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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • 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

Definitions

• This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding coactivator proteins involved in regulation of gene expression in plants and seeds. BACKGROUND OF THE INVENTION
• GCN5 In yeast, GCN5 (yGCN5) is a transcriptional coactivator that enhances the activation of transcription by acidic activators such as GCN4, Gal4-VP16, and the HAP2-HAP3-HAP4 complex (Georgakopoulos, T. and Thireos, G. (1992) EMBO J. 11: 4145-4152). yGCN5 itself is also a histone acetyltransferase (Brownell et al. (1996) Cell 84: 843-851). It is proposed that yGCN5 can be recruited to a specific gene through selective interaction with a subset of transcription factors (Wolffe and Pruss, (1996) Cell 84: 817-819).
• the instant invention relates to isolated nucleic acid fragments encoding plant histone acetyltransferase proteins that may be involved in regulation of gene expression. More particularly, this invention concerns isolated nucleic acid fragments encoding maize, rice, and wheat histone acetyltransferase proteins. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding the maize, rice and wheat histone acetyltransferase proteins.
• the instant invention relates to a chimeric gene that comprises a nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase protein, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase protein, the nucleic acid fragment being operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in transformed host cells that are altered (i.e., increased or decreased) relative to the levels produced in untransformed host cells.
• the instant invention concerns a transformed host cell comprising in its genome a chimeric gene comprising a nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase protein or a chimeric gene comprising a nucleic acid fragment that is complementary to the nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase protein, the chimeric gene being operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of protein encoded by the operably linked nucleic acid fragment 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 maize, rice, or wheat histone acetyltransferase protein in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase protein or a chimeric gene that comprises a nucleic acid fragment that is complementary to the nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase 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 protein encoded by the operably linked nucleic acid fragment in the transformed host cell.
• An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or substantially all of an amino acid sequence encoding a maize, rice, or wheat histone acetyltransferase 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 maize, rice, or wheat histone acetyltransferase protein protein, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a maize, rice, or wheat histone acetyltransferase 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 the protein encoded by the operably linked nucleic acid fragment in the transformed host cell;
• Figure 1 shows a comparison of the amino acid sequences of the Saccharomyces cerevisiae GCN5 transcriptional coactivator (X68628), a human histone acetyltransferase (U57316) and the instant maize and wheat GCN5 protein homologs (maize GCN5 and wlln.pk0003.c2, respectively).
• Figure 2 shows a comparison of the amino acid sequence of the Arabidopsis thaliana histone acetyltransferase protein and the instant histone acetyltransferase homologs encoded by clones cep7.pk0001.al l, wlln.pk0003.c2, wrl.pk0045.f4 and rlr6.pk0084.c5.
• the following sequence descriptions and sequence listings 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 the entire cDNA insert in clone cep7.pk0001.al 1 encoding cGCN5, a maize homolog of the yeast GCN5 protein.
• SEQ ID NO:2 is the deduced amino acid sequence of a portion of a of maize homolog of the yeast GCN5 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 wlln.pk0003.c2 encoding wGCN5, a wheat homolog of the yeast GCN5 protein.
• SEQ ID NO:4 is the deduced amino acid sequence of a portion of a wheat homolog of the yeast GCN5 derived from the nucleotide sequence of SEQ ID NO:3.
• SEQ ID NO: 5 is the amino acid sequence encoding the Saccharomyces cerevisiae GCN5 transcriptional coactivator having EMBL accession No. X68628.
• SEQ ID NO: 6 is the amino acid sequence encoding a human histone acetyltransferase having GenBank accession No. U57316.
• SEQ ID NOs:7, 8 and 9 are the nucleotide sequences of the 5' RACE primers GSP 1 ,
• SEQ ID NOs:10 and 11 are the nucleotide sequences of the 5' RACE Abridged Anchor primer and the Abridged Universal Amplification primer, respectively, used in the 5' RACE protocol.
• SEQ ID NO: 12 is the nucleotide sequence of the contig assembled from SEQ ID NO:l and the nucleotide sequence information obtained from the 5' RACE protocol encoding a portion of a maize homolog of the yeast GCN5 protein.
• SEQ ID NO: 13 is the deduced amino acid sequence derived from the nucleotide sequence set forth in SEQ ID NO: 13.
• SEQ ID NO: 14 is the nucleotide sequence comprising a portion of the cDNA insert in clone rlr6.pk0084.c5 encoding a rice histone acetyltransferase.
• SEQ ID NO: 15 is the deduced amino acid sequence of a histone acetyltransferase derived from the nucleotide sequence of SEQ ID NO: 14.
