JP2002543782A - Novel corn RAD51-like gene and use thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides isolated maize RAD51 nucleic acids and their encoded proteins. The present invention provides methods and compositions related to altering corn RAD51 levels in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. Controlling homologous recombination by regulating RAD51 provides a means of regulating the efficiency with which the nucleic acid of interest is integrated into the genome of the target plant cell.

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 [0002] Cellular DNA undergoes double-strand breaks during the course of many physiological events and in response to various environmental seizures (Friedburg, E., Walker, G. et al.
And Seede, W .; , DNA Repair and Mutagenes
is, ASM Press, Washington DC. , 1995; Nic
kollof J .; And Hoekstra, M .; , DNA damage a
nd Repair, Humana Press,. Totowa, NJ, 19
98). Such double-strand breaks (DSBs) that remain and are not repaired lead to mutations that can be tested for lethality to the organism. Thus, these DSBs are immediately repaired via two independent pathways: i) homologous recombination ii) non-homologous end joining (Friedburg, E., Walker, G. and Siede, W., et al.
DNA Repair and Mutagenesis, ASM Press
, Washington DC. Nickollof J., 1995; And Hoekstra, M .; , DNA Damage and Repair, Hu
mana Press, Totowa, NJ, 1998).

The first pathway involves a series of highly specific biochemical reactions catalyzed by a complex of cellular proteins (Shinohara and Ogawa, Trend).
s in Biochem. Sci. 237: 387-391.1995). Due to the large number of proteins contained in this complex, it is referred to as "recombinosomes" (Hays et al., Proc. N
atl. Acad. Sci. ScL USA 92: 6925-6929, 1995).
This pathway is the dominant mode of DSB repair in lower eukaryotes, such as yeast (Nickollof J. and Hoekstra, M., DNA Dama).
Ge and Repair, Humana Press, Totowa, NJ
, 1998). Thus, yeast has been used as a model eukaryote to study the biochemical and molecular details of double-strand break repair. RAD51 is one of the genes of the RAD52 epistasis group associated with this pathway. This gene is about 3
It encodes an 8 kDa protein (Rad51). Rad51 is a structural and functional homolog of the bacterial recombinase enzyme (also known as RecA).

Because of the critical role of the double-strand break repair pathway in maintaining genomic stability, they have been found to be conserved throughout evolution. As a result, RAD51 homologs have been discovered and characterized from a number of animal and plant sources (O
gawa et al., Cold Spring Harbor Symp. On Qua
nt. Biol. , LVIII, pp. 567-576, 1993). further,
Many eukaryotes have multiple forms of the RAD51 gene. The RAD51 family also contains structurally and functionally related genes (eg, DMC1, LI
M15, RAD57). DMC1 is involved in meiotic recombination and related double-strand breaks (Ogawa et al., Cold Spring Harbor S).
ymp. On Quant. Biol. VLIII, pp. 567-576,
1993).

[0005] All members of the RAD51 family and their bacterial counterparts (Rec
A) shares an important structural motif known as the "RecA signature sequence" or domain II. This sequence forms the ATP binding site, a key property of all these proteins. However, eukaryotic members of the RAD51 family have a RAD51 family member and a C-terminal extension of about 100 amino acids from the bacterial RecA protein (which is present in RecA, but not RAD51).
(Not present in family members). Significant differences also exist in the primary structure of eukaryotic RAAD51 and other closely related genes (RAD55 and RAD57), indicating a different physiological role for each of these genes (Johnson, R .; D.
And Symington, L .; S. , Mol. Cell. Biol. , 154
843-4850, 1995). The RAD55 and RAD57 protein products have been suggested to interact with the RAD51 gene product during double-strand break repair and genetic recombination. Germinated yeast Saccharomyces Ce
revisiae also belongs to the RAD52 epistasis group (Kans, J. et al.
And Mortimer, R.A. Gene 105: 139-140, 1991).
. All members of this group are required for double-strand break repair and genetic recombination. The RAD57 gene is derived from bacterial RecA and its eukaryotic counterpart (Rad57).
(Jeggo, P. Radiat. Res. 150: S80-S91, 1998).
). Specifically, the ATP binding motif (Walker Box A) is conserved in all known Rad57 sequences (Hays, S. et al., Proc. Natl. A).
cad. Sci. 92: 6925-6929, 1995). Functional analysis has revealed the interaction of Rad57 with Rad51, Rad52 and Rad55 to form a "recombinosome" (Joh).
nson, R .; Et al., Mol. Cell. Biol. 15: 4843-4850,
1995). Furthermore, in yeast, Rad57-Rad55 heterodimers significantly stimulate DNA strand exchange by Rad51 (Sung P. Genes).
Developer. 11: 111-121, 1997).

[0006] Biochemical studies have established that Rad51 catalyzes the strand exchange reaction between circular ssDNA and linear DNA in the presence of ATP and Mg ²⁺ (Sun
g, P. Science, 265: 1241-1243, 1994; Sung,
P. And Roberson, D .; L. Cell 82: 453-461, 1
995). These properties are very similar to RecA. However, unlike bacterial proteins, Rad51 slows strand exchange and requires the presence of complementary 3 'and 5' overhangs. Rad51 does not promote ligation molecule formation when linear DNA has blunt or concave complementary ends. As a result of this requirement for the presence of an overhang, Rad51, which catalyzes a strand exchange reaction, is polar (Sun
g, P. Science, 265: 1241-1243, 1994; Sung,
P. And Roberson, D .; L. Cell 82: 453-461, 1
995). RecA also binds to ssDNA, or partially single-stranded DNA, while Rad51 shows a similar binding affinity for ssDNA and dsDNA. These biochemical observations, combined with extensive genetic studies, indicate the involvement of additional proteins in eukaryotic recombination and double-stranded repair reactions.

[0007] Recent studies have revealed several RAD51 genes in higher eukaryotes (Vispe, S. and Defais, D. Biochimie 7).
9: 587-592, 1997). For example, at least four human RAD51 genes, RAD51 (Yoshimura, Y. Morita, T., Yamamo
to, A. And Matsushiro, A .; Mol. Cell. Biol. 2
1,1665), RAD51B / REC2 (Rice, MC, Smith,
S. T. Bullrich, F .; Havre, P .; And Kmiec, E
. B. Proc. Nalt. Acad. Sci. 94, 7414-7422
Albala, J .; S. , Thelen, M .; P. Prange, C .; , F
an, W.S. , Christensen, M .; Thompson, L .; H. And Lennon, G .; G. FIG. Gemonics 46, 476-479, 1998.
), RAD51C (Dosanjh, M., Collins, D., Fan, W
. Lennon, G .; C. , Albala, J .; Shen, Z .; And Sch
ild, D .; Nucleic Acid Res. 26, 1179-1184,
1998) and RAD51D (Pittman, D., Weinberg, L.
. And Shimenti, J. et al. Genomics, 49 103-111)
Have been cloned and characterized.

[0008] The presence of multiple isoforms of the RAD51 gene product in higher eukaryotes suggests their different functional roles in meiotic versus mitotic recombination. Interestingly, yeast RAD51 is not an essential gene,
The D51 null mutation is embryonic lethal (T
suzuki, T .; Fujii, Y .; Sakumi, K .; , Tominag
a, Y. , Nakao, K .; , Sekiguchi, M .; , Matsushir
o, A. , Yoshimura, Y .; And Morita, T .; Proc.
Natl. Acad. Sci. 93, 6236-6240, 1996). RAD
Biochemical and genetic analysis of 51 different orthologs (preferably from the same species) will help elucidate their exact function. Nevertheless, two very recent studies have shown that overexpression of Rad51 protein stimulates homologous recombination and that immortalized human cells (Xia, S., Shammas, M .;
, Shookler Reis, R .; , And Mol. Cell. Biol.
17, 7151-7158, 1997) and Chinese hamster cells (V
ispe, S .; , Cazaux, C .; , Lesca, C.E. And Defais,
M. , Nucleic Acid Res. 26,2859-2864,199
8) increase the resistance to ionizing radiation.

In view of the central role of the RAD51 gene in recombination and double-strand repair, the RAD51 gene from maize is generally used as a tool for improving maize transformation, and particularly maize gene targeting. Find out. The present invention provides a full-length cDN for a novel corn ortholog of RAD51.
A, which shows high homology to the human RAD51C gene.

[0010] Controlling homologous recombination by regulating RAD51 provides a means of regulating the efficiency with which the nucleic acid of interest is integrated into the genome of the target plant cell. Control of these processes has important implications in the production of new recombinantly engineered cereals (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 corn RAD51. 1): an antigenic fragment of the protein of the present invention,
2): a transgenic plant comprising the nucleic acid of the invention, 3): it is an object of the invention to provide a method for regulating the expression of the nucleic acid of the invention in a transgenic plant.

Thus, in one aspect, the present invention provides (a) a polynucleotide having a particular sequence identity to a polynucleotide encoding a polypeptide of the present invention;
(B) a polynucleotide that is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising a specified number of contiguous nucleotides from the polynucleotide of (a) or (b). The present invention relates to an isolated nucleic acid containing a selected member. The isolated nucleic acid can be DNA.

[0013] In another aspect, the invention relates to a recombinant expression cassette comprising a nucleic acid of the invention operably linked to a promoter.

[0014] In another aspect, the present invention relates to a host cell into which the above-mentioned recombinant expression cassette has been introduced.

[0015] 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.

[0016] In another aspect, the present invention provides an isolation comprising a polynucleotide of a specific length that selectively hybridizes under stringent conditions to a polynucleotide of the invention or a complement thereof. Related nucleic acids. 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 the above-mentioned library, wherein 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.

[0018] In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any isolated nucleic acid of the present invention.

Definitions Units, prefixes, and symbols may be indicated in SI accepted form. Unless otherwise indicated, nucleic acids are described from left to right in the 5 'to 3' direction, and amino acid sequences are described from left to right, amino to carboxy. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids are referred to herein as IUPAC-IUB Biochemical.
It may be mentioned by any of their commonly known three-letter symbols or one-letter symbols recommended by Nomenclature Commission. Nucleotides may also be referred to by their generally accepted one letter code. Unless otherwise provided, software, electrical, and electronic terms, as used herein, are used in the New IEEE Standard.
Dictionary of Electric and Electro
nics Term (5th edition, 1993). The terms defined below are more fully defined by reference to the entire specification.

By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. The amplification system includes a polymerase chain reaction (PCR) system, a ligase chain reaction (LCR) system, and amplification based on nucleic acid sequences (NASBA, Cangene, Mi).
ssissauga, Ontario), the Q-Beta lipase system, the transcription-based amplification system (TAS), and the strand displacement amplification (SDA). For example, D
iagnostic Molecular Microbiology: Pri
nipples and Applications, D.C. H. Persing
Ed., American Society for Microbiology
y, Washington, D.C. C. (1993). The product of the amplification is named amplicon.

The term “antibody” includes reference to an antigen-binding form of the antibody (eg, Fab, F (ab) ₂ ). The term “antibody” frequently refers to a polypeptide substantially encoded by an immunoglobulin gene (s) or fragment thereof that specifically binds and recognizes an analyte (antigen). But,
Various antibody fragments can be defined for digestion of intact antibodies,
One skilled in the art will recognize that such fragments can be obtained de novo, chemically or recombinantly.
It is understood that it can be synthesized either using the NA method. Thus, the term antibody, as used herein, also refers to single-chain Fvs, antibody fragments such as chimeric antibodies (ie, including constant and variable regions from different species), humanized antibodies (ie, (Comprising complementarity determining regions (CDRs) from non-human sources), and heteroconjugate antibodies (eg, bispecific antibodies).

The term “antigen” includes reference to a substance against which an antibody can be generated and / or against which the antibody is specifically immunoreactive. Specific immunoreactive sites within an antigen are known as epitopes or antigenic determinants. These epitopes can be linear sequences of monomers (eg, amino acids in proteins) in the polymeric composition, or can consist of or include more complex secondary or tertiary structures. One skilled in the art will recognize that all immunogens (ie, substances capable of eliciting an immune response) are antigens; however, some antigens (eg, haptens) bind to carrier molecules rather than immunogens. Can be made immunogenic. Antibodies immunoreactive with a particular antigen can be produced in vivo or by recombinant methods (eg, selection of a library of recombinant antibodies in phage or similar vectors). For example, Huse et al., Science 246:
1275-1281 (1989); and Ward et al., Nature 341:
544-546 (1989); and Vaughan et al., Nature Bi.
otech. 14: 309-314 (1996).

