EP1002089A1

EP1002089A1 - Plant genes encoding dr1 and drap1, a global repressor complex of transcription

Info

Publication number: EP1002089A1
Application number: EP98942037A
Authority: EP
Inventors: Stephen M. Allen; Zhan-Bin Liu
Original assignee: EI Du Pont de Nemours and Co
Current assignee: EIDP Inc
Priority date: 1997-08-15
Filing date: 1998-08-12
Publication date: 2000-05-24
Also published as: AU9017498A; WO1999009175A1; AR016819A1

Abstract

This invention relates to an isolated nucleic acid fragment encoding a protein involved in regulation of gene expression. The invention also relates to the construction of a chimeric gene encoding all or a portion of the protein involved in regulation of gene expression, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the protein involved in regulation of gene expression in a transformed host cell.

Description

TITLE

PLANT GENES ENCODING DR1 AND DRAP1, A GLOBAL REPRESSOR

COMPLEX OF TRANSCRIPTION

This application claims the benefit of U.S. Provisional Application No. 60/055,865, filed 8/15/97.

FIELD OF THE INVENTION This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding proteins involved in regulation of gene expression in plants and seeds. BACKGROUND OF THE INVENTION

Like many biological processes, transcription is controlled by both stimulatory and inhibitory proteins whose interplay regulates the overall activity of RNA polymerase II. The majority of regulatory proteins target specific genes through interaction with defined DNA elements in the proximity of or at a distance from the start site of transcription. In many instances, activators influence the activity of RNA polymerase II through direct or indirect interactions with the general transcription factors (Conaway and Conaway, (1993) Annu. Rev. Biochem. 62: 161-190; Zawel and Reinberg, (1995) Annu. Rev. Biochem. 64: 533-561). In cells, transcription is also negatively regulated by another family of factors. These factors repress transcription by different modes. Some are sequence-specific DNA binding proteins, which upon binding to specific promoters, render the gene silent (Hanna-Rose and Hansen, (1996) Trends Genet. 12: 229-234; Shi et al., (1991) Cell 67: 377-388). Other gene-specific repressors inhibit transcription by sequestering activators and preventing their translocation to the nucleus and/or preventing their association with promoter sequences (Benezra et al., (1990) Cell 61: 49-59; Baeuerle and Batimore (1988) Science 242: 540-545). Another growing family of repressors includes molecules that are tethered to promoters by interacting with sequence-specific DNA binding proteins and/or components of the basal transcription machinery (Ayer et al., (1995) Cell 80: 767-776; Inostroza et al., (1992) Cell 70: 477-489). One member of this last category is the Drl/DrAPl repressor complex.

Drl is a TATA-binding protein (TBP)-associated phosphoprotein and functions as an inhibitor of gene transcription (Inostroza et al., (1992) Cell 70: 477-489). Drl genes have been isolated from human, yeast, and Arabidopsis (Inostroza et al., (1992) Cell 70: 477-489; Kim et al, (1997) Proc. Natl. Acad Sci. USA 94: 820-825; Kuromori et al., (1994) Nucleic Acids Research 22: 5296-5301). Effective repression by Drl requires a Drl -associated polypeptide (DrAPl), a corepressor of transcription. Association of DrAPl with Drl results in higher stability of the Drl -TBP-TATA motif complex and precludes the entry of TFIIA and/or TFIIB to preinitiation complexes (Mermelstein et al., (1996) Genes & Development 10: 1033-1048). DrAPl genes have only been isolated from human and yeast (Mermelstein et al., (1996) Genes & Development 10: 1033-1048; Kim et al., (1997) Proc. Natl. Acad. Sci. USA 94: 820-825). No plant DrAPl has been reported. Accordingly, the availability of nucleic acid sequences encoding all or a portion of a Drl or DrAPl transcriptional control protein would facilitate alteration of gene expression in plants, and facilitate studies to better understand transcriptional regulation mechanisms in plants. Drl and DrAPl proteins may also provide targets to facilitate design and/or identification of inhibitors of Drl and DrAPl protiens that may be useful as herbicides.

