US20030166197A1 - Ethylene insensitive plants - Google Patents

Abstract

Nucleic acid and polypeptide sequences are described which relate to an EIN6 gene, a gene involved in the plant ethylene response. Plant transformation vectors and transgenic plants are described which display an altered ethylene-dependent phenotype due to altered expression of EIN6 in transformed plants.

Description

RELATED APPLICATIONS
• This application claims priority from U.S. Provisional Application 60/290,303 filed on May 10, 2001.[0001]
GOVERNMENTAL SUPPORT
• [0002] This work was supported in part by research grants from the National Science Foundation MCB0049003 and Department of Energy grant ER15113. The U.S. government may have certain rights to this invention.
BACKGROUND OF THE INVENTION
• 1. Field of the Invention[0003]
• The field of the invention relates to plants and plant genes, including both plant mutants and transgenic plants containing a gene that confers an ethylene insensitive phenotype. Also encompassed by the invention are methods of using the disclosed plant gene to confer an ethylene insensitive phenotype.[0004]
• 2. Description of the Related Art[0005]
• The plant hormone ethylene plays a central role in a variety of processes, including wounding, pathogen attack, cell fate and elongation, senescence, and fruit ripening. The classical triple response to ethylene, inhibited hypocotyl and root elongation, thickening of the hypocotyl, and exaggeration of the apical hook, has been used to study the signaling pathway that mediates this hormone response. Molecular genetic studies in[0006]Arabidopsis thalianahave exploited the triple response to ethylene, and resulted in the identification of mutants defective in this response. Epistasis studies with plants that are insensitive to the hormone or display a constitutive response to ethylene have suggested a signaling pathway in which one group of proteins acts to antagonize pathway activity in the absence of the hormone, and a second group functions to transmit the signal and initiate physiological responses.
• The ETR1 gene and a family of related genes, including EIN4, ETR2, ERS1, and ERS2, encode ethylene receptors. Ethylene binding is mediated by the hydrophobic amino terminus of ETR1 in a copper dependent manner. Point mutations in this domain can disrupt ethylene binding, and result in dominant ethylene insensitivity, suggesting that the proteins actively suppress pathway activity in the absence of the hormone, and that ethylene binding removes this inhibitory activity. Genetic experiments in which combining loss of function alleles of at least three family members resulted in constitutive pathway activity in the absence of ethylene confirmed the idea that the receptors negatively regulate ethylene signaling in the absence of the hormone. The important role copper plays in receptor function was underscored by the identification of the RAN1 gene, which bears significant sequence similarity to the Menkes/Wilson disease copper transporter. Disruption of RAN1 by mutation or co-suppression resulted in plants that display a constitutive triple response, suggesting that copper is required for the normal formation or proper structure of a receptor complex in addition to ethylene binding.[0007]
• Mutation of the Raf kinase homolog CTR1 also results in a constitutive triple response in the absence of ethylene. Based on epistasis studies, CTR1 functions downstream of the ethylene receptors, and represses signaling pathway activity in the absence of the hormone. The repressive roles played by both CTR1 and the receptors suggests a functional link between the two. Indeed, protein interaction studies have shown that CTR1 and ETR1 physically interact, but the manner by which ETR1 may activate CTR1 is unknown. Although the homology of CTR1 to Raf suggests a downstream MAP kinase signaling cascade, the manner by which CTR1 represses ethylene signaling is also unknown.[0008]
• Mutations of the EIN2 gene are recessive and also confer complete ethylene insensitivity. Epistasis testing indicates that EIN2 functions downstream of CTR1, and consequently is thought to play a central role in ethylene responses. EIN2 possesses an amino terminus that is predicted to contain multiple transmembrane domains, and is similar to metal transporters of the Nramp family. The exact manner in which EIN2 functions to activate the ethylene signaling pathway is unclear, however it has been shown that expression of its carboxyl end is sufficient to stimulate ethylene-dependent effects, including the triple response.[0009]
• Terminal components of the primary ethylene signaling pathway are encoded by the EIN3 gene and a related family of EIL genes. ein3 mutations partially block activation by both ctr1 mutations and the EIN2 carboxyl end, indicating that it functions downstream of EIN2 in the signaling cascade. EIN3 and EIL proteins represent a novel class of transcription factors, and have been found to bind as dimers to a unique site found in some promoters of a second class of transcription factors termed ethylene response element binding proteins (EREBP). Many of these factors bind to the GCC box found in many ethylene responsive genes that are activated several hours after ethylene treatment. An EIN3 binding site was found in the promoter of one such factor, ERF1, which was activated within fifteen minutes of ethylene treatment in an EIN3-dependent manner. Overexpression of ERF1 activated a subset of ethylene responses and bypassed an ein3 mutation, indicating that it functions downstream of EIN3 in the signaling cascade. These data indicate that a complete ethylene response is mediated by a series of transcription factors that act in sequential steps. EIN3 and EIL proteins act very early in the transcriptional cascade, whereas ERF1 and related proteins act later.[0010]
• Roman et al. described a mutant plant, ein6 (Roman, et al. (1995) “Genetic analysis of ethylene signal transduction in[0011]Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway” Genetics 139(3): 1393-1409) This mutant plant demonstrated ethylene insensitivity, which was assumed to be the result of a single recessive mutation. The EIN6 gene was not identified. Unlike etr1 and ein2 mutants, ein6 mutant plants were found to retain some sensitivity to ethylene, with the root being less sensitive to ethylene than the hypocotyl. However, the original work mischaracterized the ein6 mutant plant as the phenotype was thought to be due to a single mutation.
• In addition, the chromosomal location and DNA/Protein sequence of the EIN6 gene was not described, thus hindering in-depth studies of this mutation. However, as described below, the present application describes the isolation and characterization of the EIN6 gene, as well as the mutant allele, ein6.[0012]
SUMMARY OF THE INVENTION
• One embodiment of the invention is a mutant plant containing a mutated form of the EIN6 gene, wherein said mutant plant exhibits an altered response to ethylene. In a preferred embodiment, the altered response to ethylene includes an ethylene insensitive or an ethylene insensitive root (EIR) phenotype. In a more preferred embodiment, the mutant plant contains an ein6 gene which has the nucleotide sequence shown in SEQ ID NO: 23.[0013]
• Another embodiment of the invention is a mutant plant containing a mutated form of the EIN6 and a mutated form of the een gene. This double mutant plant was found to be ethylene insensitive throughout the plant, in contrast to the EIR phenotype found in ein6 single mutants.[0014]
• Another embodiment of the invention relates to the discovery that the ein6/een mutation was found to be epistatic to ctr1, partially suppressing the constitutive hormone response in a manner similar to ein3 mutations. In these respects, ein6/een double mutants were very similar to other ethylene insensitive mutants. Interestingly, certain phenotypes of the ein6/een double mutant distinguish it from other ethylene-related mutants, such as hypersensitivity to the microtubule-stabilizing drug taxol.[0015]
• The present application describes the isolation and characterization of the EIN6 gene, which was found to be required for normal ethylene responses. The EIN6 gene encodes a protein containing multiple zinc fingers, and additional domains that are implicated in protein-protein interactions. As such, it represents an excellent candidate for a partner of EIN3 or ERF1 in the regulation of ethylene-dependent transcription.[0016]
• Another embodiment of the invention is drawn to an isolated polynucleotide having a nucleic acid sequence that hybridizes to the sequence of SEQ ID NO: 22 under moderate stringency, wherein expression of the polynucleotide in a plant results in an altered response to ethylene. In a preferred embodiment, the isolated polynucleotide has the sequence shown in SEQ ID NO: 23. Also encompassed within the present invention is a plant expression vector containing an isolated polynucleotide that hybridizes to the sequence of SEQ ID NO: 22 under moderate stringency, operably linked to a promoter, wherein expression of the polynucleotide in a plant results in an altered response to ethylene. In one embodiment, the promoter is a constitutive promoter. In an alternate embodiment, the promoter is a tissue specific promoter. In another alternate embodiment, the promoter is an inducible promoter.[0017]
• Also included within the invention is a method for producing a transgenic plant with an altered ethylene-dependent phenotype including the steps of:[0018]
• transforming a plant cell with a plant expression vector containing the isolated polynucleotide that hybridizes to the sequence of SEQ ID NO: 22 under moderate stringency;[0019]
• regenerating a transgenic plant from the transformed plant cell;[0020]
• growing said transgenic plant under conditions such that the isolated polynucleotide is expressed in the plant; and[0021]
• selecting at least one plant having an altered ethylene-dependent phenotype.[0022]
• In one embodiment, the altered ethylene-dependent phenotype is selected from the group including ripening, flowering, senescence, browning, and sensitivity to pathogens. In a more preferred embodiment, the plant expression vector contains the nucleotide sequence of SEQ ID NO: 22. In one preferred embodiment, the polynucleotide is overexpressed in the plant. In another preferred embodiment, expression of the polynucleotide inhibits expression of a corresponding gene. In a most preferred embodiment, the polynucleotide transcribes an RNA that is complementary to that gene.[0023]
• Also encompassed within the invention is an isolated polypeptide containing the amino acid sequence of SEQ ID NOS: 20 or 21.[0024]
• Also included in embodiments of the invention is an isolated polynucleotide encoding an EIN6 gene as well as polynucleotides which have 80%, 85%, 90%, or 95% homology to SEQ ID NO: 22.[0025]
• In another embodiment, the invention includes a recombinant expression vector containing a polynucleotide sequence encoding an EIN6 gene. In a further embodiment, the present invention includes a bacterial or plant host cell containing the recombinant vector containing the EIN6 gene.[0026]
• Another embodiment of the invention includes a genetically modified plant containing at least one exogenous nucleic acid sequence encoding an EIN6 gene. Also included are embodiments of the invention wherein the exogenous nucleic acid sequence has at least 80%, 85%, 90%, or 95% homology to the sequence of SEQ ID NO: 22. In a further embodiment the exogenous nucleic acid sequence of the genetically modified plant contains a regulatory nucleic acid sequence. In a preferred embodiment, the regulatory sequence is a promoter. In a more preferred embodiment, the promoter is a constitutive promoter. In an alternate more preferred embodiment, the promoter is an inducible promoter. In another alternate preferred embodiment, the promoter is a tissue specific promoter. In a preferred embodiment, the exogenous nucleic acid sequence in the genetically modified plant contains a selectable marker. In a preferred embodiment, the genetically modified plant is a dicotyledonous plant. In an alternate preferred embodiment, the genetically modified plant is a monocotyledonous plant. Also encompassed by the present invention are seeds which germinate into a plants containing at least one exogenous nucleic acid sequence encoding an EIN6 gene.[0027]
• For purposes of summarizing the invention and the advantages achieved over the prior art, certain objects and advantages of the invention have been described above. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.[0028]
• Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.[0029]
BRIEF DESCRIPTION OF THE DRAWINGS
• These and other feature of this invention will now be described with reference to the drawings of preferred embodiments which are intended to illustrate and not to limit the invention.[0030]
• FIG. 1. Genetic and Physical Map of the EIN6 Genomic Region. Genomic region shown corresponds to the middle of[0031]chromosome 3, “proximal” and “distal” referring to the orientation relative to the centromere. The CAPS markers TOPP5 and g19397 were mapped relative to the EIN6 gene, and map positions of TOPP5 and AP3, and genetic distance between the g19397 and AP3 markers are based on published recombinant inbred mapping data. The numbers below TOPP5 and g19397 the chromosomal map reflect the number of recombinant plants identified out of populations of 124 and 350 F2 plants, respectively. The BAC contig for this interval is the ESSA sequencing contig for this region ofchromosome 3. The numbers below the markers for this map represent the number of recombinant plants identified out of a population of 496 F2 plants. The marker locations of the 71 kb interval of T29H11 were determined from the genomic sequence determined by the ESSA Sequencing Consortium. The numbers below the markers are the number of recombinant plants identified out of a population of 496 F2 plants.
• FIG. 2. Nucleotide Sequence of the EIN6 Gene (SEQ ID NO: 1). The genomic sequence of the EIN6 gene from the Landsberg erecta ecotype was determined by direct sequencing of overlapping PCR products covering the locus. Nucleotides are numbered with the first nucleotide of the cDNA being +1. Exons are listed in capital letters, whereas introns are in lowercase. Asterisks above the line indicate the 7 nucleotides deleted in the ein6 mutant, and solid boxes above the nucleotides denote an intron that is removed by alternative splicing.[0032]
• FIG. 3. Amino Acid Sequence of the EIN6 Polypeptides. This figure shows the amino acid sequence of the full length EIN6 protein (SEQ ID NO: 20). The solid box above the sequence denotes a putative coil domain. The vertically-hatched box above the sequence denotes a putative ATP/GTP-binding P-loop domain. The asterisks above the sequence denote putative nuclear localization signals. The diagonally-hatched boxes above the sequence indicate acidic domains.[0033]
• FIG. 4. This figure shows the amino acid sequence of the alternatively-spliced EIN6 gene product (SEQ ID NO: 21).[0034]
• FIG. 5. Alignment of Zinc Finger Motifs. The sequence of the EIN6 zinc finger domains (SEQ ID NO: 2) aligned with other zinc finger domain-containing proteins. GLI1 (SEQ ID NO: 3) and GLI3 (SEQ ID NO: 5) zinc finger proteins from[0035]Mus musculus. X84986 (SEQ ID NO: 4) is a putative zinc finger protein fromDrosophila melanogaster. SPH1-BF (SEQ ID NO: 6) is the SPH1 binding factor fromHomo sapiens. RPH1p (SEQ ID NO: 7) is the repressor of photolyase gene fromSaccharomyces cerevisiae. Identical residues are highlighted by black boxes, and similar residues are indicated by shading. Numbers indicate the positions of the residues within the respective polypeptides.