As used herein, “antisense orientation” includes reference to a duplex polynucleotide sequence operably linked to a promoter in the direction 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 by reference to a linear segment of DNA that it contains. This chromosomal region can be defined by reference to two unique DNA sequences (ie, 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 variant of the amino acid sequence or a conservatively modified variant. Due to the degeneracy of the genetic code, a large number of 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 an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide.
Such nucleic acid mutations are “non-phenotypic mutations” and represent one species of conservatively modified mutation. Every nucleic acid sequence herein which encodes a polypeptide also describes, by reference to the genetic code, every possible non-phenotypic variation of the nucleic acid. 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 the only codon for tryptophan) can be modified to yield functionally identical molecules. To understand the. Accordingly, each non-phenotypic variation of a nucleic acid which encodes a polypeptide of the invention is implicit in each described polypeptide sequence and is within the scope of the invention.

With respect to amino acid sequences, one of skill in the art will recognize individual substitutions, deletions in nucleic acid, peptide, polypeptide or protein sequences that alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence. Alternatively, it is understood that the addition is a “conservatively modified variant”. Here, the alteration results in the replacement of an amino acid with a chemically similar amino acid. Therefore, any number of amino acid residues selected from the group of integers consisting of 1 to 15 can be changed in this way. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 changes may be made. Conservatively modified variants typically provide a biological activity similar to the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity,
Or ligand / receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 8% of the native protein relative to its native substrate.
0% 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.

By “encode” or “encoded” with respect to a particular nucleic acid is meant to include information about translation into a particular 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 from which proteins are encoded is specified by the use of codons. Typically, amino acid sequences are encoded by nucleic acids using the "universal" genetic code. However, variants of the universal code (eg, any plant, animal, and fungal mitochondria, the bacterium Mycoplas)
ma capricolum, or those present in the cilia Macronucleus) can be used when the nucleic acid is expressed in them.

When the nucleic acid is prepared or modified synthetically, the known codon preference of the host of interest in which the nucleic acid is expressed can be used. For example, the nucleic acid sequences of the present invention can be expressed in both monocot and dicot species, but have been shown to have different codon choices (Murray et al., Nuc).
l. Acids Res. 17: 477-498 (1989)).
Monocotyledons or dicots can be modified to take into account the particular codon choice and GC content choice. Thus, the preferred corn codons for a particular amino acid may be derived from a known gene sequence from corn. 28 from corn plants
Maize codon usage for this gene is listed in Table 4 of Murray et al., Supra.

As used herein, a “full-length sequence” with reference to a particular polynucleotide or its encoded protein refers to the native (non-synthetic), endogenous, biological, Having the entire amino acid sequence in a more active form. Methods for determining whether a sequence is full length are well known in the art and include exemplary techniques such as Northern or Western blots, primer extension, S1 protection, and ribonuclease protection. For example, P
lant Molecular Biology: A Laboratory
Manual, edited by Clark, Springer-Verlag, Berlin
(1997). Known full-length homology (ortholog (ortholog)
ou and / or paralogous) sequences can also be used to identify full length sequences of the invention. Further, typically, mR
Consensus sequences present in the 5 'and 3' untranslated regions of NA aid in identifying the polynucleotide as full length. For example, the consensus sequence, ANNNN A UG G (here, the codon underlined, represents an N-terminal methionine), the polynucleotide assist in determining whether 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” with reference to a nucleic acid may refer to a nucleic acid derived from a foreign species or, if derived from the same species, by intentional human intervention, in a comparison. A nucleic acid that is substantially modified from its native form and / or locus. 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, has one or both of their native origin. Has been substantially modified. A heterologous protein can be derived from an exogenous species, or, if 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 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.

“Immunologically reactive conditions” or “immunoreactive conditions” are defined as conditions in which an antibody reactive to a particular epitope is substantially free of other antibodies in a reaction mixture containing the particular epitope. Refers to conditions that allow binding to that epitope to a detectably greater degree (eg, at least twice background) than to bind to all epitopes. 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 La.
ne, Antibodies, A Laboratory Manual, Co
ld Spring Harbor Publications, New Yo
rk (1988).

The term “introduced” in the context of the insertion of a nucleic acid into a cell means “transfection” or “transformation” or “transduction”, and prokaryotic or eukaryotic cells of the nucleic acid Contains a reference to the ingestion. Where the nucleic acid is
It can be integrated into the genome of the cell (eg, chromosome, plasmid, plastid or mitochondrial DNA), converted to an autonomously replicating replicon, or expressed transiently (eg, a transfected mRNA).

The term “isolated” refers to a substance such as a nucleic acid or 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. The isolated material optionally includes materials that are not found with the material in its natural environment. Or (2) if the substance is in its natural environment, the substance is synthetically (non-naturally) converted to a composition by intentional human intervention and / or is found in the environment. Is located at a location in the cell that is not native to (eg, a genomic or intracellular organelle). This alteration to produce the synthetic material can be performed on the material in or removed from its natural state. For example, a naturally-occurring nucleic acid becomes an isolated nucleic acid when altered or transcribed from altered DNA by human intervention performed in the cell from which it originated. For example, Co
mounds and Methods for Site Direct
d Mutagenesis in Eukaryotic Cells, Km
iec, US Patent No. 5,565,350; In Vivo Homologo.
us Sequence Targeting in Eukaryotic
See Cells; Zarling et al., PCT / US93 / 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. "Isolated" as defined herein
"Nucleic acids are also referred to as" heterologous "nucleic acids.

Unless otherwise indicated, the term “corn RAD51 nucleic acid” refers to a nucleic acid of the invention and includes nucleic acids comprising a polynucleotide of the invention encoding a corn RAD51 polypeptide (“corn RAD51 polynucleotide”). means. "Maize RAD51 gene" is the gene of the present invention,
And refers to the heterologous genomic form of the full length maize RAD51 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 Contains a reference to the continuous length of the containing chromosome.

As used herein, a “marker” includes a reference to a locus on a chromosome that helps identify a unique location on the chromosome. "Polymorphic markers"
Contains references to markers that appear in multiple forms (alleles), so that different forms of the marker allow transmission of each chromosome in that pair to be followed if they are present in a homologous pair I do. Genotype may be defined by the use of one or more markers.

As used herein, “nucleic acid” includes reference to a polymer of deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and unless otherwise specified. The term includes 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 contains and substantially represents the entire transcribed fraction of the genome of a particular organism. Exemplary nucleic acid libraries (eg, genomic libraries and cD
Construction of NA libraries) is performed using standard molecular biology references (eg, Ber
ger and Kimmel, Guide to Molecular Clon
ing Technologies, Methods in Enzymology
152, Academic Press, Inc. , San Diego
, CA (Berger); Sambrook et al., Molecular Clon.
ing-A Laboratory Manual, 2nd edition, volumes 1-3 (19
89); and Current Protocols in Molecul.
ar Biology, F.R. M. Ausubel et al., Current Pro.
tocols, Greene Publishing Associates,
Inc. And John Wiley & Sons, Inc. Joint venture with (19
94)).

As used herein, “operably linked” includes reference to an operable linkage between a promoter and a second sequence. Here, the promoter sequence initiates and mediates the transcription of a DNA sequence corresponding to the second sequence. In general, operably linked means that the nucleic acid sequences to be linked are contiguous and, if necessary to join the two protein coding regions, contiguous and in the same reading frame. .

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. But not limited to these. The plant family that can be used in the methods of the present invention are generally higher plants of the broadest family that can be subjected to transformation techniques, including both monocots and dicots. Particularly preferred plants are Zea ma
ys.

As used herein, a “polynucleotide” is a deoxyribopolynucleotide, a ribopolynucleotide, or a string of substantially identical nucleotide sequences to a naturally occurring nucleotide under stringent hybridization conditions. Includes references to those analogs that hybridize to and / or retain the essential properties of natural ribonucleotides in that they 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 specified 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 provide many useful purposes known to those of skill in the art. The term polynucleotide, as used herein, includes such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as viruses and cells, including simple and complex cells, among others. And DNA and RNA in chemical forms unique to

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, which 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 analogs of naturally occurring amino acids is that when incorporated into a protein, the antibody 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 completely 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 man-made operations 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. Further, the present invention contemplates the use of both methionine-containing and methionine-free amino-terminal variants of the proteins of the present invention.

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. "Plant promoter"
Is a promoter capable of initiating transcription in plant cells, even if the origin is not from plant cells. Exemplary plant promoters include, but are not limited to, plant promoters obtained from: plants, plant viruses, and Ag
A bacterium containing a gene expressed in a plant cell, such as Robacterium 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 is a particular cell type in one or more organs (eg,
It drives expression mainly in root or leaf vascular cells). An "inducible" or "regulated" promoter is a promoter that is under environmental control. Examples of environmental conditions that can effect transcription by an inducible promoter include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific 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 “corn RAD51 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). "
"Maize RAD51 protein" is a protein of the present invention and encompasses a corn RAD51 polypeptide.

As used herein, “recombinant” includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or 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. Native genes that are not expressed or are not expressed at all are the result of intentional artificial intervention. As used herein, the term “recombinant” refers to a naturally occurring event (eg, a spontaneous mutation, a spontaneous transformation / transduction / transposition), such as a change that occurs without intentional human intervention. ) Does not include cell or vector changes.

[0049] As used herein, a "recombinant expression cassette" is a recombinantly or synthetically produced, having a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. Nucleic acid constructs. The recombinant expression cassette comprises a plasmid,
It can be integrated into chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments. 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, “proteins”). Used interchangeably. This amino acid is
It can be a naturally occurring amino acid and, unless otherwise limited, can include non-natural analogs of the naturally occurring amino acids, which can function in a manner similar to the naturally occurring amino acids.

The term “selectively hybridizes” refers to a detectable greater extent (eg, at least twice background) than its hybridization to non-target nucleic acid sequences under stringent hybridization conditions. Higher) and the reference to hybridization of the nucleic acid sequence to a particular nucleic acid target sequence, such as to substantially eliminate non-target nucleic acids. Selectively hybridizing sequences will have sequence identity to one another, typically of at least about 80%,
Preferably 90% sequence identity, and most preferably 100% sequence identity (
That is, it is complementary).

The term “specifically reacts” includes reference to a binding reaction between an antibody and a protein having an epitope recognized by the antigen binding site of the antibody. This binding reaction determines the presence of a protein with an epitope that is recognized in the presence of a heterogeneous population of proteins and other biologicals. Thus, under certain immunoassay conditions, a particular antibody will have a substantially higher degree than substantially all other analytes lacking this epitope present in the sample (
(E.g., at least more than twice background) binds to the analyte having the recognized epitope.

[0053] Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised 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 provide 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, a solid phase ELISA immunoassay
It is routinely used to select monoclonal antibodies that are specifically immunoreactive with the protein. For a description of immunoassay formats and conditions that can be used to determine selective reactivity, see Harlow and Lane, Antibod.
ies, A Laboratory Manual, Cold Spring
See Harbor Publications, New York (1988).

The term “stringent conditions” or “stringent hybridization conditions” refers to the extent to which a probe is detectably higher than other sequences (eg,
Reference to conditions that hybridize to the target sequence at least at least twice background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow for some mismatches in the sequence, so that a lower degree of similarity is detected (heterologous probing). Generally, probes are less than about 1000 nucleotides in length, and optionally less than 500 nucleotides in length.