SUMMARY OF THE INVENTION The instant invention relates to isolated nucleic acid fragments encoding proteins involved in regulation of gene expression. Specifically, this invention concerns an isolated nucleic acid fragment encoding a Drl or DrAPl protein. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding a Drl or DrAPl protein.

An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a protein involved in regulation of gene expression selected from the group consisting of Drl or DrAPl protein. In another embodiment, the instant invention relates to a chimeric gene encoding a Drl or DrAPl protein, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a Drl or DrAPl protein, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.

In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a Drl or DrAPl protein, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method of altering the level of expression of a Drl or DrAPl protein in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a Drl or DrAPl protein; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of Drl or DrAPl protein in the transformed host cell. An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a Drl or DrAPl protein.

A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a Drl or DrAPl protein ,the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a Drl or DrAPl protein, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of Drl or DrAPl protein in the transformed host cell; (c) optionally purifying the Drl or DrAPl protein expressed by the transformed host cell; (d) treating the Drl or DrAPl protein with a compound to be tested; and (e) comparing the activity of the Drl or DrAPl protein that has been treated with a test compound to the activity of an untreated Drl or DrAPl protein, thereby selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detailed description and the accompanying drawings and sequence descriptions which form a part of this application.

Figure 1 shows a comparison of the amino acid sequences of the Arabidopsis thaliana Drl protein (D38110), the human Drl protein (M97388), and the instant soybean Drl protein (se3.08b05).

Figure 2 shows a comparison of the amino acid sequences of the human DrAPl protein (U41843) and the instant maize (csl.pk0049.al) and wheat (wll.pk0012.f3) DrAPl protiens. The following sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

SEQ ID NO:l is the nucleotide sequence comprising a portion of the cDNA insert in clone cbn2.pk0039.h8 encoding a corn DrAPl protein. SEQ ID NO:2 is the deduced amino acid sequence of a DrAPl protein derived from the nucleotide sequence of SEQ ID NO:l.

SEQ ID NO:3 is the nucleotide sequence comprising a portion of the cDNA insert in clone rlsl2.pk0015.el2 encoding a rice DrAPl protein.

SEQ ID NO:4 is the deduced amino acid sequence of a DrAPl protein derived from the nucleotide sequence of SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence comprising a portion of the cDNA insert in clone rl0n.pk0076.gl encoding a rice Drl protein.

SEQ ID NO: 6 is the deduced amino acid sequence of a Drl protein derived from the nucleotide sequence of SEQ ID NO:5. SEQ ID NO:7 is the nucleotide sequence comprising a portion of the cDNA insert in clone ses2w.pk0043.b3 encoding a soybean Drl protein.

SEQ ID NO:8 is the deduced amino acid sequence of a Drl protein derived from the nucleotide sequence of SEQ ID NO:7. SEQ ID NO: 9 is the nucleotide sequence comprising a portion of the cDNA insert in clone wleln.pk0106.gl 1 encoding a wheat Drl protein.

SEQ ID NO: 10 is the deduced amino acid sequence of a Drl protein derived from the nucleotide sequence of SEQ ID NO:9. SEQ ID NO: 11 is the nucleotide sequence comprising a portion of the cDNA insert in clone wlml.pk0016.f3 encoding a wheat DrAPl protein.

SEQ ID NO: 12 is the deduced amino acid sequence of a DrAPl protein derived from the nucleotide sequence of SEQ ID NO: 11.

SEQ ID NO: 13 is the amino acid sequence encoding the Arabidopsis thaliana Drl protein having GenBank Accession No. D38110.

SEQ ID NO: 14 is the amino acid sequence encoding the human Drl protein having GenBank Accession No. M97388.