• FIG. 6. Alignment of the EIN6 amino terminus with two interrupted domains of homology. The sequence of the EIN6 amino terminal domain (SEQ ID NO: 8) aligned with proteins whose homology extend over two non-contiguous regions. The EIN6 sequence is listed as contiguous, but only the homologous regions of related proteins are shown. The regions of homology are interrupted by a 300 amino acid domain in the homologous proteins. RBP2 is the retinoblastoma binding protein 2 of[0036]Homo sapiens(SEQ ID NO: 9). Smcx (SEQ ID NO: 10) is an X-linked protein not subject to X-linked inactivation fromMus musculus. CG9088 (SEQ ID NO: 11) is a predicted protein fromDrosophila melanogaster. AL136078 (SEQ ID NO: 12) is a predicted protein fromSchizosaccharomyces pombe. F2K11.14 (SEQ ID NO: 13) is a predicted protein fromArabidopsis thaliana. Identical residues are highlighted by black boxes, and similar residues are indicated by shading. Numbers indicate the positions of the residues within the respective polypeptides.
• FIG. 7. Alignment of the EIN6 Amino Terminus (SEQ ID NO: 14) with a Continuous Domain of Homology. The sequence of the EIN6 amino terminal domain aligned with proteins whose homology extend over a single continuous sequence. RPH1p (SEQ ID NO: 15) is the repressor of photolyase gene from[0037]Saccharomyces cerevisiae. F19I3.11 (SEQ ID NO: 16) is a predicted protein fromArabidopsis thaliana. CG4037 (SEQ ID NO: 17) is a predicted protein fromDrosophila melanogaster. Y48B6A.11 (SEQ ID NO: 18) is a hypothetical protein fromCaenorhabditis elegans. KIAA0780 (SEQ ID NO: 19) is a predicted protein fromHomo sapiens. Identical residues are highlighted by black boxes, and similar residues are indicated by shading. Numbers indicate the positions of the residues within the respective polypeptides.
DETAILED DESCRIPTION
• Embodiments of the invention are based, in part, upon the identification, isolation, cloning and sequencing of a novel gene family regulating developmental and stress responses in plants, including the classical triple response to ethylene. Described below are genomic (SEQ ID NO: 1) and cDNA (SEQ ID NO: 22) sequences of the EIN6 gene, homologues, mutants, and fragments thereof. In addition, a specific mutated sequence of ein6 with a 7 base pair deletion is specifically described (SEQ ID NO: 23). Also disclosed is a sequence for the full length polypeptide, identified in[0038]Arabidopsis thaliana(SEQ ID NO: 20). A second protein, produced by alternate splicing, is also described (SEQ ID NO: 21).
• A mutation in the novel ethylene-signaling gene Ethylene Insensitive 6 (EIN 6) was identified from a large pool of fast neutron mutagenesis-treated Arabidopsis (Landsberg ecotype) seed (Example 1). The mutagenized pool was screened for lines that were insensitive to ethylene (Roman et al., 1995). Ethylene insensitive mutants found by this screening method did not display the characteristic seedling triple response to the hormone. In addition to ein6, the screen also identified a number of other ethylene pathway mutants such as ein4, ein5, ein7 and eir1. The ein6 mutant had a moderately ethylene insensitive phenotype, weaker than that of the strong ethylene insensitive mutant ein2.[0039]
• The ein6 mutation was found to be a recessive mutation that was classified by having significantly reduced gametophytic transmission. The reduced transmission was originally attributed to the existence of possible chromosomal aberrations caused by the fast neutron-mutagenesis. The gene was originally mapped to the bottom of[0040]chromosome 3, between the markers GL1, and nga112. It was hypothesized to be a reduction of function mutant, rather than a loss of function mutant, because of its moderate ethylene insensitive phenotype. The nucleic acid sequence of the 7 bp deletion in the ein6 mutation was identified using denaturing high performance liquid chromatography as described in Example 2.
• Further genetic analysis showed that the original ein6 mutant line described by Roman et al. was actually a double mutant (see Example 3) containing a mutation in both the EIN6 gene, plus an enhancer mutation currently being called ENHANCER OF ETHYLENE INSENSITIVITY (EEN) (see Example 6). The finding of the presence of the double mutation explains the earlier finding of the reduced transmission phenotype mentioned above. Thus, the screening assays revealed a series of ein6/een double mutant plants. Unlike strong insensitive mutants, such as etr1 and ein2, the ein6/een double mutants were found to still retain some ethylene sensitivity.[0041]
• The analysis of ethylene response pathway double mutants is a useful method to elucidate EIN6 function and its relation to other ethylene pathway members. Epistasis relationships between mutations in a biochemical or regulatory pathway can provide information about the way these genes interact without any a priori knowledge of their molecular identity (Ferguson et al. 1987; Chory 1990). To define the relationship between ethylene mutants, double mutants were isolated as described in Example 12. The triple-response phenotypes of wild-type, single and double mutant strains were quantified in the presence and absence of ethylene.[0042]
• The EIN6 gene was found to encode a protein of 1359 amino acids, having three zinc fingers and three putative nuclear localization sites (see Example 5). Embodiments of the invention provide isolated nucleic acids including nucleotide sequences comprising or derived from the EIN6 genes and/or encoding polypeptides comprising or derived from the EIN6 proteins. EIN6 sequences include the specifically disclosed sequences, and splice variants, allelic variants, synonymous sequences, and homologous or orthologous variants thereof. Thus, for example, embodiments of the invention include genomic and cDNA sequences from the EIN6 gene.[0043]
• Embodiments of the invention also include allelic variants and homologous or orthologous sequences. For example, these variants are useful in allele specific hybridization screening or PCR amplification techniques. Moreover, subsets of the EIN6 sequences, including both sense and antisense sequences, and both normal and mutant sequences, as well as intronic, exonic and untranslated sequences, may be employed for these techniques. Such sequences may comprise a small number of consecutive nucleotides from the sequences which are disclosed or otherwise enabled herein but preferably include at least 8-10, and more preferably 9-25, consecutive nucleotides from an EIN6 sequence. Various nucleic acid constructs in which EIN6 sequences, either complete or subsets, are operably joined to exogenous sequences to form cloning vectors, expression vectors, fusion vectors, transgenic constructs, and the like are also contemplated.[0044]
• The location of EIN6 in the ethylene pathway has been difficult to determine because the original mutation was generated in the Ler ecotype and a majority of the other ethylene mutants are in the Col-0 ecotype. It has been determined that EIN6 is downstream of CTR1, based on a cross of ein6 to a Ler allele of ctr1. Sequence analysis of the putative polypeptide sequence encoded by the EIN6 gene has three nuclear localization signals, indicating that the EIN6 protein may be localized in the nucleus.[0045]
• Interestingly, plants having only the ein6 mutant displayed an even greater selective insensitivity to ethylene, with the root being the only part of the plant that is fully insensitive. In contrast, plants having both the ein6 and the een mutation had an insensitivity to ethylene that is distributed throughout the entire plant rather than just in the root. Further characterization of the double mutant determined that seedlings possessing the ein6/een mutations were also hypersensitive to the microtubule-stabilizing drug taxol. This phenotype differentiated it from other ein mutants, and indicated that the EIN6 gene product played a role in phenomena that were unrelated to ethylene.[0046]
• Accordingly, embodiments of the invention include the EIN6 gene and mutations thereof, as well as a characterization of the ein6/een double mutants which have been discovered in Arabidopsis. However, the disclosed methods are not limited to any particular plant type. It is expected that similar mutations in other plants will result in similar phenotypes.[0047]
• In other embodiments of the invention, genetically modified plants having decreased levels of the functional EIN6 polypeptide, and further exhibiting decreased root sensitivity to ethylene, are provided.[0048]
• In another embodiment of the invention, genetically modified plants having decreased levels of both EIN6 and EEN are provided. Such loss of function double mutant plants were found to have decreased sensitivity to ethylene throughout the entire plant. In addition, it was discovered that these double mutants had increased sensitivity to the drug taxol.[0049]
• Nucleic Acids[0050]
• The polynucleotides encoding EIN6 include the nucleotide sequences of SEQ ID NOS: 1, 22, and 23. A genomic sequence for EIN6 is shown in SEQ ID NO: 1. The corresponding cDNA sequence is shown in SEQ ID NO: 22 and one example of a mutant form of the ein6 gene is shown in SEQ ID NO: 23. Nucleic acid sequences complementary to SEQ ID NOS: 1, 22, and 23 are also encompassed within the present invention.[0051]
• As used herein, the terms “polynucleotides” and “nucleic acid sequences” refer to DNA, RNA and cDNA sequences. A complementary sequence may include an antisense nucleotide. When the sequence is RNA, the deoxyribonucleotides A, G, C, and T of SEQ ID NOS: 1, 22, or 23 are replaced by ribonucleotides A, G, C, and U, respectively. Also included in the invention are fragments (“probes”) of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the probe to selectively hybridize to DNA that encodes the protein of either of SEQ ID NOS : 20 or 21.[0052]
• Embodiments of the invention also provide an isolated polynucleotide sequence encoding a polypeptide having the amino acid sequence of either of SEQ ID NOS : 20 or 21. The term “isolated” as used herein includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode EIN6. It is understood that polynucleotides encoding all or varying portions of EIN6 are included herein, as long as they encode a polypeptide with EIN6 activity. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides as well as splice variants. For example, portions of the mRNA sequence may be altered due to alternate RNA splicing patterns or the use of alternate promoters for RNA transcription. Without being bound by any theory, SEQ ID NO: 21 is believed to represent a splice variant of SEQ ID NO: 20.[0053]
• Moreover, EIN6 polynucleotides include polynucleotides having alterations in the nucleic acid sequence which still encode a polypeptide having the ability to modify the response to ethylene. Alterations in EIN6 nucleic acid include but are not limited to intragenic mutations (e.g., point mutation, nonsense (stop), antisense, splice site and frameshift) and heterozygous or homozygous deletions. Detection of such alterations can be done by standard methods known to those of skill in the art including sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization. Embodiments of the invention also include anti-sense polynucleotide sequences. A specific alteration of EIN6 comprising a 7 base pair deletion that results in a decreased response to ethylene, is exemplified herein (SEQ ID NO: 23).[0054]
• As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a template sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the template sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired template sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded template sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the template molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired template sequence. The length of the amplified segment of the desired template sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the template sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”.[0055]
• With PCR, it is possible to amplify a single copy of a specific template sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of[0056]³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
• “Antisense” nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub (1990) Scientific American 262: 40). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. This interferes with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause non-specific interference with translation than larger molecules. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura (1988) Anal. Biochem. 172: 289). In the present case, plants transformed with constructs containing antisense fragments of the EIN6 gene would display phenotypes such as prolonged shelf due to delayed ripening, delayed abscission, delayed senescence, and reduced browning.[0057]
• “Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Suitably stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.[0058]
• For example, hybridization under high stringency conditions could occur in about 50% formamide at about 37° C. to 42° C. Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30° C. to 35° C. In particular, hybridization could occur under high stringency conditions at 42° C. in 50% formamide, 5X SSPE, 0.3% SDS, and 200 n/ml sheared and denatured salmon sperm DNA. Hybridization could occur under medium stringency conditions as described above, but in 35% formamide at a reduced temperature of 35° C. The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.[0059]
• “Selective hybridization” as used herein refers to hybridization under moderately stringent or highly stringent physiological conditions (See, for example, the techniques described in Maniatis et al., 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporated herein by reference), which distinguishes related from unrelated EIN6 nucleotide sequences.[0060]
• Also included in embodiments of the invention are nucleotide sequences that are greater than 70% homologous with any of the sequences shown in SEQ ID NOS: 1, 22, or 23, but still retain the ability to confer an ethylene-dependent phenotype. Other embodiments of the invention include nucleotide sequences that are greater than 75%, 80%, 85%, 90% or 95% homologous with any of the sequences shown in SEQ ID NOS: 1, 22, or 23, but still retain the ability to confer an ethylene-dependent phenotype.[0061]
• Specifically disclosed herein are genomic and cDNA sequences for EIN6. DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization or computer-based techniques which are well known in the art. Such techniques include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest; 4) computer searches of sequence databases for similar sequences; and 5) differential screening of a subtracted DNA library.[0062]
• Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the EIN6 sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of the amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al., Nucl. Acid Res., 9:879, 1981). Alternatively, a subtractive library is useful for elimination of non-specific cDNA clones.[0063]
• Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).[0064]
• Expression studies of EIN6 can be performed using gene chip technologies. Although EIN6 appears to be a component of the ethylene signaling pathway, expression analysis of EIN6 (utilizing an Affymetrix GeneChip and other methods) has shown that EIN6 is not regulated by ethylene. The expression of the gene is moderate and can be easily seen in all conditions analyzed. In experiments using Columbia ecotype Arabidopsis plants grown in air and grown in exogenous ethylene, there is no difference in the expression of EIN6. There is also no dramatic difference in expression level of this gene in many of the ethylene mutants, such as ein5, ein2, hls, and ctr1.[0065]
• Embodiments of the invention also include functional EIN6 polypeptides, and functional fragments thereof. As used herein, the term “functional polypeptide” refers to a polypeptide which possesses biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in the cell. The term “functional fragments of EIN6 polypeptide”, refers to all fragments of EIN6 that retain EIN6 activity, e.g., responsiveness to ethylene. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.[0066]
• The polynucleotides described herein include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of EIN6 polypeptide encoded by such nucleotide sequences retains EIN6 activity. A “functional polynucleotide” denotes a polynucleotide which encodes a functional polypeptide as described herein. In addition, embodiments of the invention also include a polynucleotide encoding a polypeptide having the biological activity of an amino acid sequence of either of SEQ ID NOS: 20 or 21 and having at least one epitope for an antibody immunoreactive with EIN6 polypeptide.[0067]
• EIN6 Polypeptides[0068]
• Many modifications of the EIN6 primary amino acid sequence may result in plants having reduced or abolished ethylene responses. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of EIN6 is present. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. This can lead to the development of a smaller active molecule which could have broader utility. For example, it may be possible to remove amino or carboxy terminal amino acids required for EIN6 activity.[0069]
• EIN6 polypeptides include amino acid sequences substantially the same as the sequence set forth in SEQ ID NOS: 20 or 21, including mutants that result in plants having altered ethylene responsiveness. The term “substantially the same” refers to amino acid sequences that provide nearly the same amino acid sequence, or retain the activity of EIN6 as described herein. The EIN6 polypeptides of the invention include conservative variations of the polypeptide sequence.[0070]
• The term “conservative variation” as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.[0071]
• Another aspect of the invention is polypeptides or fragments thereof which have at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than about 95% homology to one of the polypeptides of SEQ ID NOS: 20 or 21, and sequences substantially identical thereto, or a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof. Homology may be determined using any of the methods described herein which align the polypeptides or fragments being compared and determines the extent of amino acid identity or similarity between them. It will be appreciated that amino acid “homology” includes conservative amino acid substitutions such as those described above.[0072]
• Homologous amino acid or nucleotide sequences of the present invention preferably comprise enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool) (for a review see Altschul, et al.,[0073]Meth Enzymol.266:460, 1996; and Altschul, et al.,Nature Genet.6:119, 1994). BLAST is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx using the statistical methods of Karlin and Altschul (available at www.ncbi.nih.gov/BLAST) Altschul, et al.,J. Mol. Biol.215:403, 1990). The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. The BLAST pages offer several different databases for searching. Some of these databases, such as ecoli, dbEST and month, are subsets of the NCBI (National Center for Biotechnology Information) databases, while others, such as SwissProt, PDB and Kabat are compiled from outside sources. Protein BLAST allows one to input protein sequences and compare these against other protein sequences.
