JP2002534093A - Ribonucleotide reductase large subunit (R1) cDNA and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides isolated R1 nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering the R1 concentration and / or composition of a plant. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The objects of the present invention are as follows: 1) an antigenic fragment of the protein of the present invention; 2) a transgenic plant containing the nucleic acid of the present invention; 3) a method for regulating the expression of the nucleic acid of the present invention in the transgenic plant. , Is to provide.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates generally to plant molecular biology. More particularly, the present invention relates to nucleic acids and methods for regulating their expression in plants.

BACKGROUND OF THE INVENTION Ribonucleotide reductase (EC 1.17.4; hereinafter referred to as R
(Abbreviated as NR) catalyzes the synthesis of deoxyribonucleoside diphosphate or deoxyribonucleoside triphosphate from ribonucleoside precursors (Thee
lander L. And Reichard P.S. , Ann. Rev .. Bioc
hem. 48: 133-158, 1979). This unique and basic biochemical reaction is essential for the synthesis of DNA of all living organisms. These structures,
Based on development and cofactor needs, RNRs have been classified into at least four different groups (see Table 1).

[0003]

[Table 1] Class Ia enzymes (found in most aerobic bacteria and all eukaryotes) is organized as a heterodimer of alpha ₂ beta ₂ type, two large (lar
ge) subunit (R1) and two small subunits (R2). This enzyme is inhibited by dATP. A structurally similar enzyme (Ib) is also present in some aerobic bacteria,
Stimulated by P. Class III enzymes are found primarily in anaerobic bacteria. This enzyme is structurally similar to Class I RNR, but uses a glycyl radical for the reduction reaction. The catalytic and allosteric sites for class I and class III enzymes are located on the large subunit. Small subunits provide the tyrosyl residues required for radical generation. Class II RN
R (found in both aerobic and anaerobic eubacteria and archaebacteria) is a single subunit enzyme that can function as a homodimer (Thel
ander L. And Reichard P.S. , Ann. Rev .. Bioch
em. 48: 133-158, 1979; And Ehr
enberg, A .; , Science 260: 1173-1177, 1983.
Stubbe, J .; , Advan. Enzymol. Relat. Area.
Mol. Biol. 63: 349-417, 1990; Reichard, P .;
, Trends Biochem. Sci. 22: 81-85, 1997). However, this enzyme uses adenosylcobalamin (vitamin B12) as a cofactor and is stimulated by dATP.

[0004] Despite these structural differences between the different classes of RNRs, these RNs
The basic chemical reaction catalyzed by all of R is the same (ie, reduction of ribonucleotides to deoxyribonucleotides). This seemingly simple enzymatic reaction has great biochemical, evolutionary, and clinical significance. RNR is r
It is the only enzyme known to be able to convert NTP to dNTP, ie, RNA to DNA. Since it is generally accepted that RNA was the original primitive genetic material, the ability to synthesize dNTPs could be one of the most important properties acquired by the primordial organism. The enzymatic activity of RNR is described in the “RNA World (
RNA World) to "DNA World". Therefore, the evolutionary significance of RNR cannot be paralleled with the evolution of life, as we know. Finally, RNR has been a very attractive target for various antineoplastic, antiviral and antiparasitic drugs (
Stubbe J .; And van Der Donk, W.C. A. Chem. B
iol. 2: 793-801, 1995; Stubbe, J .; Proc. Na
tl. Acad. Sci. 95: 2723-2724, 1998). as a result,
This enzyme and cognate genes have been isolated from various animal sources (Thelan
der L. And Reichard P.S. , Ann. Rev .. Biochem
. 48: 133-158, 1979; And Ehren
berg, A .; , Science 260: 1173-1177, 1983; S
tubbe, J .; , Advan. Enzymol. Relat. Area. Mo
l. Biol. 63: 349-417, 1990; Reichard, P .; , T
rends Biochem. Sci. 22: 81-85, 1997). Plants have also been shown to contain RNR. Tobacco and Arabidopsis
The cDNA sequence of the derived small (R2) subunit has been published (Ph
ilipps, G .; Et al., FEBS Lett. 358: 67-70, 1995;
Phillips, G .; Et al., GenBank registration number X92443, 1997)
And the sequence of the large (R1) subunit from tobacco has also been published (Chaboute, ME et al., Plant Mol. Biol. 38:79).
7-806, 1998).

[0005] RNR is an enzyme essential for cell division and proliferation. R in eukaryotic cells
Decreased NR activity results in cell death. This is because cells can no longer properly synthesize DNA. Thus, if the level of RNR activity can be reduced in developing anther cells, the cells will stop developing, leading to a male fertile phenotype. Or,
When RNR expression in cell culture is regulated by use of an inducible promoter,
Cell proliferation may be induced, thereby improving transformation. Methods such as these for modulating or altering the cell cycle have important implications in the production of new recombinantly engineered crops (eg, corn). The present invention provides this and other advantages.

SUMMARY OF THE INVENTION In general, it is an object of the present invention to provide nucleic acids and proteins related to the maize ribonucleotide reductase large subunit (referred to herein as R1). The objects of the present invention are as follows: 1) an antigenic fragment of the protein of the present invention; 2) a transgenic plant containing the nucleic acid of the present invention; 3) a method for regulating the expression of the nucleic acid of the present invention in the transgenic plant. , Is to provide.

[0007] Accordingly, in one aspect, the present invention relates to the following: (a) a polynucleotide having a specific sequence identity to a polynucleotide encoding a polypeptide of the present invention; A polynucleotide selected from the group consisting of: a polynucleotide complementary to a nucleotide; and (c) a polynucleotide comprising a specified number of contiguous nucleotides from the polynucleotides of (a) and (b). Related to nucleic acids. The isolated nucleic acid can be DNA.

[0008] In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid of the present invention operably linked to a promoter.

[0009] In another aspect, the present invention relates to a host cell into which a recombinant expression cassette has been introduced.

[0010] In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specific number of contiguous amino acids encoded by the isolated nucleic acids of the present invention.

In another aspect, the present invention provides an isolated nucleic acid comprising a polynucleotide of a specific length, or a nucleic acid thereof, that selectively hybridizes to a polynucleotide of the present invention under stringent hybridization conditions. For the complement. In some embodiments, the isolated nucleic acid is operably linked to a promoter.

In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid amplified from a library as mentioned above. Here, the nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to host cells transfected with the recombinant expression cassette. In some embodiments, the invention relates to a protein of the invention produced from this host cell.

[0013] In yet another aspect, the invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any isolated nucleic acid of the invention. The present invention also provides a transgenic seed from a transgenic plant.

Definitions Units, prefixes, and symbols may be displayed in their SI approved form. Unless otherwise indicated, nucleic acids, respectively, are written left to right in a 5 'to 3'orientation; amino acid sequences are written left to right in an amino to carboxy orientation. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids are represented by their commonly known three letter symbols or IU
PAC-IUB Biochemical Nomenclature Com
It may be referred to herein by either of the single letter symbols recommended by mission. Similarly, nucleotides may be referred to by their generally accepted one-letter code. The terms defined below are more fully defined by reference to the specification as a whole.

“Amplified” uses at least one nucleic acid sequence as a template,
The construction of multiple copies of a nucleic acid sequence or of multiple copies complementary to the nucleic acid sequence is meant. As the amplification system, a polymerase chain reaction (PCR) system, a ligase chain reaction (L
CR) system, nucleic acid sequence-based amplification (NASBA, Cangene, Missis)
sauga, Ontario), Q-Beta replicase system, transcription-based amplification system (TAS), and strand displacement amplification (SDA). For example, Diag
nostic Molecular Microbiology: Princi
ples and Applications, D.C. H. Ed. Persing et al.,
American Society for Microbiology, Wa
Shinton, D.M. C. (1993). The amplification products are called amplicons.

The term “antibody” includes reference to an antigen-binding form of the antibody (eg, Fab, F (ab) ₂ ). The term “antibody” often refers to a polypeptide substantially encoded by an immunoglobulin gene or a fragment thereof that specifically binds and recognizes an analyte (antigen). However, although various antibody fragments can be defined with respect to intact antibody digestion, those skilled in the art will recognize that such fragments can be newly generated either chemically or by utilizing recombinant DNA methodology. Recognize that they can be synthesized. Thus, as used herein, the term antibody also refers to single-chain Fv, chimeric antibodies (ie, including constant and variable regions from different species), humanized antibodies (ie, non-human sourced). A complementarity determining region (CDR) from a source) and a heteroconjugate antibody (eg,
Antibody fragments such as bispecific antibodies).

The term “antigen” includes reference to a substance that is capable of producing antibodies and / or for which the antibodies are specifically immunoreactive. That specific immunoreactive site within an antigen is known as an epitope or antigenic determinant. These epitopes may be linear arrays of monomers in a polymer composition, such as amino acids in a protein, or may consist of more complex secondary or tertiary structures, or may represent such structures. May be included. One of skill in the art recognizes that all immunogens (ie, substances that can elicit an immune response) are antigens; however, some antigens, such as haptens, are not immunogens but are conjugated to carrier molecules. Can be immunogenic. Antibodies immunologically reactive with a particular antigen can be produced in vivo or by recombinant methods such as selection of a library of recombinant antibodies in phage or similar vectors. See, for example, Huse et al., Science 246: 12.
75-1281 (1989); and Ward et al., Nature 341: 54.
4-546 (1989); and Vaughan et al., Nature Biote.
ch. 14: 309-314 (1996).

As used herein, “antisense orientation” includes reference to a duplex polynucleotide sequence operably linked to a promoter in the orientation in which the antisense strand is transcribed. The antisense strand is sufficiently complementary to the endogenous transcript so that translation of the endogenous transcript is often inhibited.

As used herein, “chromosomal region” includes a reference to the length of a chromosome that can be measured with reference to the linear segment of DNA that it contains. The chromosomal region can be defined with reference to two unique DNA sequences, namely markers.

The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or conservatively modified variant of this amino acid sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed without altering the encoded polypeptide.
It can be changed to any of the corresponding codons described. Such nucleic acid variations are "silent variations," and represent a type of conservatively modified variation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid by reference to the genetic code. One skilled in the art will recognize that each codon in the nucleic acid (except AUG, which is usually the only codon for methionine; and UGG, which is usually the only codon for tryptophan) is modified to obtain a functionally identical molecule. Recognize that it can be done. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention refers to each described polypeptide sequence and is within the scope of the present invention.

With respect to amino acid sequences, one skilled in the art will recognize that one or a small percentage of amino acids in the encoded sequence may be altered, added, or deleted, to a nucleic acid, peptide, polypeptide, or protein sequence. An individual substitution, deletion, or addition is considered to be a "conservatively modified variant" if the change results in the replacement of the amino acid with a chemically similar amino acid. Therefore,
Any number of amino acid residues selected from the group of integers consisting of 1-15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 changes may be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which the variant was derived. For example, substrate specificity, enzymatic activity, or ligand / receptor binding is generally at least 30%, 40%, 50%, 60% of the native protein relative to the native substrate.
%, 70%, 80%, or 90%. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
And 6) phenylalanine (F), tyrosine (Y), tryptophan (W). Creighton (1984) Proteins W.C. H. Freeman
See also and Company.

For a specified nucleic acid, “encoding” or “encoded” means including the information for translation into the specified protein. A nucleic acid encoding a protein may include untranslated sequences (eg, introns) in the translated region of the nucleic acid, or may lack such intervening untranslated sequences (eg, as in a cDNA). The information that encodes the protein is
Specified by the use of codons. Typically, the amino acid sequence is “universal”
It is encoded by a nucleic acid using the genetic code. However, some plants, animals, and fungal mitochondria, the bacterium Mycoplasma capricolu
m, or variants of the universal code as present in the ciliate Macronucleus, can be used when expressing nucleic acids in these organisms.

When preparing or altering a nucleic acid synthetically, one can take advantage of the known codon preferences of the host that intends to express the nucleic acid. For example, the nucleic acid sequences of the invention can be expressed in both monocotyledonous and dicotyledonous species, but have been shown to have different priorities, so that the sequences are specific to monocotyledonous or dicotyledonous plants. Codon priority and G
C content can be modified to account for priority (Murray et al., Nucl. Aci.
ds Res. 17: 477-498 (1989)). Thus, the codon preferred by corn for a particular amino acid may be derived from a known gene sequence derived from corn. Maize codon usage for 28 genes from corn plants is listed in Murray et al., Supra, Table 4.

As used herein, “full-length sequence” in reference to a particular polynucleotide or its encoded protein refers to the native (non-synthetic), endogenous, catalytically active form of the particular protein. In all forms. Methods for determining whether a sequence is full length are well known in the art, and exemplary techniques include Northern or Western blot, primer extension, S1 protection, and ribonuclease protection. For example, Plant Molecular Biology: A Laboratory
ry Manual, Clark ed., Springer-Verlag, Ber
lin (1997). Known full-length homology (orthogas)
Comparison of logous and / or paralogous) sequences can also be used to identify full length sequences of the invention. In addition, consensus sequences, typically present in the 5 'and 3' untranslated regions of the mRNA, aid in identifying the polynucleotide as full length. For example, consensus sequence, ANN
NN AUG G (where the underlined codon represents the N-terminal methionine)
Assists in determining whether a polynucleotide has a complete 5 'end.
A consensus sequence at the 3 ′ end (eg, a polyadenylation sequence) helps to determine whether a polynucleotide has a complete 3 ′ end.

As used herein, “heterologous” in reference to a nucleic acid may be a nucleic acid derived from a foreign species, or, if derived from the same species, by deliberate human intervention.
A nucleic acid that is substantially modified from its native form and / or locus in a comparison. For example, a promoter operably linked to a heterologous structural gene is from a different species than the species from which the structural gene is derived, or, if from the same species, one or both are in their original form. Has been substantially modified. A heterologous protein can be derived from an exotic species, or, if derived from the same species, has been substantially modified from its original form by deliberate human intervention.

“Host cell” means a cell that contains a vector and supports the replication and / or expression of the expression vector. The host cell is E. coli. It can be a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as a yeast cell, insect cell, amphibian cell, or mammalian cell. Preferably, the host cell is a monocot or dicot plant cell. Particularly preferred monocot host cells are corn host cells.

The term “hybridization complex” includes reference to a double-stranded nucleic acid structure formed by two single-stranded nucleic acid sequences that hybridize selectively to one another.

An “immunologically reactive condition” or “immunoreactive condition” is defined as an antibody reactive to a particular epitope, wherein the antibody is substantially reduced in a reaction mixture containing the particular epitope. Conditions that are capable of binding the epitope to a detectably greater extent (eg, at least twice background) than binding to any other epitope. The conditions of immunological reactivity will depend on the type of antibody binding reaction, and will typically be those utilized in immunoassay protocols. For a description of immunoassay formats and conditions, see Harlow and Lan.
e, Antibodies, A Laboratory Manual, Col
d Spring Harbor Publications, New York
k (1988).

The term “introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction”, and eukaryotic or prokaryotic cells of the nucleic acid Includes references to incorporation into Here, the nucleic acid can be incorporated into the genome of the cell (eg, chromosome, plasmid, plastid or mitochondrial DNA) and converted to an autonomously replicating replicon or can be transiently expressed (eg, transfected). MRNA).

The term “isolated” refers to a substance such as a nucleic acid or a protein,
These substances are: (1) substantially or essentially free of components that normally accompany or interact with it, as found in its naturally occurring environment. Does the isolated material optionally include a material that is not found with the material in its natural environment; or (2) if the material is in its natural environment, Is synthetically (non-naturally) converted to a composition by the intervention of and / or placed at a locus in the cell that is not native for a substance found in its environment (eg, a genomic or intracellular organelle). You. This alteration to produce the synthetic material can be performed within the natural state or in a material that has been removed from the natural state. For example, naturally occurring nucleic acids can be modified by artificial intervention performed in the cell from which they are derived.
An isolated nucleic acid when altered or transcribed from altered DNA. For example, Compounds and Methods for
Site Directed Mutagenesis in Eukaryo
tic Cells, Kmiec, U.S. Patent No. 5,565,350; In V
Ivo Homelogous Sequence Targeting in
Eukaryotic Cells; Zarling et al., PCT / US93 /
See 03868. Similarly, a naturally-occurring nucleic acid (eg, a promoter) has been isolated when introduced by non-naturally occurring means into a genomic locus that is not native to the nucleic acid. An "isolated" nucleic acid as defined herein is also referred to as a "heterologous" nucleic acid.

