WO2001019994A2 - PLANT APOPTOSIS INDUCING FACTORS (AIFs) - Google Patents

PLANT APOPTOSIS INDUCING FACTORS (AIFs) Download PDF

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WO2001019994A2
WO2001019994A2 PCT/US2000/024859 US0024859W WO0119994A2 WO 2001019994 A2 WO2001019994 A2 WO 2001019994A2 US 0024859 W US0024859 W US 0024859W WO 0119994 A2 WO0119994 A2 WO 0119994A2
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seq
polypeptide
nucleotide sequence
ofthe
sequences
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PCT/US2000/024859
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French (fr)
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WO2001019994A3 (en
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Karlene H. Butler
Rebecca E. Cahoon
Theodore M. Klein
Gatsha Mazithulela
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E.I. Du Pont De Nemours And Company
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Priority to AU73679/00A priority Critical patent/AU7367900A/en
Publication of WO2001019994A2 publication Critical patent/WO2001019994A2/en
Publication of WO2001019994A3 publication Critical patent/WO2001019994A3/en

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    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8263—Ablation; Apoptosis

Definitions

  • This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding apoptosis inducing factors in plants and seeds.
  • Multicellular organisms have evolved elaborate signal transduction pathways for maintaining homeostasis through the control of cell proliferation and death.
  • Programmed cell death or apoptosis is a process in which unwanted cells are eliminated during growth and development (Steller, H., (1998) PNAS 95:5421-5422).
  • Mitochondria play a key part in the regulation of apoptosis. Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in apoptosis.
  • AIF mammalian mitochondria apoptosis-inducing factor
  • the mature AIF protein is normally confined to the mitochondrial intermembrane space but translocates to the nucleus in a variety of apoptosis-inducing conditions.
  • AIF induces nuclear chromatin condensation as well as large scale DNA fragmentation (Susin et al., (1999) Nature 397(6715):441-446; Daugas et al. (2000) FEBS Lett 476: 18-123).
  • the present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a second nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:30, (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a fourth nucleotide sequence
  • the isolated polynucleotide ofthe claimed invention comprises a first nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.
  • this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 and the complement of such nucleotide sequences.
  • this invention relates to a chimeric gene comprising an isolated polynucleotide ofthe present invention operably linked to at least one suitable regulatory sequence.
  • the present invention concerns an isolated host cell comprising a chimeric gene ofthe present invention or an isolated polynucleotide ofthe present invention.
  • the host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell.
  • the present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide ofthe present invention or a chimeric gene ofthe present invention.
  • the invention also relates to a process for producing an isolated host cell comprising a chimeric gene ofthe present invention or an isolated polynucleotide ofthe present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide ofthe present invention.
  • the invention concerns an isolated AIF polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:30, (c) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2, (e) a polypeptide of at least 150 amino acids having at least 80%
  • the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an AIF polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level ofthe AIF polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level ofthe AIF polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level ofthe AIF polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
  • the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of an AIF polypeptide, preferably a plant AIF polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer.
  • the amplified nucleic acid fragment preferably will encode a substantial portion of an AIF amino acid sequence.
  • this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding an AIF polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide ofthe present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide ofthe present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
  • this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide ofthe present invention.
  • this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette ofthe present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression ofthe AIF polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
  • this invention relates to a method of altering the level of expression of an AIF in a host cell comprising: (a) transforming a host cell with a chimeric gene ofthe present invention; and (b) growing the transformed host cell under conditions that are suitable for expression ofthe chimeric gene wherein expression ofthe chimeric gene results in production of altered levels ofthe AIF in the transformed host cell.
  • Figure 1 shows an alignment ofthe amino acid sequences of AIF encoded by the nucleotide sequences derived from Arabidopsis clone acs2c.pk006.pl 1 (SEQ ID NO:4), corn clone p0048.cqlaf62r (SEQ ID NO:8), rice clone rds2c.pk002.gl6 (SEQ ID NO:18), rice clone rl0n.pk0066.c9 (SEQ ID NO:22), soybean clone sdp4c.pk009.118 with additional nucleotide sequence obtained via PCR (SEQ ID NO:26), and wheat clone wlmkl.pk0007.f9 (SEQ ID NO: 34), and the monodehydroascorbate reductase-like AIF from Oryza sativa (NCBI GI No. 4666287; SEQ ID NO:41). Amino acids which are conserve
  • Figure 2 shows an alignment of the amino acid sequences of AIF encoded by the nucleotide sequences derived from a contig assembled from corn clones cbn2.pk0010.a8 and pOl 17.chclk04ra (SEQ ID NO:12), a contig assembled from rice clones rl0n.pk0025.f6 and rcaln.pk012.el 1 and additional nucleotide sequence obtained via PCR (SEQ ID NO: 14), and soybean clone ssm.pk0031.c9 with additional nucleotide sequence obtained via PCR (SEQ ID NO:28), and the monodehydroascorbate reductase-like AIF from Brassica juncea (NCBI GI No.
  • Table 1 also identifies the cDNA clones as individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs or PCR fragment sequence ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR fragment sequence ("CGS").
  • Nucleotide SEQ ID Nos:l, 5, 9, 15, 19, 23, 31, 35, and 39 correspond to nucleotide SEQ ID Nos: l, 3, 5, 7, 9, 11, 13, 15, and 17, respectively, presented in U.S. Provisional Application No.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 75:3021-3030 (1985) and in the Biochemical J. 219 (No. :345-373 (1984) which are herein incorporated by reference.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.822. DETAILED DESCRIPTION OF THE INVENTION In the context of this disclosure, a number of terms shall be utilized.
  • polynucleotide polynucleotide sequence
  • nucleic acid sequence nucleic acid sequence
  • nucleic acid fragment'V'isolated nucleic acid fragment are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • An isolated polynucleotide ofthe present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, or the complement of such sequences.
  • isolated polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as other chromosomal and extrachromosomal DNA and RNA, that normally accompany or interact with it as found in its naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides. The term "recombinant" means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.
  • sequence refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can 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 ofthe polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology.
  • Substantially similar also refers to modifications ofthe nucleic acid fragments ofthe instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties ofthe resulting transcript vis-a-vis the ability to mediate gene silencing or alteration ofthe functional properties ofthe resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.
  • the terms “substantially similar” and “corresponding substantially” are used interchangeably herein.
  • Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications ofthe nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level ofthe polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell.
  • a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell.
  • the level ofthe polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level ofthe polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
  • 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 using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed.
  • alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties ofthe encoded polypeptide are well known in the art.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
  • Nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions ofthe polypeptide molecule would also not be expected to alter the activity ofthe polypeptide.