• SEQ ID NO: 16 is the nucleotide sequence comprising a portion of the cDNA insert in clone wrl.pk0045.f4 encoding a wheat histone acetyltransferase.
• SEQ ID NO: 17 is the deduced amino acid sequence of a histone acetyltransferase derived from the nucleotide sequence of SEQ ID NO: 16.
• SEQ ID NO: 18 is the amino acid sequence encoding an Arabidopsis thaliana histone acetyltransferase set forth in GenBank Accession No. AFO31958.
• the amino acid sequence similarity between the instant maize, rice, and wheat histone acetyltransferase proteins and the Arabidopsis thaliana histone acetyltransferase and yGCN5 proteins indicates that the maize, rice, or wheat histone acetyltransferase proteins may function as transcriptional coactivators.
• the maize, rice or wheat huistone acetyltransferese proteins may be used to reduce expression of specific genes whose promoters are normally regulated by a histone acetyltransferase activity, using antisense or co-suppression technology.
• the plant proteins may also be used to enhance gene expression of those genes whose promoters are normally targeted by the transcription factors that histone acetyltransferase proteins normally interact with.
• the maize, rice, or wheat histone acetyltransferase protein coactivation function can be targeted to a novel promoter region by the addition of either a DNA binding domain or a protein-protein interaction domain.
• the instant maize, rice, or wheat histone acetyltransferase proteins can be fused to a very defined DNA-binding domain, such as, but not limited to, a bacterial lexA DNA binding domain, a yeast Gal4 DNA-binding domain or a DNA binding domain from a plant transcription factor.
• yGCN5 For example, it has also been shown that targeting yGCN5 to a promoter by fusing it to a heterologous DNA-binding domain leads to transcriptional activation in yeast, most probably due to the histone acetyltransferase activity of yGCN5 (Candau et al. (1997) EMBOJ. 7(5:555-565).
• a synthetic promoter can be designed to contain multiple copies of a target site which is necessary for the specific binding by either the lexA, Gal4 or plant DNA binding domain.
• the maize, rice, or wheat histone acetyltransferase protein can be specifically targeted to the engineered synthetic promoter to acetylate the core histones, weaken the interaction between core histones and DNA, open up chromatin structure, and increase the efficiency of transcriptional initiation, thus leading to a higher level of gene expression.
• maize, rice, or wheat histone acetyltransferase protein can be fused to a transcription factor that already includes its own DNA binding domain in order to target the coactivator.
• the maize, rice, or wheat histone acetyltransferase protein can also be fused to other transcription regulatory proteins, such as mediators.
• these mediators do not bind to DNA directly and are recruited to their target sites by interaction with other DNA-binding proteins.
• the histone acetyltransferase activity can be targeted to specific regulatory elements through the interaction between the mediators and other DNA-binding proteins.
• the maize, rice, or wheat histone acetyltransferase proteins can provide a tool to enhance trait gene expression by targeted histone acetylation.
• 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.
• "contig” refers to an assemblage of overlapping nucleic acid sequences to form one contiguous nucleotide sequence. For example, several DNA sequences can be compared and aligned to identify common or overlapping regions. The individual sequences can then be assembled into a single contiguous nucleotide sequence.
• 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.
• 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.
• 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.
• 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.
• 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. 215:403-410; 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 sequences set forth in SEQ ID NOs:2, 4, 13, 15 and 17.
• 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.
• Gene refers to a nucleic acid fragment that encodes 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.
• 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.
• 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 5:225).
• 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. Alternatively, the RNA transcript may be an RNA sequence derived from posttranscriptional processing of the primary transcript; this 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 an 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 is not translated 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.
• 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).
• This invention relates to maize cDNAs with homology to the yeast GCN5 transcritional coactivator, human histone acetyltransfease and Arabidopsis thaliana histone acetyltransferase protein.
• the invention also relates to rice and wheat cDNAs with homology to Arabidopsis thaliana histone acetyltransferase protein.
• the instant maize, rice and wheat cDNAs have been isolated and identified by comparison of random plant cDNA sequences to the GenBank database using the BLAST algorithms well known to those skilled in the art.
• the nucleotide sequence of a maize histone acetyltransferase protein is provided in SEQ ID NO:l, and the deduced amino acid sequence is provided in SEQ ID NO:2.
• the nucleotide sequence of a wheat histone acetyltransferase protein is provided in SEQ ID NO:3, and the deduced amino acid sequence is provided in SEQ ID NO:4.