Unless otherwise indicated, the term “R1 nucleic acid” is a nucleic acid of the invention and refers to a nucleic acid that includes a polynucleotide of the invention that encodes an R1 polypeptide (“R1 polynucleotide”). "R1 gene" is a gene of the present invention and refers to a heterologous genomic form of a full-length R1 polynucleotide.

As used herein, with respect to a particular marker, “defined by and localized within a chromosomal region that includes” is defined by the indicated marker, and the marker Includes references to contiguous lengths of the containing chromosome.

As used herein, “marker” includes reference to a location on a chromosome that serves to identify a unique location on the chromosome. "Polymorphic markers" occur in different forms (alleles) such that when different forms of a marker are present in a homologous pair, the marker allows the continuation of each of the chromosomes of the pair to continue. Includes references to markers. Genotype may be defined by the use of one or more markers.

As used herein, “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single-stranded or double-stranded form, and unless otherwise limited. Include known analogs that possess the essential properties of naturally occurring nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (eg, peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNA molecules that comprises and substantially represents the entire transcribed fraction of the genome of a designated organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, are described in Berger and Kimmel, Guide to
Molecular Cloning Technologies, Methods
in Enzymology, Volume 152, Academic Press,
Inc. , San Diego, CA (Berger); Sambrook et al.,
Molecular Cloning-A Laboratory Manua
1, Second Edition, Volumes 1-3 (1989); and Current Proto
cols in Molecular Biology, F.C. M. Ausube
ed., Current Protocols, Greene Publish
ing Associates, Inc. And John Wiley & Son
s, Inc. And taught in standard molecular biology references, such as the Merging Company (1994).

As used herein, “operably linked” includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence is Initiates and mediates the transcription of a DNA sequence corresponding to the second sequence. Operably linked generally means that the linked nucleic acid sequences are contiguous and, if necessary to join two protein regions, contiguous and in the same reading frame. Means

[0038] As used herein, the term "plant" includes reference to whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds, and plant cells, their progeny. As used herein, plant cells include seeds, culture suspensions, embryos, meristem regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Are not limited to these. In general, the classes of plants that can be used in the methods of the present invention are the broadest class of higher plants that can be subjected to transformation techniques, including monocots and dicots. Particularly preferred plants are Zea ma
ys.

As used herein, “polynucleotide” refers to a deoxyribopolynucleotide, ribopolynucleotide, or a string of nucleotides that is substantially the same as a naturally occurring nucleotide under stringent hybridization conditions. Includes references to those analogs that retain the essential properties of natural ribonucleotides in that they hybridize to and / or allow translation into the same amino acid as the naturally occurring nucleotide. A polynucleotide can be full length or a partial sequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specific sequence and its complement. Thus, DNA or RNA having a backbone that has been modified for stability or for other reasons is a "polynucleotide" as the term is intended herein. Furthermore, DNA or RNA containing an unusual base such as inosine or a modified base such as a tritylated base, to name just two examples,
The term is a polynucleotide as used herein. It will be appreciated that a wide variety of modifications have been made to DNA and RNA that serve a number of useful purposes known to those of skill in the art. The term polynucleotide as used herein refers to such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as viruses and cells, especially simple and complex cells. And DNA and RNA in a specific chemical form.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. This term is 1
One or more amino acid residues apply to amino acid polymers that are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The essential property of such naturally occurring amino acid analogs is that when incorporated into a protein, the protein is raised against a protein consisting of the same but all naturally occurring amino acids Is specifically reactive with The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to: glycosylation,
Lipid binding, sulphation, γ-carboxylation of glutamate residues, hydroxylation and ADP-ribosylation. As is well known and described above, it is recognized that polypeptides are not necessarily perfectly linear. For example, polypeptides can diverge as a result of ubiquitination, and polypeptides generally undergo post-translational events, including natural processing events and events caused by artificial manipulations that do not occur in nature. As a result, it may be cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides can also be synthesized by non-translated natural processes and fully synthetic methods. Furthermore, the use of both methionine-containing and methionine-free amino-terminal variants of the proteins of the invention is contemplated.

As used herein, “promoter” refers to a region of DNA that is upstream from the start of transcription and that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Including. A "plant promoter" is a promoter capable of initiating transcription in a plant cell, whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, plant promoters obtained from: plants, plant viruses, and bacteria containing genes expressed in plant cells, such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in a particular tissue (eg, leaf, root, or seed). Such a promoter is referred to as "tissue-preferred." Promoters that initiate transcription only in a particular tissue are referred to as "tissue-specific". A “cell type” -specific promoter drives mainly expression in a particular cell type in one or more organs (eg, root or leaf vascular cells). An “inducible” or “repressible” promoter is a promoter that is under environmental control. Examples of environmental conditions that can affect transcription by an inducible promoter include anaerobic conditions or the presence of light. Tissue specific promoters, tissue preferred promoters, cell type specific promoters and inducible promoters constitute a class of "non-constitutive" promoters. A "constitutive" promoter is a promoter that is active under most environmental conditions.

The term “R1 polypeptide” is a polypeptide of the invention and refers to one or more amino acid sequences in glycosylated or non-glycosylated form. The term also includes fragments, variants, homologs, alleles or precursors thereof (eg, preproprotein or proprotein). "R1 protein" is a protein of the invention and encompasses R1 polypeptides.

As used herein, “recombinant” includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the same form in a cell in its native (non-recombinant) form, or is otherwise aberrantly or under-expressed Alternatively, a native gene that is not expressed at all is expressed as a result of intentional artificial intervention. As used herein, the term “recombinant” refers to a naturally occurring event (eg, spontaneous mutation, spontaneous transformation / transduction / transposition), such as a change that occurs without intentional human intervention. Does not include cell or vector changes due to

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

The term “residue” or “amino acid residue” or “amino acid” is used herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). Is used interchangeably. Amino acids can be naturally occurring amino acids and include, unless otherwise limited, non-natural analogs of naturally occurring amino acids that can function in a manner similar to the naturally occurring amino acids.

The term “selectively hybridizes” to a degree that is detectably greater than hybridization to a non-target nucleic acid sequence under stringent hybridization conditions (eg, at least twice background). And references to the hybridization of nucleic acid sequences to designated nucleic acid target sequences and the substantial elimination of non-target nucleic acids. Selectively hybridizing sequences typically have at least about 80% sequence identity to each other, preferably 90% sequence identity, and most preferably 100% sequence identity (ie, complementary).

The term “specific reactivity” includes reference to the binding reaction between an antibody and a protein having an epitope recognized by the antigen binding site of the antibody. This binding reaction is a determinant of the presence of the protein with the recognized epitope in the presence of a heterogeneous population of proteins and other biologicals. Thus, under designated immunoassay conditions, the designated antibodies will have a substantially greater degree (e.g., back-up) than for substantially all other analytes lacking the epitope present in the sample. (At least twice above ground).

[0048] Specific binding to an antibody under such conditions may require an antibody that is selected for specificity for a particular protein. For example, antibodies raised against a polypeptide of the invention can be selected to obtain antibodies that specifically react with the polypeptide of the invention. Proteins used as immunogens can be in native conformation or can be modified to give linear epitopes.

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

The term “stringent conditions” or “stringent hybridization conditions” refers to a probe that binds its target sequence to a detectably greater extent than other sequences (eg, at least twice background). And references to conditions that hybridize to Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and / or wash conditions,
Target sequences that are 00% complementary can be identified (homology probing). Alternatively, stringent conditions can be adjusted to tolerate some mismatches in the sequence, thus detecting a lower degree of similarity (heterologous probing). Generally, probes are less than about 1000 nucleotides in length, and optionally less than 500 nucleotides in length.

[0051] Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01-1.0 M Na ion concentration (or other salt).
PH is 7.0-8.3 and temperature is short probe (eg, 10-50
Nucleotides) and at least about 60 ° C. for long probes (eg, more than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary conditions of low stringency include hybridization at 37 ° C. in a buffer solution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and 1 × to 50 ° -55 ° C.
Washing in 2 × SSC (20 × SSC = 3.0 M NaCl / 0.3 M trisodium citrate). Exemplary conditions of moderate stringency include hybridization at 37 ° C. in 40-45% formamide, 1 M NaCl, 1% SDS, and 0.5 × -1 × S at 55-60 ° C.
Washing in SC. Exemplary conditions of high stringency include:
Hybridization at 37 ° C. in 50% formamide, 1 M NaCl, 1% SDS, and washing in 0.1 × SSC at 60-65 ° C.

[0052] Specificity is typically a function of post-hybridization washes, the key factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T _m is determined by Meinkoth and Wahl, Anal. Bioc
hem. , 138: 267-284 (1984), which equation is: _Tm = 81.5 C + 16.6 (log M) + 0.41 (% GC)-0.6.
1 (% form) -500 / L; where M is the molar concentration of the monovalent cation,% GC is the percentage of guanosine nucleotides and cytosine nucleotides in DNA, and% form is the percentage in the hybridization solution. Is the percentage of formamide, and L is the length of the hybrid in base pairs. _Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. T _m is reduced by about 1 ° C. per 1% mismatch; thus, T _m , hybridization and / or washing 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 reduced by 10 ° C. Generally, stringent conditions are defined ionic strength and p
H for the specific sequence and its complement
It is selected to be about 5 ° C. less than the lting point (T _m ). However, severely stringent conditions may utilize hybridization and / or washes 1, 2, 3 or 4 ° C. below the thermal melting point (T _m ), while moderately stringent conditions may utilize a thermal melting point (T _m ). obtained using from 6, 7, 8, 9 or 10 ° C. lower hybridization and / or washing, _m), low stringency conditions, the thermal melting point (T _m) than 11,12,13,14,15 or 20 Hybridization and / or washing at <RTIgt; 0 C </ RTI> may be utilized. Using this equation, the hybridization and wash composition, and the desired T _m , one skilled in the art can:
It is understood that variations in the stringency of hybridization and / or wash solutions are essentially described. The desired degree of mismatch is 4
If it results in a T _m below 5 ° C. (aqueous solution) or 32 ° C. (formamide solution), it is preferable to increase the SSC concentration, so that higher temperatures can be used.
An extensive guide to nucleic acid hybridization is found at: Ti
jssen, Laboratory Technologies in Bioch
emistry and Molecular Biology-Hybrid
Ization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principals of hybrid."
Diversion and the strategy of nuclear
acid probe assays ", Elsevier, New Yo
rk (1993); and Current Protocols in Mol.
ecology Biology, Chapter 2, Ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, Ne
w York (1995).

As used herein, “transgenic plant” includes reference to a plant that contains a heterologous polynucleotide in its genome. Generally, a heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is passed on to successive generations. Heterologous polynucleotides can 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 whose genotype has been altered by the presence of a heterologous nucleic acid. Transgenics include those transgenics that have been so modified initially, and those that have been produced by sexual crossing or asexual reproduction from the original transgenics. As used herein, the term “transgenic” refers to conventional plant breeding methods or naturally occurring events (eg, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transfer). Or genomic (chromosomal or extrachromosomal) alterations due to natural or natural mutations).

As used herein, “vector” includes a reference to a nucleic acid used to transfect a host cell into which a polynucleotide can be inserted. Vectors are often replicons. The expression vector allows transcription of the nucleic acid inserted into the expression vector.

The following terms are used to describe the sequence relationship between two or more nucleic acids or polypeptides: (a) a “reference sequence”, (b) a “comparison window”, (c) “ "Sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".

(A) As used herein, a “reference sequence” is a defined sequence used as a basis for a sequence comparison. A reference sequence can be a subset or the whole of a specified sequence: for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence.

(B) As used herein, a “comparison window” includes reference to a contiguous and designated segment of a polynucleotide / polypeptide sequence, where the polynucleotide / polypeptide is The sequence can be compared to a reference sequence, where the portion of the polynucleotide / polypeptide sequence in the comparison window is the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. May include additions or deletions (ie, gaps) as compared to Generally, the comparison window is at least 20 contiguous nucleotides / amino acid residues in length, and can be 30, 40, 50, 100 or longer as needed. One skilled in the art will appreciate that by including gaps in the polynucleotide / polypeptide sequence,
It is understood that gap penalties are typically introduced and subtracted from the number of matches to avoid high similarity to the reference sequence.

Methods for alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison is based on Smith and Waterma.
n, Adv. Appl. Math. 2: 482 (1981), by the local homology algorithm; Needleman and Wunsch, J .; Mol. Bi
ol. 48: 443 (1970), by the homology alignment algorithm; Pearson and Lipman, Proc. Natl. Acad. Sci
. 85: 2444 (1988); can be performed by computerized implementation of the algorithm, including, but not limited to, the following: Intelligenetics, Mountain V
CLUST in PC / Gene program by IEW, California
AL; Wisconsin Genetics Software Packa
ge, Genetics Computer Group (GCG), 575
Science Dr. , Madison, Wisconsin, USA GA
P, BESTFIT, BLAST, FASTA, and TFASTA; CLUS
The TAL program is described in Higgins and Sharp, Gene 73:23.
7-244 (1988); Higgins and Sharp, CABIOS 5
: 151-153 (1989); Corpet et al., Nucleic Acids.
Research. 16: 10881-90 (1988); Huang et al., C.
omputa Applications in the Bioscience
ces 8: 155-65 (1992), and Pearson et al., Meth.
ods in Molecular Biology 24: 307-331 (
1994).

The BLAST family of programs that can be used for database similarity searches include: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; proteins BLASTP for protein query sequences against database sequences; TBLASTN for protein query sequences against nucleotide database sequences
And TBLASTX for nucleotide query sequences against nucleotide database sequences. Current Protocols in Molecu
lar Biology, Chapter 19, Ausubel et al., Greene Pu
blushing and Wiley-Interscience, New
See York (1995).

Software for performing BLAST analysis is publicly available (eg, National Center for Biotechnology).
Information (http: //www.ncbi.nlm.nih
. gov /)). The algorithm involves first identifying a high-scoring sequence pair (HSP) by identifying short words of length W in the query sequence, which aligns with words of the same length in the database sequence. If so, it either matches or satisfies some positive threshold score T. T is referred to as the neighborhood word score threshold. These starting neighbor word hits act as seeds for initiating searches to find longer HSPs containing words. The word hits are then extended in both directions along each sequence, as long as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameter M (reward for a pair of matched residues) for nucleotide sequences.
Score; always> 0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the scoring matrix (sco
(ring matrix) is used to calculate the cumulative score. The expansion of word hits in each direction is stopped if: the cumulative alignment score decreases by X amount from its maximum attainment; the cumulative score becomes zero due to the accumulation of one or more negative scoring residue alignments. When: or when the end of either sequence is reached. BLAST algorithm parameters W, T, and X
Determines the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) has 11 word lengths (W), 10 expected values (E), 1
A cutoff of 00, M = 5, N = -4, and a comparison of both strands is used as default. For amino acid sequences, the BLASTP program has a word length of 3 (W)
, An expected value (E) of 10, and a BLOSUM62 scoring matrix as defaults (Henikoff and Henikoff (1989) Proc.
Natl. Acad. Sci. ScL USA 89: 10915).

In addition to calculating percent sequence identity, the BLAST algorithm
Statistical analysis of similarity between the two sequences is also performed (eg, Karlin and Al)
tschul, Proc. Nat'l. Acad. Sci. USA 90:58
73-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which is 2
It gives an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance.

[0062] BLAST searches assume that proteins can be modeled as random sequences. However, many actual proteins contain regions of non-random sequence,
This can be an area of homopolymer, a short repeat, or a region rich in one or more amino acids. Such low complexity regions can be aligned between unrelated proteins, even if other regions of the protein are quite different. Many low complexity filter programs can be used to reduce such low complexity alignments. For example, SEG (Woten and Federhen,
Comput. Chem. , 17: 149-163 (1993)) and XNU.
(Claverie and States, Comput. Chem., 17: 1.
91-201 (1993) low complexity filters can be used alone or in combination.

