WO1999009175A1 - Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription - Google Patents

Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription Download PDF

Info

Publication number
WO1999009175A1
WO1999009175A1 PCT/US1998/016688 US9816688W WO9909175A1 WO 1999009175 A1 WO1999009175 A1 WO 1999009175A1 US 9816688 W US9816688 W US 9816688W WO 9909175 A1 WO9909175 A1 WO 9909175A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid fragment
gene
protein
regulation
Prior art date
Application number
PCT/US1998/016688
Other languages
English (en)
Inventor
Stephen M. Allen
Zhan-Bin Liu
Original Assignee
E.I. Du Pont De Nemours And Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by E.I. Du Pont De Nemours And Company filed Critical E.I. Du Pont De Nemours And Company
Priority to AU90174/98A priority Critical patent/AU9017498A/en
Priority to EP98942037A priority patent/EP1002089A1/fr
Publication of WO1999009175A1 publication Critical patent/WO1999009175A1/fr
Priority to US10/628,969 priority patent/US7288695B2/en
Priority to US11/888,497 priority patent/US7985850B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • 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
  • RNA polymerase II 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.
  • 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).
  • transcription In cells, transcription is also negatively regulated by another family of factors. These factors repress transcription by different modes.
  • 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).
  • Drl/DrAPl repressor complex 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.
  • DrAPl DrAPl -associated polypeptide
  • DrAPl 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 :20 s the nucleotide sequence comprising the cDNA insert in clone wll.pk0012.f3 encoding a portion of a wheat DrAPl protein.
  • SEQ ID NO:21 is the deduced amino acid sequence encoding a portion of a wheat
  • DrAPl protein derived from the nucleotide sequence of SEQ ID NO:20 is derived from the nucleotide sequence of SEQ ID NO:20.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • 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.
  • 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.
  • 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).
  • 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/).
  • BLAST Basic Local Alignment Search Tool
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • expression 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.
  • 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).
  • a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • genes encoding other Drl or DrAPl proteins 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).
  • 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.
  • 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.
  • 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.
  • the second primer sequence may be based upon sequences derived from the cloning vector.
  • 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).
  • RACE protocol Frohman et al., (1988) PNAS USA ⁇ 5:8998
  • 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.
  • 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.
  • 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.
  • 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 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).
  • 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.
  • the instant Drl or DrAPl proteins could be appropriate for new herbicide discovery and design.
  • 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.
  • the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers.
  • RFLP restriction fragment length polymorphism
  • 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.
  • 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).
  • 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).
  • FISH direct fluorescence in situ hybridization
  • 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.
  • 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.
  • 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.
  • 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.
  • an arbitrary genomic site primer such as that for a restriction enzyme site-anchored synthetic adaptor.
  • 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
  • cDNA libraries were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Conversion of the Uni-ZAPTM 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.
  • 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 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. ⁇ 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 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.pkOOl ⁇ . ⁇ 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.
  • 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
  • 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.
  • Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (SequenaseTM 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).
  • PAT phosphinothricin acetyl transferase
  • 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.
  • gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
  • Ten ⁇ g of plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
  • Calcium chloride 50 ⁇ L of a 2.5 M solution
  • spermidine free base (20 ⁇ L 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 ⁇ L 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 ⁇ L of ethanol.
  • An aliquot (5 ⁇ L) of the DNA-coated gold particles can be placed in the center of a KaptonTM flying disc (Bio-Rad Labs). The particles are then accelerated into the com tissue with a BiolisticTM 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.
  • 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.
  • 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.
  • a seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the ⁇ 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.
  • Nco I which includes the ATG translation initiation codon
  • Sma I which includes the ATG translation initiation codon
  • Kpn 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.
  • PCR polymerase chain reaction
  • Soybean embroys may then be transformed with the expression vector comprising sequences encoding a protein involved in regulation of gene expression.
  • 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 BiolisticTM 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water.
  • Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA).
  • 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 ⁇ g/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.
  • 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- ⁇ -galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°.
  • IPTG isopropylthio- ⁇ -galactoside, the inducer
  • 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 ").
  • GST glutathione S-transferase
  • Trx thioredoxin
  • (His) 6 ") C- and/or N-terminal hexahistidine polypeptide
  • 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 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.
  • 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.
  • a protein involved in regulation of gene expression may be expressed as a fusion protein coupled to the C-terminus of thioredoxin.
  • 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.
  • 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.
  • reagents include ⁇ -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 ThioBondTM 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.

