EP1501942A2 - Verfahren zur identifizierung evolutionsmässig signifikanter änderungen in polynukleotid- und polypeptidsequenzen in hauspflanzen und -tieren - Google Patents

Verfahren zur identifizierung evolutionsmässig signifikanter änderungen in polynukleotid- und polypeptidsequenzen in hauspflanzen und -tieren

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Publication number
EP1501942A2
EP1501942A2 EP03713258A EP03713258A EP1501942A2 EP 1501942 A2 EP1501942 A2 EP 1501942A2 EP 03713258 A EP03713258 A EP 03713258A EP 03713258 A EP03713258 A EP 03713258A EP 1501942 A2 EP1501942 A2 EP 1501942A2
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Prior art keywords
seq
polynucleotide
polypeptide
plant
domesticated
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EP03713258A
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French (fr)
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EP1501942A4 (de
Inventor
Walter Messier
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Evolutionary Genomics LLC
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Evolutionary Genomics LLC
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Priority claimed from US10/079,042 external-priority patent/US7252966B2/en
Application filed by Evolutionary Genomics LLC filed Critical Evolutionary Genomics LLC
Priority to EP08100680A priority Critical patent/EP1947201A3/de
Publication of EP1501942A2 publication Critical patent/EP1501942A2/de
Publication of EP1501942A4 publication Critical patent/EP1501942A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention comprises narrowing the search for such polynucleotides by comparing the corresponding polynucleotide sequences of domesticated and ancestor organisms to select those sequences containing nucleotide changes that are evolutionarily significant, which is typically indicated by a Ka/Ks ratio of 1.0 or greater.
  • Ka/Ks ratio of 1.0 or greater.
  • the subset of ancestor-modern plant polynucleotide pairs with Ka/Ks ratios of 1.0 should contain polynucleotides affected by neutral evolution, that is those for which the trait has not been under pressure, imposed by man or nature, to either be conserved or to change.
  • methods for identifying a polynucleotide sequence encoding a polypeptide of a domesticated organism (e.g., a plant or animal), wherein the polypeptide may be associated with a commercially or aesthetically relevant trait that is unique, enhanced or altered in the domesticated organism as compared to the ancestor of the domesticated organism, comprising the steps of: a) comparing protein-coding nucleotide sequences of said domesticated organism to protein-coding nucleotide sequences of said wild ancestor; and b) selecting a polynucleotide sequence in the domesticated organism that contains a nucleotide change as compared to a corresponding sequence in the wild ancestor, wherein said change is evolutionarily significant.
  • commercially or aesthetically relevant trait is used herein to refer to traits that exist in domesticated organisms such as plants or animals whose analysis could provide information (e.g., physical or biochemical data) relevant to the development of improved organisms or of agents that can modulate the polypeptide responsible for the trait, or the respective polynucleotide.
  • the commercially or aesthetically relevant trait can be unique, enhanced or altered relative to the ancestor.
  • altered it is meant that the relevant trait differs qualitatively or quantitatively from traits observed in the ancestor.
  • stress-resistance refers to the ability to withstand drought, disease, pests (including, but not limited to, insects, animal herbivores, and microbes), high salt levels, and other adverse stimuli, internal or external, that tend to disturb the plant's homeostasis, and may lead to disorder, disease, or death if uncorrected.
  • nucleotide change encompasses at least one nucleotide change, either a substitution, a deletion or an insertion, in a protein-coding polynucleotide sequence of a domesticated organism as compared to a corresponding sequence from the ancestor.
  • the change is a nucleotide substitution. More preferably, more than one substitution is present in the identified sequence and is subjected to molecular evolution analysis.
  • SEQ ID NO: 101 SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:l 10, SEQ ID NO:l 12, SEQ ID NO:l 13, SEQ ID NO:l 14, SEQ ID NO: 116, SEQ ID NO:l 17, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO: 145,
  • the present invention also provides a method of isolating a yield-related gene from plant cell DNA comprising a) providing a sample of plant cell DNA; b) providing a pair of oligonucleotides having sequence homology to a conserved region of an EG307 OR EGl 117 gene; c) combining the pair of oligonucleotides with the plant cell DNA sample under conditions suitable for polymerase chain reaction-mediated DNA amplification; and d) isolating the amplified yield-related gene or fragment thereof.