  • an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of an AIF polypeptide in a host cell.
  • a method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell may comprise the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.
  • substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
  • One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min.
  • a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C.
  • Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.
  • nucleic acid fragments ofthe instant invention may also be characterized by the percent identity ofthe amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.
  • Suitable nucleic acid fragments encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein.
  • Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein.
  • nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.
  • a "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification ofthe protein or gene that the amino acid or nucleotide sequence comprises.
  • Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
  • a sequence often or more contiguous amino acids or thirty or more contiguous 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 30 or more 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 or more nucleotides 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 a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
  • the instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantial portion ofthe disclosed sequences for purposes known to those skilled in this art.
  • 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.
  • the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion ofthe amino acid sequences set forth herein.
  • the skilled artisan is well aware ofthe "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid.
  • nucleic acid fragments 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 larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment.
  • “Chemically synthesized”, as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro.
  • nucleic acid fragments may be tailored for optimal gene expression based on optimization ofthe nucleotide sequence to reflect the codon bias ofthe 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 nucleotide 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 ofthe associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleotide 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 nucleotide 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 may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide 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 nucleic acid fragment 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, nucleic acid fragments of different lengths may have identical promoter activity.
  • Translation leader sequence refers to a nucleotide 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 ofthe translation start sequence.
  • the translation leader sequence may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 5:225-236).
  • 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 ofthe DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing ofthe 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 polypeptides by the cell.
  • cDNA refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I.
  • Sense-RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell.
  • Antisense RNA refers to an 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 (see U.S. Patent No. 5,107,065, incorporated herein by reference).
  • the complementarity of an antisense RNA may be with any part ofthe specific nucleotide sequence, 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 two or more nucleic acid fragments on a single polynucleotide 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 ofthe 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 ofthe 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. Patent No. 5,231,020, incorporated herein by reference).
  • a “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.
  • altered levels or “altered expression” 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.
  • Null mutant refers here to a host cell which either lacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.
  • “Mature protein” or the term “mature” when used in describing a 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” or the term “precursor” when used in describing a protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
  • chloroplast transit peptide is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made.
  • Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
  • a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53).
  • a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
  • an endoplasmic reticulum retention signal may be added.
  • any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1621-1632).
  • Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol 143:211) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Patent No. 4,945,050, incorporated herein by reference).
  • isolated polynucleotides ofthe present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • PCR or "polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Patent Nos. 4,683,195 and 4,800,159).
  • the present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a second nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:30, (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a fourth nucleotide sequence encoding a polypeptide of
  • the first nucleotide sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.
  • Nucleic acid fragments encoding at least a portion of several AIFs have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art.
  • the nucleic acid fragments ofthe 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 AIFs could be isolated directly by using all or a portion ofthe 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).
  • an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • primers can be designed and used to amplify a part or all ofthe 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 ofthe 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 ofthe other primer takes advantage ofthe presence ofthe polyadenylic acid tracts to the 3' end ofthe 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) Proc. Natl Acad. Sci. USA 55:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe 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) Proc. Natl. Acad. Sci. USA 86:5613-5611; Loh et al. (1989) Science 243:211-220).
  • Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1 : 165).
  • a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.
  • the present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of an AIF polypeptide, preferably a substantial portion of a plant AIF polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, and 39, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer.
  • the amplified nucleic acid fragment preferably will encode a portion of an AIF polypeptide.
  • Synthetic peptides representing portions ofthe 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 (1984) Adv. Immunol. 36: -34; Maniatis).
  • this invention concerns viruses and host cells comprising either the chimeric genes ofthe invention as described herein or an isolated polynucleotide ofthe invention as described herein.
  • host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.
  • the nucleic acid fragments ofthe instant invention may be used to create transgenic plants in which the disclosed polypeptides 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 apoptosis in those cells. Consequently, the nucleic acid fragments ofthe instant invention may be expressed in a tissue-specific manner to selectively kill defined cell types and therefore influence developmental pathways in plant organs. Also, the nucleic acid fragments ofthe instant invention may be useful for engineering male sterility, altering plant architecture, manipulating stress response, influencing growth of cells and tissues in culture, and improving transformation efficiency by controlling death caused by the trauma of particle bombardment.
  • Overexpression ofthe proteins ofthe instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development.
  • the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided.
  • the instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.
  • Plasmid vectors comprising the instant isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants.
  • the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:241-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.100:1621-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.
  • a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences.
  • a chimeric gene designed to express antisense RNA for all or part ofthe 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 ofthe co ⁇ esponding endogenous genes are reduced or eliminated.
  • the ability to restrict the expression of a specific phenotype to the reproductive tissues ofthe plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
  • tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
  • special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes.
  • the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
  • the present invention concerns an isolated polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80%) identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a polypeptide of at least 50 amino acids having at least 95 % identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 30, (c) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2, (e) a polypeptide of at least 150 amino acids having at least 80% identity
  • the instant polypeptides may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to these proteins by methods well known to those skilled in the art.
  • the antibodies are useful for detecting the polypeptides ofthe instant invention in situ in cells or in vitro in cell extracts.
  • Prefe ⁇ ed heterologous host cells for production ofthe instant polypeptides 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 polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression ofthe encoded AIF.
  • Example 6 An example of a vector for high level expression ofthe instant polypeptides in a bacterial host is provided (Example 6). All or a substantial portion ofthe polynucleotides ofthe instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used 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 ofthe instant invention.
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map.
  • the nucleic acid fragments ofthe 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 ofthe DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 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 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 (Trask ( 1991 ) Trends Genet. 7: 149- 154).
  • 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 (Kazazian (1989) J. Lab. Clin. Med. 77:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 75:325-332), allele- specific ligation (Landegren et al. (1988) Science 247:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet.
  • Loss of function mutant phenotypes may be identified for the instant cD ⁇ A 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 5(5:9402-9406; Koes et al. ( 1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways.
  • short segments ofthe instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on D ⁇ As prepared from a population of plants in which Mutator transposons or some other mutation-causing D ⁇ A element has been introduced (see Bensen, supra).
  • the amplification of a specific D ⁇ A fragment with these primers indicates the insertion ofthe mutation tag element in or near the plant gene encoding the instant polypeptide.
  • the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor.
  • an arbitrary genomic site primer such as that for a restriction enzyme site-anchored synthetic adaptor.
  • composition of cDNA Libraries Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various Arabidopsis (Arabidopsis thaliana), corn (Zea mays), rice (Oryza sativa), soybean (Glycine max), and wheat (Triticum aestivum) tissues were prepared. The characteristics ofthe libraries are described below.
  • cDNA libraries may be prepared by any one of many methods available.
