EP1263945A2 - Transkriptionsfaktoren aus wurzeln und ihre anwendungen - Google Patents

Transkriptionsfaktoren aus wurzeln und ihre anwendungen

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Publication number
EP1263945A2
EP1263945A2 EP01949003A EP01949003A EP1263945A2 EP 1263945 A2 EP1263945 A2 EP 1263945A2 EP 01949003 A EP01949003 A EP 01949003A EP 01949003 A EP01949003 A EP 01949003A EP 1263945 A2 EP1263945 A2 EP 1263945A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
plant
polypeptide
present
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01949003A
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English (en)
French (fr)
Inventor
Wesley B. Bruce
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pioneer Hi Bred International Inc
Original Assignee
Pioneer Hi Bred International Inc
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Filing date
Publication date
Application filed by Pioneer Hi Bred International Inc filed Critical Pioneer Hi Bred International Inc
Publication of EP1263945A2 publication Critical patent/EP1263945A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

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.
  • Cloning and molecular characterization of the HY5 gene indicate that it encodes a protein with a bZIP motif, characteristic of a transcriptional activator, and that the gene product is primarily located in the nucleus. Taken together, these data suggest that the HY5 gene functions in the nucleus as a key modulator of signal transduction pathways mediating a wide variety of stimulus responses and developmental processes in the root and hypocotyl.
  • HY5 protein is constitutively located in the nucleus and is a positive regulator of transcriptional activity by interaction with promoters containing the "G-box", a light-responsive element (Chattopadhyay, S , et al (1998) Plant Cell 10 673-683)
  • nucleic acids and proteins relating to a root transc ⁇ ptional factor it is an object of the present invention to provide transgemc plants comprising the nucleic acids of the present invention, and methods for modulating, in a transgemc plant, the expression of the nucleic acids of the present invention
  • 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 composing 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, composing 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 transgemc plant composing the recombinant expression cassette
  • the host cell and plant are optionally a maize cell or maize plant, respectively
  • nucleic acids are w ⁇ tten left to ⁇ ght m 5' to 3' o ⁇ entation
  • amino acid sequences are w ⁇ tten left to ⁇ ght in amino to carboxy o ⁇ entation, respectively
  • Nume ⁇ c ranges recited withm 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 Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5 t ⁇ edition, 1993).
  • 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
  • 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 ma ⁇ be derived from known gene sequences from maize Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al , supra
  • full-length sequence in reference to a specified polynucleotide or its encoded protein means having the entire ammo 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 m the art including such exemplary techniques as northern or western blots, p ⁇ mer extension, SI protection, and ⁇ bonuclease protection See, e g , Plant Molecular Biology A Laboratory Manual, Clark, Ed .
  • consensus sequences typically present at the 5 and 3' untranslated regions of mR ⁇ A aid in the identification of a polynucleotide as full-length
  • the consensus sequence A ⁇ AUGG where the underlined codon represents the ⁇ -termmal methionme, aids in determining whether the polynucleotide has a complete 5' end
  • Consensus sequences at the 3' end such as polv denylation sequences, aid in determining whether the polynucleotide has a complete 3' end
  • heterologous in reference to a nucleic acid is a nucleic acid that o ⁇ gmates from a foreign species, or, if from the same species, is substantially modified from its native form m composition and/or genomic locus by deliberate human mten ention
  • 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 o ⁇ ginal form
  • a heterologous protein may o ⁇ gmate 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 coh, 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
  • the term "introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (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:
  • 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.
  • root transcriptional factor nucleic acid is a nucleic acid of the present invention and means a nucleic acid comprising a polynucleotide of the present invention (a "root transcriptional factor polynucleotide”) encoding a root transcriptional factor polypeptide.