• the nucleotide sequence of a wheat histone acetyltransferse protein is provided in SEQ ID NO: 16, and the deduced amino acid sequence is provided in SEQ ID NO: 17. This sequence appears to be more 5' than SEQ ID NO:3 but overlapping regions of homology were not long enough to form a contig.
• nucleotide sequence of a rice histone acetyltransferse protein is provided in SEQ ID NO: 14, and the deduced amino acid sequence is provided in SEQ ID NO: 15.
• Homologs of histone acetyltransferase proteins from other plants can now be identified by comparison of random cDNA sequences to the maize, rice and wheat histone acetlytransferase sequences provided herein.
• the full insert of cDNA clone cep7.pk0001.al 1 encoding the maize homolog of yGCN5 has been completely sequenced. Amino acid sequence comparison indicates that there is 45.9% sequence identity between this maize homolog and yGCN5 ( Figure 1).
• the insert in EST clone wlln.pk0003.c2 appears to encode the 3'coding region of a wheat GCN5 homolog. At the amino acid level, this wheat peptide is approximately 80% identical to the maize GCN5 encoded by cDNA clone cep7.pk0001.al 1. Nucleotide identity between the wheat and maize cDNAs is approximately 62%. Sequence alignments and percent identity calculations were performed by the Jotun Hein method using the Megalign program of DNAStarTM sequence analysis software (DNASTAR Inc. 1228 South Park Street, Madison Wisconsin, 53715).
• the inserts in EST clones rlr6.pk0084.c5 and wrl.pk0045.f4 appear to encode the 3' coding regions of a rice and wheat histone acetyltransferase homolog.
• the histone acetyltransferases encoded by the cDNAs of rlr6.pk0084.c5 wrl.pk0045.f4 are 49% and 52% (respectively) identical to the Arabidopsis thaliana histone acetyltransferase peptide set forth in GenBank Accession No. AFO31958.
• the nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding other gene homologs of histone acetyltransferase 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).
• 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 plant homologs of histone acetyltransferase protein 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 primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
• specific primers can be designed and used to amplify a part of or full-length 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.
• Genomic fragments can be isolated that include the promoter region that directs expression of the maize, rice, or wheat histone acetyltransferase protein protein. This promoter may be prepared as a DNA fragment including regulatory elements with or without the untranslated leader and used in expression of other coding regions or for co-suppression.
• 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)
• 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).
• nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed maize, rice and wheat histone acetyltransferase proteins are present at higher or lower levels than normal or in cell types or developmental stages in which it is not normally found. This would have the effect of altering the level of histone acetyltransferase activity in those cells.
• Overexpression of maize, rice or wheat histone acetyltransferase proteins 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 a promoter sequence and translation leader sequence derived from the same gene.
• a 3' non- coding sequence encoding a transcription termination signal 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) EMBOJ. 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.
• chimeric genes designed for co-suppression of the instant maize, rice and wheat histone acetyltransferase genes can be constructed by linking the genes or gene fragments encoding the maize, rice, or wheat histone acetyltransferase proteins to plant promoter sequences.
• chimeric genes designed to express antisense RNA for all or part of the instant nucleic acid fragments can be constructed by linking the genes or gene fragments 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 plant histone acetyltransferase proteins may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the histone acetyltransferase proteins by methods well known to those skilled in the art.
• the antibodies are useful for detecting maize, rice and wheat histone acetyltransferase proteins in situ in cells or in vitro in cell extracts.
• Preferred heterologous host cells for production of the instant histone acetyltransferase 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 chimeric genes for production of the instant maize, rice and wheat histone acetyltransferase proteins. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of maize, rice and wheat histone acetyltransferase proteins.
• An example of a vector for high level expression of the maize, rice and wheat acetyltransferase proteins in a bacterial host is provided (Example 7).
• the instant histone acetyltransferase proteins can be used as targets to facilitate design and/or identification of inhibitors of the protein that may be useful as herbicides. This is desirable because the protein described plays a key role in regulation of gene expression. Accordingly, inhibition of the activity of the proteins described herein could lead to inhibition of gene expression sufficient to inhibit plant growth. Thus, the instant maize, rice and wheat histone acetyltransferase 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 expression of the instant maize, rice and wheat histone acetyltransferase proteins. 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.
• 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) ⁇ m.J.Hwm.Ge «et.52:314-331).
• 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).
• nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping.
• FISH direct fluorescence in situ hybridization
• 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, polymorphism of PCR-amplified fragments (CAPS), allele- specific ligation, nucleotide extension reactions, Radiation Hybrid Mapping and Happy Mapping.
• 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.