GAP can also be used to compare a reference sequence to a polynucleotide or polypeptide of the invention. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-435, 1970) to find an alignment of the two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap locations and creates an alignment with the largest number of matching bases and the smallest gap. This allows for the provision of gap creation and gap extension penalties on a matching base basis. The GAP must benefit from the number of matching gap creation penalties for each gap it inserts. If a gap extension penalty greater than zero is selected, then G
The AP must benefit for each inserted gap of (gap length) * (gap extension penalty). The default values for the gap creation penalty and the gap extension penalty are the Wisconsin Genetics Software Package for protein sequences.
In the case of version 10 (ge), the values are 8 and 2, respectively. For nucleotide sequences, the default for the gap extension penalty is 3, while the default for the gap creation penalty is 50. The gap creation penalty and gap extension penalty can be expressed as an integer selected from the group of integers consisting of 0-200. Thus, for example, the gap creation penalty and the gap extension penalty are each independently 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 65 or more.

GAP represents one member of the best aligned family. There can be many members in this family, but none of the other members have good quality. GAP displays four figures of merit for alignment: quality, proportion, identity, and similarity. Quality is a maximized metric for aligning sequences. The ratio is the quality divided by the number of bases in the shorter segment. Percent identity is the percentage of symbols that actually matched. Percent similarity is the percentage of similar symbols. Symbols across the gap are ignored. The similarity is a score matrix value for a pair of symbols.
If it is 50 or more, it will be scored. Wisconsin Genetics
The scoring matrix used in version 10 of the Software Package is BLOSUM62 (Henikoff and Henikoff (1
989) Proc. Natl. Acad. Sci. ScL USA 89: 10915).

Unless otherwise specified, sequence identity / similarity values given herein are based on the BLAST 2.0 program package (Alt) using default parameters.
Schul et al., Nucleic Acids Res. 25: 3389-340
2, 1997; Altschul et al. Mol. Bio. 215: 403-4
10, 1990) or a GAP program (Wisconsin Genetics Software) using default parameters.
Package, Genetics Computer Group (GCG
), 575 Science Dr. , Madison, Wisconsin,
USA (see USA).

(C) As used herein, in the context of the sequence of two nucleic acids or polypeptides, “sequence identity” or “identity” will give the greatest match for a designated comparison window. And references to residues in the two sequences that are identical when aligned. When the percentage of sequence identity is used in connection with proteins, it is recognized that the positions of non-identical residues often differ by conservative amino acid substitutions, wherein the amino acid residues have similar chemical properties (eg, , Charged or hydrophobic) and therefore does not change the functional properties of the molecule. If the sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are referred to as “sequence similarity” or “sequence similarity”.
Similarity ". Means for making this adjustment are well-known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial rather than total mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if the same amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution will be given a score between zero and one. Scoring of conservative substitutions is described, for example, in Meyers and Mill.
er, Computer Applic. Biol. Sci. , 4: 11-17
(1988), for example, PC / GENE (Intelli)
Genetics, Mountain View, California, US
A) Calculated to be executed in the program.

(D) As used herein, “percentage of sequence identity” means a value determined by comparing two optimally aligned sequences over a comparison window, where The portion of the polynucleotide sequence in the window may include additions or deletions (ie, gaps) relative to a reference sequence, which does not include additions or deletions, for optimal alignment of the two sequences. . The percentage here determines the number of positions where the same nucleobase or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, It is then calculated by multiplying the result by 100 to get the sequence identity percentage.

(E) (i) The term “substantial identity” of a polynucleotide sequence refers to a polynucleotide that has been identified using one of the described alignment programs using standard parameters. It is meant to include sequences that have at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% as compared to the sequence. One of skill in the art would consider codon degeneracy, amino acid similarity, reading frame positioning, etc.
It will be appreciated that these values can be adjusted appropriately to determine the corresponding identity of the proteins encoded by the two nucleotide sequences. For these purposes, substantial identity of the amino acid sequences usually means at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95% sequence identity. .

Another indicator that the nucleotide sequences are substantially identical is whether the two molecules hybridize to each other under stringent conditions. However, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when a copy of the nucleic acid is made using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is
That the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.

(E) (ii) The term “substantial identity” in the context of a peptide means that the peptide has at least 70% sequence identity to the reference sequence for the specified comparison window, preferably 80 %, More preferably 85%, most preferably at least 90% or 95% sequence identity. If necessary, optimal alignments are determined by Needleman and Wuns.
ch. Mol. Biol. 48: 443 (1970). An indication that the two peptide sequences are substantially the same is that the first peptide is immunologically reactive with antibodies raised against the second peptide. Thus, for example, where two peptides differ only by conservative substitutions, the peptides are substantially identical to the second peptide. Peptides that are "substantially similar" share a sequence as described above, except that the positions of non-identical residues may differ due to conservative amino acid changes.

DETAILED DESCRIPTION OF THE INVENTION Overview The present invention provides, inter alia, compositions and methods for modulating (ie, increasing or decreasing) the level of a polynucleotide or polypeptide of the present invention in plants. I do. In particular, the polypeptides of the present invention may be expressed temporally or spatially in developmental stages, tissues, and / or amounts that are not characteristic of non-recombinantly engineered plants. Thus, the present invention provides utility in exemplary applications, such as regulating male sterility by down-regulating expression and increasing transformation efficiency through cell cycle regulation.

The invention also includes a polynucleotide that is sufficiently long and complementary to a gene of the invention for use as a probe or amplification primer in the detection, quantification, or isolation of gene transcripts. An isolated nucleic acid is provided. For example, the isolated nucleic acids of the present invention can be used to detect mutations (eg, substitutions, deletions, or additions) in genes as probes in detecting deficiencies in mRNA levels in screening for desired transgenic plants. To monitor up-regulation or changes in the expression of enzyme activity in compound screening assays, to detect any number of allelic variants (polymorphisms) of a gene, or in eukaryotic cells. Site-directed mutagenesis (eg, US Pat. No. 5,565,565)
No. 350). The isolated nucleic acids of the invention can also be used for recombinant expression of their encoded polypeptides or for use as immunogens in antibody preparation and / or screening. The isolated nucleic acids of the invention can also be used for the use of sense suppression or antisense suppression of one or more genes of the invention in a host cell, tissue, or plant. Attachment of chemical agents that bind, insert, cleave, and / or crosslink to the isolated nucleic acids of the present invention can also be used to regulate transcription or translation.

The present invention also provides an isolated protein comprising a polypeptide of the invention (eg, a preproenzyme, proenzyme, or enzyme). The present invention also provides a protein comprising at least one epitope derived from the polypeptide of the present invention. The proteins of the invention can be used in assays for enzyme agonists or antagonists of enzyme function, or for use as immunogens or antigens to obtain antibodies specifically immunoreactive with the proteins of the invention. Such antibodies can be used for the identification and / or isolation of a nucleic acid of the invention from an expression library in an expression level assay, or for purification of a polypeptide of the invention.

[0074] The isolated nucleic acids and proteins of the present invention include, among others, Hordeum, Sec.
are, Triticum, Sorghum (eg, S. bicolor),
In monocotyledonous plants, such as species of the Gramineae family, including Zea and Zea (eg, Z. mays), they can be used across a wide range of plant types. The isolated nucleic acids and proteins of the present invention can also be used for species from the following genera: Cuc
urbita, Rosa, Vitis, Juglans, Fragaria, L
otus, Medicago, Onobrychis, Trifolium, T
rigonella, Vigna, Citrus, Linum, Geraniu
m, Manihot, Daucus, Arabidopsis, Brassic
a, Raphanus, Sinapis, Atropa, Capsicum, D
atura, Hyoscyamus, Lycopersicon, Nicoti
ana, Solanum, Petunia, Digitalis, Majora
na, Ciahorium, Helianthus, Lactuca, Brom
us, Asparagus, Antirrhinum, Heterocalli
s, Nemesis, Pelargonium, Panieum, Pennis
etum, Rununculus, Senecio, Salpilossis
, Cucumis, Browaria, Glycine, Pisum, Pha
seolus, Lolium, Oryza, and Avena.

(Nucleic Acid) The present invention provides, inter alia, RNA, DNA and their analogs and / or chimeric isolated nucleic acids, including the polynucleotides of the present invention.

The polynucleotides of the present invention include: (a) polynucleotides encoding the polypeptide of SEQ ID NO: 2, conservatively modified variants and polymorphic variants thereof (exemplary sequences of SEQ ID NO: 1) Polynucleotide sequences of the present invention also include American Type
The corn R1 polynucleotide sequence as contained in a plasmid deposited with the Culture Collection (ATCC) and assigned accession number PTA-831; A polynucleotide that is an amplification product from a Zea mays nucleic acid library using a primer pair that selectively hybridizes under stringent conditions to a locus in the polynucleotide selected from the sequences included; (C) a polynucleotide that selectively hybridizes to the polynucleotide of (a) or (b); (d) a specified sequence identity with the polynucleotide of (a), (b), or (c); (E) protota A polynucleotide encoding a protein having a specified number of contiguous amino acids from a polypeptide, wherein the protein is specifically recognized by an antiserum elicited by display of the protein, and A polynucleotide that does not detectably immunoreact with antisera fully immunosorbed to the protein; (f) (a), (b), (c), (d) or (e) And (g) at least a specified number of contiguous nucleotides from the polynucleotide of (a), (b), (c), (d), (e) or (f). , Polynucleotide.

[0077] The polynucleotide of SEQ ID NO: 1 is an American Type Culture.
reCollection (ATCC) on Oct. 8, 1999 and is contained in a plasmid assigned accession number PTA-831. Am
erican Type Culture Collection is 1080
1 University Blvd. , Manassas, VA 20110
-2209.

The ATCC deposit is maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. This deposit is provided as a convenience to those of ordinary skill in the art and is not an admission that a deposit is required under 35 USC 112. The deposited sequence, and the polypeptide encoded by the sequence,
Any inconsistencies, such as sequencing errors, are incorporated herein by reference and are controlled as described herein.

(A. Polynucleotide Encoding Polypeptide of the Present Invention, or Conservatively Modified or Polymorphic Variant) As described in (a) above, the present invention relates to the present invention. Provided herein, wherein the polynucleotide encodes a polypeptide of the invention, or a conservatively modified or polymorphic variant thereof. Accordingly, the present invention includes the polynucleotides of SEQ ID NO: 1, as well as sequences as included in ATCC Deposit Assignment Accession Number PTA-831, as well as silent variations of the polynucleotides encoding the polypeptide of SEQ ID NO: 2. The invention further provides an isolated nucleic acid comprising a polynucleotide encoding a conservatively modified variant of the polypeptide of SEQ ID NO: 2. Conservatively modified variants can be used to generate or select antibodies immunoreactive with the non-variant polypeptide. In addition, the present invention further provides one or more alleles (
(Polymorphic) An isolated nucleic acid comprising a polynucleotide encoding a variant polypeptide / polynucleotide is provided. Polymorphic variants are frequently used, for example, to track segregation of chromosomal regions in marker-assisted selection methods for crop improvement.

(B. Polynucleotide Amplified from Zea mays Nucleic Acid Library) As shown in (b) above, the present invention provides an isolated nucleic acid comprising the polynucleotide of the present invention. This polynucleotide is amplified from a Zea mays nucleic acid library. Zea mays strain B73, PHRE1, A
632, BMS-P2 # 10, W23, and Mo17 are known and publicly available. Another publicly known and available corn line is the Maize Genetics Cooperation (Urbana, Id.)
L). The nucleic acid library can 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 presentation of relatively rare cDNAs. In any of the embodiments, the cDNA library is constructed using a full-length cDNA synthesis method. An example of such a method is Oligo-Capping (
Maruyama, K .; And Sugano, S .; Gene 138: 171-
174, 1994), a biotinylated CAP trapper (Biotinylated).
CAP Trapper) (Carninci, P., Kvan, C. et al., G.
Enomics 37: 327-336, 1996), and the CAP retention procedure (
CAP Retention Procedure) (Edery, E., Ch.
u, L. L. Et al., Molecular and Cellular Biol
15: 3363-3371, 1995). cDNA synthesis is often catalyzed at 50 ° C to 55 ° C to prevent the formation of RNA secondary structure. An example of a reverse transcriptase that is relatively stable at these temperatures is SuperSc
listII Reverse Transcriptase (Life Te
channels, Inc. ), AMV Reverse Transcr
iptase (Boehringer Mannheim), and Retro
Amp Reverse Transcriptase (Epicentre)
It is. Rapidly growing tissues or rapidly dividing cells are preferably mRN
Used as A source.

The present invention also provides partial sequences of the polynucleotide of the present invention. The various subsequences may be present at at least two sites in the polynucleotide of the invention, or at two sites in the nucleic acid adjacent to and including the polynucleotide of the invention, or at one site in the polynucleotide of the invention. It can be obtained using primers that selectively hybridize under stringent conditions to one site and one site in a nucleic acid comprising a polynucleotide of the invention. Primers are selected to selectively hybridize under stringent hybridization conditions to the polynucleotides of the invention. Generally, a primer is complementary to a subsequence of the target nucleic acid to be amplified. As the skilled artisan will appreciate, the sites at which the primer pairs selectively hybridize are selected such that a single continuous nucleic acid can be formed under the desired amplification conditions.

In any embodiment, the primer encodes a carboxy-terminal or amino-terminal amino acid residue of the polynucleotide of the present invention (ie, a region encoding the 3 ′ end and a region encoding the 5 ′ end, respectively). It is constructed to selectively hybridize under stringent conditions to a sequence (or its complement) in the target nucleic acid that contains the codons of interest. Optionally, in these embodiments, the primers are constructed to selectively hybridize to the entire coding region of the target polynucleotide of the invention such that the amplification product of the cDNA target consists of the coding region of the cDNA. The nucleotide length of the primer should be at least 15 to 50
Selected from the group of integers consisting of Therefore, the primer should have at least 15
, 18, 20, 25, 30, 40, or 50 nucleotides in length. One skilled in the art will recognize that the extended primer sequence may have a binding specificity (ie,
(Annealing). 5 'of the primer
Non-annealing sequences at the ends ("tails") can be added, for example, to introduce a cloning site at the end of the amplicon.

The amplification products can be translated using expression systems well known to those of skill in the art and as described below. The resulting translation product can be, for example, assayed for appropriate catalytic activity (eg, specific activity and / or substrate specificity), or by one or more linear sequences specific for a polypeptide of the invention. The polypeptide of the present invention can be confirmed by confirming the presence of the epitope. Methods for protein synthesis from PCR-derived templates are known in the art and are commercially available. See, for example, Amersham Life Sciences, Inc. of'
See catalog, p. 354, 1997.

[0084] Methods for obtaining the 5 'and / or 3' ends of a vector insert are well known in the art. For example, Frohman, M .; A. , PCR Pro
tocols: A Guide to Methods and Applic
ations, M .; A. Innis, D .; H. Gelfand, J .; J. Sni
nsky, T .; J. White (Academic Press, Inc.,
RACE (Rapid Amplification of Complete) as described in San Diego, 1990), pages 28-38 (1990).
Mentary Ends); U.S. Patent No. 5,470,722 and Current Protocols in Molecular Bio.
, Unit 15.6, Ausubel et al., Greene Publ.
ising and Wiley-Interscience, New Yo
rk (1995); Frohman and Martin, Techniques.
1: 165 (1989).

(C. Polynucleotide that selectively hybridizes to the polynucleotide of (A) or (B)) As shown in (c) above, the present invention relates to the isolation comprising the polynucleotide of the present invention. Provided herein, wherein the polynucleotide selectively hybridizes under selective hybridization conditions to the polynucleotide of paragraph (A) or (B), as discussed above. Thus, the polynucleotide of this embodiment can be used to isolate, detect, and / or quantify a nucleic acid comprising the polynucleotide of (A) or (B). For example, the polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotide is a genomic or cDNA sequence isolated from a dicotyledonous or monocotyledonous nucleic acid library or is otherwise complementary to cDNA from such a library. It is a target. Exemplary species of monocotyledonous and dicotyledonous plants include corn, canola, soybean, cotton, wheat, sorghum, sunflower, oats, sugarcane, millet, barley, and rice,
It is not limited to these. Optionally, the cDNA library is at least 8
It comprises 0% full length sequences, preferably at least 85% or 90% full length sequences, and more preferably at least 95% full length sequences. cDNA libraries can be normalized to increase rare sequence presentation. Low stringency hybridization conditions are typically, but not exclusively, used with sequences having reduced sequence identity as compared to complementary sequences. Moderate and high stringency conditions can be used as needed for more identical sequences. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

(D. Polynucleotide Having Specific Sequence Identity to Polynucleotide of (A), (B) or (C)) As shown in (d) above, the present invention relates to the polynucleotide of the present invention. An isolated nucleic acid comprising the nucleotide is provided, wherein the polynucleotide is as defined in paragraph (A) above.
), (B) or (C) have the specified identity at the nucleotide level to a polynucleotide as disclosed above. The percentage of identity to the reference sequence is at least 60% and rounded up to the nearest integer to 6
It can be represented as an integer selected from the group of integers consisting of 0 to 99. Therefore,
For example, the percentage of identity to the reference sequence is at least 60%, 65%
%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86
%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95
%, 96%, 97%, 98%, or 99%.