  • a “root transcriptional factor gene” is a gene of the present invention and refers to a full-length root transcriptional factor 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 m standard molecular biology references such as Berger and Kimmel, Guide to Moleculat Cloning Techniques Methods in Vol 152, Academic Press, Inc , San Diego, CA (Berger), Sambrook et al Molecu ⁇ 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 transc ⁇ ption of the DNA sequence cooespondmg 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
  • the term "plant” includes reference to whole plants, plant parts or organs (e g , 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, me ⁇ stematic 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 A particularly preferred plant is Zea mays
  • polynucleotide includes reference to a deoxy ⁇ bopolynucleotide, ⁇ bopolynucleotide, or chimeras or analogs thereof that have the essential nature of a natural deoxy
  • polypeptide polypeptide
  • peptide protein
  • amino acid polymers in which one or more ammo acid residue is an artificial chemical analogue of a corresponding naturally occur ⁇ ng amino acid, as well as to naturally occurring ammo acid polymers
  • the essential nature of such analogues of naturally occur ⁇ ng amino acids is that, w hen incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occur ⁇ ng ammo acids
  • polypeptide polypeptide
  • peptide protein
  • proteins are also inclusive of modifications including, but not limited to, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ⁇ bosylation
  • this invention contemplates the use of both the methionine-containmg and the methionine-less ammo terminal variants of the
  • 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 transc ⁇ ption
  • a "plant promoter” is a promoter capable of initiating transc ⁇ ption 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 bacte ⁇ a which compnse genes expressed in plant cells such Agrobacterium or Rhizobium
  • Examples of promoters under developmental control include promoters that preferentially initiate transc ⁇ ption in certain tissues, such as leaves, roots, or seeds Such promoters are referred to as "tissue preferred” Promoters which initiate transc ⁇ ption only in certain tissue are referred to as "tissue specific"
  • 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 de ⁇ ved from a cell so modified
  • recombinant cells express genes that are not found in identical form within the native (non-recombmant) 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 occur ⁇ ng without deliberate human intervention
  • a "recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a senes of specified nucleic acid elements which permit transc ⁇ ption of a particular nucleic acid in a host cell
  • the recombinant expression cassette can be incorporated mto a plasmid, chromosome, mitochondnal 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
  • ammo acid residue or “ammo acid residue” or “amino acid” are used interchangeably herein to refer to an ammo acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein")
  • the ammo acid may be a naturally occur ⁇ ng ammo acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function m a similar manner as naturally occur ⁇ ng ammo acids
  • sequences include reference to hybndization, under stnngent hyb ⁇ dization 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 hyb ⁇ dization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids
  • hyb ⁇ dizmg sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (t e , complementary) with each other
  • Stnngent conditions or “stringent hyb ⁇ dization 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) Stnngent conditions are sequence-dependent and will be different in different circumstances
  • target sequences can be identified which are 100% complementary to the probe (homologous probing)
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of simila ⁇ ty are detected (heterologous probing)
  • a probe is less than about 1000 nucleotides m length, optionally less than 500 nucleotides in length
  • stnngent 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)
  • Stnngent conditions may also be achieved with the addition of destabilizing agents such as formamide
  • Exemplary moderate st ⁇ ngency conditions include
  • reference sequence is a defined sequence used as a basis for sequence companson with a polynucleotide/polypeptide of the present invention
  • a reference sequence may be a subset or the entrrety of a specified sequence, for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence
  • compa ⁇ son 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 m the compa ⁇ son window may comp ⁇ se additions or deletions (l e , gaps) compared to the reference sequence (which does not compnse additions or deletions) for optimal alignment of the two sequences
  • the compa ⁇ son window is at least 20 contiguous nucleotides/ammo acrds residues m length, and optionally can be 30, 40, 50, 100, or longer Those of skill in the art understand that to avoid a high simila ⁇ ty to a reference sequence due to inclusion of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically introduced and is subtracted from the number of matches
  • Optimal alignment of sequences for compa ⁇ son may be conducted by the local homology algo ⁇ thm of Smith and Waterman, Adv Appl Math 2 482 (1981), by the homology alignment algo ⁇ thm of Needleman and Wunsch, J Mol Biol 48 443 (1970), by the search for simila ⁇ ty method of Pearson and Lipman, Proc Natl Acad Sci 85 2444 (1988), by computenzed implementations of these algo ⁇ thms, including, but not limited to CLUSTAL in the PC/Gene program by Intelhgenetics, Mountain View, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr , Madison, Wisconsin, USA, the CLUSTAL program is well desc ⁇ bed by Higgins and Sharp, Gene 73 237-244 (1988), Higgins and Sharp, CABIOS 5 151-153 (1989), Corpet
  • the BLAST family of programs which can be used for database similarity searches includes BLASTN for nucleotide query sequences against nucleotide database sequences, BLASTX for nucleotide query sequences against protein database sequences, BLASTP for protein query sequences against protein database sequences, TBLASTN for protein query sequences against nucleotide database sequences, and TBLASTX for nucleotide query sequences against nucleotide database sequences See, Current Protocols in Molecular Biology , Chapter 19, Ausubel, et al , Eds , Greene Publishing and Wiley-Interscience, New York ( 1995)
  • HSPs high sconng sequence pairs
  • a sconng matnx 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-sco ⁇ ng residue alignments, or the end of either sequence is reached.