• The five BLAST programs available at Internet website:www.ncbi.nlm.nih.gov perform the following tasks:[0074]
• blastp—compares an amino acid query sequence against a protein sequence database.[0075]
• blastn—compares a nucleotide query sequence against a nucleotide sequence database.[0076]
• blastx—compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.[0077]
• tblastn—compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).[0078]
• tblastx—compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.[0079]
• Other computer program methods to determine identity and similarity between the two sequences include but are not limited to the GCG program package (Devereux, et al.,[0080]Nucl. Acids Res.12:387, 1984) and FASTA (Atschul, et al.,J Molec. Biol.215:403, 1990). By “percentage identity” is meant % of identical amino acids between the two compared proteins. By “% similarity” is meant the percentage of similar amino acids between the two compared proteins.
• The polypeptides or fragments having homology to one of the polypeptides of SEQ ID NOS: 20 or 21, and sequences substantially identical thereto, or a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may be obtained by isolating the nucleic acids encoding them using the techniques described herein.[0081]
• Alternatively, the homologous polypeptides or fragments may be obtained through biochemical enrichment or purification procedures. The sequence of potentially homologous polypeptides or fragments may be determined by proteolytic digestion, gel electrophoresis and/or microsequencing. The sequence of the prospective homologous polypeptide or fragment can be compared to one of the polypeptides of SEQ ID NOS: 20 or 21, and sequences substantially identical thereto, or a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof using any of the programs described above.[0082]
• One skilled in the art can purify a polypeptide using standard techniques for protein purification to obtain an EIN6 polypeptide that is “substantially pure”. As used herein, the term “substantially pure” refers to polypeptides which are substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. The EIN6 proteins can be analyzed by standard SDS-PAGE and/or immunoprecipitation analysis and/or Western blot analysis, for example. The purity of an EIN6 polypeptide can also be determined by amino-terminal amino acid sequence analysis.[0083]
• EIN6 Antibodies[0084]
• The invention also provides antibodies immunoreactive with any EIN6 polypeptide, or antigenic fragments thereof, where an antibody may consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations is provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al.,[0085]Nature,256:495, 1975).
• The term “antibody” as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2, and Fv capable of binding to an epitopic determinant present in EIN6 polypeptide. Such antibody fragments retain some ability to selectively bind with its antigen or receptor.[0086]
• Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).[0087]
• As used in this invention, the term “epitope” refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.[0088]
• Antibodies which bind to the EIN6 polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N- or C-terminal domains of EIN6. The polypeptide or peptide used to immunize an animal may be derived from translated cDNA or may be chemically synthesized, and may further be conjugated to a carrier protein, if desired. Commonly used carriers which are chemically coupled to an immunizing peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.[0089]
• Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and eluting from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art are familiar with various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al.,[0090]Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference).
• It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the “image” of the epitope bound by the first monoclonal antibody.[0091]
• A cDNA expression library such as lambda gt11, can be screened indirectly for EIN6 peptides using antibodies specific for EIN6 epitopes. Such antibodies may be polyclonally or monoclonally derived, and may be used to detect expression product indicative of the presence of EIN6 cDNA.[0092]
• Plants With EIN6 Gene Mutations[0093]
• Another embodiment of the invention relates to plants that have been altered to be insensitive to ethylene in their root. Such modifications include transgenic plants with decreased response to ethylene due to transformation with constructs using antisense or cosuppression technology that affect transcription or expression from the EIN6 gene. Such plants exhibit well-known ethylene insensitive phenotypes, such as delayed ripening, flowering, delayed senescence, browning, and altered sensitivity to pathogens.[0094]
• Another embodiment of the invention relates to plants that have been altered to be insensitive to ethylene throughout the plant. Such modifications include transgenic plants with decreased response to ethylene due to transformation with constructs using antisense or cosuppression technology that affect transcription or expression from the EIN6 gene and the een gene. Such plants exhibit well-known ethylene insensitive phenotypes, such as delayed ripening, flowering, delayed senescence, browning, and altered sensitivity to pathogens.[0095]
• In another embodiment, embodiments of the invention provide a method for producing a genetically modified plant characterized as having an altered ethylene-dependent phenotype as compared to a plant which has not been genetically modified (e.g., a wild-type plant). The method includes the steps of contacting a plant cell with at least one vector containing at least one nucleic acid sequence encoding an EIN6 gene or a mutant, homolog or fragment thereof, wherein the nucleic acid sequence is operably associated with a promoter, to obtain a transformed plant cell; producing a plant from the transformed plant cell; and thereafter selecting a plant exhibiting an altered ethylene-dependent phenotype.[0096]
• Transgenic plants that result in an altered ethylene-dependent phenotype may be obtained by reduced expression of the EIN6 gene, or the EIN6 gene and the een gene. Thus, one embodiment of the invention includes plants transformed with antisense polynucleotides complementary to the EIN6 gene or fragments thereof wherein production of the antisense polynucleotides results in reduced expression of the EIN6 gene. In an alternate embodiment, reduced expression of EIN6 may also be achieved by methods such as cosuppression (Hooper, C. (1991) J. NIH Res. 3: 49-54) by operatively linking a truncated form of an EIN6 gene to a promoter. In an alternate embodiment, transgenic plants overexpressing the EIN6 gene are described. Such plants might be expected to display accelerated ripening and enhanced sensitivity to pathogens.[0097]
• The term “genetic modification” as used herein refers to the introduction of one or more heterologous nucleic acid sequences, e.g., an EIN6 or an ein6 mutant encoding sequence, into one or more plant cells, which can generate whole, sexually competent, viable plants. The term “genetically modified” as used herein refers to a plant which has been generated through the aforementioned process. Genetically modified plants of the invention are capable of self-pollinating or cross-pollinating with other plants of the same species so that the foreign gene, carried in the germ line, can be inserted into or bred into agriculturally useful plant varieties. The term “plant cell” as used herein refers to protoplasts, gamete producing cells, and cells which regenerate into whole plants. Accordingly, a seed comprising multiple plant cells capable of regenerating into a whole plant, is included in the definition of “plant cell”.[0098]
• As used herein, the term “plant” refers to either a whole plant, a plant part, a plant cell, or a group of plant cells, such as plant tissue, for example. Plantlets are also included within the meaning of “plant”. Plants included in the invention are any plants amenable to transformation techniques, including angiosperms, gymnosperms, monocotyledons and dicotyledons.[0099]
• Examples of monocotyledonous plants include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats. Examples of dicotyledonous plants include, but are not limited to tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or[0100]Brassica oleracea(e.g., cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals. Woody species include poplar, pine, sequoia, cedar, oak, etc.