SEQ ID NO: 15 is the amino acid sequence encoding the human DrAPl protein having GenBank Accession No. U41843. SEQ ID NO: 16 is the nucleotide sequence comprising the cDNA insert in clone csl.pk0049.al encoding a maize DrAPl protein.

SEQ ID NO: 17 is the deduced amino acid sequence of a maize DrAPl protein derived from the nucleotide sequence of SEQ ID NO: 16.

SEQ ID NO: 18 is the nucleotide sequence comprising the cDNA insert in clone se3.08b05 encoding a soybean Drl protein.

SEQ ID NO: 19 is the deduced amino acid sequence of a soybean Drl protein derived from the nucleotide sequence of SEQ ID NO: 18

SEQ ID NO :20s the nucleotide sequence comprising the cDNA insert in clone wll.pk0012.f3 encoding a portion of a wheat DrAPl protein. SEQ ID NO:21is the deduced amino acid sequence encoding a portion of a wheat

DrAPl protein derived from the nucleotide sequence of SEQ ID NO:20.

The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 73:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2J:345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION In the context of this disclosure, a number of terms shall be utilized. As used herein, an "isolated nucleic acid fragment" is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C -terminal portions of the protein molecule would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are 80% identical to the coding sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are 90% identical to the coding sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are 95% identical to the coding sequence of the nucleic acid fragments reported herein. The percent identity used herein, can be precisely determined by the DNASTAR protein alignment protocol using the Jotun-Hein algorithm ( Hein, J.J. (1990) Unified Approach to Alignment and Phylogenies. Methods in Enzymology, vol. 183, 626-645). Default parameters for the Jotun-Hein method for multiple alignments are: GAP PENALTY=11, GAP LENGTH PENALTY=3; for pairwise alignments KTUPLE 6.

A "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

"Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the Drl or DrAPl proteins as set forth in SEQ ID NOs:2, 4, 6, 8, 10, 17, 19 and 21. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. "Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available. "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

"Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 75:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. The "translation leader sequence" refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G.D. (1995) Molecular Biotechnology 3:225). The "3' non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 7:671-680.

"RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

"Altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

"Mature" protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

A "chloroplast transit peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. "Chloroplast transit sequence" refers to a nucleotide sequence that encodes a chloroplast transit peptide. A "signal peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. ¥2:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel ( 1992) Plant Phys.100: 1627- 1632). "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:211) and particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Pat. No. 4,945,050).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis"). A plant homolog of Drl has been identified from a collection of soybean ESTs. The entire cDNA insert of the clone se3.08b05 has been fully sequenced and found to contain a complete opening reading frame that encodes a protein of 144 amino acids. Amino acid sequence comparison indicates that there is 74% identity of the soybean Drl sequence to the Arabidopsis Drl (GenBank Accession No. D38110) and 31% identity to the human Drl (GenBank Accession No. M97388). Nucleotide identity of the coding sequences between the soybean and Arabidopsis Drl genes is approximately 68%.

A plant homolog of DrAPl has been identified from a collection of maize and wheat ESTs. The enitre cDNA insert of the maize clone csl.pk0049.al has been fully sequenced and found to contain a complete opening reading frame for a protein of 159 amino acids. Amino acid sequence comparison indicates that there is a 30% identity of this maize DrAPl to the human DrAPl (GenBank Accession No. U41843). Likewise, the cDNA insert in the wheat clone wll.pk0012.f3 contains almost an entire opening reading frame, apparently missing a short segment of the nucleotide sequence sufficient to encode several N-terminal amino acids of the wheat DrAPl homolog. At the amino acid level, the wheat peptide is approximately 78% identical to the maize DrAPl encoded by cDNA clone csl.pk0001.al 1. Nucleotide identity between wheat and maize cDNAs is approximately 80%.