  • the BLAST algo ⁇ thm parameters W, T, and X determine the sensitivity and speed of the alignment
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 sconng matnx (see Hemkoff & Hemkoff ( 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 simila ⁇ ty between two sequences (see e g , Karhn & Altschul, Pi oc Nat l Acad Sci USA 90 5873-5877 (1993))
  • One measure of simila ⁇ ty provided by the BLAST algorithm is the smallest sum probability (P(N)), 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
  • GAP can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence
  • GAP uses the algoothm 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 If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty
  • Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protem sequences are 8 and 2, respectively
  • the default gap creation penalty is 50 while the default gap extension penalty is 3
  • GAP presents one member of the famrly of best alignments There may be many members of this family, but no other member has a better quality
  • GAP displays four figures of meot for alignments Quality, Ratio, Identity, and Similaoty
  • 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 Similaoty is the percent of the symbols that are similar Symbols that are across from gaps are ignored
  • a similarity is scored when the scoong matrix value for a pair of symbols is greater than or equal to 0 50, the simila ⁇ ty threshold
  • the default sconng matnces used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 for polypeptide comparisons (see Hemkoff & Hemkoff (1989) Proc Natl Acad Sci USA 89 10915) and NWSGAPDNA for polynucleotide compansons
  • sequence identity/simila ⁇ ty values provided herein refer to the value obtained using the BLAST 2 0 suite of programs using default parameters (Altschul et al , Nucleic Acids Res 25 3389-3402, 1997, Altschul et al , J Mol Bio 215 403-410, 1990) or to the value obtained using the GAP program version 10 using default parameters (see the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr , Madison, Wisconsin, USA)
  • sequence identity' or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified companson window
  • 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 simila ⁇ ty” or “simila ⁇ ty” Means for making this adjustment are well-known to those of skill m the art Typrcally this involves sconng a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity
  • percentage of sequence identity means the value determined by companng two optimally aligned sequences over a compa ⁇ son window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (1 e , gaps) as compared to the reference sequence (which does not comp ⁇ se additions or deletions) for optimal alignment of the two sequences The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs m both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions m the window of companson 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 (l 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 uncharactenstic of non-recombinantly engineered plants
  • the present invention provides utility in such exemplary applications as providing a means to control expression of genes involved in root initiation and growth, including responses to environmental or pathogenic cues
  • increased lateral root initiation and growth typical of hv5 mutants could be achieved through transformation with antisense HY5 under the control of a root-specific promoter This altered root growth could enhance root anchorage and/or drought tolerance
  • the present invention also provides isolated nucleic acids comp ⁇ sing polynucleotides of sufficient length and complementanty to a gene of the present invention to use as probes or amplification pnmers in the detection, quantitation, or isolation of gene transc ⁇ pts
  • isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA m screenings for desired transgemc plants, for detecting mutations in the gene (e g , substitutions, deletions, or additions), for momto ⁇ ng upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allehc va ⁇ ants (polymorphisms), orthologs, or paralogs of the gene, or for srte drrected 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 poly
  • the present invention also provides isolated proteins comprising a polypeptide of the present invention (e g , preproenzyme, proenzyme, or enzymes)
  • the present mventron 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 punfication 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, Tritwum, Sorghum (e g , S bicolor) and Zea (e g , Z max s)
  • the isolated nucleic acid and proteins of the present invention can also be used m species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria Lotus, Medicago, Onobrvchis Trifolium, Tngonella, Vigna, Citrus Linum, Geranium, Manihot, Daucus, Arabidopsis Brassica, Raphanus, Sinapis, Atropa Capsicum, Datura Hyoscyamus, Lycopersicon Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Hehanthus, Lactuca, Bromus, Asparagus, Antirr
  • the present invention provides, among other things, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, compnsmg a polynucleotide of the present invention
  • a polynucleotide of the present invention is inclusive of
  • a polynucleotide encoding a polypeptide of SEQ ID NOS 2, 6, or 10, including exemplary polynucleotides of SEQ ID NOS 1. 5, and 9,
  • a polynucleotide which is the product of amplification from a Zea nuns nucleic acid library using primer pairs which selectively hybndize under stringent conditions to loci withm a polynucleotide selected from the group consisting of SEQ ID NOS 1 , 5, and
  • a polynucleotide compnsmg at least a specific number of contiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e), or (f)
  • the present invention provides isolated nucleic acids compnsmg 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, descnbes every possible silent va ⁇ ation of the nucleic acid
  • each codon m a nucleic acid except AUG, which is ordinarily the only codon for methronme, and UGG , which is ordinarily the only codon for tryptophan
  • each silent va ⁇ ation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each desc ⁇ bed polypeptide sequence and is within the scope of the present invention
  • the present invention includes polynucleotides selected from the group consisting of SEQ ID NOS 1, 5, and 9, and polynucleotides
  • the present invention provides an isolated nucleic acid compnsmg 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.