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

Specificity is typically a function of post-hybridization washes,
Important factors are the ionic strength and temperature of the final wash solution. DNA-DNA hive
For lid, T_mAre described in Meinkoth and Wahl, Anal. Bi
ochem. , 138: 267-284 (1984): T_m= 81.5
° C + 16.6 (logM) + 0.41 (% GC)-0.61 (% form)-
500 / L (where M is the molar concentration of monovalent cation and% GC is the DNA
Guanosine and cytosine nucleotides
% Form is the percentage of formamide in the hybridization solution
And L is the length of the hybrid in base pairs)
Can be done. T_mIs a probe that perfectly matches 50% of the complementary target sequences.
The temperature at which hybridization occurs (under defined ionic strength and pH). T _m Decreases about 1 ° C. for every 1% of mismatch; thus, T_m, Hybridis
The washing and / or washing conditions are such that they hybridize to sequences of the desired identity.
Can be adjusted to For example, when a sequence having 90% or more identity is required,
T_mCan be reduced by 10 ° C. Generally, stringent conditions are defined
The thermal melting point (T.sub.T) for a particular sequence and its complement at strength and pH_m)
About 5 ° C. lower. However, severe stringent conditions
Thermal melting point (T_m), 2 ° C, 3 ° C, or 4 ° C lower than
Medium and / or washing may be utilized;
Solution point (T_m6), 7 ° C., 8 ° C., 9 ° C., or 10 ° C. lower than
And / or washing; low stringency conditions may include thermal fusion.
Solution point (T_m11 ° C, 12 ° C, 13 ° C, 14 ° C, 15 ° C, or 20 ° C lower than
Hybridization and / or washing may be utilized. This equation, high
Hybridization and washing compositions, and the desired T_mUsing a person skilled in the art
Is the stringency of the hybridization and / or washing solution.
Understand that the variations are essentially described. Desired degree of mistake
T below 45 ° C (aqueous solution) or 32 ° C (formamide solution)_mWith
The SSC concentration is increased so that higher temperatures can be used.
Preferably. There is a great deal of guidance on nucleic acid hybridization
, Tijssen, Laboratory Technologies in Bi
Chemistry and Molecular Biology-Hyb
authorization with Nucleic Acid Probes,
Part I, Chapter 2, "Overview of principals of hy
bridging and the strategy of nucl
eic acid probe assays ", Elsevier, New
York (1993); and Current Protocols in M.
olecular Biology, Chapter 2, Ed. Ausubel et al., Green
e Publishing and Wiley-Interscience,
Found in New 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 the next generation. 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 ( (Including transgenics so modified initially, as well as transgenics produced by sexual crossing or asexual breeding from the original transgenics). The term “transgenic” as used herein, refers to conventional plant breeding methods or naturally occurring events (eg, random allogeneic insemination, non-recombinant viral infections, non-recombinant bacterial transformation, non-recombinant bacterial transformation, It does not include genome (chromosomal or extrachromosomal) modifications due to recombination or spontaneous mutation).

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

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

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

(B) As used herein, a “comparison window” includes references to contiguous and specific segments of a polynucleotide / polypeptide sequence, where the polynucleotide / polypeptide sequence is Can be compared to a reference sequence, wherein the portion of the polynucleotide / polypeptide sequence in the comparison window is
For optimal alignment of the two sequences, it may contain additions or deletions (ie, gaps) as compared to a reference sequence, which contains no additions or deletions. Generally, the comparison window is at least 20 contiguous nucleotides / amino acid residue in length, and can be 30, 40, 50, 100 or more, as needed. One of skill in the art will appreciate that gap penalties are typically introduced and subtracted from the number of matches to avoid high similarity to the reference sequence due to inclusion of gaps in the polynucleotide / polypeptide sequence Understand that.

Methods for alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison can be performed by: Smi
th and Waterman, Adv. Appl. Math. 2: 482 (19
81) Local homology algorithm; Needleman and Wunsch, J
. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad.
Sci. 85: 2444 (1988); Computerized implementation of these algorithms, including, but not limited to:
Intelligenetics, Mountain View, Califo
PC / Gene program CLUSTAL by rnia; Wisconsi
n Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr.
, Madison, Wisconsin, USA GAP, BESTFIT, B
LAST, FASTA and TFASTA; the CLUSTAL program is more fully described as follows: Higgins and Sharp, Gene 73:
237-244 (1988); Higgins and Sharp, CABIOS.
5: 151-153 (1989); Corpet et al., Nucleic Aci.
ds Research 16: 10881-90 (1988); Huang et al., Computer Applications in the Biosci.
accesses 8: 155-65 (1992) and Pearson et al., Met.
hods 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; protein query against protein database sequences BLASTP for sequences; TBLASTN for protein query sequences against nucleotide database sequences;
And TBLASTX for nucleotide query sequences against nucleotide database sequences. Current Protocols in Mole
Cultural Biology, Chapter 19, Ausubel et al., Greene
Publishing and Wiley-Interscience, Ne
See w 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
Either it matches or satisfies some positive threshold score T. T is referred to as the neighborhood word score threshold. These initial neighbor word hits act as seeds for the start of the search to find longer HSPs containing the word. 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 for nucleotide sequences using the parameters M (reward score for a pair of matched residues; always greater than 0) and N (penalty score for mismatched residues; always less than 0). . For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The expansion of word hits in each direction is halted if: the cumulative alignment score decreases by an amount X from its maximum attainment; the cumulative score becomes zero due to the accumulation of one or more negative scoring residue alignments. If; or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) has a word length (W) of 11, an expected value (E
), A cutoff of 100, M = 5, N = -4, and a comparison of both strands is used as default. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (Henikoff and Henikoff (19).
89) 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 Altschul, Proc. Nat'l. Acad. Sci. USA 90:
5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P (N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance.

[0067] 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 polynucleotide or polypeptide of the invention to a reference sequence. GAP uses Needlem to find an alignment of the two complete sequences that maximizes the number of matches and minimizes the number of gaps.
an and Wunsch (J. Mol. Biol. 48: 443-453, 19).
70). GAP considers all possible alignments and gap locations, and creates an alignment with the largest matched bases and the smallest gap. This allows for the provision of gap creation penalties and gap extension penalties in units of matched bases. 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 chosen, then the GAP 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 Geneti for protein sequences.
8 and 2 respectively in the 10th edition of the cs Software Package
It is. For nucleotide sequences, the gap creation penalty defaults to 50, while the gap extension penalty defaults to three. 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 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. This family can have many members, but none of the other members have good quality. GAP shows four forms 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. Similarity is scored if the scoring matrix value for a pair of symbols is greater than or equal to the similarity threshold of 0.50. Wisconsin Gene
The scoring matrix used in the tenth edition of the tics Software Package is BLOSUM62 (Henikoff and Hen
ikoff (1989) Proc. Natl. Acad. Sci. USA 89
: 10915).

Unless otherwise indicated, sequence identity / similarity values provided herein are those obtained using the BLAST 2.0 suite of programs using the default parameters (Altschul et al.). , Nucleic Acids Res. 25
: 3389-3402, 1997; Altschul et al. Mol. Bio.
215: 403-410, 1990) or values obtained using a GAP program using default parameters (Wisconsin Geneti).
cs Software Package, Genetics Compute
r Group (GCG), 575 Science Dr. , Madison
, Wisconsin, USA).

(C) As used herein, “sequence identity” or “identity” is 2
In the context of two nucleic acid or polypeptide sequences, references to residues of two sequences that are the same when aligned for maximum match for a particular comparison window are included. When percentage sequence identity is used for proteins, it is recognized that non-identical residue positions often differ due to conservative amino acid substitutions. Here, an amino acid residue is replaced with another amino acid residue having similar chemical properties (eg, charge or hydrophobicity), and thus does not alter the functional properties of the molecule. If the sequences differ in 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 said to have "sequence similarity" or "similarity." Means for making this adjustment are well-known to those of skill in the art. Typically, this involves scoring conservative substitutions as part rather than overall 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 0, the conservative substitution is given a score between 0 and 1. Scoring of conservative substitutions is described, for example, in Meyers and Miller, Computer Appl.
ic. Biol. Sci. 4: 11-17 (1988), for example, the program PC / GENE (Intelligenetics, Mo.)
(Untain View, California, USA).

(D) As used herein, “percentage of sequence identity” means a value determined by comparing two sequences that are optimally aligned for a comparison window. In that, the portion of the polynucleotide sequence in the comparison window indicates that for an optimal alignment of the two sequences, when compared to a reference sequence (which does not include any additions or deletions), ). The percentage is that the same nucleobase or amino acid residue is
Determining the number of positions that appear in both sequences to obtain the number of matched positions;
It is calculated by dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to get the percentage sequence identity.

(E) (i) The term “substantial identity” of a polynucleotide sequence means that the polynucleotide is identical to the reference sequence using one of the described alignment programs using standard parameters. By comparison, it is meant to include sequences having at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity. One of skill in the art will appreciate that these values can be used to determine the corresponding identity of the proteins encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Recognize that it can be adjusted. Substantial identity of amino acid sequences for these purposes 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, if 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 It is.

(E) (ii) The term “substantially identical” in the context of a peptide means that the peptide has at least 70% sequence identity to the reference sequence, to the reference sequence for a particular comparison window And preferably contains 80%, more preferably 85%, and most preferably at least 90% or 95% sequence identity. If necessary, the optimal alignment is Needleman and Wun
sch, J. et al. Mol. Biol. 48: 443 (1970), using the homology alignment algorithm. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by conservative substitutions. Peptides that are "substantially similar" share a sequence as described above, except that residue positions that are not identical 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 levels of polynucleotides and polypeptides of the invention in plants. provide. In particular, the polynucleotides and polypeptides of the present invention can be expressed transiently or spatially in development, in tissues, and / or in amounts, which is characteristic of non-recombinantly engineered plants. is not. Thus, the present invention provides utility in exemplary applications, such as controlling recombination or transformation efficiency in plants.

The present invention also includes polynucleotides of sufficient length and complementarity to the genes of the present invention for use as probes or amplification primers in the detection, quantification, or isolation of gene transcripts. And an isolated nucleic acid. For example,
The isolated nucleic acids of the invention can be used as a probe to detect mutations (eg, substitutions, deletions, or additions) in a gene as a probe in detecting a deficiency in mRNA levels in a screen for the desired transgenic plant. In addition, for the detection of multiple allelic variants (polymorphisms), orthologs, or paralogs of a gene, for monitoring up-regulation or changes in the phenotype of enzyme activity in compound screening assays, or for eukaryotes It can be used for site-directed mutagenesis in biological cells (see, for example, US Pat. No. 5,565,350).
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 use in sense suppression or antisense suppression of one or more genes of the invention in a host cell, tissue, or plant. The attachment of chemical agents that bind, intercalate, 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 may 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 may be used for the identification and / or isolation of the nucleic acids of the invention from expression libraries in assays for expression levels, for the identification of homologous polypeptides from other species, or of the invention. It can be used for polypeptide purification.

[0079] The isolated nucleic acids and polypeptides of the present invention include those described in particular by Hordeum, Sec.
are, Triticum, Sorghum (eg, S. bicolor),
Gram including Oryza, Avena and Zea (eg, Z. mays)
In monocotyledonous plants, such as species of the ineae family, it can be used across a wide range of plant species. The isolated nucleic acids and proteins of the present invention may also be used on species from the following genera: Cucurbita, Rosa, Vitis, Juglans,
Fragaria, Lotus, Medicago, Onobrychis, T
Rifolium, Trigonella, Vigna, Citrus, Lin
um, Geranium, Manihot, Daucus, Arabidops
is, Brassica, Raphanus, Sinapis, Atropa,
Capsicum, Data, Hyoscyamus, Lycopersi
con, Nicotiana, Solanum, Petunia, Digita
lis, Majorana, Ciahorium, Helianthus, La
ctuca, Bromus, Asparagus, Antirrhinum, H
heterocallis, Nemesis, Pelargonium, Pani
eum, Pennisetum, Ranunculus, Senecio, Sa
lpilossis, Cucumis, Browaria, Glycine
, Pisum, Phaseolus, and Lolium.

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

The polynucleotides of the present invention include: (a) polynucleotides encoding the polypeptides of SEQ ID NOs: 2, 4, and 6, and conservatively modified and polymorphic variants thereof. A polynucleotide comprising the exemplary polynucleotides of SEQ ID NOs: 1, 3, and 5 (the polynucleotide sequence of the present invention also comprises an American Type Culture Collection (Ame
rice Type Culture Collection) (ATCC)
And containing the maize RAD51 polynucleotide sequence as contained in a plasmid assigned accession number 207181).

(B) SEQ ID NO: 1, 3, 5, or ATCC Deposit Assignment Registry Number 207181
An amplification product derived from a Zea mays nucleic acid library using a primer pair that selectively hybridizes under stringent conditions to a locus in a polynucleotide selected from the group consisting of sequences contained in A polynucleotide, wherein the polynucleotide is substantially sequence identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1, 3, and 5 or a sequence as included in ATCC Deposit Assignment Accession No. 207181; A polynucleotide having the property.

(C) a polynucleotide that selectively hybridizes to the polynucleotide of (a) or (b); (d) a specified sequence with the polynucleotide of (a), (b), or (c) (E) a polynucleotide encoding a protein having a specified number of contiguous amino acids from a prototype polypeptide, wherein the protein is induced by presentation of the protein. A polynucleotide that is specifically recognized by an antiserum and wherein the protein does not detectably immunoreact with an antiserum fully immunosorbed to the protein; (f) (a), (b) (C) the complementary sequence of the polynucleotide of (d) or (e); and (g) (a), (b), (c), d), (e) or (at least a certain number of consecutive nucleotides from the polynucleotide f), the polynucleotide.