• 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 56:9402; Koes et al., (1995) TVoc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell 7:75). The latter approach may be accomplished in two ways.
• 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 maize, rice, or wheat histone acetyltransferase protein gene.
• the histone acetyltransferase gene 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.
• a plant containing a mutation in the endogenous histone acetyltransferase gene can be identified and obtained.
• This mutant plant can then be used to determine or confirm the natural functon of the maize, rice, or wheat histone acetyltransferase protein gene product.
• EXAMPLE 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various corn, rice and wheat tissues were prepared. The characteristics of the libraries are described in Table 1.
• 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. Amplified insert 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 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 5:266-272) provided by the NCBI.
• BLASTX National Center for Biotechnology Information
• 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.
• SEQ ID NO:l shows the nucleotide sequence of the entire maize cDNA insert; the deduced amino acid sequence is shown in SEQ ID NO:2.
• This cDNA clone represents the first EST identified for a maize histone acetyltransferase protein with homology to yeast and human histone acetyltransferase proteins.
• 5' RACE A Rapid Amplification of cDNA Ends
• BNL commercially available 5' RACE system
• Total RNA was isolated from maize epicotyl obtained from 7 day old seedlings. Based on the nucleotide sequence of cDNA clone cep7.pk0001.al 1, three gene-specific primers were designed:
• GSPl 5' -CTTTGTGAAACCCTGCTTTACAA-3' SEQ ID NO: 7
• GSP2 5'-CGGAATTCGTTAAGAAATGTGTGAGCCCATCA-3' (SEQ ID NO: 8)
• GSP3 5' -CGGAATTCCCCGTGCATGTTGTTTCAAATGATT-3' (SEQ ID NO: 9)
• GSPl was used to prime the first strand cDNA synthesis, thus providing cDNA copies of specific mRNAs corresponding the maize homolog of the yeast GCN5.
• the first strand product was purified from unincorporated dNTPs and residual GSPl primer using the GlassMaxTM DNA Isolation Spin Cartridge Procedure (BRL). Terminal deoxynucleotidyl transferase was then used to add homopolymeric deoxycytidine tails (oligo-dC tails) to the 3' end of the cDNA.
• Tailed cDNA was then amplified by PCR using a nested, gene-specific primer (GSP2) which annealed 3' to the GSPl site, and the 5' RACE Abridged Anchor Primer as specified in the manufacturer's protocol.
• GSP2 nested, gene-specific primer
• GSP3 nested, gene-specific primer
• Abridged Universal Amplification primer An EcoRI site is encoded within the GSP2 and GSP3 primers; the anchor primers include an Spel restriction site.
• the resulting PCR product was therefore digested with EcoRI and Spel enzymes, subcloned into pBluescript, and sequenced as described in Example 1.
• One of the 5' RACE products yielded 271 bp of additional sequence beyond the original 5'-end of the cDNA insert in cDNA clone cep7.pk0001.al 1.
• a contiguous sequence of the maize homolog of the yeast GCN5 consisting of the original cDNA sequence from clone cep7.pk0001.al 1 and the additional 5' RACE sequence is provided in SEQ ID NO: 12.
• a deduced amino acid sequence encoding a portion of the maize homolog of the yeast GCN5 derived from the nucleotide sequence of SEQ ID NO:12 is provided in SEQ ID NO:13.
• the entire 5'-end sequence of the maize homolog of the yeast GCN5 can be isolated by following a similar strategy.
• the sequence of a portion of the cDNA insert from clone rlr6.0084.c5 is shown in SEQ ID NO: 14; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 15.
• the sequence of a portion of the cDNA insert from clone wrl.pk0045.f4 is shown in SEQ IDNO:16; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 17.
• the sequence of a portion of the cDNA insert from clone wlln.pk0003.c2 is shown in SEQ ID NO:3; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:4.
• BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of a histone acetyltransferase protein. These sequences represent the first rice and wheat sequences encoding homologs to an Arabidopsis thaliana histone acetlytransferase protein.
• a chimeric gene comprising a cDNA encoding a maize, rice or wheat histone acetyltransferase protein 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 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 100 uL volume in a standard PCR mix consisting of 0.4 mM of each oligonucleotide and 0.3 pM of target DNA in 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001% w/v gelatin, 200 mM dGTP, 200 mM dATP, 200 mM dTTP, 200 mM dCTP and 0.025 unit AmplitaqTM DNA polymerase. Reactions are carried out in a Perkin-Elmer Cetus ThermocyclerTM for
• the amplified DNA is then digested with restriction enzymes Ncol and Smal and fractionated on a 0.7% low melting point agarose gel in 40 mM Tris-acetate, pH 8.5, 1 mM EDTA.