  • stringent hybridization conditions refer to standard hybridization conditions under which polynucleotides, including oligonucleotides, are used to identify molecules having similar nucleic acid sequences. Such standard conditions are disclosed, for example, in Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Labs Press, 1989. Examples of such conditions are provided in the Examples section of the present application.
  • EG307 polypeptide of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology or can be produced by chemical synthesis.
  • An EG307 polypeptide of the present invention may be identified by its ability to perform the function of natural EG307 in a functional assay.
  • natural EG307 polypeptide it is meant the full length EG307 polypeptide of O. sativa, O. rufipogon, Z. mays mays, and/or Z mays parviglumis.
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, and/or SEQ ID NO: 18.
  • Inspection of EG307 genomic nucleic acid sequences indicates that the genes comprise several regions, including a first exon region, a first intron region, a second exon region, a second intron region, and a third exon region.
  • the open reading frame from nucleotide 37 through about nucleotide 2280 of SEQ ID NO:4 is represented herein as SEQ ID NO:5.
  • translation of O. sativa (strain Azucena) polynucleotide SEQ ID NO: 1 suggests an open reading frame from about nucleotide 3 to about nucleotide 2410 of SEQ ID NO:l, with the first exon spanning nucleotides 1-92 of SEQ ID NO: 1, the first intron spanning nucleotides 93-1075 of SEQ ID NO:l, the second exon spanning nucleotides 1076- 1394 of SEQ ID NO:l, the second intron spanning nucleotides 1395-1475 of SEQ ID NO:l, and the third exon spanning nucleotides 1476-2441 of SEQ ID NO : 1.
  • the open reading frame is represented herein as SEQ ID NO:2, and encodes a polypeptide represented herein as SEQ ID NO:3.
  • Such an EG307 polypeptide is encoded by a polynucleotide that hybridizes under stringent hybridization conditions with a polynucleotide having nucleic acid sequence SEQ ID NO:20, SEQ ID NO:21, SEQ ID. NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and/or SEQ ID NO:31.
  • Translation of SEQ ID NO:23 and SEQ ID NO:24 suggests an open reading frame from about nucleotide 57 of SEQ ID NO:23 to about nucleotide 1562 of SEQ ID NO:24, with the first exon spanning nucleotides 1-146 of SEQ ID NO:23, the first intron spanning nucleotides 1- 230 of SEQ ID NO:24, the second exon spanning nucleotides 231-546 of SEQ ID NO:24, the second intron spanning nucleotides 547-627 of SEQ ID NO:24, and the third exon spanning nucleotides 628-1615 of SEQ ID NO:24.
  • the open reading frame is represented as SEQ ID NO:25, and encodes a polypeptide represented herein as SEQ ID NO:26.
  • Zea mays mays EG307 polypeptide of the present invention is a polypeptide encoded by an Zea mays mays polynucleotide that hybridizes under stringent hybridization conditions with complements of polynucleotides represented by SEQ ID NO:33, SEQ ID NO:34, SEQ ID. NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,
  • SEQ ID NO:41 suggests an open reading frame from about nucleotide 61 of SEQ ID NO:40 to about nucleotide 2263 of SEQ ID NO:41, with the first exon spanning nucleotides 1-141 of SEQ ID NO:40, the first intron spanning nucleotides 142-262 of SEQ ID NO:40 and nucleotides 1-896 of SEQ ID NO:41, the second exon spanning nucleotides 897-1209 of SEQ ID NO:41, the second intron spanning nucleotides 1210-1310 of SEQ ID NO:41, and the third exon spanning nucleotides 1311-2311 of SEQ ID NO :41.