  • the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). The Uni-ZAPTM XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene.
  • cDNA inserts will be contained in the plasmid vector pBluescript.
  • the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIB CO BRL Products).
  • T4 DNA ligase New England Biolabs
  • plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences.
  • Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
  • FIS data Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol.
  • Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed Ml 3 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.
  • Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, CA) which is based upon the Saccharomyces cerevisiae Tyl transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772).
  • the in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules.
  • the transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, MD) via electroporation.
  • the transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res.
  • Phrep/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files.
  • the Phrap sequence assembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle).
  • 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 and States (1993) Nat. Genet. 5:266-272) provided by the ⁇ CBI.
  • BLASTX Gish and States (1993) Nat. Genet. 5:266-272
  • pLog the P- value (probability) of observing a match of a cD ⁇ A 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 ofthe logarithm ofthe reported P-value.
  • ESTs submitted for analysis are compared to the genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction.
  • EST Once the most 5 -prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.
  • Contig composed of: Contig 54.15 (gi 1483150) wdk2c.pk016al9 wrl.pk0123.d9 wreln.pk0034.c8 EST 79.30 (gi 4666287)
  • Table 4 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 6, 10, 16, 20, 24, 32, 36 and 40 and the Oryza sativa (gi 4666287) and (gi 4760483) , Brassica juncea and Arabidopsis thaliana sequences.
  • Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of an monodehydroascorbate reductase-like AIF. These sequences represent the first Arabidopsis, corn, rice, soybean and wheat sequences encoding monodehydroascorbate reductase-like AIF.
  • EST EST
  • FIS cDNA clones
  • Contig sequences of contigs assembled from two or more ESTs
  • Contig* sequences of contigs assembled from an FIS and one or more ESTs or PCR fragment sequence
  • CGS FIS and PCR fragment sequence
  • PSORT sequence analysis program (http://psort.nibb.ac.jp) predicts nuclear and mitochondrial targeting signals to be present in some ofthe polypeptides encoded by the instant nucleotide sequences, consistent with AIF subcellular localization.
  • Figure 1 presents an alignment ofthe amino acid sequences set forth in SEQ ID NOs:4, 8, 18, 22, 26, and 34, and the Oryza sativa sequence (NCBI GI No. 4666287; SEQ ID NO:41).
  • Figure 2 presents an alignment ofthe amino acid sequences set forth in SEQ ID NOs:12, 14, and 28, and the Brassica juncea sequence (NCBI GI No. 4704613; SEQ ID NO:42).
  • the data in Table 6 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs:4, 8, 12, 14, 18, 22, 26, 28, and 34, the Oryza sativa sequence (NCBI GI No. 4666287; SEQ ID NO:41), and the Brassica juncea sequence (NCBI GI No. 4704613; SEQ ID NO:42).
  • a chimeric gene comprising a cDNA encoding the instant polypeptide 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) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the digested vector pML103 as described below.
  • Plasmid pML103 has been deposited under the terms ofthe 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 ofthe maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end ofthe maize 10 kD zein gene in the vector pGem9Zf(+) (Promega).
  • Vector and insert DNA can be Hgated at 15°C overnight, essentially as described (Maniatis). The Hgated 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 construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, 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 ofthe 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 ofthe 35S promoter from Cauliflower Mosaic Virus (Odell et al.
  • 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 ⁇ m) 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) ofthe 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.
  • BiolisticTM PDS-1000/He Bio-Rad Instruments, Hercules CA
  • 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.
  • the tissue can be transfe ⁇ ed to N6 medium that contains bialophos (5 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 transfe ⁇ ed to fresh N6 medium containing bialophos. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialophos - 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 transfe ⁇ ed to regeneration medium (Fromm et al. (1990) Bio/Technology 5:833-839).
  • a seed-specific expression cassette composed ofthe 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. 251 :9228-9238) can be used for expression of the instant polypeptides in transformed soybean.
  • the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.
  • the cDNA fragment of this gene may be generated by polymerase chain reaction
  • PCR PCR ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.
  • Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides.
  • somatic embryos cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds ofthe 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 ⁇ 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 embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 527:70-73, U.S. Patent No. 4,945,050). A DuPont BiolisticTM PDS1000/HE instrument (helium retrofit) can be used for these transformations.
  • a selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed ofthe 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 ofthe Ti plasmid of Agrobacterium tumefaciens.
  • the seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptide and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site ofthe 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 6 Expression of Chimeric Genes in Microbial Cells
  • the cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430.
  • This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system.
  • Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a.
  • 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% low melting agarose gel. Buffer and agarose contain 10 ⁇ g/ml ethidium bromide for visualization ofthe DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies, Madison, WI) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water. Appropriate oligonucleotide adapters may be Hgated to the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly, MA).
  • T4 DNA ligase New England Biolabs (NEB), Beverly, MA.
  • the fragment containing the Hgated 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 Hgated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL).
  • Transformants can be selected on agar plates containing LB media and 100 ⁇ g/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the co ⁇ ect orientation with respect to the T7 promoter by restriction enzyme analysis.
  • a plasmid clone with the cDNA insert in the co ⁇ ect orientation relative to the T7 promoter can be transformed into E. coli strain BL21(D ⁇ 3) (Studier et al. (1986) J. Mol Biol 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio- ⁇ -galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°.
  • IPTG isopropylthio- ⁇ -galactoside, the inducer
  • Cells are then harvested by centrifugation 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 ofthe supernatant determined.
  • One ⁇ g of protein from the soluble fraction ofthe 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

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

Description

TITLE PLANT APOPTOSIS INDUCING FACTORS (AIFs) This application claims the benefit of U.S. Provisional Application No. 60/153737, filed September 13, 1999. FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding apoptosis inducing factors in plants and seeds.
BACKGROUND OF THE INVENTION Multicellular organisms have evolved elaborate signal transduction pathways for maintaining homeostasis through the control of cell proliferation and death. Programmed cell death or apoptosis is a process in which unwanted cells are eliminated during growth and development (Steller, H., (1998) PNAS 95:5421-5422).
Mitochondria play a key part in the regulation of apoptosis. Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in apoptosis. Recently, Susin et al. report the identification and cloning of a mammalian mitochondria apoptosis-inducing factor (AIF) (Susin et al., (1999) Nature 397(6718)Α41-446). The researches report that the protein appears to be a flavoprotein of relative molecular mass 57,000 and is homologous to bacterial oxidoreductases. An analysis ofthe protein, its location and function suggest that AIF is a mitochondrial effector of apoptotic cell death. The mature AIF protein is normally confined to the mitochondrial intermembrane space but translocates to the nucleus in a variety of apoptosis-inducing conditions. AIF induces nuclear chromatin condensation as well as large scale DNA fragmentation (Susin et al., (1999) Nature 397(6715):441-446; Daugas et al. (2000) FEBS Lett 476: 18-123).