• The term “exogenous nucleic acid sequence” as used herein refers to a nucleic acid foreign to the recipient plant host or, native to the host if the native nucleic acid is substantially modified from its original form. For example, the term includes a nucleic acid originating in the host species, where such sequence is operably linked to a promoter that differs from the natural or wild-type promoter. In one embodiment, at least one nucleic acid sequence encoding EIN6 or a variant thereof is operably linked with a promoter. It may be desirable to introduce more than one copy of an EIN6 polynucleotide into a plant for enhanced expression. For example, multiple copies of the gene would have the effect of increasing production of the EIN6 gene product in the plant.[0101]
• Genetically modified plants of the present invention are produced by contacting a plant cell with a vector including at least one nucleic acid sequence encoding a EIN6 or a variant thereof. Suitable methods for the creation of such modified plants are found in Examples 8-10. To be effective once introduced into plant cells, the EIN6 nucleic acid sequence are operably associated with a promoter which is effective in the plant cells to cause transcription of EIN6. Additionally, a polyadenylation sequence or transcription control sequence, also recognized in plant cells may also be employed. It is preferred that the vector harboring the nucleic acid sequence to be inserted also contain one or more selectable marker genes so that the transformed cells can be selected from non-transformed cells in culture, as described herein.[0102]
• In embodiments of the invention, EIN6 and/or EEN expression is lowered in a plant by genetic transformation methods. Any method to downregulate EIN6 and/or EEN gene expression may be used, but typical examples include antisense technology, cosuppression, RNA inhibition (RNAi), and ribozyme inhibition. In the antisense method, for example, antisense molecules are introduced into cells that contain EIN6 and/or EEN, for example, and may function by decreasing the amount of EIN6 and/or EEN polypeptide production in a cell, or may function by a different mechanism. Antisense polynucleotides useful for the present invention are complementary to specific regions of a corresponding target mRNA. An antisense polynucleotide can be introduced to a cell by introducing an expressible construct containing a nucleic acid segment that codes for the polynucleotide. Antisense polynucleotides in context of the present invention may include short sequences of nucleic acid known as oligonucleotides, usually 10-50 bases in length, as well as longer sequences of nucleic acid that may exceed the length of the gene sequence itself.[0103]
• Small Molecules[0104]
• Accordingly, embodiments of the invention include methods of screening or identifying proteins, small molecules or other compounds which are capable of inducing or inhibiting the expression of the EIN6 genes and proteins. The assays may be performed in vitro using transformed or non-transformed cells, immortalized cell lines, or in vivo using transformed plant models enabled herein. In particular, the assays may detect the presence of increased or decreased expression of EIN6 (from Arabidopsis or other plants) genes or proteins on the basis of increased or decreased mRNA expression, increased or decreased levels of EIN6 protein products, or increased or decreased levels of expression of a marker gene (e.g., beta-galactosidase, beta-glucuronidase, green fluorescent protein, alkaline phosphatase or luciferase) operably joined to an[0105]EIN6 5′ regulatory region in a recombinant construct. Cells known to express a particular EIN6, or transformed to express a particular EIN6, are incubated and one or more test compounds are added to the medium. After allowing a sufficient period of time (e.g., 0-72 hours) for the compound to induce or inhibit the expression of EIN6, any change in levels of expression from an established baseline may be detected using any of the techniques described above
• Other embodiments of the invention include methods for identifying proteins and other compounds which bind to, or otherwise directly interact with, the EIN6. The proteins and compounds include endogenous cellular components which interact with EIN6 in vivo and which, therefore, provide new targets for agricultural products, as well as recombinant, synthetic and otherwise exogenous compounds which may have EIN6 binding capacity and, therefore, are candidates for inhibition of the plant's response to ethylene. Thus, High Throughput Screening-derived proteins, DNA chip arrays, cell lysates or tissue homogenates may be screened for proteins or other compounds which bind to one of the normal or mutant EIN6 genes. Alternatively, any of a variety of exogenous compounds, both naturally occurring and/or synthetic (e.g., libraries of small molecules or peptides), may be screened for EIN6 binding capacity.[0106]
• Accordingly, assays can be conducted to detect binding between EIN6 and another moiety. The EIN6 in these assays may be any polypeptide comprising or derived from a normal or mutant EIN6 protein, including functional domains or antigenic determinants of EIN6. Binding may be detected by non-specific measures (e.g., transcription modulation, altered chromatin structure, peptide production or changes in the expression of other downstream genes which can be monitored by differential display, 2D gel electrophoresis, differential hybridization, or SAGE methods) or by direct measures such as immunoprecipitation, the Biomolecular Interaction Assay (BIAcore) or alteration of protein gel electrophoresis. The preferred methods involve variations on the following techniques: (1) direct extraction by affinity chromatography; (2) co-isolation of EIN6 components and bound proteins or other compounds by immunoprecipitation; (3) BIAcore analysis; and (4) the yeast two-hybrid systems.[0107]
• Embodiments of the invention also include methods of identifying proteins, small molecules and other compounds capable of modulating the activity of normal or mutant EIN6. Using normal cells or plants, the transformed cells and plant models of the present invention, or cells obtained from subjects bearing normal or mutant EIN6 genes, the present invention provides methods of identifying such compounds on the basis of their ability to affect the expression of EIN6, the activity of EIN6, the activity of other EIN6-regulated genes, the activity of proteins that interact with normal or mutant EIN6 proteins, the intracellular localization of the EIN6, changes in transcription activity, the presence or levels of membrane bound EIN6, or other biochemical, histological, or physiological markers which distinguish cells bearing normal and modulated EIN6 activity in plants.[0108]
• In accordance with another aspect of the invention, the proteins described herein can be used as starting points for rational chemical design to provide ligands or other types of small chemical molecules. Alternatively, small molecules or other compounds identified by the above-described screening assays may serve as “lead compounds” in design of modulators of the ethylene response in plants.[0109]
• Host Cells and Vectors[0110]
• DNA sequences encoding EIN6 can be expressed in vitro by DNA transfer into a suitable host cell. “Host cells” are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny or graft material, for example, of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.[0111]
• As part of the present invention, the EIN6 polynucleotide sequences may be inserted into a recombinant expression vector. The terms “recombinant expression vector” or “expression vector” refer to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the EIN6 genetic sequence. Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted EIN6 sequence. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.[0112]
• Methods which are well known to those skilled in the art can be used to construct expression vectors containing the EIN6 coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic techniques.[0113]
• A variety of host-expression vector systems may be utilized to express the EIN6 coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the EIN6 coding sequence; yeast transformed with recombinant yeast expression vectors containing the EIN6 coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the EIN6 coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the EIN6 coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing the EIN6 coding sequence, or transformed animal cell systems engineered for stable expression.[0114]
• Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al., 1987, Methods in Enzymology 153:516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage .gamma., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted EIN6 coding sequence.[0115]
• The EIN6 proteins produced by the method described above may be used, for example, for structural characterization studies, protein-protein interaction studies, protein-nucleic acid interaction studies, and the like. Isolation and purification of recombinantly expressed polypeptide, or fragments thereof, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.[0116]
• Vector(s) employed in the present invention for transformation of a plant cell include a nucleic acid sequence encoding EIN6, operably linked to a promoter. To commence a transformation process in accordance with the present invention, it is first necessary to construct a suitable vector and properly introduce it into the plant cell. Details of the construction of vectors utilized herein are known to those skilled in the art of plant genetic engineering.[0117]
• The term “operably linked” refers to functional linkage between a promoter sequence and a nucleic acid sequence regulated by the promoter. The operably linked promoter controls the expression of the nucleic acid sequence.[0118]
• Promoters[0119]
• The expression of structural genes may be driven by a number of promoters. Although the endogenous, or native promoter of a structural gene of interest may be utilized for transcriptional regulation of the gene, preferably, the promoter is a foreign regulatory sequence. For plant expression vectors, suitable viral promoters include the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., Nature, 310:511, 1984; Odell, et al., Nature, 313:810, 1985); the full-length transcript promoter from Figwort Mosaic Virus (FMV) (Gowda, et al., J. Cell Biochem., 13D: 301, 1989) and the coat protein promoter to TMV (Takamatsu, et al., EMBO J. 6:307, 1987). Alternatively, plant promoters such as the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al., EMBO J., 3:1671, 1984; Broglie, et al., Science, 224:838, 1984); mannopine synthase promoter (Velten, et al., EMBO J., 3:2723, 1984) nopaline synthase (NOS) and octopine synthase (OCS) promoters (carried on tumor-inducing plasmids of[0120]Agrobacterium tumefaciens) or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley, et al., Mol. Cell. Biol., 6:559, 1986; Severin, et al., Plant Mol. Biol., 15:827, 1990) may be used.
• Promoters useful in the invention include both natural constitutive and inducible promoters as well as engineered promoters. The CaMV promoters are examples of constitutive promoters. To be most useful, an inducible promoter should 1) provide low expression in the absence of the inducer; 2) provide high expression in the presence of the inducer; 3) use an induction scheme that does not interfere with the normal physiology of the plant; and 4) have no effect on the expression of other genes. Examples of inducible promoters useful in plants include those induced by chemical means, such as the yeast metallothionein promoter which is activated by copper ions (Mett, et al., Proc. Natl. Acad. Sci., U.S.A., 90:4567, 1993); In2-1 and In2-2 regulator sequences which are activated by substituted benzenesulfonamides, e.g., herbicide safeners (Hershey, et al., Plant Mol. Biol., 17:679, 1991); and the GRE regulatory sequences which are induced by glucocorticoids (Schena, et al., Proc. Natl. Acad. Sci., U.S.A., 88:10421, 1991). Other promoters, both constitutive and inducible will be known to those of skill in the art.[0121]
• The particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of structural gene product to alter an ethylene-dependent phenotype. The promoters used in the vector constructs of the present invention may be modified, if desired, to affect their control characteristics.[0122]
• Tissue specific promoters may also be utilized in the present invention. An example of a tissue specific promoter is the promoter active in shoot meristems (Atanassova, et al., Plant J., 2:291, 1992). Other tissue specific promoters useful in transgenic plants, including the cdc2a promoter and cyc07 promoter, will be known to those of skill in the art. (See for example, Ito, et al., Plant Mol. Biol., 24:863, 1994; Martinez, et al., Proc. Natl. Acad. Sci. USA, 89:7360, 1992; Medford, et al., Plant Cell, 3:359, 1991; Terada, et al., Plant Journal, 3:241, 1993; Wissenbach, et al., Plant Journal, 4:411, 1993).[0123]
• Optionally, a selectable marker may be associated with the nucleic acid sequence to be inserted. As used herein, the term “marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a plant or plant cell containing the marker. Preferably, the marker gene is an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Examples of suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phospho-transferase, thymidine kinase, xanthine-guanine phospho-ribosyltransferase and amino-[0124]glycoside 3′-O-phospho-transferase II (kanamycin, neomycin and G418 resistance). Other suitable markers will be known to those of skill in the art.
• Methods of Transforming Plants[0125]
• EIN6 and/or EEN nucleic acid sequences utilized in the present invention can be introduced into plant cells using Ti plasmids of[0126]Agrobacterium tumefaciens, root-inducing (Ri) plasmids, and plant virus vectors. (For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9, and Horsch, et al., Science, 227:1229, 1985, both incorporated herein by reference). In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, transformation using viruses or pollen and the use of microprojection.
• One of skill in the art will be able to select an appropriate vector for introducing the EIN6 and/or EEN nucleic acid sequence in a relatively intact state. Thus, any vector which will produce a plant carrying the introduced DNA sequence should be sufficient. Even use of a naked piece of DNA would be expected to confer the properties of this invention, though at low efficiency. The selection of the vector, or whether to use a vector, is typically guided by the method of transformation selected.[0127]
• The transformation of plants in accordance with the invention may be carried out in essentially any of the various ways known to those skilled in the art of plant molecular biology. (See, for example, Methods of Enzymology, Vol. 153, 1987, Wu and Grossman, Eds., Academic Press, incorporated herein by reference). As used herein, the term “transformation” means alteration of the genotype of a host plant by the introduction of an EIN6 and/or EEN gene, or a mutant form of the EIN6 and/or EEN gene.[0128]
• For example, an EIN6 and/or EEN nucleic acid sequence can be introduced into a plant cell utilizing[0129]Agrobacterium tumefacienscontaining the Ti plasmid, as mentioned briefly above. In using anA. tumefaciensculture as a transformation vehicle, it is most advantageous to use a non-oncogenic strain of Agrobacterium as the vector carrier so that normal non-oncogenic differentiation of the transformed tissues is possible. It is also preferred that the Agrobacterium harbor a binary Ti plasmid system. Such a binary system comprises 1) a first Ti plasmid having a virulence region essential for the introduction of transfer DNA (T-DNA) into plants, and 2) a chimeric plasmid. The latter contains at least one border region of the T-DNA region of a wild-type Ti plasmid flanking the nucleic acid to be transferred. Binary Ti plasmid systems have been shown effective to transform plant cells (De Framond, Biotechnology, 1: 262, 1983; Hoekema, et al., Nature, 303:179, 1983). Such a binary system is preferred because it does not require integration into the Ti plasmid of Agrobacterium, which is an older methodology.
• Methods involving the use of Agrobacterium in transformation according to the present invention include, but are not limited to: 1) co-cultivation of Agrobacterium with cultured isolated protoplasts; 2) transformation of plant cells or tissues with Agrobacterium; or 3) transformation of seeds, apices or meristems with Agrobacterium. In addition, gene transfer can be accomplished by in planta transformation by Agrobacterium, as described by Bechtold, et al., (C. R. Acad. Sci. Paris, 316:1194, 1993). This approach is based on the vacuum infiltration of a suspension of Agrobacterium cells.[0130]
• One method of introducing EIN6 and/or EEN nucleic acid sequences into plant cells is to infect such plant cells, an explant, a meristem or a seed, with transformed[0131]Agrobacterium tumefaciensas described above. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots, roots, and develop further into plants.
• Alternatively, EIN6 and/or EEN nucleic acid sequences can be introduced into a plant cell using mechanical or chemical means. For example, the nucleic acid can be mechanically transferred into the plant cell by microinjection using a micropipette. Alternatively, the nucleic acid may be transferred into the plant cell by using polyethylene glycol which forms a precipitation complex with genetic material that is taken up by the cell.[0132]
• EIN6 and/or EEN nucleic acid sequences can also be introduced into plant cells by electroporation (Fromm, et al., Proc. Natl. Acad. Sci., U.S.A., 82:5824, 1985, which is incorporated herein by reference). In this technique, plant protoplasts are electroporated in the presence of vectors or nucleic acids containing the relevant nucleic acid sequences. Electrical impulses of high field strength reversibly permeabilize membranes allowing the introduction of nucleic acids. Electroporated plant protoplasts reform the cell wall, divide and form a plant callus. Selection of the transformed plant cells with the transformed gene can be accomplished using phenotypic markers as described herein.[0133]
• Another method for introducing EIN6 and/or EEN nucleic acid into a plant cell is high velocity ballistic penetration by small particles with the nucleic acid to be introduced contained either within the matrix of such particles, or on the surface thereof (Klein, et al., Nature 327:70, 1987). Bombardment transformation methods are also described in Sanford, et al. (Techniques 3:3-16, 1991) and Klein, et al. (Bio/Techniques 10:286, 1992). Although, typically only a single introduction of a new nucleic acid sequence is required, this method particularly provides for multiple introductions.[0134]
• Cauliflower mosaic virus (CaMV) may also be used as a vector for introducing nucleic acid into plant cells (U.S. Pat. No. 4,407,956). CaMV viral DNA genome is inserted into a parent bacterial plasmid creating a recombinant DNA molecule which can be propagated in bacteria. After cloning, the recombinant plasmid again may be cloned and further modified by introduction of the desired nucleic acid sequence. The modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid, and used to inoculate the plant cells or plants.[0135]
• As used herein, the term “contacting” refers to any means of introducing EIN6 and/or EEN into the plant cell, including chemical and physical means as described above. Preferably, contacting refers to introducing the nucleic acid or vector into plant cells (including an explant, a meristem or a seed), via[0136]Agrobacterium tumefacienstransformed with the EIN6 and/or EEN nucleic acid as described above.
