.
• Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al, Ann. Rev. of Plant Phys. 38: 467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil.
• the recombinant expression cassette is stably inco ⁇ orated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
• mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use.
• mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.
• Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention.
• Transgenic plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes.
• RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention.
• in situ hybridization and immunocytochemistry can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue.
• a number of transgenic lines are usually screened for the inco ⁇ orated nucleic acid to identify and select plants with the most appropriate expression profiles.
• a preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair.
• a homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of me seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non- transgenic). Back-crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated.
• the present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or ratio of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the ratio of the polypeptides of the present invention in a plant.
• the method comprises introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention as described above to obtain a transformed plant cell, culturing the transformed plant cell under plant cell growing conditions, and inducing or repressing expression of a polynucleotide of the present invention in the plant for a time sufficient to modulate concentration and/or the ratios of the polypeptides in the plant or plant part.
• the concentration and/or ratios of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a gene to up- or down-regulate gene expression.
• the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Patent 5,565,350; Zarling et al, PCT/US93/03868.
• an isolated nucleic acid e.g., a vector comprising a promoter sequence is transfected into a plant cell.
• a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom.
• a plant or plant part altered or modified by the foregoing embodiments is grown under plant-forming conditions for a time sufficient to modulate the concentration and/or ratios of polypeptides of the present invention in the plant. Plant-forming conditions are well known in the art and discussed briefly, supra.
• concentration or the ratios of the polypeptides is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned recombinant expression cassette.
• Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development.
• Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra.
• Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound.
• inducible promoters and inducing compounds which activate expression from these promoters are well known in the art.
• the polypeptides of the present invention are modulated in monocots, particularly maize.
• UTRs and Codon Preference In general, translational efficiency has been found to be regulated by specific sequence elements in the 5' non-coding or untranslated region (5' UTR) of the RNA.
• Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al, Nucleic Acids Res. 13:7375 (1985)).
• Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing et al, Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao et al, Mol.
• the present invention provides 5' and/or 3' untranslated regions for modulation of translation of heterologous coding sequences.
• the polypepti de-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host such as to optimize the codon usage in a heterologous sequence for expression in maize.
• Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al, Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
• the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention.
• the number of polynucleotides that can be used to determine a codon usage frequency can be any integer from 1 to the number of polynucleotides of the present invention as provided herein.
• the polynucleotides will be full-length sequences.
• An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.
• sequence shuffling provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. WO 97/20078. See also, Zhang, J.- H., et al. Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo.
• the population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method.
• the characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property.
• the selected characteristic will be a decreased K m and/or increased K cat over the wild-type protein as provided herein.
• a protein or polynucleotide generated from sequence shuffling will- have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide.
• the increase in such properties can be at least 1 10%, 120%, 130%, 140% or at least 150% of the wild-type value.
• Polynucleotides and polypeptides of the present invention further include those having: (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.
• the individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phyla, or kingdoms.
• a polynucleotide having a consensus sequence from a gene family of Zea mays can be used to generate antibody or nucleic acid probes or primers to other Gramineae species such as wheat, rice, or sorghum.
• a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa.
• 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.
• a conservative amino acid substitution can be used for amino acids which differ amongst aligned sequence but are from the same conservative substitution group as discussed above.
• no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.
• Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous, or paralogous sequences as provided herein.
• similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability (P(N)).
• P(N) BLAST algorithm's smallest sum probability
• a polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001, and most preferably less than about 0.0001. or 0.00001.
• Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, WI) PILEUP software, Vector NTI's (North Bethesda, MD) ALIGNX, or Genecode's (Ann Arbor, MI) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.
• the present invention provides machines, data structures, and processes for modeling or analyzing the polynucleotides and polypeptides of the present invention.
• the present invention provides a machine having a memory comprising data representing a sequence of a polynucleotide or polypeptide of the present invention.
• the machine of the present invention is typically a digital computer.
• the memory of such a machine includes, but is not limited to, ROM, or RAM, or computer readable media such as, but not limited to, magnetic media such as computer disks or hard drives, or media such as CD-ROM.
• the present invention also provides a data structure comprising a sequence of a polynucleotide of the present invention embodied in a computer readable medium.
• the form of memory of a machine of the present invention or the particular embodiment of the computer readable medium is not a critical element of the invention and can take a variety of forms.
• the present invention provides a process for identifying a candidate homologue (i.e., an ortholog or paralog) of a polynucleotide or polypeptide of the present invention.
• a candidate homologue has statistically significant probability of having the same biological function (e.g., catalyzes the same reaction, binds to homologous proteins/nucleic acids) as the reference sequence to which it's compared.
• the polynucleotides and polypeptides of the present invention have utility in identifying homologs in animals or other plant species, particularly those in the family Gramineae 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 are generally at least 25 amino acids in length or at least 50 nucleotides in length.