Several plant proteins are identified here that have homology to the AIF reported by Susin et al. and also appear to be related to the monodehydroascorbate reductase family of proteins by amino acid sequence similarities and the presence of conserved amino acid sequence motifs. There is a great deal of interest in identifying the genes that encode proteins involved in apoptosis in plants. These genes may be used to alter apoptosis in plant cells and may eventually be used to control cell tissue culture growth. Accordingly, the availability of nucleic acid sequences encoding all or a portion of AIF would facilitate studies to better understand programmed cell death in plants, provide genetic tools to enhance cell growth in tissue culture, increase the efficiency of gene transfer and help provide more stable transformations.
SUMMARY OF THE INVENTION The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a second nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:30, (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a fourth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2, (e) a fifth nucleotide sequence encoding a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 10, (f) a sixth nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4 and 8, (g) a seventh nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 38, (h) an eighth nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:40, (i) a ninth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26, (j) a tenth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28, and (k) an eleventh nucleotide sequence comprising the complement of (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j).
In a second embodiment, it is preferred that the isolated polynucleotide ofthe claimed invention comprises a first nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 and the complement of such nucleotide sequences. In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide ofthe present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns an isolated host cell comprising a chimeric gene ofthe present invention or an isolated polynucleotide ofthe present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide ofthe present invention or a chimeric gene ofthe present invention. In a sixth embodiment, the invention also relates to a process for producing an isolated host cell comprising a chimeric gene ofthe present invention or an isolated polynucleotide ofthe present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide ofthe present invention. In a seventh embodiment, the invention concerns an isolated AIF polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:30, (c) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2, (e) a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 10, (f) a polypeptide of at least 200 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4 and 8, (g) a polypeptide of at least 200 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 38, (h) a polypeptide of at least 200 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:40, (i) a polypeptide of at least 250 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26, and (j) a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28. In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of an AIF polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level ofthe AIF polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level ofthe AIF polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level ofthe AIF polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of an AIF polypeptide, preferably a plant AIF polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of an AIF amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding an AIF polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide ofthe present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide ofthe present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone. In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide ofthe present invention.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette ofthe present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression ofthe AIF polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means. In a thirteenth embodiment, this invention relates to a method of altering the level of expression of an AIF in a host cell comprising: (a) transforming a host cell with a chimeric gene ofthe present invention; and (b) growing the transformed host cell under conditions that are suitable for expression ofthe chimeric gene wherein expression ofthe chimeric gene results in production of altered levels ofthe AIF in the transformed host cell.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS 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 an alignment ofthe amino acid sequences of AIF encoded by the nucleotide sequences derived from Arabidopsis clone acs2c.pk006.pl 1 (SEQ ID NO:4), corn clone p0048.cqlaf62r (SEQ ID NO:8), rice clone rds2c.pk002.gl6 (SEQ ID NO:18), rice clone rl0n.pk0066.c9 (SEQ ID NO:22), soybean clone sdp4c.pk009.118 with additional nucleotide sequence obtained via PCR (SEQ ID NO:26), and wheat clone wlmkl.pk0007.f9 (SEQ ID NO: 34), and the monodehydroascorbate reductase-like AIF from Oryza sativa (NCBI GI No. 4666287; SEQ ID NO:41). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.
Figure 2 shows an alignment of the amino acid sequences of AIF encoded by the nucleotide sequences derived from a contig assembled from corn clones cbn2.pk0010.a8 and pOl 17.chclk04ra (SEQ ID NO:12), a contig assembled from rice clones rl0n.pk0025.f6 and rcaln.pk012.el 1 and additional nucleotide sequence obtained via PCR (SEQ ID NO: 14), and soybean clone ssm.pk0031.c9 with additional nucleotide sequence obtained via PCR (SEQ ID NO:28), and the monodehydroascorbate reductase-like AIF from Brassica juncea (NCBI GI No. 4704613; SEQ ID NO:42). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment ofthe sequences. Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO© as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs or PCR fragment sequence ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR fragment sequence ("CGS"). Nucleotide SEQ ID Nos:l, 5, 9, 15, 19, 23, 31, 35, and 39 correspond to nucleotide SEQ ID Nos: l, 3, 5, 7, 9, 11, 13, 15, and 17, respectively, presented in U.S. Provisional Application No. 60/153737, filed September 13, 1999. Amino acid SEQ ID Nos:2, 6, 10, 16, 20, 24, 32, 36, and 40 correspond to amino acid SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, and 18, respectively, presented in U.S. Provisional Application No. 60/153737, filed September 13, 1999. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
AIFs
SEQ ID NO:
Protein (Plant Source) Clone Designation Status (Nucleotide) (Amino Acid)
Monodehydroascorbate acs2c.pk006.pl 1 EST 1 2
Reductase-Like AIF
(Arabidopsis)
Monodehydroascorbate acs2c.pk006.pl 1 CGS 3 4
Reductase-Like AIF (FIS)
(Arabidopsis)
Monodehydroascorbate p0048.cqlaf62r EST 5 6
Reductase-Like AIF (Corn)
Monodehydroascorbate p0048.cqlaf62r CGS 7 8
Reductase-Like AIF (Corn) (FIS)
Monodehydroascorbate p0117.chclk04ra FIS 9 10
Reductase-Like AIF (Corn)
Monodehydroascorbate Contig of CGS 11 12
Reductase-Like AIF (Corn) cbn2.pk0010.a8 p0117.chclk04ra
(FIS)
Monodehydroascorbate Contig of CGS 13 14
Reductase-Like AIF (Rice) rl0n.pk0025.f6 rcaln.pk012.el l
(FIS)
PCR fragment sequence
Monodehydroascorbate rds2c.pk002.gl6 EST 15 16
Reductase-Like AIF (Rice)
Monodehydroascorbate rds2c.pk002.gl6 CGS 17 18
Reductase-Like AIF (Rice) (FIS)
TABLE 1
AIFs
Monodehydroascorbate rl0n.pk0066.c9 EST 19 20
Reductase-Like AIF (Rice) Monodehydroascorbate rl0n.pk0066.c9 CGS 21 22
Reductase-Like AIF (Rice) (FIS)
Monodehydroascorbate sdp4c.pk009.118 EST 23 24
Reductase-Like AIF (Soybean)
Monodehydroascorbate Contig of CGS 25 26
Reductase-Like AIF (Soybean) sdp4c.pk009.118
(FIS)
PCR fragment sequence
Monodehydroascorbate Contig of CGS 27 28
Reductase-Like AIF (Soybean) ssm.pk0031.c9
(FIS)
PCR fragment sequence
Monodehydroascorbate wdrlf.pk003.bl8 EST 29 30
Reductase-Like AIF (Wheat)
Monodehydroascorbate wlmkl.pk0007.f9 EST 31 32
Reductase-Like AIF (Wheat)
Monodehydroascorbate wlmkl.pk0007.f9 CGS 33 34
Reductase-Like AIF (Wheat) (FIS)
Monodehydroascorbate Contig of Contig 35 36
Reductase-Like AIF (Wheat) wdk2c.pk016al9 wrl.pk0123.d9
Monodehydroascorbate Contig of Contig* 37 38
Reductase-Like AIF (Wheat) wrl.pk0123.d9
(FIS)
PCR fragment sequence
Monodehydroascorbate wreln.pk0034.c8 EST 39 40
Reductase-Like AIF (Wheat)
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 75:3021-3030 (1985) and in the Biochemical J. 219 (No. :345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822. DETAILED DESCRIPTION OF THE INVENTION In the context of this disclosure, a number of terms shall be utilized. The terms "polynucleotide", "polynucleotide sequence", "nucleic acid sequence", and "nucleic acid fragment'V'isolated nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide ofthe present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, or the complement of such sequences.