• Normally, a plant cell is regenerated to obtain a whole plant from the transformation process. The immediate product of the transformation is referred to as a “transgenote”. The term “growing” or “regeneration” as used herein means growing a whole plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).[0137]
• Regeneration from protoplasts varies from species to species of plants, but generally a suspension of protoplasts is first made. In certain species, embryo formation can then be induced from the protoplast suspension, to the stage of ripening and germination as natural embryos. The culture media will generally contain various amino acids and hormones, necessary for growth and regeneration. Examples of hormones utilized include auxins and cytokinins. It is sometimes advantageous to add glutamic acid and proline to the medium, especially for plant species such as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these variables are controlled, regeneration is reproducible.[0138]
• Regeneration also occurs from plant callus, explants, organs or parts. Transformation can be performed in the context of organ or plant part regeneration. (see Methods in Enzymology, Vol. 118 and Klee, et al., Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leaf disk-transformation-regeneration method of Horsch, et al., Science, 227:1229, 1985, disks are cultured on selective media, followed by shoot formation in about 2-4 weeks. Shoots that develop are excised from calli and transplanted to appropriate root-inducing selective medium. Rooted plantlets are transplanted to soil as soon as possible after roots appear. The plantlets can be repotted as required, until reaching maturity.[0139]
• In vegetatively propagated crops, the mature transgenic plants are propagated by utilizing cuttings or tissue culture techniques to produce multiple identical plants. Selection of desirable transgenotes is made and new varieties are obtained and propagated vegetatively for commercial use.[0140]
• In seed propagated crops, the mature transgenic plants can be self crossed to produce a homozygous inbred plant. The resulting inbred plant produces seed containing the newly introduced foreign gene(s). These seeds can be grown to produce plants that exhibit an altered response to ethylene.[0141]
• Parts obtained from regenerated plant, such as flowers, seeds, leaves, branches, roots, fruit, and the like are included in the invention, provided that these parts comprise cells that have been transformed as described. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.[0142]
• Plants exhibiting an altered ethylene-dependent phenotype as compared with wild-type plants can be selected by visual observation. For example, an altered ethylene-dependent phenotype may be detected by utilization of the “triple response.” The “triple response” consists of three distinct morphological changes in dark-grown seedlings upon exposure to ethylene: inhibition of hypocotyl and root elongation, radial swelling of the stem and exaggeration of the apical hook. Thus, a triple response displayed in the presence of ethylene inhibitors would indicate one type of altered ethylene-dependent phenotype. Altered responses to ethylene may be visualized by other methods in addition to the “triple response” phenotype. For example, since ethylene affects a wide group of other plant characteristics, such as flowering, fruit ripening, and senescence, one of skill in the art would be able to design other types of visual screens for altered responses to ethylene, depending on the plant species being tested. Types of screens that could be designed include, for example, screens for delayed leaf or flower senescence or delayed fruit ripening in response to ethylene application.[0143]
• In some embodiments of the invention, ethylene gas may be used to analyze ethylene response phenotypes. Additionally, molecules which are precursors to ethylene may be used. For example, the endogenous ethylene precursor ACC may be provided to the plant to examine the response to ethylene. Once taken up by the plant, the ACC is converted to ethylene by natural plant processes. Alternatively, synthetic compounds which degrade to produce ethylene may be sprayed on the surface of the plant. For example, ethephon (chloro-ethanephosphonic acid), is a commercially available compound that is typically sprayed on the surface of the plant, liberating ethylene once inside the plant tissue. Therefore, ethylene, ethylene analogs, or ethylene generating agents may be useful to screen and analyze ethylene response phenotypes according to the present invention.[0144]
• Several commercially available herbicides, such as Arvest, Bromeflor, Flordimex, Etherel, etc., may contain ethephon (chloro-ethanephosphonic acid) as an active ingredient. Weed growth can be slowed or stopped by the application of these herbicides because the ethylene that is liberated acts as a plant growth regulator, to slow or stop growth. Accordingly, embodiments of the present invention may include ethylene insensitive mutant or transgenic plants that are herbicide resistant or herbicide insensitive due to the altered levels of functional EIN6 and/or EEN present in these plants.[0145]
• The role of ethylene in the reduction of hypocotyl and root elongation has not yet been elucidated. One possibility relates to the theory that cortical microtubules associated with the plasma membrane may direct the cellulose synthase complex that is responsible for cell wall deposition. The drug taxol acts to stabilize microtubules by inhibiting microtubule depolymerization (Schiff and Horwitz 1981) and may inhibit the shift in cortical microtubules upon ethylene treatment (Heinstein and Chang 1994). To determine if taxol could affect the ethylene insensitivity phenotype of ein6/een mutants, wild-type and ein6/een mutants were given a 10 day taxol treatment in the dark (Example 12). Interestingly, the ein6/een mutants were found to be especially sensitive to even low levels (0.1 μM) of taxol. The mutants treated with taxol had a phenotype approaching that of the wild-type seedlings.[0146]
• Accordingly, another embodiment of the invention is a mutant or transgenic plant with altered ethylene sensitivity and taxol hypersensitivity is disclosed. The taxol hypersensitivity phenotype may be useful, for example, in assays to examine microtubule action or cytoskeletal activity, particularly in relation to cell elongation. One method of creating a plant with a taxol hypersensitivity would involve transforming a plant with an antisense construct of both the EIN6 and the EEN genes.[0147]
• Ethylene affects a vast array of agriculturally important plant processes, including fruit ripening, flower and leaf senescence and leaf abscission, in addition to the readily observable “triple response” of seedlings. For example, plants having a decreased sensitivity to ethylene may have better storage characteristics. Fruits of such plants may ripen more slowly. This could be an advantage for post-harvest handling of agricultural products, such as processing, packaging, and storage of fruit. There may be less of a loss of fruit crops due to such traditionally damaging problems as rotting, over-ripening, and degradation. It may be possible to better control the ripening rate and ripening characteristics of plants carrying these modified EIN6 and/or EEN genes. It is possible, as well, to link the modified genes to specific promoters in order to better modulate the expression of the genes so that the response to ethylene is turned off at certain times or in certain tissues, while acting normally in other parts of the plant or at other times in development.[0148]
• Plants having reduced EIN6 and/or EEN activity and thus a reduced sensitivity to ethylene to could be useful for the floral industry. Since ethylene may be involved in floral senescence, these modified plants may have a longer flower longevity. Further, it may be useful to use the present invention to create vegetative crops that do not bolt or flower easily. For example, lettuce, spinach, other leafy vegetables, or certain herbs may have higher yields due to decreased floral initiation. Because ethylene has been implicated in senescence, potted plants made according to the present invention may last longer than control plants due to reduced leaf senescence.[0149]
• One of skill in the art would appreciate that different plants or different agricultural crops may benefit from different types of promoter/EIN6 and or promoter/EEN gene modifications. For example, in some plants it may be desirable to be able to decrease the sensitivity constitutively using, for example, a CaMV35S promoter linked to the modified EIN6 gene. Other plants may benefit from decreasing the sensitivity to ethylene only at fruit ripening, for example, by linking the modified EIN6 gene to a fruit ripening-specific promoter. It may be useful to create plants that have modified ethylene sensitivity only in the vegetative parts of the plant. In another example, it may be useful to prepare plants having a modified EIN6 and/or EEN gene so that the ethylene sensitivity is decreased only at high temperatures. Such a scenario may be important in post-harvest storage and transportation of fruit, for example. It may be useful to link the gene to a promoter such that the ethylene insensitivity characteristic is brought about only when the plant is stored in the darkness by operably linking a darkness-inducible promoter to the modified modified EIN6 and/or EEN gene. This may be particularly useful for managing long term storage of certain crops between the time of harvest and the time of display for sale. Once the agricultural product is unpacked for market display in the light, for example, the modified EIN6 and/or EEN gene gene would be downregulated, the ethylene insensitivity would decrease, and ripening would resume. Many other options for modifying specific crops as desired can be designed by one of skill in the art[0150]
• Alternatively, it may be of interest to produce plants that overexpress one or more members of the EIN6 gene family. This may cause plants to have increased sensitivity to ethylene, or it may create other desirable plant phenotypes which may be of agronomic importance. The EIN6 overexpression may be either constitutive, inducible, or tissue-specific.[0151]
• Further, it may be of interest to produce plants that overexpress one or more members of the EEN gene family. This may cause plants to have increased sensitivity to ethylene, or it may create other desirable plant phenotypes which may be of agronomic importance. The EEN overexpression may be either constitutive, inducible, or tissue-specific.[0152]
• Further, since ethylene pathways involve the action of many gene products, as detailed herein, it may be useful to engineer plants having modified EIN6 and/or EEN genes in combination with other modified genes in the ethylene synthesis or response pathway. The other ethylene pathway genes may be inactivated or partially inactivated, for example, by insertional mutation, or point mutation, by antisense technology, or by molecular decoy technology. Alternatively, the other ethylene pathway genes may be conditionally overexpressed or may be preferentially expressed (such as in certain tissues, or in response to certain developmental or environmental cues).[0153]
• As describe herein, ethylene signaling processes may interact with the signaling processes of other hormones, such as auxin. Therefore, mutant or transgenic plants having altered expression of EIN6 and/or EEN genes may exhibit useful alterations in auxin-related pathways. Further, since ethylene is released upon certain types of pathogen infection, mutant or transformed plants having an altered ethylene sensitivity may be useful to modify responses to pathogens. In fact, since many genes have altered expression characteristics in response to ethylene application, mutant or transgenic plants carrying altered EIN6 and/or EEN genes to alter the sensitivity may be useful in altering a myriad of physiological roles and morphological phenotypes in plants.[0154]
• The ability to control the sensitivity of plants to ethylene could thus significantly improve the quality and longevity of many crops. The invention includes plants produced by the method of the invention, as well as plant tissue and seeds.[0155]
• In yet another embodiment, the invention provides a method for producing a genetically modified plant cell such that a plant produced from said cell has an altered ethylene-dependent phenotype compared with a wild-type plant. The method includes contacting the plant cell with an EIN6 and/or EEN nucleic acid sequence to obtain a transformed plant cell; growing the transformed plant cell under plant forming conditions to obtain a plant having an altered ethylene-dependent phenotype. Conditions such as environmental and promoter inducing conditions vary from species to species, but should be the same within a species.[0156]
• It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.[0157]
EXAMPLE 1Mapping of the ein6 Locus
• The EIN6 gene was isolated from a population of fast neutron-mutagenized plants, and was originally mapped to the bottom of[0158]chromosome 3, 17.6 cM north of nga112 (Roman, G. et al. (1995) “Genetic analysis of ethylene signal transduction inArabidopsis thaliana: five novel mutant loci integrated into a stress response pathway” Genetics 139(3): 1393-1409). Further mapping indicated that the gene was located farther north, located in a ˜12 cM interval between the markers TOPP5 and g19397 (FIG. 2). The acetolactate synthase gene is located in this interval, and was used to screen bacterial artificial chromosome (BAC) libraries to identify BAC clones in this region. Additional markers were developed from cloned genes in the region and BAC end sequences obtained from TAIL-PCR clones and the Arabidopsis Genome Initiative.
• Using these markers, EIN6 was mapped on an F2 population of 496 plants. It was found that the closest flanking marker to the north, F1K9-T7, resided on the BAC T29H11 (Accession AL049659), which was sequenced by the ESSA sequencing consortium. The lone plant recombinant at the closest flanking marker to the south of the gene, F11N9, remained recombinant at a marker made from the southern end of the BAC (29H11-End), indicating that the genomic sequence of EIN6 was located on T29H11 (FIG. 2). The genomic sequence in this 71 kb interval was exploited to design a series of markers that allowed the EIN6 locus to be fine mapped to a 44 kb region between the markers 12.1 and 29H11-End (FIG. 1).[0159]
EXAMPLE 2Identification of the ein6 Mutation
• Although the ein6 mutation was induced by fast neutrons, no evidence of a deletion was observed when genomic Southern blots of the mutant were probed with F3E7, a BAC that overlapped this region (data not shown). Using the genomic data available, a novel approach was undertaken to identify the mutation in the EIN6 gene. Denaturing High Performance Liquid Chromatography (DHPLC) is a technique that is capable of identifying single base mismatches in double stranded DNA (O'Donovan, 1998). Primers were designed to amplify overlapping ˜500 bp PCR fragments that covered the 44 kb interval between the two closest recombinant markers. PCR fragments that contain no mismatches elute from the DHPLC column as single peaks. In the event that a mismatch is present in a particular fragment, multiple peaks are observed. Multiple peaks were only observed for a single fragment contained within the interval. Sequencing of this fragment from both wild type and mutant plants identified a 7 base deletion in the putative coding region of T29H11.50, a gene that had been predicted by annotation (FIG. 2). This 7 base pair deletion (indicated by asterisks in FIG. 2) causes a frame shift and introduces a stop codon into the coding sequence. There is one homolog of EIN6 in the Arabidopsis genome, which shares 53% identity with the overall putative protein sequence. It is annotated as a gene of unknown function and is located on[0160]chromosome 5.