• the test sequence can be at least 50, 100, 150, 200, 250, ?00, or 400 amino acids in length.
• a test polynucleotide can be at least 50, 100, 200, 300, 400, or 500 nucleotides in length. Often the test sequence will be a full-length sequence.
• Test sequences can be obtained from a nucleic acid of an animal or plant.
• the test sequence is obtained from a plant species other than maize whose function is uncertain but will be compared to the test sequence to determine sequence similarity or sequence identity; for example, such plant species can be of the family Gramineae, such as wheat, rice, or sorghum.
• the test sequence data are entered into a machine, typically a computer, having a memory that contains data representing a reference sequence.
• the reference sequence can be the sequence of a polypeptide or a polynucleotide of the present invention and is often at least 25 amino acids or 100 nucleotides in length.
• the greater the sequence identity/similarity between a reference sequence of known function and a test sequence the greater the probability that the test sequence will have the same or similar function as the reference sequence.
• the machine further comprises a sequence comparison means for determining the sequence identity or similarity between the test sequence and the reference sequence.
• exemplary sequence comparison means are provided for in sequence analysis software discussed previously.
• sequence comparison is established using the BLAST or GAP suite of programs.
• a smallest 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 suite of algorithms under default parameters identifies the test sequence as a candidate homologue (i.e., an allele, ortholog, or paralog) of the reference sequence.
• a nucleic acid comprising a polynucleotide having the sequence of the candidate homologue can be constructed using well known library isolation, cloning, or in vitro synthetic chemistry techniques (e.g., phosphoramidite) such as those described herein.
• a nucleic acid comprising a polynucleotide having a sequence represented by the candidate homologue is introduced into a plant; typically, these polynucleotides are operably linked to a promoter. Confirmation of the function of the candidate homologue can be established by operably linking the candidate homolog nucleic acid to, for example, an inducible promoter, or by expressing the antisense transcript, and analyzing the plant for changes in phenotype consistent with the presumed function of the candidate homolog.
• the plant into which these nucleic acids are introduced is a monocot such as from the family Gramineae. Exemplary plants include maize, sorghum, wheat, rice, canola, alfalfa, cotton, and soybean.
• the present invention provides a process of modeling/analyzing data representative of the sequence a polynucleotide or polypeptide of the present invention.
• the process comprises entering sequence data of a polynucleotide or polypeptide of the present invention into a machine, 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.
• a variety of modeling and analytic tools are well known in the art and available from such commercial vendors as Genetics Computer Group (Version 10, Madison, WI).
• Included amongst the modeling/analysis tools are methods to: 1) recognize overlapping sequences (e.g., from a sequencing project) with a polynucleotide of the present invention and create an alignment called a "contig"; 2) identify restriction enzyme sites of a polynucleotide of the present invention; 3) identify the products of a Tl ribonuclease digestion of a polynucleotide of the present invention; 4) identify PCR primers with minimal self-complementarity; 5) compare two protein or nucleic acid sequences and identifying points of similarity or dissimilarity between them; 6) compute pairwise distances between sequences in an alignment, reconstruct phylogenetic trees using distance methods, and calculate the degree of divergence of two protein coding regions; 7) identify patterns such as coding regions, terminators, repeats, and other consensus patterns in polynucleotides of the present invention; 8) identify RNA secondary structure; 9) identify sequence motifs, isoelectric point, secondary structure, hydrophobicity
• the present invention further provides methods for detecting a polynucleotide of the present invention in a nucleic acid sample suspected of containing a polynucleotide of the present invention, such as a plant cell lysate, particularly a lysate of maize.
• a gene of the present invention or portion thereof can be amplified prior to the step of contacting the nucleic acid sample with a polynucleotide of the present invention.
• the nucleic acid sample is contacted with the polynucleotide to form a hybridization complex.
• the polynucleotide hybridizes under stringent conditions to a gene encoding a polypeptide of the present invention.
• Formation of the hybridization complex is used to detect a gene encoding a polypeptide of the present invention in the nucleic acid sample.
• an isolated nucleic acid comprising a polynucleotide of the present invention should lack cross-hybridizing 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.
• the nucleic acid sample, or a portion thereof may be assayed by hybridization formats including but not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.
• Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
• Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels.
• Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Labeling the nucleic acids of the present invention is readily achieved such as by the use of labeled PCR primers.
• Example 1 This example describes the construction of the cDNA libraries.
• RNA samples were pulverized in liquid nitrogen before the addition of the TRIzol Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.
• biotinylated oligo(dT) primers were used to hybridize to the 3' poly(A) tails on mRNA.
• the hybrids were captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand.
• the mRNA was washed at high stringency conditions and ⁇ luted by RNase-free deionized water.
• cDNA Library Construction cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the SuperscriptTM Plasmid System (Life Technologies, Inc., Rockville, MD). The first strand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by SuperscriptTM Reverse
• Transcriptase II at 45°C.