[0084] The polynucleotides of SEQ ID NOs: 1, 3 and 5 are of the American Type
Culture Collection (ATCC), March 31, 1999
Included in the plasmid deposited on the day and assigned accession number 207181. American Type Culture Collection
Is 10801 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 only as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 USC 112.

(A. Polypeptide Encoding A Polypeptide of the Present Invention, or Conservatively Modified or Polymorphic Modification) 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 relates to polynucleotides of SEQ ID NOs: 1, 3, 5 as well as ATC
C, and silent variations of the polynucleotide encoding the polypeptide of SEQ ID NOs: 2, 4, and 6. The invention further provides an isolated nucleic acid comprising a polynucleotide that encodes a conservatively modified variant of the polypeptide of SEQ ID NOs: 2, 4, and 6. Conservatively modified variants can be used to generate or select antibodies immunoreactive with the non-variant polypeptide. In addition, the present invention further provides an isolated nucleic acid comprising a polynucleotide encoding one or more allelic (polymorphic) variant polypeptide / polynucleotide. Polymorphic variants are
For example, it is frequently used 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. Here, the polynucleotide is Zea mays
Amplified from nucleic acid library. Zea mays strain B73, PHRE1,
A632, BMS-P2 # 10, W23, and Mo17 are known and publicly available. Other publicly known and available corn lines are:
Maize Genetics Cooperation (Urbana, IL)
) Can be obtained. 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 re-presentation of relatively rare cDNAs. In any of the embodiments, the cDNA library is constructed using a full-length cDNA synthesis method. Examples of such methods are described in Oligo-Capping (Maruyama, K. and Sugano, S. Gene 138: 171-174, 1994), Bio.
tinylated CAP Trapper (Carninci, P., Kv
an, C.I. Et al., Genomics 37: 327-336, 1996), and the CAP Retention Procedure (Edery, E., Chu).
, L .; L. Et al., Molecular and Cellular Biolo
gy 15: 3363-3371, 1995). cDNA synthesis is often catalyzed at 50 ° C. to 55 ° C. to prevent formation of RNA secondary structure.
An example of a reverse transcriptase that is relatively stable at these temperatures is SuperScri.
ptII Reverse Transcriptase (Life Tech
nologies, Inc. ), AMV Reverse Transcript
case (Boehringer Mannheim), and RetroAm
p Reverse Transcriptase (Epicentre). Rapidly growing tissues or rapidly dividing cells are preferably used as a source of mRNA.

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 a site and to 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. 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 199
See 7-year catalog, page 354.

[0091] 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, as described in San Diego, 1990), pages 28-38.
(Rapid Amplification of Complementar
y Ends); U.S. Patent No. 5,470,722 and Curr.
ent Protocols in Molecular Biology, U
nit 15.6, Ausubel et al., Green Publishing.
and Wiley-Interscience, New York (199
5); Frohman and Martin, Technologies 1: 165.
(1989).

(C. Polynucleotide that selectively hybridizes to the polynucleotide of (A) or (B)) As shown in the above (c), 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 complementary to a genomic or cDNA sequence isolated from a dicotyledonous or monocotyledonous nucleic acid library or otherwise cDNA from such a library. . Exemplary species of monocotyledonous and dicotyledonous plants include, but are not limited to, corn, canola, soybean, cotton, wheat, sorghum, sunflower, oats, sugarcane, millet, barley, and rice. Preferably, the cDNA library contains at least 80% full length sequences, preferably at least 85% or 90% full length sequences, and more preferably at least 95% full length sequences. The cDNA library is
Rare sequences can be normalized to increase presentation. Low stringency hybridization conditions typically employ, but are not exclusively used with, sequences having reduced sequence identity to complementary sequences. Moderate and high stringency conditions can be used as needed for more identical sequences. Low stringency conditions can 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. Providing an isolated nucleic acid comprising the nucleotide, wherein the polynucleotide comprises the sequence of paragraph (A),
It has the specified identity at the nucleotide level to a polynucleotide as disclosed above in (B) or (C). The percentage of identity to the reference sequence is at least 60% and may be rounded up to the nearest integer and expressed as an integer selected from the group of integers consisting of 60 to 99. Thus, for example, the percentage of identity to a reference sequence is at least 70%, 75%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
It can be 98% or 99%.

[0094] Optionally, the polynucleotide of this embodiment is selected from paragraph (A), (B) or (C).
) Encodes a polypeptide that shares an epitope with the polypeptide encoded by the polynucleotide of). Thus, these polynucleotides comprise (A)
, (B) or (C) encodes a first polypeptide that elicits the production of an antiserum that includes an antibody that specifically reacts with a second polypeptide encoded by the polynucleotide of (B) or (C). However, when the antiserum is completely immunosorbed with the first polypeptide, the first polypeptide does not bind to the antiserum raised against itself. Thus, the polynucleotide of this embodiment can be used, for example, to produce antibodies for use in screening an expression library of nucleic acids comprising the polynucleotide of (A), (B) or (C). Alternatively, it can be used for purification or immunoassay of the polypeptide encoded by the polynucleotide of (A), (B) or (C). The polynucleotide of this embodiment comprises:
Includes nucleic acid sequences that can be used for selective hybridization to a polynucleotide encoding a polypeptide of the invention.

[0095] Screening a polypeptide for specific binding to an antiserum comprises:
This can be conveniently achieved using a peptide display library. The method involves screening a large peptide collection 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 1
It can be as long as 00 amino acids, and often about 8 to 15 amino acids. In addition to direct chemical synthesis methods for producing peptide libraries, several recombinant DNA methods have been described. One type involves the display of peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence that encodes the particular peptide sequence displayed. Such a method is described in PCT Patent Publication No. 91/17271,
Nos. 91/18980, 91/19818 and 93/08278
No. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See PCT Patent Publication Nos. 92/05258, 92/14843 and 96/19256. U.S. Patent Nos. 5,658,754; and 5,643,76.
See also Issue 8. Peptide display libraries, vectors and screening kits are commercially available from vendors 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 the 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, 40 from a prototype polypeptide.
, 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, 1
5, 25, 50, 100, or 200 nucleotides).

The protein encoded by the polynucleotide of this embodiment, when presented as an immunogen, is, for example, but not limited to, a polynucleotide encoded by the polynucleotide of (a) or (b) above. Induces the production of polyclonal antibodies that specifically bind to the prototype polypeptide. But,
Generally, the protein encoded by the polynucleotide of this embodiment is:
If the antiserum is fully immunosorbed to the prototype polypeptide, it will not bind to the antiserum raised against the prototype polypeptide. 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.

[0098] 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. Accordingly, the proteins of the present invention may be allelic variants of a prototype polypeptide, conservatively modified variants,
And minor recombinant modifications.

A 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. The molecular weight is determined by SDS-PAGE under reducing conditions.
Can be determined more easily. Preferably, the molecular weight is within 15% of the full length polypeptide of the invention, more preferably within 10% or 5%, and most preferably 3%, 2% or 1% of the full length polypeptide of the invention. Within.

Optionally, the polynucleotide of this embodiment is a native polynucleotide of the invention.
At least 50%, 60%, 80% of cell extracts containing endogenous full-length polypeptide
% Or 90% of the protein having a particular enzymatic activity. further,
The protein encoded by the polynucleotide of this embodiment optionally has an affinity constant (K _m) substantially similar to the native endogenous full-length protein.
) And / or catalytic activity (ie, microscopic rate constant, k _cat ). One skilled in the art will recognize that the k _cat / K _m value determines the specificity for a competing substrate and is often referred to as its specificity constant. The protein of this embodiment may have a _kcat / _Km value of at least 10% of the full-length polypeptide of the invention determined using the endogenous substrate of the polypeptide. Optionally, the k _cat / K _m value is at least 20%, 30%, 4% or less of the k _cat / K _m value of the full-length polypeptide of the invention.
0%, 50%, and most preferably at least 60%, 70%, 80%, 9
0% 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 rate of initiation (ie, the first 5% or less of the reaction) can be controlled by rapid mixing and sampling techniques (eg, continuous flow, stopped flow, or rapid quenching techniques), flash photolysis, or relaxation methods (eg, , Temperature jump) in combination with exemplary methods of measurement such as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance, or radioactivity procedures. Kinetic values are measured by Lineweaver-Burk or Eadi.
It is conveniently obtained using an e-Hofstee plot.

(F. Complementary Polynucleotides to the Polynucleotides of (A) to (E)) As shown in the above (f), the present invention provides a polynucleotide complementary to the polynucleotides of the above paragraphs A to E Provided isolated nucleic acid comprising a polynucleotide complementary to One skilled in the art will recognize the polynucleotides of sections (A)-(E) and sequence base pairs complementary over their entire length (ie, having 100% sequence identity over their entire length). Complementary bases bind via double bonds in double-stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; because adenine and uracil.

(G. Polynucleotide which is a Partial Sequence of Polynucleotide from (A) to (F)) As shown in the above (g), the present invention provides the above-mentioned sections (A) to (F)
An isolated nucleic acid comprising a polynucleotide comprising at least 15 contiguous bases from the polynucleotide of the invention. The length of the polynucleotide is
The polynucleotide is given as an integer selected from the group consisting of at least 15 and up to the length of the nucleic acid sequence whose subsequence is the subsequence. Thus, for example, the polynucleotides of the present invention comprise at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 7 from the polynucleotides (A) to (F).
Polynucleotides containing 0, 75, 80, 85, 90, or 100 contiguous nucleotides in length are included. Optionally, the number of such subsequences encoded by the polynucleotide of this embodiment is any integer (eg, 2, 3, 4, or 5) selected from the group consisting of 1-20. possible. The subsequences can be separated from one by any integer number of nucleotides, the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

[0103] A subsequence of the invention may include the structural features of the sequence from which it is derived. Alternatively, the subsequence may lack certain structural features of the larger sequence from which it was derived (eg, the poly (A) tail. Optionally, the subsequence may be common to the prototype polypeptide sequence provided in (a) above). A subsequence from a polynucleotide encoding a polypeptide having at least one linear epitope can encode an epitope common to the prototype sequence, or the subsequence does not encode an epitope common to the prototype. May be, but may be used to isolate larger sequences, for example, by nucleic acid hybridization with the sequence from which it was derived.
By introducing into its subsequence compound that cleaves and / or crosslinks,
It can be used to regulate or detect gene expression. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin, or chloroethylaminoaryl conjugate.

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

[0105] The nucleic acid may conveniently comprise sequences in addition to the polynucleotide of the invention. For example, a multiple cloning site that includes one or more endonuclease restriction sites can be inserted into the nucleic acid and aid in isolating the polynucleotide. Also, translatable sequences can be inserted to assist in isolating the translated polynucleotide of the present invention. For example, a hexahistidine marker sequence provides a convenient means for purifying a protein of the invention. A polynucleotide of the invention can be linked to a vector, adapter, or linker for expression of the polynucleotide of the invention, for cloning and / or expression. Additional sequences may be used to optimize their function in cloning and / or expression, to assist in isolating the polynucleotide, or to improve the introduction of the polynucleotide into cells. It can be added to cloning and / or expression sequences. Typically, the length of a nucleic acid of the invention that is less than the length of a polynucleotide of the invention is less than a 20 kilobase pair, often less than 15 kb, and often less than 10 kb. The use of cloning vectors, expression vectors, adapters, and linkers is well known and has been extensively described in the art. For a description of various nucleic acids, see, for example, Stratage.
ne Cloning Systems, Catalogs 1995, 199
6, 1997 (La Jolla, CA); and Amersham Life
Sciences, Inc. Catalog '97 (Arlington H
rights, IL).

A. Recombinant Methods for the Construction of Nucleic Acids The isolated nucleic acid compositions of the present invention (eg, RNA, cDNA, genomic DNA,
Or hybrids thereof) can be obtained from a biological source of the plant using any number of cloning methodologies known in the art. In some embodiments, an oligonucleotide probe that selectively hybridizes to a polynucleotide of the invention under stringent hybridization conditions will have a cD
In NA or genomic DNA libraries, it can be used to identify the desired sequence. While isolation of RNA, construction of cDNA and genomic libraries is well known to those skilled in the art, the following highlights some of the methods utilized.