The term "isolated" polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as other chromosomal and extrachromosomal DNA and RNA, that normally accompany or interact with it as found in its naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides. The term "recombinant" means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.
As used herein, "contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence. As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties ofthe polypeptide encoded by the nucleotide sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology. "Substantially similar" also refers to modifications ofthe nucleic acid fragments ofthe instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties ofthe resulting transcript vis-a-vis the ability to mediate gene silencing or alteration ofthe functional properties ofthe resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. The terms "substantially similar" and "corresponding substantially" are used interchangeably herein.
Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications ofthe nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level ofthe polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level ofthe polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level ofthe polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
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 using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties ofthe encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions ofthe polypeptide molecule would also not be expected to alter the activity ofthe polypeptide. Each ofthe proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity ofthe encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of an AIF polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.
Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C. Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.
Substantially similar nucleic acid fragments ofthe instant invention may also be characterized by the percent identity ofthe amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides ofthe present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
A "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification ofthe protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantial portion ofthe 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 comprising a nucleotide sequence that encodes all or a substantial portion ofthe amino acid sequences set forth herein. The skilled artisan is well aware ofthe "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage ofthe host cell. "Synthetic nucleic acid fragments" 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 larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. "Chemically synthesized", as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments 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 nucleic acid fragments can be tailored for optimal gene expression based on optimization ofthe nucleotide sequence to reflect the codon bias ofthe 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 nucleotide 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 ofthe associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
"Promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a nucleotide 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 may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide 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 nucleic acid fragment 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, nucleic acid fragments of different lengths may have identical promoter activity.
"Translation leader sequence" refers to a nucleotide 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 ofthe translation start sequence. The translation leader sequence may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 5:225-236).
"3" non-coding sequences" refer to nucleotide 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 ofthe 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 ofthe DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing ofthe 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 polypeptides by the cell. "cDNA" refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I. "Sense-RNA" refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. "Antisense RNA" refers to an 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 (see U.S. Patent No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part ofthe specific nucleotide sequence, 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.
The term "operably linked" refers to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control ofthe promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression ofthe 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. Patent No. 5,231,020, incorporated herein by reference).
A "protein" or "polypeptide" is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.
"Altered levels" or "altered expression" 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. "Null mutant" refers here to a host cell which either lacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.
"Mature protein" or the term "mature" when used in describing a 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" or the term "precursor" when used in describing a protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
A "chloroplast transit peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. "Chloroplast transit sequence" refers to a nucleotide sequence that encodes a chloroplast transit peptide. A "signal peptide" is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1621-1632).
"Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol 143:211) and particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Patent No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides ofthe present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").
"PCR" or "polymerase chain reaction" is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Patent Nos. 4,683,195 and 4,800,159).
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a second nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:30, (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a fourth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2, (e) a fifth nucleotide sequence encoding a polypeptide of at least 150 amino acids having at least 80%) identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 10, (f) a sixth nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4 and 8, (g) a seventh nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 38, (h) an eighth nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:40, (i) a ninth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26, (j) a tenth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28, and (k) an eleventh nucleotide sequence comprising the complement of (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j).
Preferably, the first nucleotide sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39, that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. Nucleic acid fragments encoding at least a portion of several AIFs have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments ofthe instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
For example, genes encoding other AIFs, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all ofthe instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency. In addition, two short segments ofthe 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 ofthe other primer takes advantage ofthe presence ofthe polyadenylic acid tracts to the 3' end ofthe mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl Acad. Sci. USA 55:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe 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) Proc. Natl. Acad. Sci. USA 86:5613-5611; Loh et al. (1989) Science 243:211-220). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1 : 165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of an AIF polypeptide, preferably a substantial portion of a plant AIF polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, and 39, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of an AIF polypeptide.
Availability ofthe instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions ofthe 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 (1984) Adv. Immunol. 36: -34; Maniatis).
In another embodiment, this invention concerns viruses and host cells comprising either the chimeric genes ofthe invention as described herein or an isolated polynucleotide ofthe invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants. As was noted above, the nucleic acid fragments ofthe instant invention may be used to create transgenic plants in which the disclosed polypeptides 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 apoptosis in those cells. Consequently, the nucleic acid fragments ofthe instant invention may be expressed in a tissue-specific manner to selectively kill defined cell types and therefore influence developmental pathways in plant organs. Also, the nucleic acid fragments ofthe instant invention may be useful for engineering male sterility, altering plant architecture, manipulating stress response, influencing growth of cells and tissues in culture, and improving transformation efficiency by controlling death caused by the trauma of particle bombardment.
Overexpression ofthe proteins ofthe instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression. Plasmid vectors comprising the instant isolated polynucleotide (or chimeric gene) may be 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 ofthe 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.
For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:241-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.100:1621-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.
It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences.
Alternatively, a chimeric gene designed to express antisense RNA for all or part ofthe 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 ofthe coπesponding endogenous genes are reduced or eliminated.
Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Patent Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of a specific phenotype to the reproductive tissues ofthe plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed. The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.
In another embodiment, the present invention concerns an isolated polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80%) identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 22, 24, 32, and 36, (b) a polypeptide of at least 50 amino acids having at least 95 % identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 30, (c) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6 and 34, (d) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2, (e) a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 10, (f) a polypeptide of at least 200 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:4 and 8, (g) a polypeptide of at least 200 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 38, (h) a polypeptide of at least 200 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:40, (i) a polypeptide of at least 250 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26, and (j) a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28.