[0087] Optionally, the polynucleotide of this embodiment encodes a polypeptide that shares an epitope with the polypeptide encoded by the polynucleotide of paragraph (A), (B) or (C). Accordingly, these polynucleotides may comprise a second polynucleotide encoded by the polynucleotide of (A), (B) or (C).
Encodes a first polypeptide that elicits the production of an antiserum that includes an antibody that specifically reacts with the polypeptide. However, the first polypeptide does not bind to the antiserum raised against itself if the antiserum raised against itself is completely immunosorbed with the first polypeptide. Therefore, the polynucleotide of this embodiment may be, for example, (
A) to produce antibodies for use in screening expression libraries for nucleic acids comprising the polynucleotides of (B) or (C), or to produce antibodies of (A), (B) or (C). It can be used for the purification of polypeptides encoded by the polynucleotides or to raise antibodies in immunoassays for those polypeptides. The polynucleotide of this embodiment comprises a nucleic acid sequence that can be used for selective hybridization to a polynucleotide encoding a polypeptide of the invention.

Screening polypeptides for specific binding to antisera
This can be conveniently achieved using a peptide display library. The method involves screening a large collection of peptides for individual members having a desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequence is 3 to 5000 or more amino acids long, often 5 to 10 amino acids.
It can be 0 amino acids in length, and often about 8 to 15 amino acids in length. Several recombinant DNA methods have been described, in addition to direct chemical synthesis methods, for making peptide libraries. One type involves the display of peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence that codes for the particular peptide sequence displayed. Such a method is described in PCT Patent Publication Nos. 91/17271 and 91/1.
Nos. 8980, 91/19818 and 93/08278. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. PCT Patent Publication No. 92/052
Nos. 58, 92/14843 and 96/19256. See also U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors and screening kits are commercially available from suppliers such as Invitrogen (Carlsbad, CA).

E. Polynucleotides Having a Partial Sequence from a Prototype Polypeptide and Encoding a Protein That Is Cross-Reactive with the Prototype Polypeptide As shown in (e) above, the invention relates to Providing an isolated nucleic acid comprising a polynucleotide of the invention, wherein said polynucleotide comprises a partial sequence of contiguous amino acids from a prototype polypeptide of the invention as provided in (a) above. Encodes a protein having The length of contiguous amino acids from the prototype polypeptide is selected from the group of integers consisting of at least 10 and up to the number of amino acids in the prototype sequence. Thus, for example, a polynucleotide may comprise at least 10, 15, 20, 25, 30, 35, 35,
It may encode a polypeptide having a subsequence having 40, 45, or 50 contiguous amino acids. Further, the number of such subsequences encoded by the polynucleotide of this embodiment can be any integer selected from the group consisting of 1 to 20, such as 2, 3, 4, or 5. . The subsequence can be any integer number of nucleotides from 1 to the number of nucleotides in the sequence (eg, at least 5, 10 or more).
, 15, 25, 50, 100, or 200 nucleotides).

The protein encoded by the polynucleotide of this embodiment, when presented as an immunogen, is, for example, the polypeptide encoded by the polynucleotide of (a) or (b) above. Triggers the production of polyclonal antibodies that specifically bind to the non-limiting prototype polypeptide. But,
Generally, the protein encoded by the polynucleotide of this embodiment is:
If the antiserum raised against the prototype polypeptide is sufficiently immunoadsorbed to the prototype polypeptide, it will not bind to the antiserum. Methods for making antibodies and assaying for binding specificity / affinity of antibodies are well known in the art. Exemplary immunoassay formats include ELISA, competitive immunoassay, radioimmunoassay, western blot, indirect immunofluorescence assay, and the like.

In a preferred assay method, fully immunosorbed and pooled antisera raised against a prototype polypeptide can be used in a competitive binding assay to test the protein. The concentration of the prototype polypeptide required to inhibit 50% of the binding of the antiserum to the prototype polypeptide is determined. If the amount of protein required to inhibit binding is less than twice the amount of the prototype protein, the protein is said to specifically bind to antisera raised against the immunogen. Thus, the proteins of the present invention include allelic variants, conservatively modified variants, and minor recombinant variants of the prototype polypeptide.

The polynucleotide of the invention optionally encodes a protein having a molecular weight as a non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptide of the invention. Molecular weight can be easily determined by SDS-PAGE under reducing conditions. Optionally, the molecular weight is one of the full-length polypeptides of the invention.
Within 5%, more preferably 10% or 5% of the full-length polypeptide of the invention.
Within, and most preferably within 3%, 2%, or 1%.

[0093] Optionally, the polynucleotide of this embodiment is native to the invention.
At least 50%, 60%, 80% of cell extracts containing endogenous full-length polypeptide
% Or a protein having a specific enzyme activity of 90%. Further, the protein encoded by the polynucleotide of this embodiment may optionally have an affinity constant (K _m ) and / or catalytic activity (ie, microscopic) substantially similar to the native endogenous full-length protein. Kinetic constant, k _cat ). One skilled in the art will recognize that _kcat / _Km values determine specificity for competing substrates and are often referred to as specificity constants. The protein of this embodiment may have a _kcat / _Km value of at least 10% of the full length polypeptide of the invention, as determined using the endogenous substrate of the polypeptide. If necessary, the k _cat / K _m value is
At least 20%, 30%, 40% of the k _cat / K _m value of the full-length polypeptide of the invention.
, 50%, and most preferably at least 60%, 70%, 80%,
90% or 95%. The determination of k _cat , K _m , and k _cat / K _m can be determined by any number of means well known to those skilled in the art. For example, the initial rate (i.e., less than the first 5% of the reaction) may be determined by rapid mixing and sampling techniques (e.g., , Continuous flow, stop flow, or rapid quenching techniques), flash photolysis, or relaxation methods (eg, temperature jumps). Reaction rate constants are conveniently obtained using a Lineweaver-Burk plot or an Edie-Hoffstape lot.

(F. Polynucleotide Complementary to Polynucleotides of (A) to (E)) As shown in (f) above, the present invention relates to a polynucleotide complementary to the polynucleotide of paragraphs A to E above. An isolated nucleic acid comprising the polynucleotide is provided. As the skilled artisan will appreciate, the complementary sequences may be referred to as paragraphs (A)-(a) throughout the length of the complementary sequence.
Base-pair with the polynucleotide of (E) (ie, having 100% sequence identity over its entire length). Complementary bases associate by hydrogen bonding in double-stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.

(G. Polynucleotide which is a Partial Sequence of Polynucleotide of (A) to (F)) As shown in the above (g), the present invention relates to the above paragraphs (A) to (F). An isolated nucleic acid comprising a polynucleotide comprising at least 15 contiguous bases from the polynucleotide is provided. The length of the polynucleotide is given as an integer selected from the group consisting of at least 15 and the length of the nucleic acid sequence of which the polynucleotide is a subsequence. Thus, for example, the polynucleotides of the present invention comprise at least 15, 20, 2,
Polynucleotides that include 5, 30, 40, 50, 60, 75, or 100 contiguous nucleotides in length. Optionally, the number of such subsequences encoded by the polynucleotide of this embodiment is 2, 3, 4, or 5
May be any integer selected from the group consisting of 1 to 20. The subsequence may be at least 5, 10, 15, 25, 50, 100, or 200
They can be separated by any integer number of nucleotides, such as nucleotides, from 1 to the number of nucleotides in the sequence.

A subsequence of the invention may include the structural features of the sequence from which it was derived. Alternatively, a subsequence may lack certain structural features of the larger sequence from which it was derived. For example, a subsequence from a polynucleotide encoding a polypeptide having at least one linear epitope in common with the prototype polypeptide sequence as provided in (a) above may be common to the sequence of the prototype. Can encode an epitope. Alternatively, a subsequence may not encode an epitope in common with the sequence of the prototype, but can be used to isolate larger sequences, for example, by nucleic acid hybridization with the sequence from which it was derived. The subsequence can be used to regulate or detect gene expression by introducing into the subsequence a compound that binds, intercalates, cleaves, and / or crosslinks nucleic acids. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugate.

Construction of Nucleic Acids The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or a combination thereof. In some embodiments, the polynucleotides of the present invention are cloned, amplified or otherwise constructed from monocotyledonous plants. In a preferred embodiment, the monocotyledonous plant is Zea mays.

[0098] Nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the invention. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in polynucleotide isolation. Also, translatable sequences may be inserted to assist in isolating the translated polynucleotide of the present invention. For example,
The hexahistidine marker sequence provides a convenient means for purifying the proteins of the invention. A polynucleotide of the invention can be linked to a vector, adapter or linker for cloning and / or expression of the polynucleotide of the invention. Additional sequences may be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to assist in the isolation of the polynucleotide, or to add the polynucleotide to a cell. Can be improved. Typically, the length of a nucleic acid of the invention less than the length of a polynucleotide of the invention is less than 20 kilobase pairs, often less than 15 kb, and often less than 10 kb. The use of cloning vectors, expression vectors, adapters and linkers is well known in the art and has been extensively described. For a description of various nucleic acids, see, eg, Stratagene Cloni.
ng Systems, Catalogs 1995, 1996, 1997 (L
a Jolla, CA); and Amersham Life Science
s, Inc, Catalog '97 (Arlington Heights, I
See L).

A. Recombinant Methods for Constructing Nucleic Acids The isolated nucleic acid compositions of the present invention (eg, RNA, cDNA, genomic DNA or hybrids thereof) can be prepared using any of a number of cloning techniques known to those of skill in the art. It can be obtained from plant biological sources using methodology. In some embodiments, under stringent conditions, oligonucleotide probes that selectively hybridize to a polynucleotide of the invention are used to identify a desired sequence in a library of cDNA or genomic DNA. The isolation of RNA and the construction of cDNA and genomic libraries is well known to those skilled in the art.
Some of the methods used are highlighted.

(A1. MRNA Isolation and Purification) Total RNA derived from plant cells is mitochondrial RNA, chloroplast RNA, rRNA
, TRNA, hnRNA and mRNA. Total RNA preparations typically involve cell lysis and removal of organelles and proteins, followed by precipitation of nucleic acids. Extraction of total RNA from plant cells can be achieved by various means. Frequently, the extraction buffer contains a strong detergent (eg, SDS) and an organic denaturant (eg, guanidinium isothiocyanate, guanidine hydrochloride or phenol). After total RNA isolation, the poly (A) ⁺ mRNA is typically
dT) Purified from the remaining RNA using cellulose. An exemplary total RNA and mRNA isolation protocol is Plant Molecular Biolo.
gy: A Laboratory Manual, edited by Clark, Spring
er-Verlag, Berlin (1997); and Current Pr.
otocols in Molecular Biology, Ausubel
Ed., Green Publishing and Wiley-Inter
science, New York (1995). Total RNA and mRNA isolation kits are available from Stratagene (La Jolla, Calif.)
Clonetech (Palo Alto, CA), Pharmacia (Pi
commercially available from vendors such as scataway, NJ) and 5'-3 '(Paoli, Inc. PA). See also U.S. Patent Nos. 5,614,391; and 5,459,253. mRNA is about 0.5, 1.0,
It can be fractionated into populations having a size range of 1.5, 2.0, 2.5 or 3.0 kb. The cDNA synthesized for each of these fractions can be selected in size to be in the same size range as its mRNA prior to vector insertion. This method helps to eliminate truncated cDNAs formed by incompletely reverse transcribed mRNA.

(A2. Construction of cDNA Library) Construction of a cDNA library generally requires five steps. First, first strand cDNA synthesis is initiated from a poly (A) ⁺ mRNA template using a poly (dT) primer or random hexanucleotides. Second, the resulting RNA
-DNA hybrids are reacted with a combination of RNAse H and DNA polymerase I (or Klenow fragment), typically to form a double-stranded cD
Converted to NA. Third, the ends of the double-stranded cDNA are ligated to an adapter. Ligation of the adapter can create sticky ends for cloning. Fourth,
Size selection of double-stranded cDNA eliminates excess adapter and primer fragments and eliminates partial cDNA molecules due to mRNA degradation or inability of reverse transcriptase to synthesize complete first strand. Fifth, the cDNA is ligated into a cloning vector and packaged. cDNA synthesis protocols are well known to those of skill in the art, and are described in Plant Molecular Biol.
logic: A Laboratory Manual, edited by Clark, Spri
nger-Verlag, Berlin (1997); and Current
Protocols in Molecular Biology, Ausub
el et al., Green Publishing and Wiley-Int
erscience, New York (1995). The cDNA synthesis kit is available from Stratagene or Pharm.
It is available from various commercial vendors, such as acia.

A number of cDNA synthesis protocols have been described that provide a substantially pure full-length cDNA library. A substantially pure full-length cDNA library is constructed that contains at least 90%, and more preferably at least 93% or 95%, of the full-length insert among clones that contain the insert. The length of the inserts in such a library can be 0-8, 9, 10, 11, 12, 13 or more kilobase pairs. Vectors for receiving inserts of these sizes are known in the art and are commercially available. For example, Stratagene's λ
See ZAP Express (a cDNA cloning vector with a cloning capacity of 0-12 kb).

An exemplary method for constructing a full-length cDNA library of greater than 95% purity is as follows:
Carninci et al., Genomics, 37: 327-336 (1996). In that protocol, the cap structure of eukaryotic mRNA is
Labeled chemically with biotin. By using streptavidin-coated magnetic beads, only the full-length first-strand cDNA / mRNA hybrid is selectively recovered after RNaseI treatment. This method uses the starting mRNA
It provides a high yield library with unbiased presentation of the population. Other methods for generating full length libraries are known in the art. See, eg, Edery et al., Mol. Cell Biol. , 15 (6): 3363-33.
71 (1995); and PCT application WO 96/34981.

(A3. Normalized cDNA library or subtracted (subtrac)
ted) cDNA library) A non-normalized cDNA library represents the mRNA population of the tissue from which it was made. Unique clones can be difficult to isolate because they outnumber clones from highly expressed genes. cDNA library normalization is the process of creating a library in which each clone is more equally represented.

A number of approaches for normalizing cDNA libraries are known in the art. One approach is based on hybridization to genomic DNA. Each hybridized cDNA in the resulting normalized library
Is proportional to the frequency of each corresponding gene in genomic DNA.
Another approach is based on kinetic. If the cDNA reannealing follows second order kinetics, the rarer species will anneal more slowly, and the cDNA in the remaining single-stranded fraction will gradually and more normalize during the course of hybridization. Will be done. No specific loss of cDNA of any species occurs at any Cot value, regardless of its amount. Construction of a normalized library is described in Ko, Nulc. Acids. Res. , 18 (19): 57
05-5711 (1990); Patanjari et al., Proc. Natl. A
cad. U. S. A. , 88: 1943-1947 (1991); U.S. Patent No. 5
, 482,685 and 5,637,685. Soare
In the exemplary method described by S. et al., normalization resulted in a decrease in the amount of clones from a range of four orders of magnitude to a narrow range of only one order of magnitude. P
rc. Natl. Acad. Sci. USA, 91: 9228-9232 (1
994).