• the second strand of cDNA was labeled with alpha- 32 P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes.
• cDNA molecules smaller than 500 base pairs and unligated adapters were removed by Sephacryl-S400 chromatography.
• the selected cDNA molecules were ligated into pSPORTl vector in between of Not I and Sal I sites.
• Example 2 This example describes cD ⁇ A sequencing and library subtraction.
• D ⁇ A was prepared either by PCR with Ml 3 forward primers and Ml 3 reverse primers, or by plasmid isolation. All the cD ⁇ A clones were sequenced using Ml 3 reverse primers.
• Q-bot Subtraction Procedure cD ⁇ A libraries subjected to the subtraction procedure were plated out on 22 x 22 cm agar plate at density of about 3,000 colonies per plate. The plates were incubated in a 37°C incubator for 12-24 hours. Colonies were picked into 384-well plates by a robot colony picker, Q-bot (GE ⁇ ETIX Limited). These plates were incubated overnight at 37°C. Once sufficient colonies were picked, they were pinned onto 22 x 22 cm 2 nylon membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864 colonies. These membranes were placed onto agar plate with appropriate antibiotic. The plates were incubated at 37°C for overnight.
• Colony hybridization was conducted as described by Sambrook ., Fritsch, E.F. and Maniatis, T., (in Molecular Cloning: A laboratory Manual, 2 nd Edition). The following probes were used in colony hybridization:
• a Sal-A20 oligo nucleotide TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.
• the image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold- colonies from 384 well plates to 96 well plates was conducted using Q-bot.
• This example describes identification of the gene from a computer homology search.
• Gene identities were determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also • www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases).
• BLAST Basic Local Alignment Search Tool
• the cDNA sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr” database using the BLASTN algorithm.
• the DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr” database using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-212 (1993)) provided by the NCBI.
• the sequencing data from two or more clones containing overlapping segments of DNA were used to construct contiguous DNA sequences.
• This example provides an analysis of the transit peptides of the present invention in comparison to other plant alternative oxidase genes.
• AtAOXla_ AWIWTRA-PTIGGMRF j ASTITLGEKTPMKEEDANQKKTENESTGGDA-A GGNNKGD
• GmAO 3 A TSYWGIT--RPKVRREDGTEWPWNCFMPWDSYHSDVSIDVTKHHTPKSLTDKVAFRA
• This example describes the profiling of ZmAOX3 mRNA in Zea mays GS3 cell suspension cultures following exposure to spores of the fungus Fusarium moniliforme or to chito-oligosaccharides.
• Maize GS3 (HYII) cells were grown as suspension cultures to mid-log phase, when they were treated with either 1 ml water (control), 1 ml Fusarium moniliforme spores to give a final concentration of 100,000 spores/ml, or 1 ml chito-oligosaccharide mixture to give a final concentration of 100 ⁇ g/ml.
• the chito-oligosaccharide mixture was a pa ⁇ ial hydrolysate of crab shell chitin from CarboMer, Inc. (Westborough, MA). (See Yalpani, M. and D. Pantaleone (1994) Carbohydrate Research 256:159-175 for details of preparation of the chito-oligosaccharides.)
• RNA extraction and polyA+RNA isolation were performed as described in Example 1.
• Double-stranded cDNA was synthesized using the SuperscriptTM Plasmid System (Life Technologies, Inc., Rockville, MD).
• In- vitro transcription labeling of cRNA with biotin conjugated ribonucleotides was performed with the MEGAscript TM T7 kit (Ambion, Inc., Austin, TX), followed by the QIAGEN, Inc. (Valencia, CA) RNeasy® mini protocol for RNA cleanup.
• the resulting cRNA was fragmented and hybridized for 16 hours to a customized GeneChip® array of Zea mays oligonucleotides, then washed and stained with streptavidin, R-phycoerythrin conjugate, using the Affymetrix GeneChip Fluidics Station.
• the Hewlett-Packard G2500A Gene Array Scanner and Affymetrix GeneChip Analysis Suite software were used to analyze the results.
• Polidoros, A.N. et al. (1997) GenBank Direct submission. Accession AF040566. Submitted (30-DEC- 1997).
• Rhoades D.M. et al. (1993) Plant Mol. Bio. Int. J. Mol. Biol. Biochem. Genet. Eng.
• polynucleotides of SEQ ID NOS: 1, 4, and 7 are contained in a deposit made to the American Type Culture Collection (ATCC) on January 14, 2000, and assigned Accession Number PTA-1209.
• ATCC American Type Culture Collection
• PTA-1209 American Type Culture Collection is located at 10801 University Boulevard., Manassas, VA 201 10-2209.
• the ATCC deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
• the deposit is provided as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. Section 112.
• the deposited sequences, as well as the polypeptides encoded by the sequences, are incorporated herein by reference and control in the event of any conflict, such as a sequencing error, with the description in this application.

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