The amino acid sequence similarity between the instant soybean Drl, the maize DrAPl, and the wheat DrAPl proteins and the human Drl/DrAPl and Arabidopsis Drl proteins indicates that the plant Drl/DrAPl may function as a transcription repressor complex. Although the Drl/DrAPl is a non-specific, global repressor for transcription, the plant proteins may still be used specifically to down-regulate expression of specific genes in plants by specific targeting of this repression complex. Accordingly, the nucleotide sequences encoding the instant plant Drl/DrAPl proteins can be fused to a very defined DNA-binding domain of a plant endogenous transcription factor, which is required to bind to a specific target site in a plant promoter to result in specific gene activation. The chimeric fusion protein comprising the plant Drl/DrAPl complex and the DNA-binding domain of the plant transcription factor results in the specific transcription repression. By using this approach, the plant Drl/DrAPl repressors can be specifically targeted to the native plant promoter, leading to the specific silencing of the native gene expression at the transcription level.

Nucleic acid fragments encoding at least a portion of several proteins involved in regulation of gene expression have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. Table 1 lists the proteins that are described herein, and the designation of the cDNA clones that comprise the nucleic acid fragments encoding these proteins. TABLE 1 Proteins Involved In Regulation Of Gene Expression Enzyme Clone Plant

Drl rl0n.pk0076.gl Rice ses2w.pk0043.b3 Soybean se3.08b05 wleln.pk0106.gl l Wheat

DrAPl cbn2.pk0039.h8 Corn csl.pk0049.al rlsl2.pk0015.el2 Rice wlml.pk0016.β Wheat wll.pk0012.f3

The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other Drl or DrAPl proteins, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., (1988) PNAS USA §5:8998) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., (1989) PNAS USA 86:5613; Loh et al, (1989) Science 243:211). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman, M.A. and Martin, G.R., (1989) Techniques 7:165).

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner, R.A. (1984) Adv. Immunol. 36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed Drl or DrAPl proteins are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of altering transcriptional regulation of genes controlled by Drl and DrAPl in those cells.

Overexpression of the Drl and DrAPl proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. For reasons of convenience, the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al, (1989) Mol. Gen. Genetics 275:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis. For some applications it may be useful to direct the instant proteins involved in regulation of gene expression to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by altering the coding sequence to encode a Drl or DrAPl protein with appropriate intracellular targeting sequences such as transit sequences (Keegstra, K. (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel, N. (1992) Plant Phys.100:1621-1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genes encoding Drl or DrAPl proteins in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant proteins involved in regulation of gene expression can be constructed by linking a gene or gene fragment encoding a Drl or DrAPl protein to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

The instant Drl or DrAPl proteins (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting Drl or DrAPl proteins in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant Drl or DrAPl proteins are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant Drl or DrAPl proteins. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded proteins involved in regulation of gene expression. An example of a vector for high level expression of the instant Drl or DrAPl proteins in a bacterial host is provided (Example 7). Additionally, the instant Drl or DrAPl proteins can be used as a targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the Drl or DrAPl proteins described herein catalyze various steps in global transcriptional regulation. Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition plant growth. Thus, the instant Drl or DrAPl proteins could be appropriate for new herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at., (1987) Genomics 7:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein, D. et al., (1980) Am. J. Hum. Genet. 52:314-331).

The production and use of plant gene-derived probes for use in genetic mapping is described in R. Bernatzky, R. and Tanksley, S. D. (1986) Plant Mol. Biol. Reporter 4(1) :31 -41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art. Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel, J. D., et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan, M. et al. (1995) Genome Research 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes. A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian, H. H. (1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield, V. C. et al. (1993) Genomics 76:325-332), allele-specific ligation (Landegren, U. et al. (1988) Science 241:1011-1080), nucleotide extension reactions (Sokolov, B. P. (1990) Nucleic Acid Res. 18:3611), Radiation Hybrid Mapping (Walter, M. A. et al. (1997) Nature Genetics 7:22-28) and Happy Mapping (Dear, P. H. and Cook, P. R. (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods. Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer, (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al., (1995) Proc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell 7:15). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the Drl or DrAPl protein.

Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding a Drl or DrAPl protein can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the Drl or DrAPl protein gene product.

EXAMPLES The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

EXAMPLE 1 Composition of cDNA Libraries: Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various corn, rice, soybean and wheat tissues were prepared. The characteristics of the libraries are described below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat

Library Tissue Clone cbn2 Corn developing kernel two days after pollination cbn2.pk0039.h8 csl Corn leaf sheath from 5 week old plant csl.pk0049.al rlOn Rice 15 day old leaf* rl0n.pk0076.gl rlsl2 Rice leaf 15 days after germination, 12 hours after infection rlsl2.pk0015.el2 of strain Magaporthe grisea 4360-R-67 (AVR2-YAMO) se3 Soybean embryo, 17 days after flowering se3.08b05 ses2w Soybean embryogenic suspension 2 weeks after subculture ses2w.pk0043.b3 wl 1 Wheat leaf from 7 day old seedling wl 1.pkOO 12.f3 wleln Wheat leaf from 7 day old etiolated seedling* wleln.pk0106.gl 1 wlml Wheat seedlings 1 hour after inoculation with Erysiphe wlml.pkOOlό.β graminis f. sp tritici

*These libraries were normalized essentially as described in U.S. Pat. No. 5,482,845

cDNA libraries were prepared in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Conversion of the Uni-ZAP™ XR libraries into plasmid libraries was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells. Amplified insert DNAs or plasmid DNAs were sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams, M. D. et al., (1991) Science 252:1651). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

EXAMPLE 2 Identification of cDNA Clones ESTs encoding proteins involved in regulation of gene expression were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993)

J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish, W. and States, D. J. (1993) Nature Genetics 3:266-212) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins.

EXAMPLE 3 Characterization of cDNA Clones Encoding Drl

The BLASTX search using the EST sequence of clone se3.08b05 revealed similarity of the protein encoded by the cDNA to the Drl protein homolog from Arabidopsis thaliana (Genbank Accession No. D38110) and the human Drl protein (Genbank Accession No. M97388). The sequence of the entire cDNA insert in clone se3.08b05 was determined and is set forth in SEQ ID NO: 18 the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 19. The entire cDNA insert in clone se3.08b05 was reevaluated by BLAST, yielding an even higher pLog value vs. the Arabidopsis (D38110; pLog = 71.92) and human (M97388; pLog = 20.96) sequences. Sequence alignments and BLAST scores and probabilities indicate that the instant nucleic acid fragment encodes the entire soybean Drl protein. This is the first soybean EST identified that encodes a Drl homolog.

Additional BLASTX searching resulted in the identification of further EST sequences from clones ses2w.pk0043.b3 and wleln.pk0106.gl 1 wherein the sequence of the proteins encoded by the cDNAs was similar to Drl from Arabidopsis thaliana (Swiss-Prot, Accession No. P49592). The BLASTX search using the EST sequence from clone rl0n.pk0076.gl revealed similarity of the protein encoded by the cDNA to Drl from

Arabidopsis thaliana (PIR, Accession No. S53582). The BLAST results for each of these ESTs are shown in Table 3:

TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous to Drl Clone BLAST pLog Score rl0n.pk0076.gl 10.00 ses2w.pk0043.b3 56.30 wleln.pk0106.gl l 49.70

The sequence of a portion of the cDNA insert from clone rl0n.pk0076.gl is shown in SEQ ID NO:5; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:6. The sequence of a portion of the cDNA insert from clone ses2w.pk0043.b3 is shown in SEQ ID NO:7; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:8. The sequence of a portion of the cDNA insert from clone wleln.pk0106.gl 1 is shown in SEQ ID NO:9; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 10. BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of a Drl protein. These sequences represent the first rice, soybean and wheat sequences encoding a Drl protein.