The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides ofthe instant invention in situ in cells or in vitro in cell extracts. Prefeπed heterologous host cells for production ofthe instant polypeptides 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 polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression ofthe encoded AIF. An example of a vector for high level expression ofthe instant polypeptides in a bacterial host is provided (Example 6). All or a substantial portion ofthe polynucleotides ofthe instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments ofthe instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments ofthe 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 ofthe DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 52:314-331). The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:31-4 . Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; .see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask ( 1991 ) Trends Genet. 7: 149- 154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 77:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 75:325-332), allele- specific ligation (Landegren et al. (1988) Science 247:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DΝA sequence differences between the parents ofthe mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
Loss of function mutant phenotypes may be identified for the instant cDΝA 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 5(5:9402-9406; Koes et al. ( 1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments ofthe instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DΝAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DΝA element has been introduced (see Bensen, supra). The amplification of a specific DΝA fragment with these primers indicates the insertion ofthe mutation tag element in or near the plant gene encoding the instant polypeptide. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptide can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function ofthe instant polypeptides disclosed herein.
EXAMPLES The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating prefeπed embodiments ofthe invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications ofthe invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope ofthe appended claims.
The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
EXAMPLE 1
Composition of cDNA Libraries: Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various Arabidopsis (Arabidopsis thaliana), corn (Zea mays), rice (Oryza sativa), soybean (Glycine max), and wheat (Triticum aestivum) tissues were prepared. The characteristics ofthe libraries are described below.
TABLE 2 cDNA Libraries from Arabidopsis, Corn, Rice, Soybean, and Wheat
Library Tissue Clone acs2c Arabidopsis Landsberg erecta Fertilized Carpel With acs2c.pk006.pl 1 Developing Seeds 6-7 Days After Fertilization cbn2 Corn Developing Kernel Two Days After Pollination cbn2.pk0010.a8 p0048 Corn Embiyo (Axis + Scuttelum) One Day After p0048.cqlaf62r Germination p0117 Corn Expanding Internode: Plants Sampled at the VI 01 Stage. p0117.chclk04ra Internodes 5-9 (the Upper 4-5 Expanding Internodes)2 rcaln Rice Callus2 rcaln.pk012.el l rds2c Rice Developing Seeds From Middle ofthe Plant rds2c.pk002.gl6 rlOn Rice 15 Day Old Leaf2 rl0n.pk0025.f6 rl0n.pk0066.c9 sdp4c Soybean Developing Pod (10-12 mm) sdp4c.pk009.118 ssm Soybean Shoot Meristem ssm.pk0031.c9 wdk2c Wheat Developing Kernel, 7 Days After Anthesis wdk2c.pk016al9 wdrlf Wheat Developing Root wdrlf.pk003.bl8 wlmkl Wheat Seedling 1 Hour After Inoculation With Erysiphe wlmkl. pk0007.f9 graminisf sp tritici and Treatment With Herbicide3 wrl Wheat Root From 7 Day Old Light Grown Seedling wrl.pk0123.d9 wreln Wheat Root From 7 Day Old Etiolated Seedling2 wreln.pk0034.c8
Corn developmental stages are explained in the publication "How a corn plant develops" from the Iowa State University Coop. Ext. Service Special Report No. 48 reprinted June 1993.
2These libraries were normalized essentially as described in U.S. Patent No. 5,482,845, incorporated herein by reference. application of 6-iodo-2-propoxy-3-propyl-4(3/7)-quinazolinone; synthesis and methods of using this compound are described in USSN 08/545,827, incorporated herein by reference. cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIB CO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed Ml 3 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.
Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, CA) which is based upon the Saccharomyces cerevisiae Tyl transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, MD) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 77:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI Prism dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.
Sequence data is collected (ABI Prism Collections) and assembled using Phred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle).
EXAMPLE 2 Identification of cDNA Clones cDNA clones encoding AIF were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release ofthe SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 5:266-272) provided by the ΝCBI. For convenience, the P- value (probability) of observing a match of a cDΝA 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 ofthe logarithm ofthe reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDΝA sequence and the BLAST "hit" represent homologous proteins. ESTs submitted for analysis are compared to the genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5 -prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.
EXAMPLE 3 Characterization of cDΝA Clones Encoding
Monodehydroascorbate Reductase-Like AIF The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity ofthe polypeptides encoded by the cDΝAs to monodehydroascorbate reductase- like AIF from Oryza sativa (ΝCBI GenBank Identifier (GI) No. 4666287),Oryzα sativa (NCBI GI No. 4760483), Brassica juncea (NCBI GI No. 4704613) and Arabidopsis thaliana (NCBI GI No. 1483150). Shown in Table 3 are the BLAST results for individual ESTs ("EST"), the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones ("FIS"), or contigs assembled from two or more ESTs ("Contig"): TABLE 3
BLAST Results for Sequences Encoding Polypeptides Homologous to Oryza sativa, Brassica juncea and Arabidopsis thaliana Monodehydroascorbate Reductase-Like AIF
Clone Status BLAST pLog Score acs2c.pk006.pl 1 EST 42.70 (gi 4666287) p0048.cqlaf62r EST 36.00 (gi 4760483) p0117.chclk04ra FIS >254.00(gi 4704613) rds2c.pk002.gl6 EST 32.00 (gi 4760483) rl0n.pk0066.c9 EST 34.52 (gi 4666287) sdp4c.pk009.118 EST 41.22 (gi 4666287) wlmkl. pk0007.f9 EST 10.22 (gi 4666287)
Contig composed of: Contig 54.15 (gi 1483150) wdk2c.pk016al9 wrl.pk0123.d9 wreln.pk0034.c8 EST 79.30 (gi 4666287)
The data in Table 4 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 6, 10, 16, 20, 24, 32, 36 and 40 and the Oryza sativa (gi 4666287) and (gi 4760483) , Brassica juncea and Arabidopsis thaliana sequences.
TABLE 4
Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Oryza sativa, Brassica juncea and Arabidopsis thaliana Monodehydroascorbate Reductase-Like AIF
SEQ ID NO. Percent Identity to
2 60% (g 4666287)
6 60% (g: 4760483)
10 72% (g: 4704613)
16 54% (g.4760483)
20 55% (g 4666287)
24 44% (g 4666287)
32 60% (g 4666287)
36 58% (g: 1483150)
40 86% (g: 4666287)
Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WTNDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of an monodehydroascorbate reductase-like AIF. These sequences represent the first Arabidopsis, corn, rice, soybean and wheat sequences encoding monodehydroascorbate reductase-like AIF.