  • the nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcnpts at any stage of intron processing cDNA hbranes 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 Ohgo-Cappmg (Maruyama, K and Sugano, S Gene 138 171-174, 1994), Biotmylated CAP Trapper (Carmnci, 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
  • the present invention also provides subsequences of the polynucleotides of the present invention
  • a va ⁇ ety of subsequences can be obtained using pnmers which selectively hybndize under stnngent conditions to at least two sites within a polynucleotide of the present invention, or to two sites with the nucleic acid which flank and comp ⁇ se a polynucleotide of the present invention, or to a site withm a polynucleotide of the present invention and a site within the nucleic acid which comp ⁇ ses it
  • Pnmers are chosen to selectively hybndize, under stnngent hybndization 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
  • 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 venfying the presence of one or more epitopes which are specific to a polypeptide of the present invention
  • catalytic activity e g , specific activity and/or substrate specificity
  • 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
  • the present invention provides isolated nucleic acids compnsmg polynucleotides of the present invention, wherein the polynucleotides selectively hybndize, 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 compnsmg 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 genomtc 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,
  • 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 algo ⁇ thms 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 compnsmg 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 m, for example, the screening of expression hbranes for nucleic acids comprising polynucleotides of (A), (B), or (C), or for purification of, or in tmmunoassays for, polypeptides encoded by the polynucleotides
  • the present invention provides isolated nucleic acids compnsmg 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 ammo acids from the prototype polypeptide is selected from the group of integers consisting of from at least 10 to the number of ammo 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
  • 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), abo ⁇ e
  • 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 lmmunofluorescent assays and the like
  • the proteins of the present invention embrace allehc vanants, conservatively modified vanants, and minor recombinant modifications to a prototype polypeptide
  • a polynucleotide of the present invention optionally encodes a protein having a molecular weight as the non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptides of the present invention Molecular weight can be readily determined by SDS-PAGE under reducing conditions Optionally, the molecular weight is with 15% of a full length polypeptide of the present invention, more preferably withm 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%, or 90% of a cellular extract compnsmg the native, endogenous full-length polypeptide of the present invention
  • proteins encoded by polynucleotides of this embodiment will optionally have a substantially similar affinity constant (K m ) and/or catalytic activity (l e , the microscopic rate constant, k cat ) as the native endogenous, full-length protein
  • K m affinity constant
  • l e the microscopic rate constant
  • k cat the specificity constant
  • Proteins of this embodiment can have a k 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 ⁇ /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
  • nucleic acids compnsmg 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) (l e , have 100% sequence identity over their entire length)
  • Complementary bases associate through hydrogen bonding in double stranded nucleic acids
  • the following base pairs are complementary guanine and cytosine, adenine and thymine, and adenine and uracil
  • the present invention provides isolated nucleic acids compnsmg polynucleotides which comp ⁇ se 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 compnsmg 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 selected from the group consisting
  • the subsequences of the present invention can comprise structural charactenstics of the sequence from which it is de ⁇ ved Alternatively, the subsequences can lack certain structural charactenstics of the larger sequence from which it is de ⁇ ved such as a poly (A) tail
  • a subsequence from a polynucleotide encoding a polypeptide having at least one epitope m common with a prototype polypeptide sequence as provided in (a), abo ⁇ e 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 hyb ⁇ dization with the sequence from which it's de ⁇ ved
  • Subsequences can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, clea ⁇ e and/or crosslink to 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 m addition to a polynucleotide of the present invention
  • a multi-cloning site compnsmg 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- histidme marker sequence provides a convenient means to pu ⁇ fy the proteins of the present invention