(A1. MRNA Isolation and Purification) Total RNA from plant cells was mitochondrial RNA, chloroplast RNA, rRNA
, TRNA, hnRNA and mRNA. Total RNA preparations typically involve lysis of cells 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 strong detergents (eg, SDS).
) And organic modifiers such as guanidine isothiocyanate, guanidine HC
l or phenol). After isolation of total RNA, poly (A) ⁺ mRNA
Is typically purified from the remaining RNA using oligo (dT) cellulose. An exemplary total RNA and mRNA isolation protocol is Plant Mo
rectangular Biology: A Laboratory Manual,
Clark, eds., Springer-Verlag, Berlin (1997).
); And Current Protocols in Molecular
Biology, Ausubel ed., Green Publishing a
nd Wiley-Interscience, New York (1995)
It is described in. Total RNA and mRNA isolation kits are available from Stratagene (
LaJolla, CA), Clonetech (Palo Alto, CA),
Pharmacia (Piscataway, NJ) and 5'-3 '(Pao
li Inc. , PA). US Patent 5,
614,391; and 5,459,253. mRNA has a size range of about 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 kb.
Can be fractionated into populations. The cDNA synthesized for each of these fractions can be size-selected to the same size range as the mRNA before inserting the vector. This method is based on the truncated cD formed by incomplete reverse transcribed mRNA.
Helps eliminate NA.

(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 hybrid is converted to double-stranded cDNA, typically by reaction with a combination of RNAse H and DNA polymerase I (or Klenow fragment). 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. I do. Fifth, cDNA
Is ligated into a cloning vector and packaged. cDNA
Synthetic protocols are well known to those skilled in the art, and Plant Molecular
Biology: A Laboratory Manual, edited by Clark,
Springer-Verlag, Berlin (1997); and Curr.
ent Protocols in Molecular Biology, A
edited by ussubel et al., Green Publishing and Wiley
-Described in standard references such as Interscience, New York (1995). The cDNA synthesis kit is available from Stratagene or P
It is available from a variety of commercial sources such as H. marcia.

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 insert-containing clones. 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, λZ of Stratagene
See AP Express (cDNA cloning vector with cloning ability 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 eukaryotic mRNA cap structure is chemically labeled 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 provides a high yield library with unbiased presentation of the starting mRNA population. Other methods for generating full length libraries are known in the art. For example, Ed
ery et al., Mol. Cell Biol. , 15 (6): 3363-3371 (
1995); and PCT application WO 96/34981.

(A3. A homogenized cDNA library or a subtracted cDNA library) [0111] A non-homogenized cDNA library is obtained from the mRN of the tissue from which it was generated.
Group A is presented. Isolation of unique clones can be difficult because unique clones are inferior in number to clones derived from highly expressed genes. Homogenization of a cDNA library is the process of creating a library in which each clone is more equally represented.

Many approaches to homogenizing a cDNA library are known in the art. One approach is based on hybridization with genomic DNA. Each hybridized c in the resulting homogenized library
The frequency of the DNA is proportional to the frequency of each corresponding gene in its genomic DNA. Another approach is based on reaction rates. If the cDNA reannealing follows a second order kinetics, the less species, the less rapid the annealing and the remaining cDN
The single-stranded fraction of A becomes progressively more homogenous during hybridization. The specific deletion of any species of cDNA, regardless of its amount,
It does not occur even at the ot value. Construction of a homogenized library is described in Ko, Nucl. A
cids. Res. , 18 (19): 5705-5711 (1990); Pat.
anjari et al., Proc. Natl. Acad. U. S. A. , 88: 194
3 to 1947 (1991); U.S. Patent Nos. 5,482,685 and 5,6;
37, 685. In the illustrative method described by Soares et al., Homogenization reduced clone content from a range of four orders of magnitude to a narrow range of only one order of magnitude. Proc. Natl. Acad. Sci.
USA, 91: 9228-9232 (1994).

[0113] Subtracted cDNA libraries are another means to increase the proportion of non-abundant cDNA species. In this procedure, sequences present in a second pool of mRNA are deleted from the cDNA prepared from one pool of mRNA by hybridization. The cDNA: mRNA hybrid is removed and the remaining unhybridized cDNA pool is enriched for sequences unique to that pool. Foote et al., Plant Molecular Bi
ology: A Laboratory Manual, edited by Clark, Spr
inger-Verlag, Berlin (1997); Kho and Zarb.
1, Technique, 3 (2): 58-63 (1991); John, Nucl. Acids Res. , 16 (22): 10937.
(1988); Current Protocols in Molecula
r Biology, edited by Ausubel et al., Greene Publishin
g and Wiley-Interscience, New York (19
95); and Swaroop et al., Nucl. Acids Res. , 19)
8): 1954 (1991). cDNA subtraction kits are commercially available. For example, PCR-Select (Clontech, Palo Alt)
o, CA).

A4. Construction of Genomic Library To construct a genomic library, a large segment of genomic DNA
Produced by fragmentation (eg, using restriction endonucleases)
It is then ligated with the vector DNA to form a concatemer that can be packaged in a suitable vector. Methodologies for achieving these objects, and methods for determining the sequence of a nucleic acid, are well known in the art. Examples of suitable molecular biology techniques and instructions sufficient to direct one of skill through many construction, cloning, and screening methodologies are found below: Sambrook
Et al., Molecular Cloning: A Laboratory Man.
ual, 2nd edition, Cold Spring Harbor Laboratory
y, Volumes 1-3 (1989), Methods in Enzymology,
Volume 152: Guide to Molecular Cloning Tec
Hniques, Berger and Kimmel, Ed., San Diego: A.
Cademic Press, Inc. (1987), Current Pro
Tocols in Molecular Biology, edited by Ausubel et al., Green Publishing and Wiley-Inters.
science, New York (1995); Plant Molecula
r Biology: A Laboratory Manual, edited by Clark, Springer-Verlag, Berlin (1997). Kits for constructing 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, the degree of complementarity between the probe and target for duplex formation must be higher. The degree of stringency can be controlled by temperature, ionic strength, pH and the presence of partially denaturing solvents 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.

[0116] A nucleic acid of interest can also be amplified from a nucleic acid sample using amplification techniques. For example, polymerase chain reaction (PCR) technology can be used to amplify the polynucleotide sequences of the present invention and to amplify related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods are also
For example, a nucleic acid sequence encoding the protein to be expressed is cloned and the nucleic acid for use in detecting the presence of the desired mRNA in a sample, for sequencing the nucleic acid, or as a probe for other purposes. It can be useful for making. Examples of techniques that are sufficient to guide one skilled in the art through in vitro amplification methods include Be
rger, Sambrook, and Ausubel, and Mullis et al., US Pat. No. 4,683,202 (1987); and PCR Protocol
ols A Guide to Methods and Applicati
ons, Innis et al., eds., Academic Press Inc. , San
Diago, CA (1990). Commercially available kits for genomic PCR amplification are known to those skilled in the art. For example, Advantage-GC
See Genomic PCR Kit (Clontech). T4 g
The ene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[0117] PCR-based screening methods have also been described. Wilfinge
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. BioTechniques, 22 (3): 481-486 (1997).
In that method, a primer pair is synthesized with one primer annealing to the 5 'end of the sense strand of the desired cDNA and the other annealing to the vector. Clones are pooled to allow for large-scale screening. By this procedure, the longest possible clone is identified among the candidate clones. Further, the PCR product is used solely to diagnose the presence of the desired cDNA, and not the PCR product itself. Such a method is based on the full length cDNA described above.
It is particularly effective when combined with the construction methodology of

B. Synthetic Methods for Nucleic Acid Construction The isolated nucleic acids of the present invention can also be prepared as described, for example, in Narang et al., Meth. Enz
ymol. 68: 90-99 (1979) phosphotriester method; Brown
Et al., Meth. Enzymol. 68: 109-151 (1979); Beaucage et al., Tetra. Lett. 22: 1859-1
862 (1981), the diethylphosphoramidite method; Beaucage and Caruthers, Tetra. Lett. 22 (20): 1859-186
2 (1981) based on the solid phase phosphoramidite triester method (eg, Needham-Van Devanter et al., Nucleic Acid).
s Res. , 12: 6159-6168 (1984)), and by direct chemical synthesis by methods such as the solid support method of U.S. Patent No. 4,458,066. Can be prepared. Chemical synthesis generally produces single-stranded oligonucleotides. It can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerizing with a DNA polymerase using single-stranded as a template. Those of skill in the art understand that chemical synthesis of DNA is best used for sequences of about 100 bases or less, and longer sequences can be obtained by ligation of shorter sequences.

(Recombinant Expression Cassette) The present invention further provides a recombinant expression cassette containing the nucleic acid of the present invention. A nucleic acid sequence encoding a desired polypeptide 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. The recombinant expression cassette represents a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell (eg, a transformed plant tissue). Contains.

For example, a plant expression vector may comprise (1) a plant gene cloned under the transcriptional control of 5 ′ and 3 ′ regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also include, if desired, a promoter regulatory region (eg, a region that confers inducible or constitutive, environmental or developmental regulation, or cell or tissue-specific / selective expression). ), Transcription initiation site, ribosome binding site, RNA processing signal, transcription termination site, and / or polyadenylation signal.

A plant promoter fragment that directs the expression of a polynucleotide of the present invention can be used in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and conditions of growth or cell differentiation. Examples of constitutive promoters include the 35S transcription initiation region of cauliflower mosaic virus (CaMV), Agro
1 ′ or 2 ′ promoter derived from T. DNA of B. tumefaciens, ubiquitin 1 promoter, Smas promoter, cinnamic acid 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 corn embryos or embryogenic calli.

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 precise 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 include the Adh1 promoter induced by hypoxic or cold stress, the Hsp70 promoter induced by hot stress, and the PPDK promoter induced by light.

Examples of promoters under developmental control include promoters that initiate transcription only or dominantly initiate transcription only in a particular tissue (eg, leaf, root, fruit, seed, or flower). No. A typical promoter is anther specific promoter 55126 (U.S. Pat. Nos. 5,689,049 and 5,689,05).
No. 1), the glob-1 promoter and the gamma-zein promoter. The operation of a promoter can also vary depending on its position in the genome. Thus, an inducible promoter may be completely or partially inducible at certain positions.

[0124] Both heterologous promoters and non-heterologous promoters (eg, endogenous promoters) can be used to direct expression of the nucleic acids of the invention. These promoters can also drive expression of the antisense nucleic acid, for example, in a recombinant expression cassette to reduce the concentration and / or composition of a protein of the invention in a desired tissue,
It can be used to increase or change. Thus, in some embodiments, the nucleic acid construct comprises a promoter operably linked to a polynucleotide of the invention, which is functional in a plant cell, such as, for example, Zea mays. Promoters useful in these embodiments include endogenous promoters that drive expression of the polypeptides of the invention.

In some embodiments, an isolated nucleic acid that acts as a promoter or enhancer element is capable of up-regulating or down-regulating the expression of a polynucleotide of the present invention. It can be introduced at a suitable site in the mold (generally upstream). For example, an endogenous promoter is
Mutations, deletions and / or substitutions (Kmiec, US Pat. No. 5,565,350)
No .; Zarling et al., PCT / US93 / 03868), or an isolated promoter can be used to control expression of a gene in an appropriate direction and at an appropriate distance from a gene of the present invention. It can be introduced into plant cells. To alter the total concentration and / or composition of a polypeptide of the invention in plant cells, gene expression may be regulated under conditions suitable for plant growth. 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, developmental stage, and / or environmental conditions are well known to those of skill in the art. For example, The Maize Handbook, Chapters 114-115
Chapter, Freeling and Walbot, Springer, New Yo
rk (1994); Corn and Corn Improvement, 3rd edition, Chapter 6, Sprague and Dudley eds., American So
city of Agronomy, Madison, Wisconsin (
1988). A typical step in promoter isolation is the identification of a gene product that is expressed with some specificity in the target tissue. Methodologies include: differential hybridization of cDNA libraries; subtractive hybridization; differential display; differential two-dimensional protein gel electrophoresis; DNA probe arrays; and expressed with some specificity in target tissues. There is isolation of proteins known to be. Such methods are well-known to those skilled in the art. Commercially available products for identifying promoters are known in the art, for example, the Universal Genome Walker Kit from Clontech (Palo Alto, CA).

As a protein-based method, the amino acid sequence of at least a part of the identified protein has been obtained, and then genomic DNA is directly identified from a library prepared from the target tissue, or It is effective to use protein sequences as a basis for preparing nucleic acids that can be used as probes to identify cDNA clones. Once such a cDNA clone has been identified, its sequence can be used to identify the sequence at the 5 'end of the transcript of the indicated gene. 5 'of the transcript of the gene indicated nucleic acid sequence identified as abundant in the target tissue for differential, subtractive hybridization and differential display
Used to identify terminal sequences. Once such sequences have been identified, starting with either the protein or nucleic acid sequences, any of these sequences identified as being derived from gene transcripts have been prepared from genomic libraries prepared from target organisms. Can be used to screen Methods for identification and confirmation of the transcription start site are well known in the art.