Abstract

L'invention concerne un fragment isolé d'acide nucléique de codage d'une protéine impliquée dans la régulation de l'expression de gènes. L'invention concerne également la construction d'un gène chimérique de codage de la totalité ou d'une partie de la protéine impliquée dans la régulation de l'expression de gènes, en sens codant ou non codant. L'expression du gène chimérique entraîne la production de niveaux altérés de la protéine impliquée dans la régulation de l'expression de gènes dans une cellule hôte transformée.
PCT/US1998/016688 1997-08-15 1998-08-12 Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription WO1999009175A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU90174/98A AU9017498A (en) 1997-08-15 1998-08-12 Plant genes encoding dr1 and drap1, a global repressor complex of transcription
EP98942037A EP1002089A1 (fr) 1997-08-15 1998-08-12 Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription
US10/628,969 US7288695B2 (en) 1997-08-15 2003-07-28 Plant genes encoding Dr1 and DRAP1, a global repressor complex of transcription
US11/888,497 US7985850B2 (en) 1997-08-15 2007-07-31 Plant genes encoding Dr1 and DRAP1, a global repressor complex of transcription

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5586597P 1997-08-15 1997-08-15
US60/055,865 1997-08-15

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09485558 A-371-Of-International 2000-02-11
US09/789,054 Continuation-In-Part US20020184659A1 (en) 1997-08-15 2001-02-20 Plant genes encoding Dr1 and DRAP1, a global repressor complex of transcription

Publications (1)

Publication Number Publication Date
WO1999009175A1 true WO1999009175A1 (fr) 1999-02-25

Family

ID=22000668

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/016688 WO1999009175A1 (fr) 1997-08-15 1998-08-12 Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription

Country Status (4)

Country Link
EP (1) EP1002089A1 (fr)
AR (1) AR016819A1 (fr)
AU (1) AU9017498A (fr)
WO (1) WO1999009175A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7288695B2 (en) 1997-08-15 2007-10-30 E.I. Dupont De Nemours And Company Plant genes encoding Dr1 and DRAP1, a global repressor complex of transcription
EP2258725A3 (fr) * 2000-06-26 2014-09-17 ZymoGenetics, L.L.C. Récepteur de la cytokine zcytor17
US20140357549A1 (en) * 2011-11-15 2014-12-04 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Novel inhibitors of nox1

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0291893A1 (fr) * 1987-05-19 1988-11-23 The Du Pont Merck Pharmaceutical Company Lignes de cellules humaines stables exprimant un produit de gène indicateur sous des contrôles génétiques specifiques à un virus
WO1993015213A1 (fr) * 1992-01-24 1993-08-05 Zeneca Limited Regulation de la transcription d'un gene

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0291893A1 (fr) * 1987-05-19 1988-11-23 The Du Pont Merck Pharmaceutical Company Lignes de cellules humaines stables exprimant un produit de gène indicateur sous des contrôles génétiques specifiques à un virus
WO1993015213A1 (fr) * 1992-01-24 1993-08-05 Zeneca Limited Regulation de la transcription d'un gene