  • a polynucleotide of the present invention can be attached to a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell
  • RNA, cDNA, genomic DNA, or a hybnd thereof can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art
  • ohgonucleotide probes which selectively hybridize, under stnngent 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 hbranes is well known to those of ordinary skill in the art See, e g , Plant Molecular Biology A Laboratory Manual, Clark, Ed , Spnnger-Verlag, Berlin (1997), and, Current Protocols in Molecular Biology, Ausubel, et al , Eds , Greene Publishing and Wiley- Interscience, New York (1995)
  • a number of cDNA synthesis protocols have been desc ⁇ bed which provide substantially pure full-length cDNA hbranes
  • Substantially pure full-length cDNA hbranes are constructed to compnse at least 90%, and more preferably at least 93% or 95% full-length inserts amongst clones containing inserts
  • the length of insert in such hbranes can be from 0 to 8, 9, 10, 11, 12, 13, or more kilobase pairs Vectors to accommodate inserts of these sizes are known in the art and available commercially See, e g , Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12 kb cloning capacity)
  • An exemplary method of constructing a greater than 95% pure full-length cDNA library is descnbed by Carnmci et al , Genomics, 37 327-336 (1996)
  • Other methods for producing full-length hbranes are known m the art See, e g , Edery
  • a non-normalized cDNA library represents the mRNA population of the tissue it was made from Since unique clones are out-numbered by clones denved from highly expressed genes their isolation can be labonous 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- 571 1 (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 degiees of stnngencv of hybndization can be employed in the assay, and either the hybridization or the ash 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 polymerase chain reaction
  • other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes
  • the T4 gene 32 protein (Boehnnger Mannheim) can be used to improve yield of long PCR products
  • PCR-based screening methods have been desc ⁇ bed Wilfinger et al descnbe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study BioTechmques, 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 phosphotn ester method of Narang et al , Meth Enzvmol 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 tnester method desc ⁇ bed by Beaucage and Caruthers, Tetra Letts 22(20) 1859-1862 (1981), e g using an automated synthesizer, e g , as descnbed in Needham-VanDevanter et al Nucleic Acids Res , 12
  • 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 compnse a polynucleotide of the present invention operably linked to transcnptional initiation regulatory sequences which will direct the transc ⁇ ption of the polynucleotide the intended host cell, such as tissues of a transformed plant
  • plant expression vectors may include (1) a cloned plant gene under the transc ⁇ ptional 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 mducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transc ⁇ ption initiation start site, a ⁇ bosome binding site, an RNA processing signal, a transcnption 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 refened 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 transc ⁇ ption initiation region, the 1'- or 2'- promoter denved from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cmnamyl 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 transc ⁇ ption initiation regions from vanous plant genes known to those of skill
  • the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control
  • Such promoters are referred to here as "mducible" promoters
  • mducible promoters Environmental conditions that may effect transcnption by mducible promoters include pathogen attack, anaerobic conditions, or the presence of light
  • Examples of mducible promoters are the Adhl promoter which is mducible by hypoxia or cold stress, the Hsp70 promoter which is mducible by heat stress, and the PPDK promoter which is mducible by light
  • promoters under developmental control include promoters that initiate transc ⁇ ption only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers
  • Exemplary promoters include the root cdc2a promoter (Doerner, P , et al (1996) Nature 380 520-523) or the root peroxidase promoter from wheat (Hertig, C , et al (1991) Plant Mol Biol 16 171-174)
  • the operation of a promoter may also vary depending on its location in the genome Thus, an mducible promoter may become fully or partially constitutive in certain locations
  • heterologous and non-heterologous (I e , endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention
  • These promoters can also be used, for example, in recombinant expression cassettes to dnve expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a
  • isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the approp ⁇ ate 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, Zarhng et al , PCT US93/03868), or isolated promoters can be introduced into a plant cell in the proper o ⁇ entation 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