The sequence ofthe entire cDNA insert in most ofthe clones listed in Table 3 was determined. Further sequencing and searching ofthe DuPont proprietary database allowed the identification of other corn, rice, soybean and/or wheat clones encoding monodehydroascorbate reductase-like AIF. Sequence analysis indicated that the entire cDNA inserts in clones sdp4c.pk009.118, ssm.pk0031.c9, and wrl.pk0123.d9, and the contig assembled from the entire cDNA insert in clone pOl 17.chclk04ra and the cDNA insert in clone cbn2.pk0010.a8 did not encode entire AIF. To obtain additional nucleotide sequence, PCR-based methods well-known in the art were employed.
The BLASTX search using the EST sequences from clones listed in Table 5 revealed similarity ofthe polypeptides encoded by the cDNAs to monodehydroascorbate reductase- like AIF from Arabidopsis thaliana (NCBI GI No.6358782), Oryza sativa (NCBI GI No. 4666287), and Brassica juncea (NCBI GI No. 4704613). Shown in Table 5 are the BLAST results for individual ESTs ("EST"), the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones ("FIS"), the sequences of contigs assembled from two or more ESTs ("Contig"), sequences of contigs assembled from an FIS and one or more ESTs or PCR fragment sequence ("Contig*"), or sequences encoding an entire protein derived from an FIS, a contig, or an FIS and PCR fragment sequence ("CGS"):
TABLE 5
BLAST Results for Sequences Encoding Polypeptides Homologous to Monodehydroascorbate Reductase-Like AIF
BLAST Results
Clone Status NCBI GI No. pLog Score acs2c.pk006.pl 1 CGS 4666287 135.00
(FIS) p0048.cqlaf62r CGS 4666287 135.00
(FIS)
Contig of CGS 4704613 >180.00 cbn2.pk0010.a8 p0117.chclk04ra
(FIS)
Contig of CGS 4704613 >180.00 rl0n.pk0025.f6 rcaln.pk012.el l
(FIS)
PCR fragment sequence rds2c.pk002.gl6 CGS 4666287 128.00
(FIS) rl0n.pk0066.c9 CGS 4666287 132.00
(FIS)
Contig of CGS 4666287 135.00 sdp4c.pk009.118
(FIS)
PCR fragment sequence
TABLE 5
BLAST Results for Sequences Encoding Polypeptides Homologous to Monodehydroascorbate Reductase-Like AIF
Contig of CGS 6358782 > 180.00 ssm.pk0031.c9
(FIS)
PCR fragment sequence wdrlf.pk003.bl8 EST 4666287 49.52 wlmkl. pk0007.f9 CGS 4666287 131.00
(FIS)
Contig of Contig* 4704613 >180.00 wrl.pk0123.d9
(FIS)
PCR fragment sequence
In addition, PSORT sequence analysis program (http://psort.nibb.ac.jp) predicts nuclear and mitochondrial targeting signals to be present in some ofthe polypeptides encoded by the instant nucleotide sequences, consistent with AIF subcellular localization. Figure 1 presents an alignment ofthe amino acid sequences set forth in SEQ ID NOs:4, 8, 18, 22, 26, and 34, and the Oryza sativa sequence (NCBI GI No. 4666287; SEQ ID NO:41). Figure 2 presents an alignment ofthe amino acid sequences set forth in SEQ ID NOs:12, 14, and 28, and the Brassica juncea sequence (NCBI GI No. 4704613; SEQ ID NO:42). The data in Table 6 represents a calculation ofthe percent identity ofthe amino acid sequences set forth in SEQ ID NOs:4, 8, 12, 14, 18, 22, 26, 28, and 34, the Oryza sativa sequence (NCBI GI No. 4666287; SEQ ID NO:41), and the Brassica juncea sequence (NCBI GI No. 4704613; SEQ ID NO:42).
TABLE 6
Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Monodehydroascorbate Reductase-Like AIF
SEQ ID NO. NCBI GI No. Percent Identity
4 4666287 51.7
8 4666287 52.2
12 4704613 69.6
14 4704613 71.0
18 4666287 51.3
22 4666287 52.0
26 4666287 52.6
28 4704613 76.6
34 4666287 51.7
Sequence alignments and percent identity calculations were performed using the Megalign program ofthe LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment ofthe sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a monodehydroascorbate reductase-like AIF. EXAMPLE 4
Expression of Chimeric Genes in Monocot Cells A chimeric gene comprising a cDNA encoding the instant polypeptide 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) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation ofthe 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 ofthe plasmid pML103. Plasmid pML103 has been deposited under the terms ofthe Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment ofthe maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end ofthe maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be Hgated at 15°C overnight, essentially as described (Maniatis). The Hgated DNA may then be used to transform E. coli XLl-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, 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 ofthe 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). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control ofthe 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region ofthe nopaline synthase gene from the T-DNA ofthe Ti plasmid of Agrobacterium tumefaciens. The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 φm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) ofthe DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
For bombardment, the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
Seven days after bombardment the tissue can be transfeπed to N6 medium that contains bialophos (5 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 transfeπed to fresh N6 medium containing bialophos. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialophos - 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 transfeπed to regeneration medium (Fromm et al. (1990) Bio/Technology 5:833-839).
EXAMPLE 5 Expression of Chimeric Genes in Dicot Cells
A seed-specific expression cassette composed ofthe promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 251 :9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites. The cDNA fragment of this gene may be generated by polymerase chain reaction
(PCR) ofthe cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation ofthe DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.
Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds ofthe 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 φm, 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 (Klein et al. (1987) Nature (London) 527:70-73, U.S. Patent No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed ofthe 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 ofthe Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptide and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site ofthe vector carrying the marker gene.
To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL ofthe DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
EXAMPLE 6 Expression of Chimeric Genes in Microbial Cells The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.
Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% low melting agarose gel. Buffer and agarose contain 10 μg/ml ethidium bromide for visualization ofthe DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies, Madison, WI) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be Hgated to the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly, MA). The fragment containing the Hgated 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 Hgated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the coπect orientation with respect to the T7 promoter by restriction enzyme analysis.