EXAMPLE 3The Original ein6 Mutation is Two Genes
• The original mutation ein6 had been characterized as a recessive mutant, which conferred, reduced fertility. Backcrosses of ein6 mutant plants to the parental line Lansberg Erecta (Ler) yielded interesting results. Upon careful analysis of the F2 seedlings grown in an ethylene precursor, three phenotypes were seen. Two were those that had been seen before, wild type and ethylene insensitive (ein-like). The third was a weak ethylene insensitive phenotype, with only an ethylene insensitive root (eir-like). The numbers of each class were counted in the F2 population. They corresponded to the following ratio: 12:3:1, with {fraction (12/16)} of the plants being of the wild type (triple response) phenotype, {fraction (3/16)} of the plants being of the eir (ethylene insensitive root) phenotype, and {fraction (1/16)} of the plants being ein (ethylene insensitive) phenotype. Each of these classes were genotyped for the ein6 mutation. All of the ein-like and eir-like plants turned out to have the ein6 mutation. This indicates that the original mutation is actually two recessive genes, one conferring the eir phenotype, and the other not having a visible phenotype as a seedling.[0161]
EXAMPLE 4Complementation With an EIN6 Genomic Fragment Rescues the ein6/een Phenotype
• Agrobacterium transformation was used to transform Arabidopsis double mutant ein6/een plants. These were transformed with a 6.2 kb genomic fragment containing EIN6 plus 0.9 kb of its promotor. The plants were tested for kanamycin resistance in the T1 generation. The T2 generation of the kanamycin resistant plants was plated on media containing a precursor to ethylene to see if they would have an ethylene sensitive or insensitive phenotype. All of the transformed lines showed sensitivity to ethylene, some even displaying a complete triple response. A control experiment which only transformed Arabidopsis with a vector without any EIN6 genomic DNA, showed ethylene insensitivity, like the parental plants. This experiment demonstrated that the ein6/een double mutant could be made sensitive to ethylene by transformation with the EIN6 genomic DNA.[0162]
EXAMPLE 5Isolation and Characterization of the ein6 Gene
• Annotation predicted that T29H11.50 genomic clone would encode a ˜4.5 kb cDNA, so the fragment containing the ein6 mutation was used to probe a size selected Col-0 cDNA library to isolate the gene. A single clone was identified out of more than 300,000 plaques, and contained a partial cDNA of 3.4 kb. The 5′ end of the cDNA was isolated by direct PCR from the cDNA library, and the two fragments were ligated to yield a cDNA of 4580 bp.[0163]
• Because the ein6 mutation was generated in a different ecotype, the genomic sequence of the EIN6 locus in the L(er) ecotype was determined. The L(er) genomic sequence differed from the Col-0 sequence only slightly, lacking two nucleotides in the 5′ untranslated region, changing a single nucleotide at a wobble base that did not alter the identity of the encoded amino acid, and a three base in-frame deletion that eliminated a single residue from a stretch of glutamic acids near the carboxyl terminus of the predicted polypeptide. Based on the sequence of the Col-0 cDNA, the intron-exon boundaries were determined for the EIN6 locus in L(er), yielding a cDNA of 4575 bp (SEQ ID NO: 22).[0164]
• The EIN6 cDNA encodes a protein of 1359 amino acids with a predicted molecular weight of 152.5 kDa (FIG. 3; SEQ ID NO: 20). Without being bound by any theory, the deduced amino acid sequence provides some indications of function. The most striking structural feature is the presence of three complete zinc fingers at the extreme carboxyl terminus of the polypeptide. The fingers fall into the GLI1 class of DNA-binding C[0165]₂H₂zinc fingers (FIG. 5).
• Interestingly, an incomplete fourth zinc finger is located adjacent to the three complete fingers, but lacks the second histidine required for zinc coordination (FIG. 3). Without intending to be bound by any theory, a potential role for EIN6 in DNA binding is supported by the presence of four putative nuclear localization signals in the carboxyl half of the protein (FIG. 3). Moreover, the presence of two acidic stretches of aspartic acid and glutamic acid adjacent to the zinc fingers (FIG. 3) is reminiscent of eukaryotic transcriptional activators.[0166]
• The amino terminal portion of the EIN6 protein bears significant homology to the retinoblastoma binding protein 2 (RBP2; FIG. 6). RBP2 was isolated based on its ability to interact with the retinoblastoma (RB) tumor suppressor in in vitro assays (Defeo-Jones, 1991). The similarity to RBP2 is found over an approximately 350 residue stretch that does not contain the motifs that are known to be required for interaction between RBP2 and RB (Kim, 1994). Interestingly, homologous proteins fall into two distinct classes. The first class contains RBP2, and is characterized by approximately 50% identity over a 40 amino acid region at the amino terminus of EIN6, a gap, and 35% identity and 50% similarity over approximately 250 residues. The non-homologous stretch between these two domains is comprised of 60 amino acids in EIN6, and more than 300 amino acids in its homologs. The second class of homologous proteins are similar over the entire stretch of the first 350 amino acids at the amino terminus of EIN6, possessing about 26% identity and 42% similarity. This class contains the yeast repressor of photolyase gene transcription, RPH1p (Jang, 1999), which is also homologous to EIN6 in the zinc finger domain.[0167]
• Unlike RPH1p, EIN6 possesses an interval of more than 500 amino acids between the RBP2 homology domain and the carboxyl terminal zinc fingers. This region is not homologous to any protein in the database, but does contain two prominent structural features. Immediately following the RBP2 homology domain, a coiled coil motif is predicted, suggesting that EIN6 is capable of protein-protein interaction. In addition, an ATP/GTP binding P-loop is also predicted (FIG. 3).[0168]
• While isolating 5′ end clones by PCR directly from the cDNA library, clones lacking nucleotides 2220-2322 were obtained. Without being bound to any theory, these data suggest that the primary EIN6 transcript undergoes alternative splicing. Removal of this intron introduces a stop codon immediately after the last codon preceding the intron. Translation of this splice variant is predicted to yield a protein that possesses the complete RBP2 homology domain, but lacks the predicted coiled coil region and the zinc fingers (FIG. 4). The 7 bp deletion in the ein6 mutant introduces a frame shift approximately 75[0169]nucleotides 5′ of the alternatively-spliced intron, but the translated mutant protein still retains the RBP2 homology domain (data not shown). These data suggest that the form of EIN6 required for normal ethylene responses is the full length protein containing the coiled coil domain and zinc fingers.
EXAMPLE 6Genetic Mapping of the Second Enhancer Mutation een
• The mapping of the second enhancer mutation een and the future cloning of the EEN gene is an important goal, in view of the fact that een is currently the only known enhancer in the ethylene pathway. Mapping of the second enhancer mutation (een) using twenty-six markers distributed evenly across the five chromosomes of the genome identified a new area of linkage in the ein6/een background to chromosome four. A small mapping population between the strains ein6/een in Landsberg and Columbia was generated to begin the mapping, with this population of only sixteen plants linkage was seen to markers nga1139 on chromosome four, and nga1107 on chromosome four. Using a larger population of 180 chromosomes four recombinants were seen with the marker nga1139, and no recombinants were seen with the marker nga1107. A large mapping population of ein6/een in the Landsberg background crossed to Nd-0 had been previously generated for the mapping of the ein6 mutation. This population is useful for fine mapping of the een mutation. To date, 360 chromosomes from this cross have been analyzed for recombinants in this region. The een mutation has been narrowed to a region containing eight BACs at the end of chromosome four using SSLP markers.[0170]
EXAMPLE 7Isolation of Other ein6 Alleles to Assist in Characterization of the Role of EIN6 in the Ethylene Pathway
• Alleles of ein6 in the Columbia ecotype can be isolated so that EIN6 can be better classified in the ethylene signaling pathway. Currently four alleles have been isolated, from the Salk Institute Genomic Analysis Laboratory T-DNA collection (ein6-2 through ein6-5). These alleles do not display an obvious ethylene insensitive root like ein6-1 in Ler. Further analysis using homozygous insertional lines of these alleles can be performed to determine whether a difference exists between the Landsberg and Columbia ecotypes that may account for the lack of a noticeable phenotype.[0171]
EXAMPLE 8Creation of Ethylene Insensitive Plants Using EIN6 Complementary DNA
• As described in the paragraphs above, the ein6 mutant Arabidopsis plants were found to exhibit increased root insensitivity to ethylene, whereas the double mutant ein6/een plants exhibited increased whole plant insensitivity to ethylene. This finding can be of use, for example, in designing a strategy to create other plant species with altered root or whole plant ethylene sensitivity. For example, an EIN6 complementary DNA clone containing a section of the coding region is subcloned into a plant transformation vector. The plant transformation vector contains a cauliflower mosaic virus 35S promoter and a polyadenylation site to allow for expression of the gene in plants (Schardl, C. L. et al. (1987) “Design and construction of a versatile system for the expression of foreign genes in plants” Gene, 61: 1-11). The plasmid is then introduced into[0172]Agrobacterium tumefacienscells by electroporation, and the bacterial transformants are selected using a kanamycin selection marker. Agrobacterium cells carrying the EIN6 complementary DNA are then used to infect tomato plants by the leaf disk transformation method of Horsch et al (Science (1985) 227: 1229). The same plasmid, lacking the EIN6 DNA, is used as a control. Disks are cultured on media containing kanamycin, followed by shoot formation. The shoots are then transplanted to root-inducing selective medium. Rooted plantlets are transplanted to soil.
• In order to study the effect of the EIN6 complementary DNA clone on the ethylene response in the transformed plants, transformed plantlets are examined for the presence of the ethylene-mediated triple response phenotype (Guzman and Ecker (1990) “Exploiting the triple response of Arabidopsis to identify ethylene-related mutants”, Plant Cell 2, 513-523). Plantlets are grown in the dark for 3 days in the presence or absence of 10 μl of ethylene/liter of air. The number of plants displaying an ethylene insensitive phenotype is then determined. These plants are further selected and grown to maturity. Plants transformed with the EIN6 antisense construct are expected to have a ethylene insenstive root phenotype.[0173]
• Following the above method, but transforming with both the EIN6 and EEN antisense constructs would also be possible. Plants transformed with antisense constructs of both genes would be expected to have an ethylene insensitive whole plant phenotype.[0174]
EXAMPLE 9Overexpression of EIN6 in Plants
• A plasmid is constructed to place EIN6 coding sequences downstream of the CaMv 35S promoter, to result in high level expression of EIN6 when transformed into tomato plants. The resulting plasmid is transformed into[0175]Agrobacterium tumefaciensas described in Example 8. Agrobacterium cells carrying the EIN6 coding sequence are used to transform tomato plants by the leaf disk method as described above. The same plasmid, lacking the EIN6 coding sequences, is used as a control.
• The transformed plantlets are tested for an altered response to ethylene by exposure to air or ethylene as described in Example 8. Plants which display an increased sensitivity to ethylene, particularly in the root, are obtained.[0176]
EXAMPLE 10Creation of Ethylene-insensitive Plants Using Cosuppression
• A truncated EIN6 DNA fragment corresponding to a section of the coding region is subcloned into a plant transformation vector. The plant transformation vector contains the cauliflower mosaic virus 35S promoter and a polyadenylation site to allow for expression of the gene in plants as described in Example 8. The plasmid is introduced into[0177]Agrobacterium tumefacienscells by electroporation as described above, and the bacterial transformants are selected using a selection marker such as kanamycin. Agrobacterium cells carrying the EIN6 fragment are used to infect plants using the leaf disk method as described above. The same plasmid, lacking the EIN6 DNA, is used as a control.
• The transformed plantlets are examined for the presence of the ethylene-mediated triple response phenotype as described above. Plantlets are grown in the dark for 3 days in the presence or absence of 10 μl of ethylene/liter of air. The number of plants displaying an ethylene-insensitive phenotype are determined. These plants are grown to maturity. Plant lines are obtained which display an insensitivity to ethylene, particularly in the root.[0178]
• Alternatively, both a truncated EIN6 DNA and a truncated EEN DNA fragment may be subcloned into the vector, following the method described above. Plant lines obtained from a cosuppression of both EIN6 and EEN genes may be expected to display an insensitivity to ethylene and hypersensitivity to taxol application (see Example 12).[0179]
EXAMPLE 11Determination of Epistasis and Isolation of Double Mutants
• Analysis of gene function is greatly assisted by the creation of double mutants. Accordingly, ethylene pathway double mutants were utilized to further examine the role of EIN6 in the ethylene pathway. To prepare the double mutants, strains were backcrossed at least twice to remove potential background effects on the ethylene phenotype before double mutant construction. Chi-square analysis was performed on the F[0180]₂segregation ratios to examine possible epistasis relationships. Double mutants then were isolated to demonstrate the genetic interaction between the mutant phenotypes.