  • polypeptide expression it is generally desirable to include a polyadenylation region at the 3 '-end of a polynucleotide coding region
  • the polyadenylation region can be de ⁇ ved 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 nopahne synthase or octopme 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 sphceable mtron in the transcnption umt 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), CaWxs 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 transcnption unit
  • Use of maize introns Adhl-S intron 1, 2, and 6, the Bronze-1 intron are known in the art See generally, The Maize Handbook, Chapter 116, Freelmg and Walbot, Eds , Spnnger, New York (1994)
  • the vector comprising the sequences from a polynucleotide of the present invention will typically compns
  • 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 charactenstics
  • 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 transcnbed The construct is then transformed into plants and the antisense strand of RNA is produced In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, 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
  • ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
  • the ribozyme 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 Knooe, 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 compnse any number of contiguous ammo 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 ammo 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 algo ⁇ thms
  • 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 70%, and most preferably at least 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide
  • the substrate specificity (k cat /K m ) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide
  • the K m will be at least 30%,
  • nucleic acids of the present invention may express a protein of the present invention in a recombmantly engineered cell such as bactena, yeast, msect, mammalian, or preferably plant cells
  • a recombmantly engineered cell such as bactena, yeast, msect, 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
  • 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 cDN A to a promoter (which is either constitutive or regulatable), followed by incorporation into an expression vector
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes
  • Typical expression vectors contain transcnption and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention
  • To obtain high level expression of a cloned gene it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transc ⁇ ption, a ⁇ bosome binding site for translatronal initiation, and a transcnption/translation terminator
  • modifications can be made to a protein of the present invention without diminishing its biological activity Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a
  • A. Plant Transformation A DNA sequence coding for the desired polypeptide of the present invention, for example a cDNA or a genomic sequence encoding a full length protein, will be used to construct a recombinant expression cassette which can be introduced into the desired plant.
  • Isolated nucleic 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 cell is infected by the bacteria. See, U.S. Patent No. 5,591 ,616.
  • the introduction of DNA constructs using PEG precipitation is described in
  • Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium.
  • Agrobacterium transformation of maize is described in U.S. Patent No. 5,550,318.
  • Other methods of transfection or transformation include (1 ) Agr obacterium rhizogenes-mediaied transfonnation (see, e g , Lrchtenstem and Fuller In Genetic Engineering, vol 6 PWJ Rtgby, Ed , London, Academic Press, 1987, and Ltchtenstem, C P , and Draper, J, h DNA Cloning, Vol II, D M Glover, Ed , Oxford, IRI Press, 1985), Application PCT/US87/02512 (WO 88/02405 published Apr 7, 1988) describes the use of A rhizogenes strain A4 and its Ri plasmid along wrth A tumefaciens vectors pARC8 or pARCl ⁇ (2) hposome-mediated DNA uptake (see
  • Animal and lower eukaryotic (e g , yeast) host cells are competent or rendered competent for transfectron by vanous means
  • eukaryotic (e g , yeast) host cells are competent or rendered competent for transfectron by vanous 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 hposomes containing the DNA, DEAE dextran, electroporation, biohstics, and mrcro-mjection 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, Hutchmson 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-termmal ammo acid of the sequence to an insoluble support followed by sequential addrtion of the remaining ammo acids in the sequence
  • Techniques for solid phase synthesis are desc ⁇ bed by Barany and Memfield, Solid-Phase Peptide Synthesis, pp 3-284 m
  • the Peptides Analysis Synthesis Biology Vol 2 Special Methods in Peptide Synthesis Part A Merofield, 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 (1984)
  • Proteins of greater length may be synthesized by condensation of the ammo and carboxy termini of shorter fragments Methods of formmg peptrde bonds by activation of a carboxy terminal end (e g , by the use of the coupling rea
  • the proteins of the present invention may be purified by standard techniques well known to those of skill in the art