A subtracted cDNA library is another means for increasing the proportion of less abundant cDNA species. In this procedure, cDNA prepared from mRNA in one pool depletes sequences present in mRNA in a second pool by hybridization. The cDNA: mRNA hybrid is removed,
The remaining unhybridized cDNA pool is then enriched for sequences unique to that pool. Foote et al., Plant Molecular
Biology: A Laboratory Manual, edited by Clark,
Springer-Verlag, Berlin (1997); Kho and Z
arbl, Technique, 3 (2): 58-63 (1991);
And St. John, Nucl. Acids Res. , 16 (22): 10
937 (1988); Current Protocols in Molec.
edited by Ultra Biology, Ausubel et al., Greene Publis
ing and Wiley-Interscience, New York
(1995); and Swaroop et al., Nucl. Acids Res. , 1
9 (8): 1954 (1991). cDNA subtraction kits are commercially available. For example, PCR-Select (Clontech, P
alo Alto, CA).

(A4. Construction of Genomic Library) To construct a genomic library, a large segment of genomic DNA is
Created by random fragmentation (eg, using a restriction endonuclease) and ligated with vector DNA to form a concatemer that can be packaged into a suitable vector. Methodology to achieve these results,
And sequencing methods for confirming the sequence of nucleic acids are well known in the art. Examples of instructions sufficient to direct one of skill in the art by appropriate molecular biology techniques and many construction, cloning and screening methodologies can be found in Sambrook et al., M.
olecular Cloning: A Laboratory Manual
, Second Edition, Cold Spring Harbor Laboratory Vol. 1-3 (1989), Methods in Enzymology, No. 15.
Volume 2: Guide to Molecular Cloning Techni
Ques, Berger and Kimmel, Ed., San Diego: Acad
emic Press, Inc. (1987), Current Protocol
ols in Molecular Biology, Ausubel et al., G.
reene Publishing and Wiley-Intersie
nce, New York (1995); Plant Molecular B
iology: A Laboratory Manual, edited by Clark, Sp
Ringer-Verlag, found in Berlin (1997). Kits for the construction of genomic libraries are also commercially available.

A5. Methods for Screening and Isolating Nucleic Acids A cDNA or genomic library can be screened using a probe based on the sequence of a polynucleotide of the invention as disclosed herein.
Probes can be used to hybridize to genomic DNA or cDNA sequences and to isolate homologous genes in the same or different plant species. One skilled in the art will appreciate that varying degrees of stringency of hybridization may be used in this assay,
And it is understood that either the hybridization or the wash medium can be stringent. As hybridization conditions become more stringent, complementarity between the probe and target for duplex formation must occur to a greater extent. The degree of stringency depends on temperature, ionic strength, pH
And the presence of a partially denaturing solvent such as formamide. For example,
The stringency of hybridization can be conveniently varied by changing the polarity of the reaction solution by manipulating the formamide concentration in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary according to the stringency of the hybridization and / or wash media. The degree of complementarity is optimally 100%; however, it is understood that small amounts of sequence alterations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or wash media. Should be.

A nucleic acid of interest can also be amplified from a nucleic acid sample using amplification techniques. For example, the sequence of a polynucleotide of the present invention and related genes can be amplified directly from a genomic DNA or cDNA library using the polymerase chain reaction (PCR) technique. PCR and other in vitro amplification methods also include cloning a nucleic acid sequence encoding the protein to be expressed, e.g., for nucleic acid sequencing, or for other purposes, to obtain the desired mRNA in a sample. It may be useful to generate nucleic acids for use as probes to detect the presence. Examples of techniques sufficient to direct one skilled in the art to in vitro amplification methods are described in Berger, Sambro.
ok, and Ausubel, and Mullis et al., US Patent No. 4,683.
No. 202 (1987); and PCR Protocols A Guide.
Academic Press Inc., eds. to Methods and Applications, Innis et al. , San Diego, CA (19
90). Commercially available kits for genomic PCR amplification are known in the art. For example, Advantage-GC Genomic PCR K
See it (Clonetech). T4 gene 32 protein (Boe
(Hringer Mannheim) can be used to improve the yield of long PCR products.

[0110] PCR-based screening methods have also been described. Wilfin
Ger et al. describe a PCR-based method in which the longest cDNA is identified in the first step, so that incomplete clones can be removed from the study. B
ioTechniques, 22 (3): 481-486 (1997). In this method, a primer pair is synthesized, one primer annealing to the 5 'end of the sense strand of the desired cDNA, and the other primer annealing to the vector. Clones are pooled, allowing for large-scale screening. By this procedure, the longest possible clone is identified among the candidate clones. further,
The PCR product is used only as a diagnostic for the presence of the desired cDNA, and does not utilize the PCR product itself. Such a method involves the use of full-length cDNA
It is particularly effective in combination with the construction method described above.

B. Synthetic Methods for Constructing Nucleic Acids The isolated nucleic acids of the present invention can also be prepared as described in Narang et al., Meth. Enzymol
. 68: 90-99 (1979) phosphotriester method; Brown et al., Me.
th. Enzymol. 68: 109-151 (1979); phosphodiester method; Beaucage et al., Tetra. Lett. 22: 1859-1862 (
1981) diethylphosphoramidite method; for example, Needham-Van
Devanter et al., Nucleic Acids Res. , 12: 6159.
See, e.g., Beaucage and Caruthers, Tetra. Letts. 22
(20): prepared by direct chemical synthesis by methods such as the solid-phase foramidite triester method described by 1859-1862 (1981); and the solid support method of U.S. Pat. No. 4,458,066. Can be done. Generally, chemical synthesis produces single-stranded oligonucleotides. This can be converted to double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using this single strand as a template. One skilled in the art will recognize that chemical synthesis of DNA is most used for sequences of about 100 bases or less, but longer sequences can be obtained by ligation of shorter sequences.

(Recombinant Expression Cassette) The present invention further provides a recombinant expression cassette comprising the nucleic acid of the present invention. A nucleic acid sequence encoding a desired polynucleotide of the invention, for example, a cDNA or genomic sequence encoding a full-length polypeptide of the invention, is used to construct a recombinant expression cassette that can be introduced into a desired host cell. obtain. Typically, a recombinant expression cassette comprises a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence, wherein the regulatory sequence is contained in an intended host, such as a transformed plant tissue. Directs transcription of this polynucleotide in the cell.

For example, a plant expression vector can include (1) a cloned plant gene that is under the transcriptional control of 5 ′ and 3 ′ regulatory sequences, and (2) a dominant selectable marker. Such plant expression vectors can also be promoter-regulated, if desired (eg, inducible or constitutive, environmentally or developmentally regulated,
Or regions that provide 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
Or it may include a polyadenylation signal.

[0114] In all tissues of the regenerated plant, a plant promoter fragment that directs the expression of the polynucleotide of the present invention may be used. Such promoters
It is referred to herein as a "constitutive" promoter and is active under most environmental conditions and conditions of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, Agrobacter
Rim tumefaciens T-DNA-derived 1'- or 2 'promoter, ubiquitin 1 promoter, Smas promoter, cinnamon alcohol dehydrogenase promoter (US Pat. No. 5,683,439), Nos promoter, pEmu promoter, Rubisco promoter, GRP1- 8 promoter and other transcription initiation regions from various plant genes known to those of skill in the art. One exemplary promoter is the ubiquitin promoter, which can be used to drive expression of the present invention in embryos or embryogenic calli, especially in maize.

Alternatively, a plant promoter may direct the expression of a polynucleotide of the present invention in a particular tissue, or may be otherwise under more detailed environmental or developmental control. Such a promoter is referred to herein as an "inducible" promoter. Environmental conditions that can effect transcription by an inducible promoter include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the AdhI promoter inducible by hypoxia or cold stress, the Hsp70 promoter inducible by heat stress, and the PPDK promoter inducible by light.

Examples of promoters under developmental control include promoters that initiate transcription only or preferentially in certain tissues, such as leaves, roots, fruits, seeds, or flowers.
Exemplary promoters include anther-specific promoter 5126 (US Pat.
, 689,049 and 5,689,051), the glob-1 promoter, and the γ-zein promoter. The operation of a promoter can also vary depending on its position in the genome. Thus, an inducible promoter may be fully or partially constitutive at certain locations.

[0117] Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of the invention. These promoters can also be used, for example,
The concentration and / or composition of a protein of the invention in a desired tissue can be used in a recombinant expression cassette to drive expression of an antisense nucleic acid to decrease, increase or alter. Thus, in some embodiments, the nucleic acid construct comprises a promoter operably linked to a polynucleotide of the present invention, such as in corn, that is functional in a plant cell. Promoters useful in these embodiments include endogenous promoters that drive expression of the polypeptides of the invention.

In some embodiments, the polynucleotide of the invention is isolated such that the isolated nucleic acid provided as a promoter or enhancer element up-regulates or down-regulates expression of the polynucleotide of the invention. A non-heterologous form can be introduced at a suitable location (generally upstream). For example, can the endogenous promoter be modified in vivo by mutation, deletion, and / or substitution (Kmi
ec, US Pat. No. 5,565,350; Zarling et al., PCT / US93.
Or an isolated promoter can be introduced into plant cells in the proper orientation and distance from the gene of the invention so as to control the expression of the gene. Gene expression can be regulated under conditions appropriate for plant growth to alter the total concentration of the polypeptide of the invention and / or alter its composition in plant cells. Accordingly, the invention provides compositions and methods for making a heterologous promoter and / or enhancer operably linked to a native, endogenous (ie, non-heterologous) form of a polynucleotide of the invention. I do.

Methods for identifying a promoter having a particular expression pattern, for example, in terms of tissue type, cell type, stage of development, and / or environmental conditions are well known in the art. For example, The Maize Handbook, Chapters 114-115,
Freeling and Walbot, Springer, New York
(1994); Corn and Corn Improvement, 3rd edition, Chapter 6, Sprague and Dudley eds., American Soci.
ety of Agronomy, Madison, Wisconsin (19
88). A typical step in a promoter isolation method is the identification of a gene product that is expressed with some specificity in the target tissue. Some of the methods range:
Differential hybridization to cDNA libraries; subtractive hybridization; differential display; differential 2-D gel electrophoresis; DNA probe arrays; and isolation of proteins known to be expressed with some specificity in target tissues. . Such methods are well-known to those skilled in the art. Commercially available products for identifying promoters are available from Univers of Clontech (Palo Alto, CA).
It is known in the art, such as the al Genome Walker Kit.

For protein-based methods, one obtains the amino acid sequence of at least a portion of the identified protein and then identifies genomic DNA, either directly or, preferably, a cDNA clone from a library prepared from the target tissue. Either way, it is beneficial to use this protein sequence as a basis for preparing a nucleic acid that can be used as a probe to identify. Once such a cDNA clone has been identified, this sequence can be used to identify the sequence at the 5 'end of the transcript of the indicated gene. Differential Hybridization; For subtractive hybridization and differential display, the sequence of the 5 'end of the transcript of the indicated gene is identified using the nucleic acid sequence identified as enriched in the target tissue. Once such sequences have been identified, genomic prepared from the target organism using either of these sequences identified as being from gene transcripts, starting either from protein or nucleic acid sequences Libraries can be screened. Methods for identifying and confirming a transcription start site are well known in the art.

In the process of isolating a promoter that is expressed under specific environmental conditions or stress, or at a specific tissue, or at a specific developmental stage, it is expressed under the desired environment, in the desired tissue, or at the desired stage. Many genes are identified. Further analysis indicates the expression of each particular gene in one or more other tissues of the plant. Promoters that are active in the desired tissue or condition, but not in any other normal tissue, may be identified.

To identify a promoter sequence, the 5 'portions of the clones described herein are analyzed for characteristic sequences of the promoter sequence. For example, a promoter sequence element comprises a TATA box consensus sequence (TATAAT), which is an AT-rich stretch of 5-10 bp, usually located about 20-40 base pairs upstream of the transcription start site. Identification of TATA boxes is well known in the art. For example, one way of estimating the location of this element is to identify the transcription start site using standard RNA mapping techniques, such as primer extension, S1 analysis, and / or RNase protection. To confirm the presence of the AT-rich sequence, a structure-function analysis can be performed, including mutagenesis of the putative region, and quantification of the effect of the mutation on the expression of the linked downstream reporter gene. See, for example, The Maize Handbook, Chapter 114, Fre.
Elling and Walbot, Springer, New York, (1
994).

In plants, typically at positions −80 to −100 further upstream from the TATAbox, there is a promoter element with a series of adenines surrounding the trinucleotide G (or T) NG (ie, the CAAT box). J. Messaging
Et al., Genetic Engineering in Plants, Kosa
ge, Meredith and Hollaender, pp. 221-227 1
983. In corn, there is no well-conserved CAAT box, but TAT
There are several short, conserved protein binding motifs upstream of the A box. These include motifs for trans-acting transcription factors involved in light regulation, anaerobic induction, hormonal regulation, or anthocyanin biosynthesis, appropriate for each gene.

Once the promoter and / or gene sequences are known, an appropriately sized region is selected from genomic DNA 5 ′ to the transcription or translation start site, and then such sequences are Linked to an array. Where a transcription initiation site is used as a point of fusion, any of a number of possible 5 'untranslated regions may be used between the transcription initiation site and the partial coding sequence. 3 'of a specific promoter
If a terminal translation initiation site is used, it may be linked directly to the methionine start codon of the coding sequence.

Where polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3 ′ end of the polynucleotide coding region. The polyadenylation region may be derived from a natural gene, various other plant genes, or T-DNA.
The 3 'terminal sequence to be added may be derived, for example, from a nopaline or octopine synthase gene, or another plant gene, or less preferably any other eukaryotic gene.

Intron sequences may be added to the coding sequence of the 5 ′ untranslated region or partial coding sequence to increase the amount of mature message that accumulates in the cytosol. In both plant and animal expression constructs, inclusions of splicable introns in the transcription unit have been shown to increase gene expression at both mRNA and protein levels by up to 1000-fold. Buchman and Berg,
Mol. Cell Biol. 8: 4395-4405 (1988); Call
is 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. Corn Intron Adh1-S Intron 1
The use of the Bronze-1 intron is known in the art.
In general, The Maize Handbook, Chapter 116, Freeelin
g and Walbot, Ed., Springer, New York (1994).

Vectors containing sequences from the polynucleotides of the present invention typically include a marker gene that confers a selectable phenotype on plant cells. Usually, this selectable marker gene encodes antibiotic resistance, and suitable genes include genes encoding resistance to the antibiotic spectinomycin (eg, the aada gene), streptomycin phosphotransferase (S) encoding streptomycin resistance.
(PT) gene, neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, hygromycin phosphotransferase (HPT) gene encoding hygromycin resistance, and acts to inhibit the action of acetolactate synthase (ALS). A gene encoding resistance to a herbicide (particularly a sulfonylurea type herbicide) (eg, an acetolactate synthase (ALS) gene containing a mutation leading to such resistance, particularly an S4 and / or Hra mutation), phosphinothricin or Includes genes that encode resistance to herbicides that act to inhibit the action of glutamine synthase, such as busta (eg, the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide busta and has nptI
The I gene encodes resistance to the antibiotics kanamine and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfon.

Representative vectors useful for the expression of genes in higher plants are well known in the art and are described in Rogers et al., Meth. In Enzymol. , 1
53: 253-277 (1987).
um tumefaciens containing a vector derived from the tumor induction (Ti) plasmid. These vectors are plant integration vectors, and in transformation, the vector integrates a portion of the vector DNA into the genome of the host plant. Exemplary A.S. useful herein. tumefaciens vector is Schard
1 et al., Gene, 61: 1-11 (1987) and Berger et al., Proc.
. Natl. Acad. Sci. U. S. A. , 86: 8402-8406 (1
989) plasmids pKYLX6 and pKYLX7. Another useful vector herein is Clontech Laboratories, Inc.
Plasmid pBI101.2 available from (Palo Alto, CA)
It is.

The polynucleotides of the present invention can be expressed as desired in either a sense or antisense orientation. It is understood that modulation of gene expression in either the sense or antisense orientation can have a direct impact on observable plant characteristics. Antisense technology can be conveniently used to alter gene expression in plants. To accomplish this, the nucleic acid segment of the desired gene is cloned and operably linked to a promoter so that the antisense strand of the RNA is transcribed. This construct is then transformed into a plant and the antisense strand of the RNA is generated. In plant cells, antisense RNA has been shown to inhibit gene expression by preventing the accumulation of mRNA encoding the enzyme of interest (see, eg, Shehyy et al.
rc. Nat'l. Acad. Sci. (USA) 85: 8805-8809.
(1998); and Hitta et al., US Pat. No. 4,801,340).