• the appropriate band can be excised from the gel, melted at 68°C and combined with a 4.9 kb Ncol-Smal fragment of the plasmid pML103.
• Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard, 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.
• 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 maize, rice, or wheat histone acetyltransferase protein, and the 10 kD zein 3' region.
• the chimeric gene described above can then be introduced into corn cells by the following procedure.
• Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn 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 corn 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 rupture 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.
• 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).
• 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 maize, rice and wheat histone acetyltransferase proteins 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.
• a nucleic acid fragment encoding a maize, rice or wheat acetyltransferase proteins may be generated by polymerase chain reaction (PCR) of an appropriate 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, 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 maize, rice, or wheat histone acetyltransferase protein.
• 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 be 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) 527:70, U.S. Patent No. 4,945,050).
• a Du Pont BiolisticTM PDS 1000/HE instrument helium retrofit
• 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 575:810-812), 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 maize, rice, or wheat histone acetyltransferase protein homolog 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.
• 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.
• Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the histone acetyltransferase protein. This fragment may then be purified on a 1% NuSieve GTGTM low melting agarose gel (FMC). Buffer and agarose contain 10 ⁇ g/ml ethidium bromide for visualization of the DNA fragment. 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.
• GELaseTM Epicentre Technologies
• 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 pET24d is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as decribed above.
• the prepared vector pET24d 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 2xYT media and 50 ⁇ g/mL kanamycin. Transformants containing the gene are then screened for the correct orientation with respect to pET24d T7 promoter by restriction enzyme analysis.
• Clones in the correct orientation with respect to the T7 promoter can be transformed into BL21(DE3) competent cells (Novagen) and selected on 2xYT agar plates containing 50 ⁇ g/ml kanamycin. A colony arising from this transformation construct can be grown overnight at 30°C in 2xYT media with 50 ⁇ g/mL kanamycin. The culture is then diluted two fold with fresh media, allowed to re-grow for 1 h, and induced by adding isopropyl- thiogalactopyranoside to 1 mM final concentration.
• Cells are then harvested by centrifugation after 3 h and re-suspended in 50 ⁇ L 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 ⁇ g 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 Maize.
• Rice and Wheat Histone Acetyltransferase Proteins The maize, rice and wheat acetyltransferase proteins 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 6, or expression in eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines.
• the instant polypeptides 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)g”).
• GST glutathione S-transferase
• Trx thioredoxin
• (His)g) 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 polypeptides.
• proteases include thrombin, enterokinase and factor Xa.
• any protease can be used which specifically cleaves the peptide connecting the fusion protein and the maize, rice, or wheat histone acetyltransferase polypeptide.
• Purification of the instant polypeptides 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 polypeptide or an affinity resin containing ligands which are specific for the polypeptide.
• polypeptide may be expressed as a fusion protein coupled to the C-terminus of thioredoxin.
• a (His) 6 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. These 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 maize, rice and wheat histone acetyltransferase proteins 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 polypeptide, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit the activity of the maize, rice and wheat histone acetyltransferase proteins disclosed herein.
• Assays may be conducted under well known experimental conditions which permit optimal activity.
• An example of an in vitro assay for histone acteyltransferase activity may be found in Brownell, J.E. and Allis, CD. (1995) Proc. Natl. Acad. Sci. USA 92:6364-6368.
• the skilled artisan is well aware of simple modifications that could be made to the published protocol that would afford detection of inhibitors of histone acteyltransferase activity.
• Val Arg Leu Val Met Asp Arg Thr His Lys Ser Met Met Val lie 1 5 10 15
• CTGTCGNATG TNTAAAATTA TATTGGCCCA CTGGNNGGAC ACTACTGCCN ATNTGTAAAC 420
• MOLECULE TYPE other nucleic acid
• GAC TAT AAT ACT TAC AGG CAA CAG CTT ACT ACC CTT ATG CAG ACA GCG 911 Asp Tyr Asn Thr Tyr Arg Gin Gin Leu Thr Thr Leu Met Gin Thr Ala 290 295 300
• AGTTTAGCAT TATTTTAACC AGGAGGGACA CTGATTGATC TTTACATTTC GGTCTCAACC 1434 TGGCCGGCCT AATAATATAG ATTGAGGAGA TTCTTCAGTT TCTAAAAAAA AAAAAAAAAA 1494 AAA 1497
• AAANCCCGNG GTCCACTTAT TTCCCAATTA TTNANGAAAT TACCNAATCC NATGATTTTN 420

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