In the process of isolating a promoter that is expressed in a particular environmental condition or stress, or in a particular tissue, or under a particular developmental stage, the process can be performed under a desired environment, in a desired tissue, or in a desired tissue. Many genes that are expressed at the stage are identified. Further analysis reveals the expression of each particular gene in one or more other tissues of the plant. Promoters that have activity in a desired tissue or condition, but have no activity in any normal tissue can be identified.

To identify the promoter sequence, 5 ′ of the clone described herein.
Portions were analyzed for sequences characteristic of the promoter sequence. For example, the promoter sequence element is a consensus sequence of the TATA box (TATAAT).
Which are usually 5-10 bp AT-rich stretches 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 method for predicting the location of this element is to identify the transcription start site using standard RNA mapping techniques such as, for example, primer extension, S1 analysis, and / or RNase protection. It is. To confirm the presence of AT-rich sequences, structure-function analysis can be performed, including mutagenesis of the putative region and quantification of the effect of the mutagenesis on the expression of the linked downstream reporter gene. For example, The Maize Handb
book, Chapter 114, Freeling and Walbot, Sprinte
r, New York (1994).

In plants, further upstream from the TATA box, a promoter element with a series of adenines surrounding the three nucleotides G (or T) NG at positions −80 to −100 (ie, the CAAT box) is typical. Exists. J. Me
ssing et al., Genetic Engineering in Plants
, Kosage, Meredith and Hollaender, eds. 221-2
27 (1983). In maize, there is no fully conserved CAAT box, but there are several short conserved protein binding motifs upstream of the TATA box. These include trans-acting transcription factor motifs involved in light regulation, anaerobic induction, hormonal regulation, or anthocyanin biosynthesis that are appropriate for each gene.

Once the promoter and / or gene sequence is known, a region of appropriate size is selected from the genomic DNA 5 ′ to the transcription start or translation start site, and such a sequence is then ligated to the coding sequence. You. When a transcription start site is used as a point of fusion, any number of possible 5 'untranslated regions may also be used between the transcription start site and the partially encoding sequence. If a translation initiation site at the 3 'end of the specific promoter is used, it is 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 coding region of the polynucleotide. The polyadenylation region can be derived from a natural gene, various other plant genes, or T-DNA. The sequence at the 3 'terminus added can be derived, for example, from a nopaline synthase gene or an octopine synthase gene, or alternatively from another plant gene, or less preferably, from any other eukaryotic gene. .

Intron sequences can 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 cytoplasm.
In both plant and animal expression constructs, including splicable introns in the transcription unit would result in gene expression of both mRNA and protein of 1000
It has been shown to increase by a factor of two. Buchman and Berg
, Mol. Cell Biol. 8: 4395-4405 (1988); Cal.
lis et al., Genes Dev. 1: 1183-1200 (1987). The enhancement of gene expression of such introns is typically greatest when placed near the 5 'end of the transcription unit. The use of the maize introns Adh1-S introns 1, 2, and 6, the Bronze-1 intron, is known in the art. In general, The Maize Handbook, Chapter 116, edited by Freelig and Walbot, Spriger, New York (
1994).

Vectors containing sequences derived from the polynucleotides of the present invention typically contain a marker gene that confers a selectable phenotype on plant cells. Usually, the selectable marker gene encodes antibiotic resistance and the antibiotic spectinomycin (eg, a
ada gene), streptomycin phosphotransferase (SPT) gene encoding streptomycin resistance, neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, hygromycin phosphotransferase (HPT) encoding hygromycin resistance ) Genes, genes encoding resistance to herbicides that act to suppress the action of acetolactase synthase (ALS), especially sulfonylurea-type herbicides (eg especially S4 and / or Hra
Acetolactate synthase (ALS) containing mutations that lead to such resistance
) Gene), for example, a gene encoding resistance to a herbicide that acts to suppress the action of glutamine synthase such as phosphinothricin or basta (eg, the bar gene), or other genes known in the art. Have suitable genes, including such genes. The bar gene encodes the herbicide resistance to basta, the nptII gene encodes the resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes the herbicide resistance to chlorsulfuron.

Exemplary vectors useful for higher plant gene expression are well known in the art and are described in Rogers et al., Meth. in Enzymol. , 153: 2
53-277 (1987).
vector derived from the tumor induction (Ti) plasmid of Umefaciens. These vectors are plant integration vectors, in that the vectors integrate a portion of the vector DNA into the genome of the host plant upon transformation. Typical A.I. useful herein. Tumefaciens vectors are described in Schardl et al., Gene, 61: 1-11 (1987) and Berger et al., Proc. N
atl. Acad. Sci. U. S. A. , 86: 8402-8406 (198).
9) The plasmids pKYLX6 and pKYLX7. Another useful vector herein is Clontech Laboratories, Inc. (
PBI 101.2, available from Palo Alto, CA).

The polynucleotides of the present invention can be expressed in either sense or antisense orientation as desired. It is understood that regulation of gene expression in either sense or antisense orientation can have a direct effect on observable plant characteristics. Antisense technology can be conveniently used to inhibit plant gene expression.
To accomplish this, a nucleic acid segment from the desired gene is cloned,
The antisense strand RNA is operably linked to a promoter so that it is transcribed. The construct is then transformed into a plant to generate antisense strand RNA. 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., Proc. Nat'l. Acad. Sci. (USA) 85
: 8805-8809 (1988); and Hiatt et al., US Patent No. 4,80.
See 1,340.

Another suppression method is suppression of sense. Introduction of the nucleic acid formed in the sense orientation
It has been shown that blocking transcription of target genes can be an effective means. For an example of the use of this method of regulating endogenous gene expression, see Nap.
See Oli et al., The Plant Cell 2: 279-289 (1990) and U.S. Pat. No. 5,034,323.

[0138] Catalytic RNA molecules or ribosomes can also be used to inhibit the expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at specific locations, thereby rendering the target RNA functionally inactive Become In effecting this cleavage, the ribozyme does not change in itself, and therefore recycles,
Other molecules can be cleaved, which makes ribozymes a real enzyme. Including the ribozyme sequence in the antisense RNA allows them to
Of the construct, thereby increasing the activity of the construct. Target R
The design and use of NA-specific ribozymes is described in Haseloff et al., Natur.
e 334: 585-591 (1988).

[0139] Various crosslinkers, alkylating agents and radical-producing species as accessory groups of the polynucleotides of the present invention can be used to bind, label, detect and / or cleave nucleic acids. For example, Vlasov, V .; V. Et al., Nucleic
Acids Res (1986) 14: 4065-4076 describes the covalent attachment of a single-stranded DNA fragment with an alkylated derivative of a nucleotide complementary to the target sequence. A report of a similar study conducted by the same group was published by Knorr.
e, D. G. FIG. Et al., Biochimie (1985) 67: 785-789. Iverson and Dervan also report that sequence-specific cleavage of single-stranded DNA mediated by incorporation of modified nucleotides capable of activating cleavage (J Am Chem Soc (1987) 109: 124.
1-1243). Meyer, R .; B. Et al., J Am Chem Soc (19
89) 111: 8517-8519, which has been shown to effect covalent attachment to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleic acid sequence. Photoactivated cross-linking to single-stranded oligonucleotides mediated by psoralen is described in Lee, B. et al. L. Et al., Biochemistry (1988) 27: 3.
197-3203. The use of cross-linking with triple helix forming probes has also been described by Home et al., J Am Chem Soc (1990) 112: 243.
5-2437. The use of N4, N4-ethanocytosine as an alkylating agent to crosslink single-stranded oligonucleotides has also been described by Webb and M.
atteucci, J Am Chem Soc (1986) 108: 2768.
-2765; Nucleic Acids Res (1986) 14: 7661.
-7674; Feteritz et al. Am. Chem. Soc. 113: 40
00 (1991). Various compounds that bind, detect, label, and / or cleave nucleic acids are known in the art. For example, U.S. Patent Nos. 5,543,507; 5,672,593; 5,484,90.
No. 8, No. 5,256,648; and No. 5,681,941.

Protein The isolated protein of the present invention is a polypeptide having at least 10 amino acids encoded by any one of the polynucleotides of the present invention, or a polypeptide thereof, as discussed more fully above. Includes polypeptides that are conservatively modified variants. A protein or variant thereof of the invention 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 Optionally, this subsequence of contiguous amino acids has at least 15, 20,
25, 30, 35 or 40 amino acids long, often at least 50, 60
, 70, 80 or 90 amino acids in length. Furthermore, the number of such subsequences can be any integer selected from the group consisting of 1-20 (eg, 2, 3, 4 or 5
).

The invention further provides a protein comprising a polypeptide having a specified sequence identity to a polypeptide of the invention. The percentage of sequence identity is an integer selected from the group consisting of 50-99. An exemplary sequence identity value is 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, using the GAP or BLAST algorithm.

As the skilled artisan will appreciate, the present invention relates to catalytically active polypeptides of the invention (including
That is, an enzyme). The catalytically active polypeptide has at least 20%, 30% or 40% of the specific activity of the native (non-synthetic) endogenous polypeptide,
And preferably it has a specific activity of at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95%. In addition, the substrate specificity (k _cat / K _m ) is optionally substantially similar to the native (non-synthetic) endogenous polypeptide. Typically, K _m is at least 30% K _m of the native (non-synthetic) endogenous polypeptide is 40% or 50%; and more preferably at least 60%, 70%, 80%, or 90 %. Methods for assaying and quantifying measures of enzyme activity and substrate specificity ( _kcat / _Km ) are well known to those skilled in the art.

In general, the proteins of the invention, when presented as an immunogen, elicit the production of antibodies specifically reactive with a polypeptide of the invention. In addition, the proteins of the invention do not bind to antisera raised against the polypeptides of the invention that have been sufficiently 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 can be used as immunogens for constructing antibodies immunoreactive with the proteins of the invention for exemplary uses, such as immunoassays or protein purification techniques.

(Expression of Protein in Host Cell) The nucleic acid of the present invention is used to transform the protein of the present invention into a recombinantly engineered cell (eg, a bacterial, yeast, insect, mammalian or, preferably, a plant cell). Can be expressed. The cell produces the protein in non-native conditions (eg, for amount, composition, location, and / or time). Because they have been genetically altered to do so by human intervention.

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.

In brief, the expression of an isolated nucleic acid encoding a protein of the invention is typically driven, for example, by driving a DNA or cDNA into a promoter, which is either constitutive or inducible. It is achieved by ligating as possible, followed by incorporation into an expression vector. Vectors may be suitable for replication and integration in either prokaryotes or eukaryotes. Representative expression vectors are transcription and translation terminators useful for regulating the expression of the DNA encoding the protein of the present invention;
Includes initiation sequence as well as promoter. To obtain high levels of expression of the cloned gene, it may be desirable to construct an expression vector that includes at least a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator. . Those skilled in the art will recognize that without diminishing biological activity,
Recognize that modifications can be made to the proteins of the present invention. Some modifications can be made to facilitate cloning, expression, or incorporation of the target molecule into a fusion protein. Such modifications are well known to those skilled in the art, and include, for example,
Includes a methionine added to the amino terminus to provide a start site, or an additional amino acid (eg, poly-His) positioned at either terminus to create a conveniently located purified sequence. 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. E. coli, represented by various strains; 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 include β-lactamase (penicillinase) and lactose (lac). ) Promoter system (Chang et al., Nature 198: 10).
56 (1977)), tryptophan (trp) promoter system (Goedde
1, 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)). The selectable marker is E.C. It is also useful to include in a DNA vector that is transfected into E. coli. 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, the 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: 543-545 (1983)).

B. Expression in Eukaryotes Various eukaryotic expression systems are known to those skilled in the art, such as yeast, insect cell lines, plants 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 used as an expression system for the production of a protein of the invention, as discussed below.

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 describing 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 an origin of replication, termination sequences, and the like, as desired.

[0151] Once expressed, the proteins of the invention 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 can 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 producing peptides 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, BHK
21 cell lines and the CHO cell line. 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. 89:
49 (1986)), as well as necessary processing information sites (eg, ribosome binding sites, RNA splice sites, polyadenylation sites (eg, SV40
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, Ameri
Available from the can 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 larvae, silkworms, armyworms, moths and Drosophila cell lines (eg, Schne
ider cell line (Schneider, J. Embryol. Exp. Morp
hol. 27: 353-365 (1987)).