Another method of suppression is suppression of sense. Introduction of a nucleic acid formed in a sense orientation has been shown to be an effective means for blocking transcription of a target gene. For one example of the use of this method to regulate expression of endogenous genes, see Napoli et al., The Plant Cell 2: 279-289 (19).
90) and US Patent 5,034,323.

[0131] Catalytic RNA molecules, ie ribozymes, can also be used to inhibit the expression of plant genes. It is possible to design a ribozyme that specifically pairs with virtually any target RNA and cleaves the phosphodiester backbone at specific sites, thereby functionally inactivating that target RNA. In performing this cleavage, ribozymes are true enzymes because they do not change themselves and can reuse and cleave other molecules. Inclusion of ribozyme sequences in the antisense RNA confers on them RNA-cleaving activity, thereby increasing the activity of the construct. The design and use of RNA-specific ribozymes is described in Haselo
ff et al., Nature 334: 585-591 (1988).

As ancillary groups on the polynucleotides of the present invention, various cross-linking agents, alkylating agents, and radical-generating species can be used to bind, label, detect, and / or cleave nucleic acids. For example, Vlasov, V .; V. Et al., Nucleic Ac
ids Res (1986) 14: 4065-4076 describes the covalent attachment of an alkylated derivative of a nucleotide complementary to a target sequence to a single-stranded DNA fragment. A report of a similar study by the same group is given by Knorre, D .; G. FIG. Et al.,
Biochimie (1985) 67: 785-789. Iv
erson and Dervan have also shown sequence-specific cleavage of single-stranded DNA mediated by incorporation of modified nucleotides that can activate the cleavage (J Am
Chem Soc (1987) 109: 1241-1243). Meyer,
R. B. Et al., J Am Chem Soc (1989) 111: 8517-85.
19 achieves covalent crosslinking to the target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. Photo-activated cross-linking to single-stranded oligonucleotides mediated by psoralens has been described by Lee, B. et al. L. Et al., Biochemis
try (1988) 27: 3197-3203. The use of crosslinks in triple helix forming probes has also been described by Home et al., J Am Chem Soc (
1990) 112: 2435-2437. The use of N4, N4-ethanocytosine as an alkylating agent for cross-linking single-stranded oligonucleotides has also been described by Webb and Matteucci, J Am Chem Soc (
1986) 108: 2764-2765; Nucleic Acids Res.
(1986) 14: 7661-7674; Feteritz et al. Am. Ch
em. Soc. 113: 4000 (1991). Various compounds for binding, detecting, labeling, and / or cleaving nucleic acids are known in the art (eg, US Pat. Nos. 5,543,507; 5,672,593; 5,484,908, 5,256,648, and 5,68
See 1,941.

Protein The isolated protein of the present invention is a variant of any one of the polynucleotides of the present invention, or a conservatively modified polypeptide thereof, as discussed more fully above. Includes polypeptides having at least 10 amino acids encoded by a polypeptide. A protein of the invention or a variant thereof may comprise any number of contiguous amino acid residues from the polypeptide of the invention, wherein the number is from 10 to the number of residues in the full length polypeptide of the invention. Selected from the group of integers consisting of up to the number Optionally, the contiguous amino acid sequence is at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length. Further, the number of such sequences can be any integer selected from the group consisting of 1-20 (eg, 2, 3, 4, or 5).

The present invention further provides a protein comprising a polypeptide having specific sequence identity to a polypeptide of the present invention. The percentage of sequence identity ranges from 50 to
It is an integer selected from the group consisting of 99. Exemplary sequence identity values include:
60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, and 99%. Sequence identity can be determined, for example, by GAP or BL
It can be determined using the AST algorithm.

As will be appreciated by those skilled in the art, the present invention includes the catalytically active polypeptides (ie, enzymes) of the present invention. The catalytically active polypeptide is at least 20%, 30%, 40%, and preferably at least 50%, 60%, or 70% of the polypeptide of the native (non-synthetic) endogenous polypeptide, and most preferably, It has at least 80%, 90%, or 95% relative activity. In addition, the substrate specificity (k _cat / K _m ) is substantially similar to the native (non-synthetic) endogenous polypeptide, as appropriate. Typically, the K _m is at least 30%, 40%,
50% K _m native (non-synthetic) endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90%. Methods for assaying and quantifying measurements of enzyme activity and substrate specificity (k _cat / K _m ) are well known to those skilled in the art.

In general, a protein of the present invention is represented by a polynucleotide that, when presented as an immunogen, specifically reacts with a polypeptide of the present invention encoded by a polynucleotide of the present invention as described above. Be evoked. Furthermore, the proteins of the invention do not specifically bind to antiserum raised against a polypeptide of the invention that has been completely immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those skilled in the art. Preferred immunoassays are competitive immunoassays as discussed below. Thus, the proteins of the invention may be used as immunogens for constructing immunoreactive antibodies against the proteins of the invention for exemplary utilities such as immunoassays or protein purification techniques.

(Expression of Protein in Host Cell) Using the nucleic acid of the present invention, the protein of the present invention can be recombinantly engineered into cells (eg, bacterial cells, yeast cells, insect cells, mammalian cells or preferably plants). cell)
Can be expressed. The cell produces the protein in non-native conditions (eg, in amount, composition, location, and / or time). This is because these cells have been genetically altered by human intervention to produce in this way.

One of skill in the art is expected to be familiar with the many expression systems available for expressing nucleic acids encoding the proteins of the invention. No attempt has been made to detail the various methods known for the expression of proteins in prokaryotes or eukaryotes.

[0139] Briefly summarized, expression of an isolated nucleic acid encoding a protein of the invention typically involves, for example, driving a DNA or cDNA into a promoter, which is either constitutive or inducible. It is achieved by operably linking and subsequently incorporating into an expression vector. Vectors may be suitable for replication and integration in either prokaryotes or eukaryotes. Representative expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of DNA encoding the proteins of the invention. To obtain high levels of expression of the cloned gene, it is desirable to construct an expression vector that contains at a minimum a strong promoter that directs transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator. . One skilled in the art will recognize that modifications can be made to a protein of the invention without reducing the biological activity of the protein of the invention. Some modifications can be made to facilitate cloning, expression, or incorporation of the target molecule into the fusion protein. Such modifications are well known to those of skill in the art and may be located at either end, for example, methionine added to the amino terminus to provide a start site, or a conveniently located purified sequence. And further amino acids (eg, poly-His). Restriction sites or stop codons can also be introduced.

A. Expression in Prokaryotes Prokaryotic cells can be used as hosts for expression. Prokaryotes are most often E. coli. described by various strains of E. coli; however, other microbial strains can also be used. Commonly used prokaryotic control sequences, defined herein to include a promoter for transcription initiation (optionally with an operator) along with a ribosome binding site sequence, are β-lactamases (penicillinases) and Lactose (lac) promoter system (Chang et al., Nature 198: 10).
56 (1977)), tryptophan (trp) promoter system (Goedde
l, Nucleic Acids Res. 8: 4057 (1980)) and the λ-derived PL promoter and N gene ribosome binding site (Shima
Take et al., Nature 292: 128 (1981)). E. FIG. The inclusion of a selectable marker in the DNA vector transfected into E. coli is also useful. Examples of such markers include genes that specify resistance to ampicillin, tetracycline or chloramphenicol.

This vector is selected to allow for introduction into a suitable host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. When using a plasmid vector, bacterial cells are transfected with the plasmid vector DNA. An expression system for expressing the protein of the present invention is described in Bacillus sp. And Salmonella (Palva et al., Gene 22).
: 229-235 (1983); Mosbach et al., Nature 302: 5.
43-545 (1983)).

B. Expression in Eukaryotes Various eukaryotic expression systems are known to those of skill in the art, such as yeast, insect cell lines, plant cells and mammalian cells. As described briefly below, the polynucleotides of the present invention can be expressed in these eukaryotic systems. In some embodiments, the transformed / transfected plant cells are, as discussed below,
It is used as an expression system for producing the protein of the present invention.

The synthesis of heterologous proteins in yeast is well known. Sherman, F .; Et al.
Methods in Yeast Genetics, Cold Spring
g Harbor Laboratory (1982) is a well-recognized study that describes the various methods available for producing proteins in yeast. Two widely used yeasts for the production of eukaryotic proteins are S.
accaromyces cerevisiae and Pichia pas
toris. Vectors, strains and protocols for expression in Saccharomyces and Pichia are known in the art and are available from commercial suppliers (eg, Invitrogen). Suitable vectors usually have expression control sequences (eg, a promoter comprising 3-phosphoglycerate kinase or alcohol oxidase) and, if desired, an origin of replication, termination sequences, and the like.

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

The sequences encoding the proteins of the invention may also be ligated into various expression vectors for use in transfecting cell cultures of, for example, mammalian, insect or plant origin. An example of a cell culture useful for the production of a peptide is a mammalian cell. Mammalian cell lines are often in the form of monolayer cells, but mammalian cell suspensions can also be used. Many suitable host cell lines capable of expressing the intact protein have been developed in the art, and the HEK293 cell line,
Includes BHK21 and CHO cell lines. Expression vectors for these cells include expression control sequences (eg, an origin of replication, a promoter (eg, a CMV promoter, an HSV tk promoter or a pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol. Rev. 8).
9:49 (1986)), as well as necessary processing information sites (eg, ribosome binding sites, RNA splice sites, polyadenylation sites (eg, SV
40 large T Ag polyA addition site) and a transcription terminator sequence). Other animal cells useful for the production of the proteins of the invention include, for example, Ame
rican Type Culture Collection.

Suitable vectors for expressing the proteins of the invention in insect cells are usually derived from SF9 baculovirus. Suitable insect cell lines include mosquito larva cell lines, silkworm cell lines, caterpillar cell lines, moth cell lines and Drosophila cell lines (eg, the Schneider cell line (Schneider, J. Embr).
yol. Exp. Morphor. 27: 353-365 (1987)).

As with yeast, when higher animal or plant host cells are used, the polyadenylation or transcription terminator sequence is typically incorporated into a vector. An example of a terminator sequence is a polyadenylation sequence from the bovine growth hormone gene.
Sequences for correct splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Am.
Virol. 45: 773-781 (1983)). In addition, gene sequences for controlling replication in a host cell can be incorporated into vectors such as those found in bovine papillomavirus-type vectors. Saveria-C
ampo, M .; , Bovine Papilloma Virus DNA a
Eukaryotic Cloning Vector, DNA Cloni
ng Volume II, a Practical Approach, D.E. M. Glo
ver., IRL Press, Arlington, Virginia 21
3-238 (1985).

Transfection / Transfection of Cells The method of transformation / transfection is not critical to the invention; various methods of transformation or transfection are currently available. If newer methods are available to transform crops or other host cells,
These can be applied directly. Thus, inserting the DNA sequence into the genome of the host cell,
A wide variety of methods have been developed to achieve transcription and / or translation of this sequence and to effect a phenotypic change in the organism. Thus, any method that provides for efficient transformation / transfection can be used.

(A. Transformation of Plant) A set that can be introduced into a desired plant using a DNA sequence encoding a desired polypeptide of the present invention (eg, a cDNA sequence or a genomic sequence encoding a full-length protein). Construct a recombinant expression cassette.

The isolated nucleic acids of the present invention can be introduced into plants according to techniques known in the art. Generally, a recombinant expression cassette as described above and a recombinant expression cassette suitable for transforming plant cells are prepared. The isolated nucleic acids of the invention can then be used for transformation. In this manner, genetically modified plants, plant cells, plant tissues, seeds, etc. can be obtained. Transformation protocols can vary depending on the type of plant cell targeted for transformation (ie, monocot or dicot). Suitable methods for transforming plant cells include microinjection (Crossway et al., (1986) Biotechn.
IQs 4: 320-334), electroporation (Riggs et al.,
(1986) Proc. Natl. Acad. Sci. USA 83: 5602
-5606), Agrobacterium-mediated transformation (Hinchee et al.,
(1988) Biotechnology 6: 915-921), direct gene transfer (Paszkowski et al., (1984) EMBO J. 3: 2717-2).
722), and ballistic particle acceleration (ballistic particle ac)
celeration (eg, Sanford et al., US Pat. No. 4,945,945).
No. 050; Tomes et al., "Direct DNA Transfer int.
o Intact Plant Cells via Microproject
Tile Bombardment ", Gamburg and Phillips
(Ed.), Plant Cell, Tissue and Organic Cult
ure: Fundamental Methods, Springer-Ver
lag, Berlin (1995); and McCabe et al., (1998) Bi.
otechnology 6: 923-926). Also, Weis
Singer et al., (1988) Annual Rev. Genet. 22:42
1-477; Sanford et al., (1987) Particulate Sci.
ence and Technology 5: 27-37 (Onion); Ch
listou et al., (1988) Plant Physiol. 87: 671-6
74 (soy); McCabe et al., (1988) Bio / Technology.
6: 923-926 (soy); Datta et al., (1990) Biotech.
nology 8: 736-740 (rice); Klein et al., (1988) Pr.
oc. Natl. Acad. Sci. USA 85: 4305-4309 (maize); Klein et al., (1988) Biotechnology 6: 5.
59-563 (maize); Tomes et al., "Direct DNA Tr.
answer into Into Plant Cells via M
microprojectile Bombardment ", Gamburg and Phillips (eds.), Plant Cell, Tissue and
Organ Culture: Fundamental Methods, Sp
ringer-Verlag, Berlin (1995) (maize); K
Lein et al., (1988) Plant Physiol. 91: 440-444
(Corn); Fromm et al., (1990) Biotechnology.
8: 833-839 (maize); Hooykaas-Van Slogt
eren and Hoykaas (1984) Nature (London) 3
11: 763-764; Bytebier et al., (1987) Proc. Natl
. Acad. Sci. USA 84: 5345-5349 (lily); De We
et al., (1985) The Experimental Manipulati.
on of Ovuture Tissues, G. P. Chapman et al., 1
97-209 Longman, NY (pollen); Kaeppler et al., (19
90) Plant Cell Reports 9: 415-418; and K
aeppler et al., (1992) Theor. Appl. Genet. 84: 5
60-566 (whisker-mediated transformation); D'Hallu
in et al., (1992) Plant Cell 4: 1495-1505 (electroporation); LI et al., (1993) Plant Cell Reporter.
ts 12: 250-255 and Christou and Ford (1995)
) Annals of Botany 75: 745-750 (Agrobac
corn via terium tumefaciens);
All of which are incorporated herein by reference.

[0151] The transformed cells can be grown into plants according to conventional methods. For example, M
cCormick et al., (1986) Plant Cell Reports, 5
: 81-84. These plants can then be grown and pollinated, either with a homologous transformed strain or with a different strain, to produce a hybrid with the identified characteristics of the desired phenotype. Two or more generations can be grown to ensure that the subject's phenotypic characteristics are stably maintained and inherited, and then the seeds are harvested to ensure the desired phenotype or other characteristics acquired .

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells Animal and lower eukaryote (eg, yeast) host cells are competent for transfection by various means. Or be made competent. There are several well-known methods for introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of recipient cells with bacterial protoplasts containing DNA, treatment of recipient cells with liposomes containing DNA, DEAE dextran, electroporation, biolistics.
stic), and microinjection of DNA directly into cells. The transfected cells are cultured by means well known in the art. K
uchler, R .; J. , Biochemical Methods in C
cell Culture and Virology, Dowden, Hutc
Hinson and Ross, Inc. (1977).

(Synthesis of Protein) The protein of the present invention can be constructed using a non-cellular synthesis method. Solid phase synthesis of proteins of less than about 50 amino acids in length can be achieved by coupling the C-terminal amino acid of this sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid-phase synthesis are described in Barany and Merrifie.
ld, Solid-Phase Peptide Synthesis, 3-2
84, The Peptides: Analysis, Synthesis,
Biology. Volume 2: Special Methods in Pepti
de Synthesis, Part A; Merrifield et al. Am. Ch
em. Soc. 85: 2149-2156 (1963), and Stewart.
Et al., Solid Phase Peptide Synthesis, 2nd edition,
Pierce Chem. Co. Rockford, Ill. (1984). Longer proteins can be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods for forming a peptide bond by activation of the carboxy terminus (eg, by using the coupling reagent N, N'-dicyclohexylcarbodiimide) are known to those skilled in the art.