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GOPPELT A. ET AL: "Characterization of the basal inhibitor of class II transcription NC2 from Saccharomyces cerevisiae.", NUCLEIC ACIDS RESEARCH, (1996) 24/22 (4450-4455). ISSN: 0305-1048 CODEN: NARHAD, United Kingdom, XP002046735 *
INOSTROZA J. ET AL.: "Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II transcription", CELL, vol. 70, no. 3, 7 August 1992 (1992-08-07), pages 477 - 489, XP002087077 *
KIM S. ET AL: "The Dr1/DRAP1 heterodimer is a global repressor of transcription in vivo.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1997) 94/3 (820-825). REFS: 46 ISSN: 0027-8424 CODEN: PNASA6, United States, XP002046733 *
KUROMORI T ET AL: "Cloning of cDNAs from Arabidopsis thaliana that encode putative protein phosphatase 2C and a human Dr1-like protein by transformation of a fission yeast mutant.", NUCLEIC ACIDS RESEARCH, (1994 DEC 11) 22 (24) 5296-301. JOURNAL CODE: O8L. ISSN: 0305-1048., ENGLAND: United Kingdom, XP002087076 *
MERMELSTEIN F ET AL: "Requirement of a corepressor for Dr1-mediated repression of transcription.", GENES AND DEVELOPMENT, (1996 APR 15) 10 (8) 1033-48. JOURNAL CODE: FN3. ISSN: 0890-9369., United States, XP002087078 *
SASAKI T. ET AL.: "Rice cDNA from shoot, AC D39607", EMBL DATABASE, 14 November 1994 (1994-11-14), Heidelberg, XP002087075 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7288695B2 (en) 1997-08-15 2007-10-30 E.I. Dupont De Nemours And Company Plant genes encoding Dr1 and DRAP1, a global repressor complex of transcription
US7985850B2 (en) 1997-08-15 2011-07-26 E. I. Du Pont De Nemours And Company Plant genes encoding Dr1 and DRAP1, a global repressor complex of transcription
EP2258725A3 (fr) * 2000-06-26 2014-09-17 ZymoGenetics, L.L.C. Récepteur de la cytokine zcytor17
US20140357549A1 (en) * 2011-11-15 2014-12-04 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Novel inhibitors of nox1
US9187528B2 (en) * 2011-11-15 2015-11-17 University of Pittsburgh—of the Commonwealth System of Higher Education Inhibitors of Nox1
US9770481B2 (en) 2011-11-15 2017-09-26 University of Pittsburgh—of the Commonwealth System of Higher Education Inhibitors of NOX1

Also Published As

Publication number Publication date
AU9017498A (en) 1999-03-08
AR016819A1 (es) 2001-08-01
EP1002089A1 (fr) 2000-05-24

Similar Documents

Publication Publication Date Title
US7605247B1 (en) Nucleic acid molecules encoding a wheat sucrose transporter
US8637732B2 (en) Plant MYB transcription factor homologs
EP1071793A2 (fr) Enzyme de biosynthese du tryptophane
EP1068334A2 (fr) Proteines de regulation du cycle cellulaire cdc-16, dp-1, dp-2 et e2f tirees de plantes
EP1062355A1 (fr) Inhibiteurs de proteines d'apoptose dans les plantes
WO2000005386A2 (fr) Enzymes impliquees dans la biosynthese du chorismate
WO1999028446A2 (fr) Enzymes biosynthetiques a acides amines ramifies
US6849783B2 (en) Plant biotin synthase
US20050148765A1 (en) Plant cell cyclin genes
WO2000003026A2 (fr) Coactivateurs de transcription
EP1034274A2 (fr) Homologues vegetaux de pad1 et de crm1 de levure et de jab1 humaine: regulateurs de l'activite du facteur de transcription de type ap-1
EP1002089A1 (fr) Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription
US6911331B2 (en) Chorismate biosynthesis enzymes
US6294658B1 (en) Factors involved in gene expression
EP1025210A2 (fr) Enzymes vegetales de biosynthese a chaine ramifiee d'acide amine
US7659450B2 (en) Lcb1 subunit of serine palmitoyltransferase
US6653531B1 (en) Chorismate synthase from plants
US6624343B1 (en) Hexose carrier proteins
US20050059047A1 (en) Plant SUGI homologs
EP1338652A2 (fr) Génés de cycline de cellules végétales
WO1999004004A1 (fr) Homologue vegetal de l'ada2 de la levure, un adaptateur de transcription
WO1999049053A1 (fr) Sous-unite lcb1 de serine palmitoyltransferase
WO1999049021A1 (fr) Sous-unite lcb2 de serine palmitoyltransferase
WO1999002689A1 (fr) Homologues des proteines sug 1 vegetales

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AU AZ BA BB BG BR BY CA CN CU CZ EE GE HR HU ID IL IS JP KG KP KR KZ LC LK LR LT LV MD MG MK MN MX NO NZ PL RO RU SG SI SK SL TJ TM TR TT UA US UZ VN YU

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1998942037

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: KR

WWP Wipo information: published in national office

Ref document number: 1998942037

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1998942037

Country of ref document: EP