  • Recombmantly produced protems of the present mvention can be directly expressed or expressed as a fusron protem
  • the recombmant protein is punfied by a combination of cell lysis (e g , somcation, French press) and affinity chromatography
  • cell lysis e g , somcation, French press
  • affinity chromatography For fusion products, subsequent digestion of the fusron protem with an approp ⁇ ate proteolytic enzyme releases the desired recombinant protem
  • the proteins of this invention may be punfied to substantial pu ⁇ ty by standard techniques well known in the art, including detergent solubihzation, selective precipitation with such substances as ammonium sulfate, column chromatography, lmmunopurrfication methods, and others See, for mstance, R Scopes, Protein Purification Principles and Practice, Sp ⁇ nger-Verlag New York (1982), Deutscher, Guide to Protein Purification, Academic Press (1990)
  • antibodies may be raised to the proteins as descnbed herein Pu ⁇ fication from E coli can be achieved followmg procedures descnbed in U S Patent No 4,511,503
  • the protem may then be isolated from cells expressmg the protem and further punfied by standard protem chemistry techniques as descnbed herein Detection of the expressed protem is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation
  • Transformed plant cells whrch are de ⁇ ved by any of the above transformatron technrques can be cultured to regenerate a whole plant whrch possesses the transformed genotype
  • Such regeneration techniques often rely on manipulation of certain phytohormones m 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 vanous 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)
  • the regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be ach ⁇ e ⁇ ed as described by Horsch et al , Science, 227 1229-1231 (1985) In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots m the plant species being transformed as desc ⁇ bed by Fraley et al , Proc Natl Acad Sci (USA ), 80 4803 (1983) This procedure typically produces shoots within two to four weeks and these transformant shoots are then transfeoed to an approp ⁇ ate root-mducmg medium containing the selective agent and an antibiotic to prevent bactenal growth Transgemc 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 descnbed generally in Klee et al Ann Rev of Plant Phys 38 467-486 (1987)
  • the regeneration of plants from erther srngle plant protoplasts or vanous explants is well known in the art See, for example, Methods for Plant Molecular Biology, A Werssbach 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, rootrng the transformant shoots and growth of the plantlets m soil
  • Maize Handbook Freehng and Walbot, Eds , Sponger, New York (1994), Corn and Corn Improvement, 3 r edition, Sprague and Dudley Eds , American Society of Agronomy, Madison, Wisconsin (1988)
  • the recombinant expression cassette is stably incorporated m transgemc 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 transgemc plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants Selection of desirable transgemcs is made and new varieties are obtained and propagated vegetattvely for commercial use
  • mature transgemc plants can be self crossed to produce a homozygous mbred 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 composing the isolated nucleic acid of the present invention Progeny and vanants, and mutants of the regenerated plants are also included withm the scope of the invention, prov ided that these parts compnse the introduced nucleic acid sequences
  • Transgemc plants expressing the selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard
  • the present invention further provides a method for modulatmg (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 mcreasmg or decreasing the concentration and/or the the ratio of the polypeptides of the present invention in a plant
  • the method composes introducing into a plant cell a recombinant expression cassette composing a polynucleotide of the present invention as descobed above to obtain a transformed plant cell, culturmg 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 trme suffrcrent to modulate concentratron 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 alternativeng, in vivo or in vitr o 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, Zarlmg et al , PCT/US93/03868
  • an isolated nucleic acid (e g , a vector) compnsmg a promoter sequence is transfected into a plant cell
  • a plant cell compnsmg the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill m the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using pnmers specific to the promote
  • 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.
  • 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,
  • Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing et al, Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao et al, Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present invention provides 5' and/or 3' untranslated regions for modulation of translation of heterologous coding sequences.
  • polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage.
  • Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host such as to optimize the codon usage in a heterologous sequence for expression in maize.
  • Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference" available from the University of Wisconsin Genetics Computer Group (see Devereaux et al, Nucleic Acids Res. 12: 387-395 (1984)) or
  • 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 a means for generatmg hbranes of polynucleotides having a desired characte ⁇ stic which can be selected or screened for Lrbra ⁇ es of recombinant polynucleotrdes are generated from a populatron of related sequence polynucleotides which compnse sequence regions which have substantial sequence identity and can be homologously recombmed in vitro or in v ⁇ o
  • the populatron of sequence-recombmed polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous charactenstics and which can be selected by a suitable selection or
  • the selected characterrstrc wrll be a decreased K m and/or mcreased K cat over the wrld-type protem as provrded herern
  • a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide
  • the increase m such properties can be at least 110%, 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 genenc 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 genenc sequence of the present invention comp ⁇ ses each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide sequence, respectively
  • the individual species encompassed by a polynucleotide having an ammo 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, famrlres, orders, classes, phyla, or kingdoms
  • a polynucleotide having a consensus sequence from a gene family of Zea mays can be used to generate
  • Similar sequences used for generation of a consensus or genenc sequence include any number and combination of allehc variants of the same gene, orthologous, or paralogous sequences as provided herein
  • similar sequences used m generating a consensus or genenc sequence are identified using the BLAST algo ⁇ thm's smallest sum probability (P(N)) Vaoous suppliers of sequence-analysts software are listed m chapter 7 of Current Protocols in Molecular Biology, F M Ausubel et al , Eds , Cmrent Protocols, a joint venture between Greene Publishing Associates, Inc and John Wiley & Sons, Inc (Supplement 30)
  • a polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability m a compaoson 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 Sr
  • the present invention provides machines, data structures, and processes for modeling or anal zmg the polynucleotides and polypeptides of the present invention
  • the present invention provides a machine having a memory composing 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 composing a sequence of a polvnucleotide 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 cotical element of the invention and can take a variety of forms
  • the present invention provides a process for rdentifying 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 protems/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 famrly Gramineae such as, but not limited to, sorghum, wheat, or oce
  • 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, 300, or 400 ammo acids in length
  • a test polynucleotide can be at least 50, 100, 200, 300, 400, or 500 nucleotides in length
  • 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 polypeptide test sequence
  • sequence compaoson means for determining the sequence identity or similarity between the test sequence and the reference sequence
  • sequence comparison means are provided for m sequence analysis software discussed previously
  • sequence compaoson 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 algonthms under default parameters identifies the test sequence as a candidate homologue (l 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 compnsmg 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 oper
  • the present mvention provides a process of modeling/analyzing data representative of the sequence a polynucleotide or polypeptide of the present invention
  • the process composes enteong 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 vaoety 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 ⁇ bonuclease digestion of a polynucleotide of the present
  • 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 p ⁇ or 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 hyb ⁇ dization complex
  • the polynucleotide hybridizes under stringent conditions to a gene encoding a polypeptide of the present invention Formation of the hyb ⁇ dization complex is used to detect a gene encoding a polypeptide of the present invention in the nucleic acid sample
  • an isolated nucleic acid compnsmg a polynucleotide of the present invention should lack cross-hyb
  • 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 m the present invention include biotm for staining with labeled streptavrdm conjugate, magnetic beads, fluorescent dyes, radtolabels, enzymes, and colonmet ⁇ c labels
  • Other labels include hgands 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
  • TRIzolTM Reagent Life Technologies, Inc , Rockville, MD
  • plant tissue samples were pulvenzed m liquid nitrogen before the addition of the TRIzol Reagent, and then were further homogenized with a mortar and pestle Addition of chloroform followed by cent ⁇ fugatron was conducted for separatron of an aqueous phase and an organic phase
  • cDNA Library Construction cDNA synthesis as performed and umdrrectronal cDNA libraries were constructed usrng the SuperscriptTM Plasmid System (Life Technologies, Inc , Rockvtlle, MD)
  • the first strand of cDNA was synthesized by p ⁇ mmg an ohgo(dT) pnmer containing a Not I site
  • the reaction was catalyzed by SuperScnptTM Reverse Transc ⁇ ptase 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 unhgated adapters were removed by Sephacryl-S400 chromatography
  • the selected cDNA molecules were hgated into pSPORTl vector in between of Not I and Sal I sites
  • This example descnbes cDNA sequencing and library subtraction
  • the filters were then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution was removed by placing the filters on dry filter papers for one minute, the colony side of the filters were place into Proteinase K solution, incubated at 37°C for 40- 50 minutes. The filters were placed on dry filter papers to dry overnight. DNA was then cross-linked to nylon membrane by UN light treatment.
  • 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: 1. First strand cD ⁇ A from the same tissue as the library was made from to remove the most redundant clones.
  • 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 cD ⁇ A sequences were analyzed for similarity to all publicly available D ⁇ A 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 protem 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.

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