(Purification of Protein) The protein of the present invention can be purified by standard techniques well known to those skilled in the art. The recombinantly produced proteins of the present invention can be expressed directly or as a fusion protein. Recombinant proteins are purified by a combination of cell lysis (eg, 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.

Recombinant or synthetic proteins of the present invention can be prepared by standard techniques well known in the art, including detergent solubilization, selective precipitation with materials such as ammonium sulfate, column chromatography, immunopurification and the like. It can be purified to considerable purity by techniques. For example, R. Scopes, Protein Purification: P
rinciples and Practice, Springer-Verl
ag: New York (1982); Deutscher, Guide to
Protein Purification, Academic Press
(1990). For example, antibodies can be raised against a protein as described herein. E. FIG. Purification from E. coli is described in US Pat.
This can be achieved according to the procedure described in US Pat. The protein can then be isolated from cells that express the protein, and can be further purified by standard protein chemistry techniques as described herein. Detection of the expressed protein is accomplished by methods known in the art, and
Including radioimmunoassay, Western blotting techniques or immunoprecipitation.

Regeneration of Transgenic Plants Plant cells transformed with a plant expression vector can be regenerated, for example, 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 whole plants. Cultured protoplast-derived plant regenerates are described in Evans et al., Protoplasts Iso.
Lation and Culture, Handbook of Plant
Cell Culture, Macmillilan Publishing
Company, New York, pages 124-176 (1983); and Binding, Generation of Plants, Plant.
Protoplasts, CRC Press, Boca Raton, 21
-73 (1985).

Regeneration of plants containing foreign genes introduced by Agrobacterium from leaf explants is described in Horsch et al., Science, 227: 1229-1231.
(1985). In this procedure, transformants are transformed in the presence of a selection agent and in Fraley et al., Proc. Natl. Aca
d. Sci. (U.S.A.), 80: 4803 (1983). Grow in a medium that induces shoot regeneration in transformed plant species. This procedure typically produces shoots within 2-4 weeks, and the shoots of these transformants are then transferred to a suitable root-inducing medium containing selective drugs and antibiotics to prevent bacterial growth. The transgenic plants of the present invention can be fertile or sterile.

Regeneration can also be obtained from plant calli, explants, organs, or parts thereof. Such regeneration techniques are described in Klee et al., Ann. Rev .. of Plant
Phys. 38: 467-486 (1987). Regenerated plants from either single plant protoplasts or various explants are well known in the art. For example, Methods for Plant Mole
cultural Biology, A. et al. Weissbach and H.W. Weissb
ach, Academic Press, Inc. , San Diego, C
arif. (1988). The regeneration and growth processes include the steps of selecting transformant cells and shoots, rooting the transformant shoots and growing the seedlings in soil. For corn cell culture and regeneration,
The Maize Handbook, Freeling and Walbot
Ed., Springer, New York (1994); Corn and C
orn Improvement, Third Edition, Sprague and Dudley
Hen, American Society of Agronomy, Madis
See generally, on, Wisconsin (1988).

[0159] One of skill in the art will appreciate that after confirming that a recombinant expression cassette is stably integrated and operable in a transgenic plant, the recombinant expression cassette can be introduced into other plants by sexual crossing. Recognize that. Any of a number of standard breeding techniques can be used depending on the species to be crossed.

In asexually propagated crops, mature transgenic plants can be propagated by performing cuttings or by tissue culture techniques to produce a plurality of identical plants. Desired transgenic selections are made, and new varieties are obtained and asexually propagated for commercial use. In seed-bred crops, mature transgenic plants can be self-crossed to produce homozygous inbred plants. Inbred plants produce seed that contains the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that produce the selected phenotype.

[0161] Parts obtained from regenerated plants (eg, flowers, seeds, leaves, branches, fruits, etc.) are included in the present invention. However, these parts include cells containing the isolated nucleic acids of the present invention. Progeny and variants, and variants of the regenerated plants are also included within the scope of the invention. However, these parts include the introduced nucleic acid sequence.

[0162] Transgenic plants expressing a selectable marker can be screened for transfer of the nucleic acids of the invention, for example, by standard immunoblot and DNA detection techniques. Transgenic strains are also typically evaluated for the level of expression of the heterologous nucleic acid. Expression at the RNA level can be initially determined to identify and quantify positive expression plants. Standard techniques for RNA analysis can be used, and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA template and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then be analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be performed using a heterologous nucleic acid-specific polynucleotide probe and a heterologous nucleic acid-specific antibody, respectively, to localize the site of expression in the transgenic tissue. . In general, many transgenic strains are usually screened for incorporated nucleic acids to identify and select plants with the most appropriate expression profile.

A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; that is, a transgenic plant containing two added nucleic acid sequences, wherein one gene is Transgenic plants that are the same locus on each chromosome of a chromosome pair. Homozygous transgenic plants sexually cross (self-pollinate) heterozygous transgenic plants containing a single added heterologous nucleic acid, germinate some of the produced seeds, and control plants (ie, (Native, non-transgenic) as compared to native plants) for the altered expression of the polynucleotides of the present invention. Backcrossing to the parent plant and outcrossing with non-transgenic plants are also contemplated.

Modulation of Polypeptide Levels and / or Compositions The invention further provides for modulating (ie, increasing or decreasing) the concentration or composition of a polypeptide of the invention in a plant or part thereof. Provide a way. Modulation may be achieved by increasing or decreasing the concentration and / or composition (ie, the proportion of the polypeptide of the invention) in the plant. This method comprises the steps of introducing a recombinant expression cassette containing the polynucleotide of the present invention into a plant cell to obtain a transformed plant cell, and culturing the transformed plant cell under plant cell growth conditions. And inducing or suppressing the expression of a polynucleotide of the invention in the plant for a time sufficient to regulate the concentration and / or composition in the plant or plant part.

In some embodiments, the content of a polypeptide of the invention in a plant and / or
Alternatively, the composition can be regulated by altering the promoter of the gene in vivo or in vitro to up-regulate or down-regulate gene expression. In some embodiments, the coding region of a native gene of the invention can be altered through substitutions, additions, insertions or deletions to reduce the activity of the encoded enzyme. See, for example, Kmiec, US Pat.
No. 5,350; Zarling et al., PCT / US93 / 03868. And, in some embodiments, an isolated nucleic acid (eg, a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, plant cells containing a promoter operably linked to the polynucleotide of the present invention can be prepared by means known to those skilled in the art (eg, Southern blot, DNA sequencing, or by using primers specific for this promoter and this gene). And PCR assays that detect and detect amplicons produced therefrom. Plants or plant parts altered or modified according to the above embodiments are grown under plant-forming conditions for a time sufficient to regulate the concentration and / or composition of the polypeptide of the present invention in the plant. Plant formation conditions are well known in the art and are briefly discussed above.

In general, the concentration or composition is at least 5% compared to a native control plant, plant part, or cell lacking the recombinant expression cassette described above.
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 9
0% increase or decrease. Modulation in the present invention can occur during and / or subsequent to the growth of the plant to a desired stage of development. Modulating nucleic acid expression over time and / or modulating nucleic acid expression in a particular tissue can be performed, for example, in a sense orientation or an antisense orientation, as discussed in more detail above. It can be controlled by using a suitable promoter operably linked to Induction of expression of the polynucleotides of the present invention can also be controlled by exogenous administration of an effective amount of the inducing compound. Inducible promoters and inducing compounds that activate expression from these promoters are:
It is well known in the art. In a preferred embodiment, the polypeptide of the invention is regulated in monocotyledonous plants, especially corn.

(Molecular Markers) The present invention provides a method for genotyping a plant containing the polynucleotide of the present invention (genot).
yping) is provided. Optionally, the plant is a monocotyledonous plant (eg,
Corn or sorghum). Genotyping provides a means of identifying homologs of chromosome pairs and can be used to identify segregants in a population of plants. Molecular marker methods are used for phylogenetic studies, to characterize genetic relationships between crop varieties, to identify hybrids or somatic cell hybrids, to map chromosomal segments that result in single genetic traits, to use map-based cloning, And for quantitative genetic studies. For example, Plan
t Molecular Biology: A Laboratory Man
ual, Chapter 7, Clark, Springer-Verlag, Berlini
n (1997). For molecular marker methods, generally And And
rew H. Paterson 1996 (Chapter 2), Genome Map
ing in Plants (Andrew H. Paterson) (Ac
ademic Press / R. G. FIG. Landis Company, Aust
In, Texas, pp. 7-21) The DNA Revolution.
See n.

The particular methods of genotyping in the present invention may use any number of molecular marker analysis techniques, such as, but not limited to, restriction fragment length polymorphism (RFLP). RFLP is a DNA sequence caused by nucleotide sequence variability.
The product of allelic differences between the A restriction fragments. As is well known to those skilled in the art,
RFLP is typically detected by extraction of genomic DNA and digestion with restriction enzymes. Generally, the resulting fragments are separated according to size, and hybridized with a probe, preferably a single copy of the probe. Restriction fragments from homologous chromosomes are indicated. Differences in fragment size between alleles represent RFLP. Accordingly, the present invention further provides for the isolation of genes or nucleic acids of the invention and chromosomal sequences genetically linked to these genes or nucleic acids by R
Provides a means for tracking using techniques such as FLP analysis. The linked chromosomal sequence is within 50 centimorgans (cM) of the gene of the invention, often 40
It is present within cM or within 30 cM, preferably within 20 cM or within 10 cM, more preferably within 5 cM, within 3 cM, within 2 cM, or within 1 cM.

In the present invention, nucleic acid probes used for molecular marker mapping of the nuclear genome of a plant selectively hybridize under selective hybridization conditions to a gene encoding a polynucleotide of the present invention. In a preferred embodiment, a probe is selected from a polynucleotide of the invention. Typically, these probes are cDNA probes or restriction enzyme treated (eg, PstI) genomic clones. Probe length is discussed in more detail above,
Typically, it is at least 15 bases in length, more preferably at least 20, 25
, 30, 35, 40, or 50 bases in length. However, generally, probes are less than about 1 kilobase in length. Preferably, the probe is a single copy probe that hybridizes to a unique locus in the complement of the haploid chromosome. Some exemplary restriction enzymes used for RFLP mapping are EcoRI.
RI, EcoRv, and SstI. As used herein, the term `` restriction enzyme '' refers to a composition that recognizes a particular nucleotide sequence and cleaves that particular nucleotide sequence, alone or in combination with another composition. including.

The method of detecting RFLP involves the following steps: (a) Genomic DN of a plant
Digesting A with a restriction enzyme; (b) under selective hybridization conditions,
A step of hybridizing a nucleic acid probe to the polynucleotide sequence of the present invention in the genomic DNA; (c) a step of detecting RFLP therefrom. Other methods of identifying polymorphic (allelic) variants of the polynucleotides of the present invention can be obtained by using molecular marker techniques well known to those skilled in the art, including the following techniques: 1) Single-strand conformation analysis (SSCA); 2) Denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assay; 4) Allele-specific oligonucleotide (ASO); Use of proteins that recognize (eg, the mutS protein of E. coli); and 6) Allele-specific PCR. Other approaches based on mismatch detection between two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CM
C). Accordingly, the present invention further provides a method for genotyping, comprising the step of contacting a nucleic acid probe with a sample suspected of containing the polynucleotide of the present invention under stringent hybridization conditions.
Generally, the sample is a plant sample; preferably, the sample is suspected of containing a corn polynucleotide (eg, a gene, mRNA) of the present invention. A nucleic acid probe will selectively hybridize under stringent conditions to a subsequence of a polynucleotide of the invention that includes a polymorphic marker. Selective hybridization of a nucleic acid probe to a polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex
2 shows the presence of the polymorphic marker in the sample. In a preferred embodiment, the nucleic acid probe comprises a polynucleotide of the invention.

(UTR and Codon Preference) Generally, translation efficiency is determined by the 5 ′ non-coding or untranslated region (5 ′ U
TR) was found to be regulated by specific sequence elements. The positive sequence motif includes a translation initiation consensus sequence (Kozak, Nucle
ic Acids Res. 15: 8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al., Nucleic Acids R).
es. 13: 7375 (1985)). As negative elements,
Stable intramolecular 5'UTR stem-loop structure (Muesing et al., Cell 48
: 691 (1987)) and the appropriate AU in the AUG sequence or 5'UTR.
A short open reading frame behind G (Kozak, supra, Rao et al.
Mol. and Cell. Biol. 8: 284 (1988)). Accordingly, the present invention provides 5 'and / or 3' UTR regions for regulating translation of a heterologous coding sequence.

Further, the polypeptide coding segments of the polynucleotides of the present invention may be modified to alter codon usage. The altered codon usage can be used to alter translation efficiency and / or optimize the coding sequence for expression in a desired host (eg, for expression in maize, codon usage in a heterologous sequence). Frequency can be optimized). Codon usage in the coding regions of the polynucleotides of the present invention can be determined using commercially available software packages (eg, Universal).
site of Wisconsin Genetics Computer
"Codon Preference" available from the Group (Dever
eaux et al., Nucleic Acids. Res. 12: 387-395 (1
984)) or MacVector 4.1 (Eastman K).
Odak Co. New Haven, Conn. )) Can be used to analyze statistically. Accordingly, the present invention provides a characteristic codon usage for the coding region of at least one polynucleotide of the present invention. The number of polynucleotides that can be used to determine codon usage can be any integer from 1 to the number of polynucleotides of the invention as provided herein. If necessary,
A polynucleotide is a full-length sequence. Exemplary numbers of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.

(Sequence Shuffling) The present invention provides a method for sequence shuffling using the polynucleotide of the present invention,
And compositions resulting therefrom. Sequence shuffling is described in PCT Publication No. WO 97/20078. (Zhang, JH, et al., Proc.
Natl. Acad. Sci. USA 94: 4504-4509 (1997).
See also). In general, sequence shuffling provides a means for generating a library of polynucleotides having desired characteristics that can be selected or screened. Libraries of recombinant polynucleotides are generated from a population of polynucleotides of related sequences that have substantial sequence identity and include sequence regions that can be homologously recombined in vitro or in vivo. A population of polynucleotides that have recombined sequences include a subpopulation of polynucleotides that possess the desired or advantageous characteristics, and can be selected by a suitable selection or screening method. A feature can be any property or attribute that can be selected or detected in the screening system and can include the following properties:
Encoded protein, transcription element, transcription, RNA processing, RNA
Sequences, replication elements, protein binding elements, etc. that control stability, chromatin conformation, translation, or other expression characteristics of the gene or transgene (eg, any feature that confers selectable or detectable properties). . In some embodiments, the selected features, as provided herein, an increase in the reduction and / or K _cat of K _m for wild-type protein. In other embodiments, the proteins or polynucleotides generated from sequence shuffling have a higher ligand binding affinity than unshuffled wild-type polynucleotides. Such an increase in properties is at least 110% greater than the wild-type value.
%, 120%, 130%, 140%, or at least 150%.

Generic and Consensus Sequences The polynucleotides and polypeptides of the present invention further include those comprising: (a) the inclusion of at least two homologous polynucleotides or polypeptides of the present invention, respectively. And (b) consensus sequences of at least three homologous polynucleotides or polypeptides of the invention, respectively. The generic sequences of the present invention include each species of polypeptide or polynucleotide encompassed by a general polypeptide sequence or polynucleotide sequence, respectively.
Individual species encompassed by a polynucleotide having a consensus sequence of amino acids or nucleic acids can be used to produce antibodies for screening homologs in other species, genus, families, orders, classes, phyla, or kingdoms Alternatively, a nucleic acid probe or primer may be generated. For example, using a polynucleotide having a consensus sequence from the Zea mays gene family, wheat, rice,
Alternatively, antibodies or nucleic acid probes or primers to other Gramineae species such as sorghum may be generated. Alternatively, a polynucleotide having a consensus sequence generated from an orthologous gene may be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence will have at least 9, 10, 15, 20, 25,
It is 30, or 40 amino acids in length, or 20, 30, 40, 50, 100, or 150 nucleotides in length. As the skilled artisan will know, conservative amino acid substitutions may be used for amino acids that differ between the aligned sequences but are derived from the same conservative substituents discussed above. If necessary, no more than one or two conservative amino acids are substituted for each 10 amino acid length of the consensus sequence.