  • the open reading frame is represented as SEQ ID NO:42 encoding a polypeptide represented herein as SEQ ID NO:43.
  • the Zea luxurians EG307 polynucleotide includes an open reading frame.
  • the reading frame encodes an Zea luxurians EG307 polypeptide of about 448 amino acids, the deduced amino acid sequence of which is represented herein as SEQ ID NO:86, assuming an open reading frame having an initiation (start) codon spanning from about nucleotide 5 through about nucleotide 7 of SEQ ID NO: 84 and a termination (stop) codon spanning from about nucleotide 2365 through about nucleotide 2367 of SEQ ID NO:84, with the first exon spanning nucleotides 1 -85 of SEQ ID NO:84, the first intron spanning nucleotides 86-998 of SEQ ID NO:84, the second exon spanning nucleotides 999-1311 of SEQ ID NO:84, the second intron spanning nucleotides 1312-1414 of SEQ ID NO:84, and the
  • an O. sativa EGl 117 gene includes the nucleic acid sequence SEQ ID NO:4.
  • Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of a cDNA (complementary DNA) polynucleotide, the production of which is disclosed in the Examples. It should be noted that since nucleic acid sequencing technology is not entirely error-free, SEQ ID NO:4 (as well as other sequences presented herein), at best, represents an apparent nucleic acid sequence of the polynucleotide encoding an O. sativa EG307 polypeptide of the present invention.
  • an O. sativa EGl 117 gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 92 and/or SEQ ID NO: 93.
  • O. rufipogon EGl 117 polypeptide of the present invention is a polypeptide encoded by an O. rufipgon polynucleotide that hybridizes under stringent hybridization conditions with complements of polynucleotides represented by SEQ ID NO:92,
  • Polynucleotide homologues can be selected from a mixture of modified nucleic acids by screening for the function of the polypeptide encoded by the nucleic acid (e.g., ability to elicit an immune response against at least one epitope of an EG307 polypeptide, ability to increase yield in a transgenic plant containing an EG307 gene) and/or by hybridization with an O. sativa EG307 gene, with an O. rufipogon EG307 gene, with a Z. mays mays EG307 gene, with a Z mays parviglumis EG307 gene, a Z diploperennis EG307 gene and/or a Z luxurians EG307 gene.
  • the function of the polypeptide encoded by the nucleic acid e.g., ability to elicit an immune response against at least one epitope of an EG307 polypeptide, ability to increase yield in a transgenic plant containing an EG307 gene
  • a preferred polynucleotide of the present invention when expressed in a suitable plant, is capable of increasing the yield of the plant.
  • a polynucleotide can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based compound.
  • One embodiment of the present invention is an isolated plant polynucleotide that hybridizes under stringent hybridization conditions with at least one of the following genes: an O. sativa EGl 117 gene, an O. rufipogon EGl 117 gene, a Z mays mays EGl 117 gene, and a Z mays parviglumis EGl 117 gene.
  • a polynucleotide of the present invention can include an isolated natural plant EGl 117 gene or a homologue thereof.
  • a polynucleotide of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of a polynucleotide of the present invention is the minimal size that can form a stable hybrid with one of the aforementioned genes under stringent hybridization conditions. Suitable and preferred plants are disclosed above.
  • sativa EGl 117 gene to a O. rufipogon EGl 117 gene, to a Z mays mays EGl 117 gene, and/or to a Z mays parviglumis EGl 117 gene, to a gene of the present invention, as well as a polynucleotide that is an allelic variant of any of those polynucleotides.
  • a preferred category of promoters is that which is induced by the physiological state of the plant (i.e. wound inducible, water-stress inducible, salt-stress inducible, disease inducible, and the like). Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the EG307 or EGl 117 polypeptides only accumulate in cells in which the accumulation is desired.
  • Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200- 208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1 :
  • a recombinant molecule of the present invention is a molecule that can include at least one of any polynucleotide heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the polynucleotide(s) in the cell to be transformed, examples of which are disclosed herein.
