EP1056863A1 - FACTEUR DE TRANSLATION eIF-4E DE VEGETAUX - Google Patents

FACTEUR DE TRANSLATION eIF-4E DE VEGETAUX

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Publication number
EP1056863A1
EP1056863A1 EP99907028A EP99907028A EP1056863A1 EP 1056863 A1 EP1056863 A1 EP 1056863A1 EP 99907028 A EP99907028 A EP 99907028A EP 99907028 A EP99907028 A EP 99907028A EP 1056863 A1 EP1056863 A1 EP 1056863A1
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EP
European Patent Office
Prior art keywords
nucleic acid
amino acid
acid fragment
acid sequence
seq
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EP99907028A
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German (de)
English (en)
Inventor
Joan T. Odell
Emil M. Orozco, Jr.
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EIDP Inc
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EI Du Pont de Nemours and Co
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Publication of EP1056863A1 publication Critical patent/EP1056863A1/fr
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    • 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
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology

Definitions

  • This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding translation factors (proteins that function with ribosomes in translation) in plants and seeds.
  • translation factors proteins that function with ribosomes in translation
  • BACKGROUND OF THE INVENTION The initiation of protein synthesis in eukaryotic cells is a complicated process requiring ten or more initiation factors, mRNA, ATP, GTP, and 40S and 60S ribosomal subunits.
  • Three of the eukaryotic initiation factors, eIF-4A, eIF-4B, and eIF-4F catalyze the ATP-dependent unwinding of the 5 '-untranslated region of mRNA prior to the binding of the 40 S ribosomal subunit.
  • eIF-4F has been isolated from mammalian tissues and contains two subunits, p220 and p25; this p25 subunit has been referred to as eIF-4E, a m 7 G cap-binding protein.
  • An eIF-4F has also been isolated from wheat germ and has been shown to contain two subunits, p220 and p26; the p26 subunit is also referred to as eIF-4E.
  • an antigenically distinct form of eIF-4F referred to as eIF-(iso)4F, has also been isolated from wheat germ and wheat sprouts (Allen, M. et al., (1992) J. Biol. Chem. 267(32):23232-23236).
  • eIF-(iso)4F contains two subunits, p82 and p28; the p28 subunit is also referred to as eIF-4E.
  • a characteristic structural feature of most eIF-4E proteins is the presence of eight tryptophan residues in the same relative region of the proteins. It has been suggested that some of these conserved amino acids are involved in binding the m 7 G functional group (Rudd. K. et al. (1998) J. Biol. Chem. 273 //7J10325-10330). It has recently been shown in an Arabidopsis thaliana eIF-4E that other aromatic amino acids can substitute for two of the eight conserved tryptophan residues (Rudd, K. et al. (1998) J. Biol. Chem.
  • the instant invention relates to isolated nucleic acid fragments encoding a translation factor from corn, rice and soybean. Specifically, this invention concerns an isolated nucleic acid fragment encoding an eIF-4E translation factor. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding said translation factor.
  • an additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an eIF-4E selected from the group consisting of corn, rice and soybean eIF-4E translation factor proteins.
  • the instant invention relates to a chimeric gene encoding a corn, rice or soybean eIF-4E, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a corn, rice or soybean eIF-4E, 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 corn, rice or soybean eIF-4E, 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 corn, rice or soybean eIF-4E in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a corn, rice or soybean eIF-4E; 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 a corn, rice or soybean eIF-4E 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 corn, rice or soybean eIF-4E.
  • BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.
  • Figure 1 shows a comparison of various the eIF-4E amino acid sequences.
  • el 1 and ssl.pk0034.g7 is compared to the eIF-4E amino acid sequences o ⁇ Zea mays (gi # 3342823), Zea mays (gi # 3342821), Arabidopsis thaliana (gi # 3108209), Oryza sativa (gi # 1352440), Triticum aestivum (gi # 170753) and
  • SEQ ID NO: 1 is the nucleotide sequence comprising the entire cDNA insert in clone cebl.pk0102.fl encoding a com eIF-4E protein.
  • SEQ ID NO:2 is the deduced amino acid sequence of a com eIF-4E derived from the nucleotide sequence of SEQ ID NO:l.
  • SEQ ID NO: 3 is the nucleotide sequence comprising a contig assembled from the cDNA inserts m clones cen5.pk0032.cl l, crln.pk0051.c5 and cen3n.pk0076.cl0 encoding a com eIF-4E protein.
  • SEQ ID NO:4 is the deduced amino acid sequence of a com eIF-4E derived from the nucleotide sequence of SEQ ID NO:3.
  • SEQ ID NO:5 is the nucleotide sequence comprising the entire cDNA insert in clone cepe7.pk0021.dl encoding a com eIF-4E protein.
  • SEQ ID NO:6 is the deduced amino acid sequence of a com eIF-4E 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 crl.pk0018.hl encoding approximately 70% of a com eIF-4E protein.
  • SEQ ID NO: 8 is the deduced amino acid sequence of a com eIF-4E derived from the nucleotide sequence of SEQ ID NO:7.
  • SEQ ID NO: 9 is the nucleotide sequence comprising the entire cDNA insert in clone rls6.pk0084.dl encoding a rice eIF-4E protein.
  • SEQ ID NO: 10 is the deduced amino acid sequence of a rice eIF-4E derived from the nucleotide sequence of SEQ ID NO:9.
  • SEQ ID NO: 11 is the nucleotide sequence comprising the entire cDNA insert in clone sfll.pk0043.a2 encoding a soybean eIF-4E protein.