[0175] Similar sequences used for the generation of consensus or global sequences include any number of allelic modifications of the same gene, orthologous or paralogous sequences, as provided herein. Body and combinations thereof. Optionally, similar sequences used in generating a consensus or global sequence are identified using the minimum total probability (P (N)) of the BLAST algorithm. Various suppliers of sequence analysis software are available from Current P
rotocols in Molecular Biology, F.R. M. Au
Subel et al., Current Protocols (Greene Pub)
lishing Associates, Inc. And John Wiley & S
ons, Inc. (Supplement 30). In comparing a test nucleic acid to a reference nucleic acid, the minimum sum probability is less than about 0.1, more preferably less than about 0.01 or less than about 0.001, and most preferably less than about 0.0
If less than 001 or less than about 0.00001, the polynucleotide sequence is:
Consider similar to the reference sequence. Similar polynucleotides can be aligned and are available from a number of commercial suppliers (eg, Genetics Computer Group).
p (Madison, WI) PILEUP software, Vector NT
ALINX of I (North Bethesda, MD) or Genec
Consensus sequences or generic sequences can be generated using multiple sequence alignment software available from SEQUENCER, Inc., Ann Arbor, MI. Conveniently, such software default parameters can be used to generate a consensus or global sequence.

(Homology Search) The present invention provides: 1) a device comprising a storage device containing data representing the sequence of the polynucleotide or polypeptide of the present invention; 2) embodied in a computer-readable medium. A data structure comprising the sequence of the polynucleotide of the present invention; and 3) a process for identifying candidate homologs of the polynucleotide of the present invention. Candidate homologs have a statistically significant probability of having the same function (eg, catalyze the same reaction) as the compared reference sequence. Unless otherwise given,
Software terms, electrical terms, and electronics terms used herein are based on The New IEEE Standard Dictionary of
Electrical and Electronics Terms (5th
Edition, 1993).

[0177] The device of the present invention is typically a digital computer. Storage devices for such devices include, but are not limited to, ROM or RAM, or computer readable media (eg, magnetic media such as a computer disk or hard drive, or media such as a CD-ROM). Not done)
But not limited thereto. As will be appreciated by those skilled in the art, the form of storage of the apparatus of the present invention is not a critical element of the present invention and may take various forms.

The process of the invention involves obtaining data representing a test sequence of a polynucleotide or polypeptide. Test sequences are generally at least 25 amino acids in length, or at least 50 nucleotides in length. Optionally, the test sequence is at least 50, 100, 150, 200, 250, 300, or 4
It can be as long as 00 amino acids. The test polynucleotide has at least 50, 1
It can be 00, 200, 300, 400, or 500 nucleotides in length.
Often, the test sequence is a full-length sequence. Test sequences can be obtained from animal or plant nucleic acids. Optionally, the test sequence is obtained from a plant species other than corn whose function is uncertain, but is compared to the test sequence to determine sequence similarity or sequence identity; for example, such a plant species It can be a species of the Gramineae family (eg, wheat, rice, or sorghum). The test sequence data is input to an instrument (typically, a computer) provided with a storage device containing data representing the reference sequence. The reference sequence can be the sequence of a polypeptide or polynucleotide of the invention, and is often at least 25 amino acids or 100 nucleotides in length. As those skilled in the art know, the higher the sequence identity / similarity between a reference sequence of known function and a test sequence, the greater the likelihood that the test sequence will have the same or similar function as the reference sequence.

The device further comprises sequence comparison means for determining sequence identity or sequence similarity between the test sequence and the reference sequence. Exemplary sequence comparison means are provided in the sequence analysis software discussed above. If necessary, sequence comparisons are established using the program's BLAST package.

The results of a comparison between a test sequence and a reference sequence can be displayed. Generally, using the algorithm's BLAST 2.0 package under default parameters, less than 0.1, or less than 0.01, less than 0.001, less than 0.0001,
Alternatively, a value of the minimum total probability (P (N)) of less than 0.00001 identifies the test sequence as a candidate homolog (ie, an allele, ortholog, or paralog) of the reference sequence. Nucleic acids, including polynucleotides having the sequence of a candidate homolog, can be constructed using well-known library isolation, cloning, or in vitro synthetic chemistry techniques (eg, phosphoramidites) as described herein. . In a further embodiment, a nucleic acid comprising a polynucleotide having a sequence represented by a candidate homolog is introduced into a plant; typically, these polynucleotides are
Operably linked to a promoter. Confirmation of the function of the candidate homolog can be achieved, for example, by operably linking the candidate homolog nucleic acid to an inducible promoter, or expressing the antisense transcript and in the phenotype.
This can be achieved by analyzing the plant for changes consistent with the putative function of the candidate homolog. If necessary, the plant into which these nucleic acids are introduced may be Gram.
Monocotyledonous plants, such as those from the family Ineae. Exemplary plants include corn, sorghum, wheat, rice, canola, alfalfa, cotton, and soybean.

Assays for Compounds that Modulate Enzyme Activity or Expression The present invention also provides for binding (eg, a substrate) to a catalytically active polypeptide of the invention and / or increasing or decreasing its enzymatic activity. It provides a means to identify compounds that are (ie, modulate). The method includes the step of contacting the polypeptide of the invention with a compound whose ability to bind or to modulate enzymatic activity is to be determined. The polypeptide used will be at least 20%, preferably at least 30% or 40% of the native full-length polypeptide of the invention (eg, an enzyme).
%, More preferably at least 50% or 60%, and most preferably at least 70% or 80%. Generally, polypeptides will be present in a range sufficient to determine the effect of the compound (typically about 1 nM to 10 μM). Similarly, compounds are present at a concentration of about 1 nM to 10 μM. One skilled in the art will recognize enzyme concentrations, ligand concentrations (ie, substrates, products, inhibitors, activators).
It is understood that factors such as pH, ionic strength, and temperature are controlled to obtain useful kinetic data and to determine the presence or absence of compounds that bind or modulate polypeptide activity. Methods for measuring enzyme reaction rates are well known in the art. For example, Segel, Biochemical Calc
ulations, 2nd edition, John Wiley and Sons, New Y
ork (1976).

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be made within the scope of the appended claims. It is clear.

Example 1 This example describes the construction of a cDNA library.

(Isolation of Total RNA) Total RNA was isolated from Chomczynski and Sacchi (Chomczyn).
ski, P .; And Sacchi, N .; Anal. Biochem. 162,
156 (1987)) using a modified version of the guanidine isothiocyanate / acid-phenol procedure described in the TRIZOL reagent (Life Technology).
Inc. Gaitersburg, MD). Briefly, plant tissue samples were triturated in liquid nitrogen prior to the addition of TRIZOL reagent, and then further homogenized in a mortar and pestle. Chloroform addition was performed before centrifugation for separation of the aqueous and organic phases. Total RNA
Was recovered by precipitation from the aqueous phase with isopropyl alcohol.

(Isolation of Poly (A) + RNA) Selection of poly (A) + RNA from total RNA was performed using the POLYATTRACT system (m
RNA isolation system Promega Corporation. Madison, W
Performed using I). Briefly, biotinylated oligo (dT) primers were used to hybridize to the 3 'poly (A) tail of the mRNA.
Using streptavidin conjugated to paramagnetic particles and a magnetic separation stand,
The hybrid was captured. The mRNA was washed under high stringency conditions and eluted with RNase-free deionized water.

Construction of cDNA Library cDNA synthesis was performed and SUPERSCRIPT Plasmid S
system (Life Technology Inc. Gaithersbu)
rg, MD) was used to construct a unidirectional cDNA library. cDNA
Was synthesized by priming an oligo (dT) primer containing a NotI site. The reaction was performed using a SUPERSCRIPT Reverse T
Catalyzed at 45 ° C. by transcriptase II. cDNA second
The strand was labeled with α- ³² P-dCTP, and a portion of the reaction was analyzed by agarose gel electrophoresis to determine the size of the cDNA. CDN less than 500 base pairs
A molecule and unligated adapter were removed by Sephacryl-S400 chromatography. The selected cDNA molecule is converted into pSPOR
Ligation was performed between the NotI and SalI sites in the T1 vector.

Example 2 This example describes cDNA sequencing and library subtraction.

Preparation of Sequencing Templates Individual colonies were picked and DNA prepared either by PCR using M13 forward and M13 reverse primers or by plasmid isolation. All cDNA clones were sequenced using the M13 reverse primer.

(Q-bot Subtraction Procedure) The cDNA library subjected to the subtraction procedure was plated on a 22 × 22 cm ² agar plate at a density of about 3,000 colonies per plate. The plate was incubated for 12-24 hours in a 37 ° C incubator. A colony is picked up by a robot-type colony picker Q-bot (GENET).
IX Limited) in a 384-well plate. These plates were incubated overnight at 37 ° C.

Once sufficient colonies have been picked up, they can be
It was fixed on a 2 × 22 cm ² nylon membrane. Each membrane is 9,21
Six or 36,864 colonies were included. These membranes were placed on agar plates with the appropriate antibiotic. The plate was incubated overnight at 37 ° C.

After collecting the colonies on day 2, the filters were placed on filter paper pre-moistened with denaturing solution for 4 minutes, and then incubated for another 4 minutes on top of a boiling water bath. The filters were then placed on filter paper pre-wetted with neutralizing solution for 4 minutes. After removing the excess solution by placing the filter on dry filter paper for 1 minute, the colony side of the filter was placed in Proteinase K solution,
Incubated at 37 ° C for 40-50 minutes. The filter was placed on dry filter paper and dried overnight. The DNA was then cross-linked to a nylon membrane by UV light treatment.

Colony hybridization was performed as described by Sambrook, J. et al. , Fritsc
h. F. And Maniatis, T .; (Molecular Cloni
ng: A Laboratory Manual, 2nd edition). The following probes were used in colony hybridization: 1. First-strand cDNA from the same tissue from which the library was made, to remove most duplicate clones. 2. 48-192 most duplicated cDNA clones from the same library, based on previous sequencing data; 192 most duplicated cDNs in the entire corn sequence database
A clone, 4. Sal-A20 oligonucleotide listed in SEQ ID NO: 3: TCG AC
C CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA
4. AAA (except for clones containing a poly A tail but no cDNA); cDNA clone derived from rRNA. The images of the autoradiography were scanned with a computer and the signal intensity of each colony and the location of the non-radioactive colonies (cold colony addr)
ess) was analyzed. Relocation of non-radioactive colonies from 384-well plates to 96-well plates was performed using Q-bot.

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

The identity of the gene was determined using the BLAST “nr” database (all non-redundant Ge
nBank CDS translation product, three-dimensional structure Brookhaven Proteini
under the default parameters, for similarities to sequences contained in sequences from the Data Bank (including the latest major publications in the SWISS-PROT protein sequence database, EMBL, and the DDBJ database).
LAST (Basic Local Alignment Search To
ol; Altschul, S .; F. (1990); Mol. Biol. 2
15: 403-410; www. ncbi. nlm. nih. gov / BLAS
(See also T /) A search was performed to determine. The cDNA sequence was converted to BLAS
The TN algorithm was used to analyze similarities to all publicly available DNA sequences contained in the "nr" database. The DNA sequence is translated in all open reading frames and the BLASTX algorithm provided by NCBI (Gish, W. and States, DJ Nature Geneti).
cs 3: 266-272 (1993)) was used to compare similarities to all publicly available protein sequences contained in the "nr" database. In some cases, sequencing data from two or more clones containing overlapping segments of DNA was used to construct a contiguous DNA sequence.

Example 4 This example demonstrates the relevant features of the large subunit (R1) polypeptide of corn ribonucleotide reductase (SEQ ID NO: 2).

[0196]

Embedded image Characteristic sequences for class Ia ribonucleotide reductase are shown in bold. Amino acid residues shown in italics are conserved in all higher eukaryotes. These residues highlighted are aligned with amino acids known to be important for the substrate or subunit (R1-R2) interaction of this Class Ia enzyme.

The above examples are provided to illustrate the present invention, but not to limit the scope of the invention. Other modifications of the present invention will be readily apparent to one skilled in the art, and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

[Sequence list]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference ) 2B030 AA07 CA15 CA17 CA19 4B024 AA08 BA08 CA04 DA01 EA04 FA02 GA11 GA17 4B050 CC03 DD13 EE10 LL10 4B065 AA88X AA88Y AC14 BA02 CA28 CA53

Claims (21)

[Claims] 1. An isolated ribonucleotide reductase large subunit R1 nucleic acid, comprising: (a) a polynucleotide having at least 80% sequence identity to the polynucleotide of SEQ ID NO: 1; Wherein the sequence identity is determined by the GAP algorithm under default parameters; (b) a polynucleotide encoding a polypeptide of SEQ ID NO: 2; (c) under stringent hybridization conditions, A polynucleotide amplified from a Zea mays nucleic acid library using primers that selectively hybridize to loci within the polynucleotide of SEQ ID NO: 1; (d) stringent hybridization conditions and 0.1 × SSC
A polynucleotide that selectively hybridizes to the polynucleotide of SEQ ID NO: 1 under washing at medium 60 ° C .; (e) a polynucleotide of SEQ ID NO: 1; and (f) (a), (b), ( c) a polynucleotide complementary to the polynucleotide of (d) or (e), a nucleic acid comprising a member selected from the group consisting of: 2. A recombinant expression cassette comprising the member of claim 1 operably linked to a promoter in a sense or antisense orientation. 3. A plant host cell comprising the recombinant expression cassette according to claim 2. 4. A transgenic plant comprising the recombinant expression cassette according to claim 2. 5. The transgenic plant according to claim 4, wherein said plant is a monocotyledonous plant. 6. The transgenic plant according to claim 4, wherein said plant is a dicotyledonous plant. 7. The plant, wherein the plant is corn, soybean, sunflower, sorghum,
The transgenic plant according to claim 4, which is selected from the group consisting of canola, wheat, alfalfa, cotton, rice, barley and millet. 8. A transgenic seed derived from the transgenic plant according to claim 4. 9. A method for regulating the level of corn ribonucleotide reductase large subunit R1 in a plant, comprising: (a) the corn R according to claim 1, operably linked to a promoter.
(B) culturing the plant cells under conditions in which the plant cells proliferate; (c) transforming the plant having the transformed genotype into a plant cell. Regenerating; and (d) inducing expression of said polynucleotide for a period of time sufficient to modulate the level of corn R1 in said plant. 10. The method according to claim 9, wherein said plant is corn. 11. An isolated ribonucleotide reductase large subunit R1 protein, comprising: (a) a polypeptide of SEQ ID NO: 2; (b) at least 90% sequence relative to the polypeptide of SEQ ID NO: 2. A polypeptide having identity and having at least one linear epitope in common with the polypeptide of SEQ ID NO: 2; and (c) at least one polypeptide encoded by a member of claim 1; A protein comprising a member selected from the group consisting of: 12. A method for increasing transformation efficiency, comprising the steps of: converting at least one ribonucleotide reductase large subunit R1 polynucleotide or at least one ribonucleotide reductase large subunit R1 polypeptide and a polynucleotide of interest into a plant host cell. To produce transformed cells, and growing the transformed cells under conditions that allow the cells to grow, wherein each of the polynucleotides is operably linked to a promoter. ,Method. 13. The method according to claim 12, wherein said plant cells are derived from monocotyledonous plants or dicotyledonous plants. 14. The method of claim 12, wherein said plant cell is a corn cell. 15. A method of producing male sterility in a plant, comprising using the appropriate promoter under developmental control. 16. The ribonucleotide reductase large subunit R
16. The method of claim 15, wherein one polynucleotide is operably linked to said promoter in a sense orientation. 17. The method of claim 15, wherein said ribonucleotide reductase large subunit polynucleotide is operably linked to said promoter in an antisense orientation. 18. A transgenic plant according to claim 15. 19. The transgenic plant according to claim 16, wherein said plant is a monocotyledonous or dicotyledonous plant. 20. The transgenic plant of claim 16, wherein said plant is selected from the group consisting of corn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet. 21. A transgenic seed derived from the transgenic plant according to claim 16.