For yeast, when using higher animal or plant host cells, the polyadenylation or transcription terminator sequence will typically be incorporated into the 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. 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 Vol. II practical means, M. Glover, IRL Press,
Arlington, Virginia 213-238 (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 obtain transcription and / or translation of this sequence to effect a phenotypic change in the organism. Thus, any method that provides for efficient transformation / transfection can be used.

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

[0157] 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 is prepared as described above and suitable for transformation of plant cells. The isolated nucleic acids of the invention can then be used for transformation. In this manner, genetically modified plants, plant cells, plant tissues,
Seeds and the like can be obtained. Transformation protocols can vary depending on the type of plant cell targeted for transformation (ie, monocot or dicot). Suitable methods for transformation of plant cells include microinjection (Cros
sway et al. (1986) Biotechniques 4: 320-334),
Electroporation (Riggs et al. (1986) Proc. Natl. Ac
ad. Sci. USA 83: 5602-5606), Agrobacteri.
um-mediated transformation (eg, Zhao et al., US Pat. No. 5,981,840; H
inche et al. (1988) Biotechnology 6: 915-921.
), Direct gene transfer (Paszkowski et al. (1984) EMB).
OJ. 3: 2717-2722), and ballistic particle acceleration (eg, Sanford et al., US Pat. No. 4,945,050; Tomes).
"Direct DNA Transfer into Intact Pl
ant Cells via Microprojectile Bombbar
dment ", Gamburg and Phillips (ed.) Plant Ce
ll, Tissue and Organic Culture: Fundamen
tal Methods, Springer-Verlag, Berlin (1
995); and McCabe et al. (1988) Biotechnology 6
: 923-926). See also: We
issinger et al. (1988) Annual Rev. Genet. 22: 4
21-377; Sanford et al. (1987) Particulate Sci.
ence and Technology 5: 27-37 (Onion); Ch
listou et al. (1988) Plant Physiol. 87: 671-67
4 (soy); McCabe et al. (1988) Bio / Technology 6
: 923-926 (soybean); Datta et al. (1990) Biotechnol.
8: 736-740 (rice); Klein et al. (1988) Proc. N
atl. Acad. Sci. USA 85: 4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-56.
3 (maize); Tomes et al., "Direct DNA Transfer
into Intact Plant Cells via Micropr
object Bombardment "Plant Cell, Tiss
use and Organ Culture: Fundamental Met
Hods, Gamburg and Phillips, Springer-Ve
rlag, Berlin (1995) (maize); Klein et al. (198).
8) Plant Physiol. 91: 440-444 (corn) Fr
(1990) Biotechnology 8: 833-839 (maize); Hooykaas-Van Slogteren and Hooyk.
aas (1984) Nature (London) 311: 763-764; B
ytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84: 5345-5349 (Liliaceae); De Wet et al. (1985) The.
Experimental Manipulation of Ovule T
issues, G .; P. Edited by Chapman et al., Pp. 197-209 Longma.
n, NY (pollen); Kaeppler et al. (1990) Plant Cell R.
reports 9: 415-418; and Kaeppler et al. (1992) T
heor. Appl. Genet. 84: 560-566 (whisker (whi
(Sker) -mediated transformation); D'Hallulin et al. (1992) Plant C
ell 4: 1495-1505 (electroporation); LI et al. (1992).
3) Plant Cell Reports 12: 250-255 and C
hristou and Ford (1995) Annals of Botany
75: 745-750 (corn (Agrobacterium tun)
efaciens))); all of which are incorporated herein by reference.

The transformed cells can be grown into plants according to conventional methods. For example,
McCormick et al. (1986) Plant Cell Reports, 5
: 81-84. These plants can then be grown and pollinated with either the same transformant or a different strain, and the resulting hybrid with the desired phenotypic characteristics identified. Two or more generations can be grown to confirm that the desired phenotypic trait is stably maintained and inherited, and then the seed is harvested to achieve the desired phenotype or other trait Can be confirmed.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells Are animal and lower eukaryote (eg, yeast) host cells competent for transfection by various means? Or 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.
cs), and microinjection of DNA directly into cells. The transfected cells are cultured by means well known in the art. Ku
chler, R .; J. , Biochemical Methods in Ce
II Culture and Virology, Dowden, Hutch
inson 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 the sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. The technique for solid phase synthesis is described by: Barany
And Merrifield, Solid-Phase Peptide Sy
nthesis, pp. 3-284, The Peptides: Analysis.
, Synthesis, Biology. Volume 2: Special Metho
ds in Peptide Synthesis, Part A; Merrifie
ld et al. Am. Chem. Soc. 85: 2149-2156 (1963)
And Stewart et al., Solid Phase Peptide Syn.
thesis, 2nd edition, Pierce Chem. Co. , Rockford,
Ill. (1984). Longer proteins can be synthesized by condensation of the amino and carboxy termini of shorter fragments. A method for forming a peptide bond by activating the carboxy terminus (for example, a coupling reagent N, N'-dicyclohexylcarbodiimide (N, N'-dicycyclohexylcar)
is 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 protein is purified by a combination of cell lysis (eg, sonication, French press) and affinity chromatography. For the fusion product, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein.

Recombinant or synthetic proteins of the invention can be prepared by standard techniques well known in the art, including detergent solubilization, selective precipitation with substances such as ammonium sulfate, column chromatography, immunopurification and the like. It can be purified to considerable purity. For example, R. Scopes, Protein Purification:
Principles and Practice, Springer-Ver
lag: New York (1982); Deutscher, Guidet.
o Protein Purification, Academic Pres
s (1990). For example, antibodies can be raised against a protein as described herein. E. FIG. Purification from E. coli is described in US Pat.
511,503. The protein can then be isolated from cells expressing the protein, and further purified by standard protein chemistry techniques as described herein. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassay, Western blotting techniques or immunoprecipitation.

Transgenic Plant Regeneration Transformed plant cells derived by any of the above transformation techniques can be cultured to regenerate whole plants having the transformed genotype. Such regeneration techniques often rely on the manipulation of specific plant hormones in tissue culture growth media, and typically rely on biocide and / or herbicide markers introduced with the polynucleotides of the invention. For transformation and regeneration of corn, see Gordon-Kamm et al., The Plant Ce.
11, 2: 603-618 (1990).

[0164] Plant cells transformed with a plant expression vector can be regenerated, for example, from single cells, callus tissue or leaf disks by standard plant tissue culture techniques. It is well known in the art that various cells, tissues and organs from most of any plant can be successfully cultured to regenerate the whole plant. Plant regenerated from cultured protoplasts are described below: Evans et al., Pr.
otoplasts Isolation and Culture, Hand
book of Plant Cell Culture, Macmillill
an Publishing Company, New York, 124-1
Page 76 (1983); and Binding, Generation of
Plants, Plant Protoplasts, CRC Press,
Boca Raton, 21-73 (1985).

Regeneration from leaf explants of plants containing foreign genes introduced by Agrobacterium is described by Horsch et al., Science, 227: 1229-12-1.
31 (1985). In this procedure,
Transformants were cultured in the presence of a selection agent and in Fraley et al., Proc. Natl.
Acad. 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 shoots of these transformants are then transformed into a suitable root-inducing medium containing a selective agent and an antibiotic to prevent bacterial growth. ). The transgenic plants of the present invention can be fertile or sterile.

[0166] 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 P
hys. 38: 467-486 (1987). Regeneration of plants from either single plant protoplasts or various explants is well known in the art. For example, Methods for Plant Molecu
lar Biology, A .; Weissbach and H.W. Weissbac
h, Academic Press, Inc. , San Diego, Cal
if. (1988). This regeneration and growth process involves the steps of selecting transformant cells and shoots, rooting the transformant shoots and growing plantlets in soil. For general information on corn cell culture and regeneration, see: The Maize Handbook, Freeling.
And Walbot eds., Springer, New York (1994); C
orn and Corn Improvement, 3rd edition, Sprague
And Dudley, American Society of Agron
omy, Madison, Wisconsin (1988).

[0167] One of skill in the art will appreciate that once a recombinant expression cassette has been confirmed to be stably integrated and operable in a transgenic plant, the recombinant expression cassette can be introduced into other plants by sexual crossing. Recognize. Any of a number of standard breeding techniques
It can be used depending on the species to be crossed.

In vegetatively propagated crops, mature transgenic plants can be propagated to produce large numbers of identical plants by performing cuttings or by tissue culture techniques. Desired transgenic selections are made, and new varieties are obtained and vegetatively propagated for commercial use. In seed propagated 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.

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.

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 lines are also typically evaluated for heterologous nucleic acid expression levels. Expression at the RNA level can be first determined to identify and quantify expression-positive 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 polynucleotide probes and antibodies, respectively, specific for heterologous nucleic acids to localize expression sites within the transgenic tissue. In general, many transgenic lines are usually screened for integrated 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 that contains two added nucleic acid sequences, wherein one gene is a chromosome pair. Are transgenic plants located at the same locus on each chromosome. 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)
It can be obtained by analyzing the resulting plant produced for altered expression of a polynucleotide of the invention. Backcrossing to parent plants and outcrossing with non-transgenic plants are also contemplated.

Modulating Polypeptide Levels and / or Polypeptide Composition The present invention further provides for modulating (ie, increasing or decreasing) the concentration or ratio of a polypeptide of the present invention in plants or parts thereof. To provide a way. Regulation can be affected by increasing or decreasing the concentration and / or ratio of a polypeptide of the invention in a plant. The method comprises the steps of: introducing a recombinant expression cassette comprising a polynucleotide of the present invention into a plant cell as described above to obtain a transformed plant cell; under plant cell growth conditions, Culturing the transformed plant cells, and inducing or suppressing expression of the polynucleotide of the present invention in the plant for a time sufficient to regulate the concentration and / or ratio of the polypeptide in the plant or plant part. Process.

In some embodiments, the content and / or ratio of a polypeptide of the invention in a plant is regulated by altering the promoter of the gene in vivo or in vitro to up-regulate or down-regulate gene expression. Can be done. 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 Patent No. 5,565,350;
See ing 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). PCR analysis to detect and amplicons produced from this gene, but are not limited to these. 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 ratio of a polypeptide of the invention in the plant. Plant formation conditions are well known in the art and are discussed briefly above.

In general, the concentration or ratio of the polypeptide will be at least 5%, 10%, 20%, 30%, as compared to a native control plant, plant part, or cell lacking the recombinant expression cassette described above. 40%, 50%, 60%, 70%, 80%
, Or 90% increased or decreased. Modulation in the present invention can occur during and / or following growth of the plant to the desired developmental stage. Modulation of nucleic acid expression in time and / or in a particular tissue is operably linked to a polynucleotide of the invention, for example, in a sense or antisense orientation, as discussed in more detail above. It can be controlled by using an appropriate promoter. Induction of expression of a polynucleotide of the invention can also be controlled by exogenous administration of an effective amount of an inducing compound. Inducible promoters and inducing compounds that activate expression from these promoters are well known in the art. In a preferred embodiment, the polypeptide of the invention is regulated in monocots, especially corn.

(Molecular Markers) The present invention relates to genotyping of a plant containing the polynucleotide of the present invention.
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 based on maps for phylogenetic studies, to characterize genetic relationships between crop varieties, to identify hybrids or somatic cell hybrids, to locate chromosomal segments that affect monogenic traits, Cloning, and for quantitative genetic studies. For example, Pla
nt Molecular Biology: A Laboratory Ma
Nual, Chapter 7, Clark, Springer-Verlag, Berlin
in (1997). For molecular marker methods, generally, An
draw 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.

Certain 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 genomic clones that have been treated with restriction enzymes (eg, PstI). Probe lengths are discussed in more detail above, but are typically at least 15 bases long, more preferably at least 20 bases long.
, 25, 30, 35, 40, or 50 bases in length. But in general,
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, 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 comprises the following steps: (a) Genomic DN of plant
Digesting A with a restriction enzyme; (b) under selective hybridization conditions,
A step of hybridizing a nucleic acid probe to the sequence of the polynucleotide 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 utilizing 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. Another approach based on detecting mismatches between two complementary DNA strands includes clamped denaturing gel electrophoresis (C
DGE); Heteroduplex analysis (HA); and Chemical mismatch cleavage (CMC)
Is mentioned. 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 produces a hybridization complex. Detection of the hybridization complex indicates 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 Selection) In general, translation efficiency depends on 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.