  • polypeptides of the present invention can be purified using a variety of standard polypeptide purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Polypeptides of the present invention are preferably retrieved in "substantially pure" form.
  • Yield response is measured by techniques well known to those skilled in the art. In the cereals yield response is determined, for example, by one or more of the following metrics, grain weight, grain length, grain weight/ 1000 grains, size of panicle, number of panicles, and number of grains/panicle.
  • the treated and untreated cells are then compared by any suitable phenotypic criteria, including but not limited to microscopic analysis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, the interactions of the cells when exposed to infectious agents, and the ability of the cells to interact with other cells or compounds. Differences between treated and untreated cells indicate effects attributable to the candidate agent. Optimally, the agent has a greater effect on experimental cells than on control cells.
  • Appropriate host cells include, but are not limited to, eukaryotic cells, preferably plant or animal cells. The choice of cell will at least partially depend on the nature of the assay contemplated.
  • EXAMPLE 8 Study of Protein Function using a Transgenic Plant
  • the functional roles of a positively selected maize gene obtained according to the methods of Examples 4-7 can be assessed by conducting assessments of each allele of the gene in a transgenic maize plant.
  • a transgenic plant can be created using an adaptation of the method described in Peng et al. (1999) Nature 400:256-261. Physiological, morphological and/or biochemical examination of the transgenic plant or protein extracts thereof will permit association of each allele with a particular phenotype.
  • genomic DNA was isolated from several different strains of O. sativa (acquired from the National Small Grains Collection, U.S.D.A., Aberdeen, Idaho), using Qiagen's protocol (DNeasy Plant Mini Kit: cat #69103).
  • EG307 was then sequenced in genomic DNA from six different O. sativa strains: Nipponbare, Lemont, IR64, Teqing, Azucena, and Kasalath. The K A /Ks ratios for each of these strains varied when compared to O. rufipogon. Table 1 shows results for the entire 1344 bases of coding region. Table 1. Full CDS Ka/Ks ratios for O. rufipogon (strain IRGC105491) vs. all O. sativa strains examined.
  • the DIG protocol (BMB-Roche PCR DIG Probe Synthesis Kit cat #1636090) successfully labeled a unique EG307 494bp PCR product (primers: 5'-
  • Rice contig 58 is on chromosome 3 and contains 181 BACs and 15 markers.
  • EGl 117 maps to the same BACs as markers CDO1387, C236, C875, R2778 and R2015. These all map to 35.8 cM on map 3RJ98.
  • This marker is mapped to several different rice maps, as accessed through the RiceGenes or Gramene website . There are also several QTLs mapped to this region.
  • One well-documented QTL for 1000-grain weight is in this region of chromosome 3 and is associated with marker RZ672 (McCouch, S.R. et al. Genetics 150:899-
  • GenBank EST entries come from clone S20126 that maps to YACs Y2533 and Y5488. These YACs are anchored with SI 0968, which maps to Chromosome 3 at 33.5 cM.
  • EGl 117 codes for a protein homologous to a family of peptide transporters. Other members of this family have been shown in plants to influence growth, flowering and seed development. EGl 117 is also linked to the QTL for grain weight. It is highly unlikely that this is a coincidence. These are ideal genes to use in the aims of this proposal to both validate the genes as agriculturally relevant.
  • Example 19 Determination of the function of gene candidate EG307.
  • Standard reagents, yeast strains, vectors and DNA isolation/sequencing specific for the HybriZAP YTH system will be obtained from Stratagene.
  • the coding region of EG307 will be cloned using an RT-PCR amplification of O. sativa shoot mRNA.
  • the PCR amplified insert will be cloned into a linearized pBD-GAL4 Cam phagemid vector and transformants carrying inserts will be selected on chloramphenicol plates to create the "bait" plasmid.
  • the cloning junctions and coding region of EG307 will be sequenced using standard sequencing techniques at EG to ensure usage of the proper reading frame and that no mutations have been introduced during amplification of EG307.