For high level expression, a plasmid clone with the cDNA insert in the coπect orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DΕ3) (Studier et al. (1986) J. Mol Biol 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration ofthe supernatant determined. One μg of protein from the soluble fraction ofthe culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Claims

CLAIMS What is claimed is:
1. An isolated polynucleotide comprising:
(a) a first nucleotide sequence encoding a first polypeptide comprising at least 50 amino acids, wherein the amino acid sequence ofthe first polypeptide and SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:32, or SEQ ID NO:36 have at least 80% identity based on the Clustal alignment method,
(b) a second nucleotide sequence encoding a second polypeptide comprising at least 50 amino acids, wherein the amino acid sequence ofthe second polypeptide and SEQ ID NO:30 have at least 95% identity based on the Clustal alignment method,
(c) a third nucleotide sequence encoding a third polypeptide comprising at least 100 amino acids, wherein the amino acid sequence ofthe third polypeptide and SEQ ID NO:6 or SEQ ID NO:34 have at least 80% identity based on the Clustal alignment method,
(d) a fourth nucleotide sequence encoding a fourth polypeptide comprising at least 100 amino acids, wherein the amino acid sequence ofthe fourth polypeptide and SEQ
ID NO:2 have at least 95% identity based on the Clustal alignment method,
(e) a fifth nucleotide sequence encoding a fifth polypeptide comprising at least 150 amino acids, wherein the amino acid sequence ofthe fifth polypeptide and SEQ ID NO: 10 have at least 80% identity based on the Clustal alignment method, (f) a sixth nucleotide sequence encoding a sixth polypeptide comprising at least 200 amino acids, wherein the amino acid sequence ofthe sixth polypeptide and SEQ ID NO:4 or SEQ ID NO:8 have at least 80% identity based on the Clustal alignment method,
(g) a seventh nucleotide sequence encoding a seventh polypeptide comprising at least 200 amino acids, wherein the amino acid sequence ofthe seventh polypeptide and SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:38 have at least 85% identity based on the Clustal alignment method,
(h) an eighth nucleotide sequence encoding an eighth polypeptide comprising at least 200 amino acids, wherein the amino acid sequence ofthe eighth polypeptide and SEQ ID NO:40 have at least 90% identity based on the Clustal alignment method, (i) a ninth nucleotide sequence encoding a ninth polypeptide comprising at least 250 amino acids, wherein the amino acid sequence ofthe ninth polypeptide and SEQ ID NO:26 have at least 80% identity based on the Clustal alignment method, or
(j) a tenth nucleotide sequence encoding a tenth polypeptide comprising at least 250 amino acids, wherein the amino acid sequence ofthe tenth polypeptide and SEQ ID NO:28 have at least 85% identity based on the Clustal alignment method.
2. The isolated polynucleotide of Claim 1 , wherein the first and second polypeptides comprise 100 amino acids.
3. The isolated polynucleotide of Claim 1 , wherein the first polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:32 or SEQ ID NO:36, wherein the second polypeptide comprises SEQ ID NO:30, wherein the third polypeptide comprises SEQ ID NO:6 or SEQ ID NO:34, wherein the fourth polypeptide comprises SEQ ID NO:2, wherein the fifth polypeptide comprises SEQ ID NO: 10, wherein the sixth polypeptide comprises SEQ ID NO:4 or SEQ ID NO:8, wherein the seventh polypeptide comprises SEQ ID NO:12, SEQ ID NO: 14, or SEQ ID NO:38, wherein the eighth polypeptide comprises SEQ ID NO:40, wherein the ninth polypeptide comprises SEQ ID NO:26, and wherein the tenth polypeptide comprises SEQ ID NO:28.
4. The isolated polynucleotide of Claim 1 , wherein the first nucleotide sequence comprises SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:31 or SEQ ID NO:35, wherein the second nucleotide sequence comprises SEQ ID NO:29, wherein the third nucleotide sequence comprises SEQ ID NO:5 or SEQ ID NO:33, wherein the fourth nucleotide sequence comprises SEQ ID NO: l, wherein the fifth nucleotide sequence comprises SEQ ID NO:9, wherein the sixth nucleotide sequence comprises SEQ ID NO:3 or SEQ ID NO:7, wherein the seventh nucleotide sequence comprises SEQ ID NO: l 1, SEQ ID NO: 13, or SEQ ID NO:37, wherein the eighth nucleotide sequence comprises SEQ ID NO:39, wherein the ninth nucleotide sequence comprises SEQ ID NO:25, and wherein the tenth nucleotide sequence comprises SEQ ID NO:27.
5. The isolated polynucleotide of Claim 1, wherein the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth polypeptides are AIFs.
6. The complement ofthe polynucleotide of any of Claims 1-5, wherein the complement and the polynucleotide consist of the same number of nucleotides and are 100% complementary.
7. The isolated polypeptide encoded by the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth nucleotide sequence comprised by the polynucleotide of any of Claims 1-5.
8. A method for transforming a cell comprising introducing the polynucleotide of any of Claims 1-5 into a cell.
9. The cell produced by the method of Claim 8.
10. A method for transforming a cell comprising introducing the complement of Claim 6 into a cell.
11. The cell produced by the method of Claim 10.
12. A method for producing a polynucleotide fragment comprising (a) selecting a nucleotide sequence comprised by the polynucleotide of any of Claims 1-5, wherein the selected nucleotide sequence contains at least 30 nucleotides, and (b) synthesizing a polynucleotide fragment containing the selected nucleotide sequence.
13. The method of Claim 12, wherein the selected nucleotide sequence comprises at least 40 nucleotides.
14. The method of Claim 12, wherein the selected nucleotide sequence comprises at least 60 nucleotides.
15. A method for producing a polynucleotide fragment comprising (a) selecting a nucleotide sequence comprised by the complement of Claim 6, wherein the selected nucleotide sequence contains at least 30 nucleotides, and (b) synthesizing a polynucleotide fragment containing the selected nucleotide sequence.
16. The method of Claim 15, wherein the selected nucleotide sequence comprises at least 40 nucleotides.
17. The method of Claim 15, wherein the selected nucleotide sequence comprises at least 60 nucleotides.
18. The polynucleotide fragment produced by the method of any of Claims 12-17.
19. A polynucleotide fragment comprising a nucleotide sequence comprised by the polynucleotide of any of Claims 1 -5, wherein the nucleotide sequence contains at least
30 nucleotides.
20. The polynucleotide fragment of Claim 19, wherein the nucleotide sequence contains at least 40 nucleotides.
21. The polynucleotide fragment of Claim 19, wherein the nucleotide sequence contains at least 60 nucleotides.
22. A polynucleotide fragment comprising a nucleotide sequence comprised by the complement of Claim 6, wherein the nucleotide sequence contains at least 30 nucleotides.
23. The polynucleotide fragment of Claim 22, wherein the nucleotide sequence contains at least 40 nucleotides.
24. The polynucleotide fragment of Claim 22, wherein the nucleotide sequence contains at least 60 nucleotides.
PCT/US2000/024859 1999-09-13 2000-09-11 PLANT APOPTOSIS INDUCING FACTORS (AIFs) WO2001019994A2 (en)

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