EXAMPLE 4 Characterization of cDNA Clones Encoding DrAPl The BLASTX search using the EST sequences of clones csl.pk0049.al and wll.pk0012.f3 revealed similarity of the protein encoded by the cDNAs to the DrAPl protein homolog from human (Genbank Accession No. U41843). The sequences of the entire cDNA inserts in clones csl.pk0049.al and wll.pk0012.β were determined and are set forth in SEQ ID NO: 16 and SEQ ID NO:20, repectively; the deduced amino acid sequences of these cDNAs are shown in SEQ ID NO:17 and SEQ ID NO:21, repectively. The cDNA inserts in clone csl.pk0049.al and wll.pk0012.f3 were reevaluated by BLAST, yielding even higher pLog values (pLog for csl.pk0049.al = 24.11; pLog for wll.pk0012.f3 = 25.75) versus the human DrAPl (U41843) sequence. Sequence alignments and BLAST scores and probabilities indicate that the cDNA insert in clone csl.pk0049.al encodes the entire corn DrAPl protein, and the cDNA insert in clone wll.pk0012.β encodes a substantial portion of a wheat DrAPl protein. These are the first plant ESTs identified that encode a DrAPl homolog.

Additional BLASTX searching resulted in the identification of further EST sequences from clones cbn2.pk0039.h8, rlsl2.pk0015.el2 and wlml.pk0016.β wherein the sequence of the proteins encoded by the cDNAs was similar to DrAPl from Homo sapiens (GenBank,

Accession No. X96506). The BLAST results for each of these ESTs are shown in Table 4:

TABLE 4 BLAST Results for Clones Encoding Polypeptides Homologous to DrAPl Clone BLAST pLog Score cbn2.pk0039.h8 25.20 rlsl2.pk0015.el2 21.00 lml.pk0016.f3 26.20

The sequence of a portion of the cDNA insert from clone cbn2.pk0039.h8 is shown in SEQ ID NO:l; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:2. The sequence of a portion of the cDNA insert from clone rlsl2.pk0015.el2 is shown in SEQ ID NO:5; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:6. The sequence of a portion of the cDNA insert from clone wlml.pkOOlό.β is shown in SEQ ID NO:l 1; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 12. BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of a DrAPl protein. These sequences represent the first com, rice, and wheat sequences encoding a DrAPl protein.

EXAMPLE 5 Expression of Chimeric Genes in Monocot Cells A chimeric gene comprising a cDNA encoding a protein involved in regulation of gene expression in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes Ncol and Smal and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb Ncol-Smal fragment of the plasmid pML 103. Plasmid pML 103 has been deposited under the terms of the Budapest

Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding a protein involved in regulation of gene expression, and the 10 kD zein 3' region.

The chimeric gene described above can then be introduced into com cells by the following procedure. Immature com embryos can be dissected from developing caryopses derived from crosses of the inbred com lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27°C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al., (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the com tissue with a Biolistic™ PDS-1000 He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a mpture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., (1990) Bio/Technology 5:833-839). EXAMPLE 6 Expression of Chimeric Genes in Dicot Cells A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261 :9228-9238) can be used for expression of the instant proteins involved in regulation of gene expression in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

Soybean embroys may then be transformed with the expression vector comprising sequences encoding a protein involved in regulation of gene expression. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26°C on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium. Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Kline et al. (1987) Nature (London) 327:10, U.S. Patent No. 4,945,050). A Du Pont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al.(1985) Nature 573:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5' region, the fragment encoding the protein involved in regulation of gene expression and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

EXAMPLE 7 Expression of Chimeric Genes in Microbial Cells The cDNAs encoding the instant proteins involved in regulation of gene expression can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430. Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as decribed above. The prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding a protein involved in regulation of gene expression are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight. EXAMPLE 8

Evaluating Compounds for Their Ability to Inhibit the Activity of Proteins Involved in Regulation of Gene Expression The proteins involved in regulation of gene expression described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 7, or expression in eukaryotic cell culture, in planta, and using viral expression systems in suitably infected organisms or cell lines. The instant proteins involved in regulation of gene expression may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase ("GST"), thioredoxin ("Trx"), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide ("(His)₆"). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.