  • GAL4 transcription activation sequence and causes activation of the reporter systems. If this occurs, two bait constructs expressing the two halves of the EG307 protein independently will be constructed. These constructs would be tested for direct activation of GAL4 reporters in
  • This technique should allow for the identification of the interacting proteins as long as the affinity of the interaction is specific and strong enough to ensure a tight binding between the EG307 protein and the potential interacting protein. These data would then allow for the identification of the interacting protein if that protein is homologous to other proteins. It is clearly possible that no proteins will be identified by this method because of a lack of affinity for the EG307 protein. Alternatively, it is possible that no interacting proteins are present in the lysates generated by the methods outlined above.
  • a second assay that will be performed is a inhibition assay.
  • the B Y4742- ptr2 EG307 transfectants as well as the BY4742 parental and BY4742-ptr2 deletion mutants as controls will be plated as a lawn on YPG (yeast extract, peptone, galactose) plates and the toxic ethionine-peptide derivatives will be spotted onto membrane discs and placed on the yeast lawns (Steiner, et al., 1994). Zones of clearing around the disc would then indicate that the yeast expressed a functional transport protein the allowed the yeast to transport the toxic peptide into the cell, killing the cell.
  • YPG yeast extract, peptone, galactose

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EP03713258A 2002-01-16 2003-01-16 Verfahren zur identifizierung evolutionsmässig signifikanter änderungen in polynukleotid- und polypeptidsequenzen in hauspflanzen und -tieren Withdrawn EP1501942A4 (de)

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EP08100680A EP1947201A3 (de) 2002-01-16 2003-01-16 Verfahren zur Bestimmung von evolutionär signifikanten Veränderungen in Polynucleotid- und Polypeptidsequenzen bei domestizierten Pflanzen und Tieren

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US34966102P 2002-01-17 2002-01-17
US349661P 2002-01-17
US10/079,042 US7252966B2 (en) 1999-01-29 2002-02-19 EG307 polynucleotides and uses thereof
US79042 2002-02-19
US36854102P 2002-03-29 2002-03-29
PCT/US2003/001460 WO2003062382A2 (en) 2002-01-16 2003-01-16 Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals
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AU2006230352A1 (en) * 2005-03-29 2006-10-05 Evolutionary Genomics Llc EG1117 and EG307 polynucleotides and uses thereof
AU2006287239A1 (en) * 2005-09-02 2007-03-08 Evolutionary Genomics, Inc. EG8798 and EG9703 polynucleotides and uses thereof
CN102888398B (zh) * 2011-07-22 2014-03-05 中国农业科学院生物技术研究所 转基因水稻品系Bar68-1的外源插入片段的旁侧序列及其应用
CN102934548B (zh) * 2011-08-15 2014-07-02 东北农业大学 辅助鉴定抗旱玉米的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011088A2 (en) * 1999-08-05 2001-02-15 Evolutionary Genomics, Llc Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6245969B1 (en) * 1997-06-24 2001-06-12 Joanne Chory Receptor kinase, Bin1
AU769931B2 (en) * 1998-01-30 2004-02-12 Evolutionary Genomics Llc Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
US6274319B1 (en) * 1999-01-29 2001-08-14 Walter Messier Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001011088A2 (en) * 1999-08-05 2001-02-15 Evolutionary Genomics, Llc Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI WEN-HSIUNG: "Unbiased estimation of the rates of synonymous and nonsynonymous substitution" JOURNAL OF MOLECULAR EVOLUTION, vol. 36, no. 1, 1993, pages 96-99, XP008058643 ISSN: 0022-2844 *
See also references of WO03062382A2 *
WHITE S E ET AL: "The molecular evolution of terminal ear1, a regulatory gene in the genus zea" GENETICS, GENETICS SOCIETY OF AMERICA, AUSTIN, TX, US, vol. 153, November 1999 (1999-11), pages 1455-1462, XP002169167 ISSN: 0016-6731 *

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CA2473555A1 (en) 2003-07-31
AU2003217221B2 (en) 2008-11-27
WO2003062382A2 (en) 2003-07-31
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