In addition, 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 coding sequences for expression in a desired host, for example, codon usage in heterologous sequences for expression in maize. Can be optimized. The codon usage in the coding region of the polynucleotide of the present invention can be determined by using a commercially available software package (for example, Universalsite).
y of Wisconsin Genetics Computer Gro
"Codon Preference" (Deverau
x et al., Nucleic Acids Res. 12: 387-395 (1984)
)) Or MacVector 4.1 (Eastman Koda).
k Co. New Haven, Conn. )) Can be used to analyze statistically. Thus, the present invention provides a unique 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. Optionally, the polynucleotide is a full-length sequence. An exemplary number of sequences for statistical analysis is at least 1
5, 5, 10, 20, 50, or 100.

(Sequence Shuffling) The present invention provides a method of 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, J .; -H. 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. The population of polynucleotides that have recombined sequences include a subpopulation of polynucleotides that retain 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 stability,
Sequences, replication elements, protein binding elements, etc. that control the conformation, translation, or other expression properties 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% of the wild-type value,
It can be 0%, 130%, 140%, or at least 150%.

General Sequences and Consensus Sequences The polynucleotides and polypeptides of the present invention further include the following: (a) general sequences of at least two homologous polynucleotides or polypeptides of the present invention, respectively; b) Include polynucleotides and polypeptides, each having a consensus sequence of at least three homologous polynucleotides or polypeptides of the invention. The general sequences of the present invention include various polypeptides or polynucleotides, each branched by a general polypeptide or polynucleotide sequence. Using individual species contained by a polynucleotide having a consensus sequence of amino acids or nucleic acids, other species, genera, families, orders,
Antibodies can be produced to screen for homologs in the class, phylum, or kingdom, or nucleic acid probes or primers can be generated. For example, Zea
Polynucleotides having a consensus sequence from the gene family of mays can be used to generate antibodies or nucleic acid probes or primers to other Gramineae species such as wheat, rice, or sorghum. Or,
Polynucleotides having a consensus sequence generated from an orthologous gene can be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence has at least 9
A length of 10, 15, 20, 25, 30, or 40 amino acids, or 20, 3,
0, 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 from the same group of conservative substitutions as 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.

[0183] Similar sequences used for the generation of consensus or general sequences include, as provided herein, any number and combination of identical genes, orthologous sequences, or paralogous sequences. Allelic variants are included.
Optionally, similar sequences used in creating consensus or general sequences are identified using the minimum sum probability (P (N)) of the BLAST algorithm. Various suppliers of sequence analysis software are available from Current Proto
cols in Molecular Biology, F.C. M. Ausube
ed., Current Protocols (Green Publish)
ing Associates, Inc. And John Wiley & Sons,
Inc. (Supplement 30). The minimum sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.1.
A polynucleotide sequence is considered similar to a reference sequence if it is less than 01 or less than about 0.001 and most preferably less than about 0.0001 or less than about 0.00001. Similar polynucleotides can be aligned and are available from a number of commercial suppliers, such as the Genetics Computer Group (Ma
disson, WI) PILEUP software, Vector NTI (No
rign Bethesda, MD) ALINX, or Genecode (
Consensus or generic sequences can be generated using multiple sequence alignment software available from SEQUENCER of Ann Arbor, MI). Conveniently, using the default parameters of such software,
Consensus or general sequences can be created.

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 a polypeptide of the invention with a compound whose ability to bind or modulate enzyme activity is to be determined. The polypeptide used may be at least 20%, preferably at least 30% or 40%, more preferably at least 50% or 60%, and most preferably at least 70% of the native full-length polypeptide (eg, an enzyme) of the present invention. % Or 80% specific activity. Generally, polypeptides
It is 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 of skill in the art would control factors such as enzyme concentration, ligand concentration (ie, substrate, product, inhibitor, activator), pH, ionic strength, and temperature to obtain useful kinetic data, and It is understood that the presence or absence of a compound that binds to or modulates polypeptide activity is determined. Methods for measuring enzyme reaction rates are well known in the art. For example, Segel, Biochem
Ial Calculations, 2nd Edition, John Wiley and
See Sons, New York (1976).

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood that certain changes and modifications can be practiced within the scope of the appended claims. Is clear.

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

Isolation of Total RNA The RNA of SEQ ID NO: 1 was sampled at night in stages V8-V12 and collected from pooled stem tissue of the B73 corn strain collected from 4-5 nodules internal to the tuft. Isolated. Tissue collected from B73 corn strain seedlings after 10 days of drought and 24 hours of heat shock was the source of RNA of SEQ ID NO: 3. The poly A RNA used to prepare the library of SEQ ID NO: 5 was prepared from a petiole dissected 20 days after pollination of the B73 corn strain. Total RNA was analyzed using Chomczyns.
ki and Sacchi (Chomczynski, P. and Sacchi,
N. Anal. Biochem. 162, 156 (1987)), using a modified version of the guanidine isothiocyanate / acid-phenol procedure, using the TRIZOL reagent (Life Technology Inc. Gaithe).
rsburg, MD). Briefly, plant tissue samples are ground in liquid nitrogen prior to the addition of TRIZOL reagent,
Further homogenized with a mortar and pestle. For the separation of the aqueous and organic phases,
Chloroform addition was performed before centrifugation. 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 (mRNA isolation system, Promega Corporation. Mad).
ion, WI). Briefly, biotinylated oligo (dT) primers were used to hybridize to the 3 'poly (A) tail of the mRNA. Hybrids were captured using streptavidin conjugated to paramagnetic particles and a magnetic separation stand. The mRNA was washed under high stringency conditions and eluted with RNase-free deionized water.

Construction of cDNA Library cDNA synthesis was performed and the SUPERSCRIPT plasmid system (Lif
e Technology Inc. Gaithersburg, MD) was used to construct a unidirectional cDNA library. The first strand of the cDNA is
It was synthesized by priming an oligo (dT) primer containing an I site. The reaction was performed with SUPERSCRIPT reverse transcriptase II for 4 hours.
Catalyzed at 5 ° C. The second strand of the cDNA 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. CDNA molecules of less than 500 base pairs and unligated adapters
Removed by SEPHACRYL-S400 chromatography. The selected cDNA molecules were ligated into the pSPORT1 vector between the NotI and SalI sites.

Example 2 This example describes cDNA sequencing and library subtraction.

Preparation of Sequencing Template Individual colonies were picked and DNA was 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 enough colonies have been picked up, Q-bots are used to
It was fixed on a 2 × 22 cm ² nylon membrane. Each membrane is 9,21
It contained 6 or 36,864 colonies. These membranes
Placed on agar plates with appropriate antibiotics. 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 on top of a boiling water bath for another 4 minutes. The filter was then placed on filter paper pre-wetted with a 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 and 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 in Sambrook, J. et al. , Fritsc
h. F. And Maniatis, T .; (Molecular Cloni
ng: A Laboratory Manual, 2nd ed.). 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 oligonucleotides listed in SEQ ID NO: 7, excluding clones containing a poly A tail but no cDNA: TCG ACC CAC GC
4. GTCC GAA AAA AAA AAA AAA AAA AAA AAA; 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 non-radioactive colonies (cold colony add)
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 the identification of a gene from a computer homology search.

The identity of the gene was stored in 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 a novel corn Rad51 ortholog (SEQ ID NO: 2) and Ara
bidopsis thaliana Rad51C-like ortholog (ortho
logue) (SEQ ID NO: 8, accession number AC002387, Locus AAB8)
2635, GI: 2583126). The ATP binding motif (Walker A Box) is highlighted.

[0200] Sequence comparisons were performed using the BestFit program in GCG. The upper row is the Arabidopsis sequence shown in SEQ ID NO: 8. The lower line is the corn sequence shown in SEQ ID NO: 2.

The parameters of the BestFit program for this calculation are as follows: Ga
p Weight: 8; Average Match: 2.912; Lengt
h Weight: 2; Average Mismatch: -2.003; R
atio: 3.623; Gaps: 2; Percent Similarity
: 78.388; Percent Identity: 67.033.

[0202]

Embedded image The above examples are provided to illustrate the 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, patent applications and computer programs 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, ZW (72) Invention , Jinlui United States Iowa 50131, Johnston, Greendale Circle 6022, Apartment 205 F-term (reference) 2B030 AA02 AA07 CA15 CA17 CA19 4B024 AA08 AA11 AA20 CA04 DA01 EA04 GA11 HA01 4B050 CC01 DD13 LL10 4B065 AA88 CA13

Claims (23)

[Claims] 1. An isolated RAD51 polynucleotide, comprising: (a) determined by the GAP algorithm under default parameters;
For a polynucleotide selected from the group consisting of SEQ ID NOs: 1, 3, and 5,
A polynucleotide having at least 80% sequence identity over its entire length; (b) a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, and 6; (c) a Zea Mays nucleic acid using primers A polynucleotide amplified from a library, wherein said primers selectively hybridize under stringent hybridization conditions to a locus within a polynucleotide selected from the group consisting of SEQ ID NOs: 1, 3, and 5. A polynucleotide soybean; (d) 0 under stringent hybridization conditions and at 60 ° C.
. A polynucleotide that selectively hybridizes under washing with 1 × SSC to a polynucleotide selected from the group consisting of SEQ ID NOs: 1, 3, and 5; (e) selected from the group consisting of SEQ ID NOs: 1, 3, and 5; (F) a polynucleotide complementary to the polynucleotide of (a), (b), (c), (d) or (e); and (g) (a), (b) , (C), (d), (e) or (f) a polynucleotide comprising at least 30 contiguous nucleotides from the polynucleotide of the polynucleotide 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 orientation or an 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 transgenic plant of claim 4, wherein said plant is: corn, soybean, sunflower, sugar corn, rape, wheat, alfalfa, cotton, rice, barley, and millet. A transgenic plant selected from the group consisting of: 8. Transgenic seed from the transgenic plant according to claim 4. 9. A method for regulating the level of RAD51 in a plant, comprising:
The method according to claim 1, wherein the method comprises the steps of: (a) introducing a recombinant expression cassette into a plant cell, wherein the recombinant cassette is operably linked to a promoter.
(B) culturing the plant cells under plant cell growth conditions; (c) regenerating the whole plant carrying the transformed genotype; and (d) Inducing expression of said polynucleotide in a plant for a time sufficient to modulate the level of RAD51. 10. The method according to claim 9, wherein the plant is corn. 11. An isolated RAD51 protein, which comprises: (a) a polypeptide of at least 20 contiguous amino acids from a polypeptide selected from the group consisting of SEQ ID NOs: 2 and 4. b) a polypeptide selected from the group consisting of SEQ ID NOs: 2 and 4; (c) a polypeptide, which has a full length relative to a polypeptide selected from the group consisting of SEQ ID NOs: 2 and 4. And at least one linear epitope in common with a polypeptide selected from the group consisting of SEQ ID NOs: 2 and 4, wherein the sequence identity is Determined using the GAP program under the default parameters,
A protein comprising: a polypeptide; and a member selected from the group consisting of: (d) at least one polypeptide encoded by the member of claim 1. 12. A method for increasing transformation efficiency, comprising the steps of: (a) introducing a polynucleotide of interest and a RAD51 polynucleotide into a plant cell to produce a transformed plant cell; (B) culturing the plant cells under cell growth conditions; and (c) inducing the expression of the RAD51 polynucleotide for a time sufficient to increase the efficiency of transformation of the polynucleotide of interest. A method comprising: 13. The method according to claim 12, wherein the plant cells are derived from monocotyledonous plants or dicotyledonous plants. 14. The method of claim 13, wherein said plant cells are from the group consisting of: corn, soybean, sunflower, sorghum, rape, wheat, alfalfa, cotton, rice, barley, and millet. The method chosen. 15. A transformed plant cell produced by the method according to claim 12. 16. The plant cell of claim 15, wherein the plant cell comprises: corn, soybean, sunflower, sweet corn, rape, wheat, alfalfa, cotton, rice, barley, and millet. A plant cell selected from the group consisting of: 17. The method of claim 12, wherein said transformed plant cells are grown under conditions sufficient to produce a transformed plant. 18. A transformed plant produced by the method according to claim 17. 19. The plant according to claim 18, wherein said plant is a monocotyledonous or dicotyledonous plant. 20. The plant according to claim 19, wherein the plant is selected from the group consisting of: corn, soybean, sunflower, sweet corn, rape, wheat, alfalfa, cotton, rice, barley, and millet. Be a plant. 21. A transgenic seed produced by the plant of claim 18. 22. The method of claim 12, wherein the RAD5
A method, wherein one polynucleotide and the polynucleotide of interest are simultaneously introduced into the plant cell. 23. The method of claim 12, wherein said RAD51 polynucleotide is introduced into said plant cell prior to introduction of said polynucleotide of interest.