• For example, ctr1-3 ctr1-1, ein3-2 ctr1-1, ein5-1 ctr1-1, ein7 ctr1-1, ein2-1 eir1-1, ein3-1 eir1-1, ein5-1 eir1-2, ctr1-1 eir1-1, ctr1-1 aux1-21, ein2-1 eto1-1, ein2-1 hls1-1 and eir1-1 aux1-21 double mutants were obtained by progeny testing F[0181]₂plants of each parental mutant phenotype to find a plant of the genotype m1/m1, +m2; the double mutants were identified as seedlings with a new phenotype in the F₃generation. The genotype of each double mutant was verified by failure to complement the hypostatic mutation. The einctr1-1 hls1-1, eto1-1 hls1-1 and eir1-1 hls1-1 double mutants were identified in F₂progenies as seedlings expressing both mutant phenotypes. The ctr1-5 allele was created by the insertion of a kanamycin-resistant marked T-DNA into the CTR1 gene (Kieber et al. 1993). This selectable marker was used to isolate recombinants between ein2-1 and ctr1-5. F₂seeds from a ctr1-5 by ein2-1 cross were examined for ethylene insensitive seedlings in the presence of 50 μg/ml kanamycin. Kanamycin-resistant (kan^r) F₂Ein⁻ plants were progeny tested for the presence of both the kan^rmarker and Ein⁻ phenotype. The double mutant was then verified by the failure to complement ctr1-1.
• Following this method, plants having the single mutations ein6 or een can be crossed to form double mutations with any suitable ethylene pathway mutant, such as those listed above.[0182]
EXAMPLE 12The Ethylene Insensitive ein6/een Double Mutants are Sensitive to Taxol
• The double mutants ein6/een were found to be sensitive to the microtubule stabilizer taxol. Wild-type Landsberg seedlings or ein6/een mutant seedlings were grown in the presence of either 0 μM, 0.1 μM, or 1 μM taxol for 10 days in the dark, following the method of Roman and Ecker (Phil. Trans. R. Soc. Lond. B. 350: 75-81, 1995, which is herein incorporated by reference in its entirety). The main effect of the addition of 1 μM taxol on both wild-type and ein6/een mutant dark grown seedlings was an inhibition of elongation. Hypocotyl cells of the taxol-treated tissue appeared shorter and rounder than wild-type cells. The ein6/een mutant seedlings treated with 1 μM taxol were significantly shorter than non-treated ein6/een mutant seedlings, displaying a phenotype very similar to the wild-type seedlings treated with 1 μM taxol. The ein6/een mutant seedlings treated with only 0.1 μM taxol also showed a shortened phenotype, while the wild-type seedlings with the same level of taxol did not have an altered size as compared to untreated seedlings. From these observations, it has been concluded that the ein6/een double mutation, (or possibly the een single mutation) is responsible for the taxol sensitivity phenotype.[0183]
• Because the ein6/een double mutants are hypersensitive to taxol, it may be desirable to create other plant species with this trait. This could be done, for example, by preparing plants that have reduced expression of both the EIN6 and the EEN gene, following the instructions in Examples 8 and 10, or using any method of reducing expression of a gene, such as antisense, cosuppression, RNA interference, molecular decoy methods, and the like.[0184]
• While the present invention, including preferred embodiments, has been described fully and completely herein, it is to be understood that modifications will occur to those skilled in the art without departing from the spirit of the invention. The scope of the invention is therefore to be determined solely by the appended claims.[0185]
• 12316829DNAArabidopsis thaliana 1 atggtcgggg aatcccattg gaaagtgagt ggatactaca cattttagtt ttatggcttg 60 gagtccagaa cctccgcatg tttctaaagc tatatattga ttttttgttt tggttgccat 120 aggtaccggg gaaagaggat gaggatcgac tattttctgg tctctgaaca actcaaagac 180 cgtatagttt cttgtaagat gcacggtcgt gggatagaac ttgaaggtgc catagataaa 240 cctgttcatc acacagattc atagaagatt attattagct ctttttgcaa caatttggtt 300 cttgatttgt ttcaggtttc catgggagtg atcattgtcc ggttacactc gagctttcga 360 agccatcttc agaaatggaa cagaaccagg tgtcaaacta aaatcatctt cttctgtatt 420 agaacataag gccttcttag gaacaagcca aagcttattt tatcagagaa tttaattttt 480 actttgccga gagatgcatt gaagatttga ttgaggttat tgagacactt atttagcctt 540 gttactctga tgaactctca aatgtactta taattattgt tgtgcttatc ttaatatgtt 600 ttattttcct aagaaatgtt gatattttta attgtgaatc ttcttcattt aattattttt 660 atttattaac ttttgagtca tttctaaaga tttagagctt ttgttggtgt attttaagca 720 tattataccc tttgctttat tgtcttagtc agtaggtaga ggcaacattc gttaaaccaa 780 tggctcattc aaattctttt ttttgggctc attcatattc ataaaaatca aatttggatt 840 cagcataaca aacacaataa taaatgtaga catttatatc atacattaat ttctactaat 900 ttcatgaact accaaagact aaaaatgttt aatgagaaat atctttttta aaaccatcaa 960 aaattttact cttaaaattg ggatttatgt ttggttttaa aaaggagttt ttgaatgtgc 1020 aaacggattt acagagagga caacaacaaa tgtgtgttag aaatggcaga atcgtaaata 1080 ataagaaaat catacccttt taaaaaccat ttctctctct aactaagtgt tctttacttg 1140 ttttaataca gagatatcac aatctcttgc tctgcccaaa cccatcccaa ccccctaaaa 1200 aaaatacaat tttttcctct gtttctttaa tccaataaaa acagagagac ctaagaaacg 1260 agatcaacaa agcccgtcgt ttttttcgag gaaaagtgaa tgacccttta agaaaataac 1320 tctattggcc ggagaaatgg tggagactcg tcgattctct taaagtttgt ttttttttcc 1380 tctcttcaaa atttttgttc tgtttatact ctgtaatggc gttttaattt gatacctaga 1440 agttaaagtg aacaaatttt gagagaaagt ctcttttttt tgttcttccc tgtgtgtgtg 1500 agagagagag agatatggcg gtttcagagc agagtcaaga tgtgtttcca tggcttaaat 1560 cgttaccggt tgctccagag ttcagaccta ctctagcaga gtttcaagat ccgatcgctt 1620 acattttgaa gattgaagaa gaagcttcta gatatggaat ctgtaaaatt ctgcctccac 1680 tgcctcctcc ttccaaaaaa acctcgattt ctaatctcaa ccgttctctg gcggcaagag 1740 cggcggcgag ggttcgtgac ggcggctttg gcgcgtgtga ttatgacggt ggtcccacat 1800 tcgccacgcg ccagcagcag atcgggtttt gccctaggaa acagcgtcca gtgcagagac 1860 ctgtgtggca gagtggagag gagtactctt ttggtgagtt cgagtttaaa gcgaagaact 1920 ttgagaagaa ttatctcaag aaatgtggta aaaagagtca actctctgcc cttgaaattg 1980 aaacacttta ctggagagcc actgtggata aacccttctc tgttgagtat gccaatgaca 2040 tgcctggctc ggcttttatc cctctgagtc tggctgctgc gaggaggaga gagtctggtg 2100 gtgaaggagg aacagttggt gagaccgctt ggaacatgag ggcaatgtct agagccgaag 2160 gatcattgct taagttcatg aaggaagaga tccctggagt tacatcacca atggtgtatg 2220 ttgctatgat gtttagttgg tttgcttggc atgtggagga ccatgacctt catagtctca 2280 attacttgca tatgggtgct ggtaagactt ggtacggtgt gccaaaggat gctgctctgg 2340 cttttgagga ggttgttagg gttcatggtt acggtgaaga gctcaatcct cttggtgagt 2400 atgataagtg ttatgtgtta gtgactcata gttactcaat gtatcaattc attgttctga 2460 ttgttggctt tgttgtgtct ctgttctttt tgttgctttt aaatgctttt ctttccctcc 2520 aaaagttgtc aggggtagaa gctgtatgca agcaaactaa ttggttatgc ctcttacatt 2580 gttgtcttat tagtgacatt ttctactctt ggtgagaaga caactgtgat gtctcctgaa 2640 gtatttgtta aagccggaat accgtgttgc aggtaatggt tttttatgtt tgtgctctgt 2700 ttatctcatc atcttgttgt ttgcatgtct cttctttggt ctcttataat tggcacactt 2760 tttcttcttc tggtttttca cgcaagtggt ttttttgtat caggttagtg caaaatcctg 2820 gagagtttgt cgtcaccttt ccgggagctt atcattcggg atttagtcat ggtgagtaag 2880 cgacctatat ttagtatctt taagtcaacc attcaaaggc ctagtatgtt gaaataactt 2940 atattatcca taaaaacaat cgtgggggtt gtacaggatt taattttgga gaagcatcta 3000 acattgccac tcccgaatgg ttgagaatgg ctaaagatgc tgctatccgg cgagctgcta 3060 taaattaccc tccaatggtt tctcatctcc agctacttta tgactttgta ttggctctag 3120 gttctaggta ctctttcttt tctatagaca cactcaaact ttttaagctg cttgaagggc 3180 tcatataagg tatgtttcac tcattgcaga gtgccaacaa gcatcaatcc caaaccacgg 3240 agttctagat taaaagataa ggcaagaagc gaaggagaaa gattgaccaa aaagctattt 3300 gtgcaaaaca ttatccacaa caacgaattg ctttcttctc tcggaaaagg atccccagtg 3360 gcccttctcc cacagagttc ctcagatata tcagtttgtt ctgacctgcg aattggatcc 3420 catttgataa ccaaccagga aaacccaatc cagttaaagt gtgaggactt aagttctgat 3480 agtgttgtgg ttgatctcag taacggttta aaggatacag tttcagtgaa agaaaaattt 3540 acatctttat gtgaaaggag cagaaatcac ctagcaagca cggagaagga cactcaagaa 3600 actctgtctg atgctgaaag gaggaagaat gatgcagctg ttgcgctttc ggatcaaagg 3660 cttttctctt gtgttacatg tggagtctta agctttgatt gtgtagctat cgttcaacct 3720 aaagaagcag ctgctagata tctcatgtct gcagattgta gcttcttcaa tgattggaca 3780 gctgcttctg gatctgcaaa tcttggtcag gctgcaagat cacttcatcc tcgtaagtta 3840 caggtcgcac tttcggttat tggctgttat ttagcattta ctaacttttt ttcatcatgc 3900 agaaagcaaa gagaagcatg atgtaaatta cttctacaat gttcctgttc aaactatgga 3960 tcattcagtg aagactggcg atcaaaaaac ttcaacaact tccccgacaa tagcgcataa 4020 agataatgat gttcttggga tgttagcttc agcatatgga gactcttctg attccgagga 4080 agaagatcaa aaaggcttag ttacccctag ttccaaaggg gaaacaaaaa cgtatgatca 4140 agaaggttca gatggccatg aggaggctag agatggtaga acttctgatt ttaactgcca 4200 gagactaacc agcgaacaga atgggttaag caaaggcgga aaatcatcac