  • SEQ ID NO: 12 is the deduced amino acid sequence of a soybean eIF-4E derived from the nucleotide sequence of SEQ ID NO: 11.
  • SEQ ID NO: 13 is the nucleotide sequence comprising a portion of the cDNA insert in clone srm.pk0004.el 1 encoding a soybean eIF-4E protein.
  • SEQ ID NO: 14 is the deduced amino acid sequence of a soybean eIF-4E derived from the nucleotide sequence of SEQ ID NO: 13.
  • SEQ ID NO: 15 is the nucleotide sequence comprising the entire cDNA insert in clone ssl.pk0034.g7 encoding a soybean eIF-4E protein.
  • SEQ ID NO: 16 is the deduced amino acid sequence of an eIF-4E derived from the nucleotide sequence of SEQ ID NO: 15.
  • SEQ ID NO: 17 is the amino acid sequence comprising the Triticum aestivum (gi # 170753) eIF-4E protein.
  • SEQ ID NO: 18 is the amino acid sequence comprising the Arabidopsis thaliana (gi # 3108209) eIF-4E protein.
  • SEQ ID NO: 19 is the amino acid sequence comprising the Homo sapiens (gi # 3172339) eIF-4E protein.
  • SEQ ID NO:20 is the amino acid sequence comprising the Zea mays (gi).
  • SEQ ID NO:21 is the amino acid sequence comprising the Zea mays (gi).
  • SEQ ID NO:22 is the amino acid sequence comprising the Triticum aestivum (gi # 547713) eIF-4E protein.
  • SEQ ID NO:23 is the amino acid sequence comprising the Oryza sativm (gi # 1352440) eIF-4E protein.
  • 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.
  • 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.
  • 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 encode amino acid sequences that are 90% similar to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are 95% similar to the amino acid sequences reported herein.
  • the percent similarity used herein can be precisely determined by the DNASTAR protein alignment protocol using the Clustal algorithm (Higgins, D.. et al., (1989)
  • 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)
  • 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).
  • 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 eIF-4E proteins as set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 and 16.
  • the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
  • “Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • Native gene refers to a gene as found in nature with its own regulatory sequences.
  • Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
  • Regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non- coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns. and polyadenylation recognition sequences.
  • Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an "enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
  • Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 15: 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.
  • RNA transcript refers to the product resulting from RNA polymerase- catalyzed transcription of a DNA sequence.
  • RNA transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
  • Sense RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell.
  • Antisense RNA refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065, incorporated herein by reference). 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 sense RNA, 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, incorporated herein by reference).
  • 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.
  • 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. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).
  • 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 eIF-4E 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 protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
  • 11 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 55:8998) 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.
  • 3' RACE or 5' RACE systems BBL
  • specific 3' or 5' cDNA fragments can be isolated (Ohara et al, (1989) PNAS USA 86:5673; Loh et al., (1989) Science 243:217).
  • 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).
  • 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 (Lemer, R. A. (1984) Adv. Immunol. 36: ⁇ ; Maniatis).
  • nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed eIF-4E 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 gene expression in those cells.
  • Overexpression of the com, rice or soybean eIF-4E proteins of the instant invention may be accomplished by first constmcting 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.
  • a chimeric gene designed for co-suppression of the instant eIF-4E proteins can be constructed by linking a gene or gene fragment encoding an eIF-4E 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 com, rice or soybean eIF-4E 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 com, rice or soybean eIF-4E in situ in cells or in vitro in cell extracts.
  • Preferred heterologous host cells for production of the instant com, rice or soybean eIF-4E 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 com, rice or soybean eIF-4E.
  • This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded com, rice or soybean eIF-4E.
  • An example of a vector for high level expression of the instant com, rice or soybean eIF-4E in a bacterial host is provided (Example 7).
  • 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. 114(2):95-96), polymorphism of PCR-amplified fragments (CAPS;
  • Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer, (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al., (1995) Proc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell 7:75). 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 plant gene encoding the com, rice or soybean eIF-4E.
  • 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.