Purification of the instant proteins involved in regulation of gene expression, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the proteins involved in regulation of gene expression are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, a protein involved in regulation of gene expression may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)g peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin. Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activition of the protein involved in regulation of gene expression disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity.

Claims

CLAIMS What is claimed is:

1. An isolated nucleic acid fragment encoding all or a substantial portion of a rice, soybean or wheat Drl protein comprising a member selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:6, 8, 10, and 19;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:6, 8, 10, and 19; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

2. The isolated nucleic acid fragment of Claim 1 wherein the nucleotide sequence of the fragment comprises all or a portion of the sequence set forth in a member selected from the group consisting of SEQ ID NO:5, 7, 9, and 18.

3. A chimeric gene comprising the nucleic acid fragment of Claim 1 operably linked to suitable regulatory sequences.

4. A transformed host cell comprising the chimeric gene of Claim 3.

5. A Drl polypeptide comprising all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:6, 8, 10 and 19.

6. An isolated nucleic acid fragment encoding all or a substantial portion of a plant DrAPl protein comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, 4, 12, 17 and 21;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, 4, 12, 17 and 21; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

7. The isolated nucleic acid fragment of Claim 6 wherein the nucleotide sequence of the fragment comprises all or a portion of the sequence set forth in a member selected from the group consisting of SEQ ID NO:l, 3, 11, 16 and 20.

8. A chimeric gene comprising the nucleic acid fragment of Claim 6 operably linked to suitable regulatory sequences.

9. A transformed host cell comprising the chimeric gene of Claim 8.

10. A DrAPl polypeptide comprising all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, 4, 12, 17 and 21.

11. A method of altering the level of expression of a protein involved in regulation of gene expression in a host cell comprising:

(a) transforming a host cell with the chimeric gene of any of Claims 3 or 8 ; and

(b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of a protein involved in regulation of gene expression in the transformed host cell.

12. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a protein involved in regulation of gene expression comprising: (a) probing a cDNA or genomic library with the nucleic acid fragment of any of Claims 1 and 6;

(b) identifying a DNA clone that hybridizes with the nucleic acid fragment of any of Claims 1 and 6;

(c) isolating the DNA clone identified in step (b); and (d) sequencing the cDNA or genomic fragment that comprises the clone isolated in step (c) wherein the sequenced nucleic acid fragment encodes all or a substantial portion of the amino acid sequence encoding a protein involved in regulation of gene expression.

13. A method of obtaining a nucleic acid fragment encoding a substantial portion of an amino acid sequence encoding a protein involved in regulation of gene expression comprising:

(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in any of SEQ ID NOs:l, 3, 5, 7, 9, 11, 16, 18, and 20; and (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector wherein the amplified nucleic acid fragment encodes a substantial portion of an amino acid sequence encoding a protein involved in regulation of gene expression.

14. The product of the method of Claim 12.

15. The product of the method of Claim 13.

16. A method for evaluating at least one compound for its ability to inhibit the activity of a protein involved in regulation of gene expression, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a protein involved in regulation of gene expression, operably linked to suitable regulatory sequences;

(b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the protein involved in regulation of gene expression encoded by the operably linked nucleic acid fragment in the transformed host cell;

(c) optionally purifying the protein involved in regulation of gene expression expressed by the transformed host cell;

(d) treating the protein involved in regulation of gene expression with a compound to be tested; and

(e) comparing the activity of the protein involved in regulation of gene expression that has been treated with a test compound to the activity of an untreated protein involved in regulation of gene expression, thereby selecting compounds with potential for inhibitory activity.