  • a plant containing a mutation in the endogenous gene encoding a com, rice or soybean eIF-4E can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the com, rice or soybean eIF-4E gene product.
  • EXAMPLE 1 Composition of cDNA Libraries: Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various com, rice and soybean tissues were prepared. The characteristics of the libraries are described below.
  • 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.
  • Example 1 The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr” database using the BLASTX algorithm (Gish, W. and States, D. j. (1993) Nature Genetics 3:266-272 and Altschul, Stephen F., et al. (1997) Nucleic Acids Res. 25:3389-3402) provided by the NCBI.
  • NCBI 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.
  • EXAMPLE 3 Characterization of cDNA Clones Encoding eIF-4E The BLASTX search using the EST sequences from clones cebl.pk0102.fl, cen3n.pk0076.cl0, cen5. ⁇ k0032.cl l, cepe7.pk0021.dl, crl.pk0018.hl, crln.pk0051.c5, rls6.pk0084.dl, sfll.pk0043.a2, srm.pk0004.el 1, and ssl.pk0034.g7 revealed similarity of the proteins encoded by the cDNAs to eIF-4E proteins from a variety of different organisms in the BLAST database.
  • the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 10, 12. 14 and 16 appear to represent full length eIF-4F proteins.
  • Figure 1 presents an alignment of SEQ ID NOs:2, 4, 6, 10, 12, 14 and 16 and the amino acid sequence of eIF-4F proteins from Zea mays, Arabidopsis thaliana, Oryza sativa and Triticum aestivum.
  • the data in Table 5 represents a calculation of the percent similarity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 10. 12, 14 and 16 and the eIF-4F proteins from Zea mays, Arabidopsis thaliana, Oryza sativa and Triticum aestivum.
  • Sequence similarities between all of the full length eIF-4E proteins of the instant invention range from 28% to 83%; clones cepe7.pk0021.dl and rls6.pk0084.dl are the most similar at 83%.
  • the sequence similarity between com clones cebl.pk0102.fl, the contig composed of clones cen5.pk0032.cl 1, crln.pk0051.c5 and cen3n.pk0076.cl0 and cepe7.pk0021.dl ranges from 30% to 46%.
  • sequence similarity between each of the soybean clones sfll.pk0043.a2, srm.pk0004.el 1 and ssl.pk0034.g7 ranges from 21% to 43%. Sequence alignments were performed and percent similarities calculated by the
  • a chimeric gene comprising a cDNA encoding an eIF-4E 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 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 pMLl 03 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a
  • Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XLl-Blue (Epicurian Coli XL-1 BlueTM; Stratagene). 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 constmct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding an elF- 4E, 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
  • 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,
  • 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 Vims (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
  • 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. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.
  • Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., (1990) Bio/Technology 5:833-839).
  • 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 eIF-4E 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.
  • 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 eIF-4E.
  • 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
  • Soybean emoryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker. 150 ⁇ m, 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 embryo igenic suspension cultures may then be transformed by the method of particle gun bombardment (Kline et al. (1987) Nature (London) 327:70, U.S. Patent No. 4,945,050).
  • a DuPont BiolisticTM PDS1000/HE instmment (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 iOdell et al.(1985) Nature 373:810-812), 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 eIF-4E 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 eIF-4E proteins can be inserted into the T7 E. coli expression vector pBT430.
  • This vector is a derivative of pET-3a
  • Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis.
  • Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve 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 instmctions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water.
  • GELaseTM Epicentre Technologies
  • oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA).
  • the fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above.
  • the vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described 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
  • Transformants can be selected on agar plates containing LB media and 100 ⁇ g/mL ampicillin. Transformants containing the gene encoding the eIF-4E are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis. For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 759:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C.
  • IPTG isopropylthio- ⁇ -galactoside, the inducer
  • IPTG isopropylthio- ⁇ -galactoside, the inducer
  • incubation can be continued for 3 h at 25°.
  • Cells are then harvested by centrifugation and resuspended 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.

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Abstract

L'invention porte sur un fragment isolé d'acide nucléique codant pour la protéine eIF-4E, et sur la construction d'un gène chimérique codant pour tout ou partie de la protéine eIF-4E à orientation sens ou antisens, l'expression dudit gène chimérique entraînant la production de niveaux modifiés de protéines eIF-4E dans une cellule transformée.
EP99907028A 1998-02-25 1999-02-16 FACTEUR DE TRANSLATION eIF-4E DE VEGETAUX Withdrawn EP1056863A1 (fr)

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