ttctggaaat 4260 agctttacca tttattccaa gatctgatga cgattcatgt cggttgcacg tgttttgtct 4320 tgagcatgct gcggaagtgg aacagcaact tcgtcctttt ggggggatta acttaatgtt 4380 actgtgccat ccaggtaaca tcaaaactac aagattcatt ttattagtat ttctgtatca 4440 gttatgcaat tctttagtct ttttcatcat tttgacgcac aacatttgca gagtacccca 4500 ggatagaggc tgaagcaaag atagttgccg aagagctcgt catcaatcac gaatggaatg 4560 atactgaatt caggaatgtg acccgagagg atgaggaaac gattcaggca gcgttggata 4620 atgttgaagc taagggtggg aacagtgatt ggaccgtaaa attgggtgtt aacctttctt 4680 acagcgctat tctcagtcgc tctcctctgt acagtaagca gatgccgtat aactccatca 4740 tatacaaggc gttcggtcgc agctctccag tagcgagctc accctcgaaa cccaaagtct 4800 ctggtaaaag atcgtccaga cagaggaaat atgttgttgg aaaatggtgt ggtaaggttt 4860 ggatgtcaca tcaggtacta ttgtctaacc tgaatccata ctaattcaga cattagtaca 4920 cttctgtttg gttcaatttg cttttctttc ttcaggtgca tccctttttg ctggagcagg 4980 acttagaggg ggaagaatct gaaagaagtt gtcatcttcg agttgctatg gatgaggatg 5040 ccactggaaa gagatcgttt cctaataatg tttccaggga ttcgacaaca atgtttggaa 5100 gaaagtattg taggaagaga aagataagag caaaggcggt gccacgcaag aagcttactt 5160 cttttaagag ggaagatgga gtttctgatg acacatcaga agatcattct tataagcagc 5220 aatggagggc ttccgggaat gaggaagagt cttattttga gacagggaac acagcttctg 5280 gtgattcatc aaatcaaatg tctgatccgc acaagggaat tatcagacat aaaggttata 5340 aagaatttga gtcagatgat gaggtttcag accgttcact tggggaagag tatactgtac 5400 gggcatgtgc agcttcagag agctcaatgg agaatggctc tcagcattca atgtatgacc 5460 atgatgatga tgatgatgat atcgacaggc agcctagggg gattccaagg agccaacaga 5520 caagagtttt taggaatcca gtttcatatg agtcagaaga taatggcgtt tatcagcaaa 5580 gcggaagaat atccataagt aataggcaag ctaatcgaat ggttggtgaa tatgattcag 5640 cagagaattc tttggaggaa cgaggctttt gcagtacagg gaaaaggcaa accaggtcaa 5700 cagccaaacg aatagcaaaa accaagacag ttcagagttc gagagacaca aaaggtcgct 5760 ttttgcaaga atttgcatct ggaaagaaga atgaagaatt ggattcatac atggagggac 5820 ctagcacacg gcttagggtg agacatcaga agccgtcgag agggtcttta gaaacaaaac 5880 caaagaagat tggtaagaag agaagtggta atgcttcctt ctccagagtt gcaactgaaa 5940 aagatgtgga ggaaaaagaa gaagaagaag aagaagagaa tgaggaagag gaatgtgcag 6000 cataccaatg taacatggag ggttgcacga tgagtttcag ttcggaaaaa cagttgatgt 6060 tacacaaaag aaacatatgc ccaattaaag gctgtggtaa aaacttcttc tcacacaagt 6120 atttggttca acaccagcgt gttcactcag acgaccgtcc tctgaaatgt ccatggaagg 6180 gatgtaagat gacgttcaag tgggcttggt ctagaaccga gcacataagg gttcacacag 6240 gcgctaggcc ttatgtttgc gctgaaccgg attgtggtca aacattcagg tttgtctctg 6300 acttcagccg gcataaaagg aagaccggtc attcggttaa gaagaccaac aaaaggtgat 6360 ttaggctcta acatggaatc tatcatgaag aagattactc cattgatcta ttagaagaaa 6420 attggattgt acgaatatta ggggttttct aagtgtgtat gtctctctgg tcctctgtca 6480 attcgggatt ataaagtcaa agtcttaaca tttggaatag ccattgtttg ttgcagacgt 6540 tttcactttc agacagattc tctctttgtt gtattctcta tttttgcagt tgaatgaatc 6600 tgtaacaaca acaaatcctg gtgtaggaaa agcattgtgt tactcaagta tttgacatat 6660 atggttgtat acgtggctta tcctggttgg ttgacagtca gagttcatcg ccgtggactt 6720 catttgactt tgataaagac tcaaacctac caaattgcta atgaatgaca ttacctgtgt 6780 caactaggat gtaaaactct ctgacttcag cacacggcac aaacgaaag 6829293PRTArabidopsis thalianaZN_FING(1)...(93) 2 Lys Arg Asn Ile Cys Pro Ile Lys Gly Cys Gly Lys Asn Phe Phe Ser 1 5 10 15 His Lys Tyr Leu Val Gln His Gln Arg Val His Ser Asp Asp Arg Pro 20 25 30 Leu Lys Cys Pro Trp Lys Gly Cys Lys Met Thr Phe Lys Trp Ala Trp 35 40 45 Ser Arg Thr Glu His Ile Arg Val His Thr Gly Ala Arg Pro Tyr Val 50 55 60 Cys Ala Glu Pro Asp Cys Gly Gln Thr Phe Arg Phe Val Ser Asp Phe 65 70 75 80 Ser Arg His Lys Arg Lys Thr Gly His Ser Val Lys Lys 85 90395PRTMus musculusZN_FING(1)...(95) 3 Lys Glu Phe Val Cys His Trp Gly Gly Cys Ser Arg Glu Leu Arg Pro 1 5 10 15 Phe Lys Ala Gln Tyr Met Leu Val Val His Met Arg Arg His Thr Gly 20 25 30 Glu Lys Pro His Lys Cys Thr Phe Glu Gly Cys Arg Lys Ser Tyr Ser 35 40 45 Arg Leu Glu Asn Leu Lys Thr His Leu Arg Ser His Thr Gly Glu Lys 50 55 60 Pro Tyr Met Cys Glu Gln Glu Gly Cys Ser Lys Ala Phe Ser Asn Ala 65 70 75 80 Ser Asp Arg Ala Lys His Gln Asn Arg Thr His Ser Asn Glu Lys 85 90 95490PRTDrosophila melanogasterZN_FING(1)...(90) 4 Arg Pro Phe Lys Cys Pro Phe Glu Gly Cys Gly Arg Ser Phe Thr Thr 1 5 10 15 Ser Asn Ile Arg Lys Val His Ile Arg Thr His Thr Gly Glu Arg Pro 20 25 30 Tyr Tyr Cys Ser Glu Pro Gly Cys Gly Arg Ala Phe Ala Ser Ala Thr 35 40 45 Asn Tyr Lys Asn His Val Arg Ile His Thr Gly Glu Lys Pro Tyr Val 50 55 60 Cys Thr Val Pro Gly Cys Asp Lys Arg Phe Thr Glu Tyr Ser Ser Leu 65 70 75 80 Tyr Lys His His Val Val His Thr His Ser 85 90596PRTMus musculusZN_FING(1)...(96) 5 Lys Lys Glu Phe Val Cys Arg Trp Leu Asp Cys Ser Arg Glu Gln Lys 1 5 10 15 Pro Phe Lys Ala Gln Tyr Met Leu Val Val His Met Arg Arg His Thr 20 25 30 Gly Glu Lys Pro His Lys Cys Thr Phe Glu Gly Cys Thr Lys Ala Tyr 35 40 45 Ser Arg Leu Glu Asn Leu Lys Thr His Leu Arg Ser His Thr Gly Glu 50 55 60 Lys Pro Tyr Val Cys Glu His Glu Gly Cys Asn Lys Ala Phe Ser Asn 65 70 75 80 Ala Ser Asp Arg Ala Lys His Gln Asn Arg Thr His Ser Asn Glu Lys 85 90 95691PRTHomo sapiensZN_FING(1)...(91) 6 Glu Arg Pro Phe Lys Cys Pro Phe Glu Gly Cys Gly Arg Ser Phe Thr 1 5 10 15 Thr Ser Asn Ile Arg Lys Val His Val Arg Thr His Thr Gly Glu Arg 20 25 30 Pro Tyr Tyr Cys Thr Glu Pro Gly Cys Gly Arg Ala Phe Ala Ser Ala 35 40 45 Thr Asn Tyr Lys Asn His Val Arg Ile His Thr Gly Glu Lys Pro Tyr 50 55 60 Val Cys Thr Val Pro Gly Cys Asp Lys Arg Phe Thr Glu Tyr Ser Ser 65 70 75 80 Leu Tyr Lys His His Val Val His Thr His Ser 85 90753PRTSaccharomyces cerevisiaeZN_FING(1)...(53) 7 Gly Lys Asn Lys Ile Tyr Ile Cys Lys Glu Cys Gln Arg Lys Phe Ser 1 5 10 15 Ser Gly His His Leu Thr Arg His Lys Lys Ser Val His Ser Gly Glu 20 25 30 Lys Pro His Ser Cys Pro Lys Cys Gly Lys Arg Phe Lys Arg Arg Asp 35 40 45 His Val Leu Gln His 508397PRTArabidopsis thaliana 8 Met Ala Val Ser Glu Gln Ser Gln Asp Val Phe Pro Trp Leu Lys Ser 1 5 10 15 Leu Pro Val Ala Pro Glu Phe Arg Pro Thr Leu Ala Glu Phe Gln Asp 20 25 30 Pro Ile Ala Tyr Ile Leu Lys Ile Glu Glu Glu Ala Ser Arg Tyr Gly 35 40 45 Ile Cys Lys Ile Leu Pro Pro Leu Pro Pro Pro Ser Lys Lys Thr Ser 50 55 60 Ile Ser Asn Leu Asn Arg Ser Leu Ala Ala Arg Ala Ala Ala Arg Val 65 70 75 80 Arg Asp Gly Gly Phe Gly Ala Cys Asp Tyr Asp Gly Gly Pro Thr Phe 85 90 95 Ala Thr Arg Gln Gln Gln Ile Gly Phe Cys Pro Arg Lys Gln Arg Pro 100 105 110 Val Gln Arg Pro Val Trp Gln Ser Gly Glu Glu Tyr Ser Phe Gly Glu 115 120 125 Phe Glu Phe Lys Ala Lys Asn Phe Glu Lys Asn Tyr Leu Lys Lys Cys 130 135 140 Gly Lys Lys Ser Gln Leu Ser Ala Leu Glu Ile Glu Thr Leu Tyr Trp 145 150 155 160 Arg Ala Thr Val Asp Lys Pro Phe Ser Val Glu Tyr Ala Asn Asp Met 165 170 175 Pro Gly Ser Ala Phe Ile Pro Leu Ser Leu Ala Ala Ala Arg Arg Arg 180 185 190 Glu Ser Gly Gly Glu Gly Gly Thr Val Gly Glu Thr Ala Trp Asn Met 195 200 205 Arg Ala Met Ser Arg Ala Glu Gly Ser Leu Leu Lys Phe Met Lys Glu 210 215 220 Glu Ile Pro Gly Val Thr Ser Pro Met Val Tyr Val Ala Met Met Phe 225 230 235 240 Ser Trp Phe Ala Trp His Val Glu Asp His Asp Leu His Ser Leu Asn 245 250 255 Tyr Leu His Met Gly Ala Gly Lys Thr Trp Tyr Gly Val Pro Lys Asp 260 265 270 Ala Ala Leu Ala Phe Glu Glu Val Val Arg Val His Gly Tyr Gly Glu 275 280 285 Glu Leu Asn Pro Leu Val Thr Phe Ser Thr Leu Gly Glu Lys Thr Thr 290 295 300 Val Met Ser Pro Glu Val Phe Val Lys Ala Gly Ile Pro Cys Cys Arg 305 310 315 320 Leu Val Gln Asn Pro Gly Glu Phe Val Val Thr Phe Pro Gly Ala Tyr 325 330 335 His Ser Gly Phe Ser His Gly Phe Asn Phe Gly Glu Ala Ser Asn Ile 340 345 350 Ala Thr Pro Glu Trp Leu Arg Met Ala Lys Asp Ala Ala Ile Arg Arg 355 360 365 Ala Ala Ile Asn Tyr Pro Pro Met Val Ser His Leu Gln Leu Leu Tyr 370 375 380 Asp Phe Val Leu Ala Leu Gly Ser Arg Val Pro Thr Ser 385 390 3959301PRTHomo sapiens 9 Pro Glu Cys Pro Val Phe Glu Pro Ser Trp Glu Glu Phe Thr Asp Pro 1 5 10 15 Leu Ser Phe Ile Gly Arg Ile Arg Pro Leu Ala Glu Lys Thr Gly Ile 20 25 30 Cys Lys Ile Arg Pro Pro Gln Ala Val Arg Glu Tyr Thr Leu Gln Ser 35 40 45 Phe Gly Glu Met Ala Asp Asn Phe Lys Ser Asp Tyr Phe Asn Met Pro 50 55 60 Val His Met Val Pro Thr Glu Leu Val Glu Lys Glu Phe Trp Arg Leu 65 70 75 80 Val Ser Ser Ile Glu Glu Asp Val Ile Val Glu Tyr Gly Ala Asp Ile 85 90 95 Ser Ser Lys Asp Phe Gly Ser Gly Phe Pro Val Lys Asp Gly Arg Arg 100 105 110 Lys Ile Leu Pro Glu Glu Glu Glu Tyr Ala Leu Ser Gly Trp Asn Leu 115 120 125 Asn Asn Met Pro Val Leu Glu Gln Ser Val Leu Ala His Ile Asn Val 130 135 140 Asp Ile Ser Gly Met Lys Val Pro Trp Leu Tyr Val Gly Met Cys Phe 145 150 155 160 Ser Ser Phe Cys Trp His Ile Glu Asp His Trp Ser Tyr Ser Ile Asn 165 170 175 Tyr Leu His Trp Gly Glu Pro Lys Thr Trp Tyr Gly Val Pro Ser His 180 185 190 Ala Ala Glu Gln Leu Glu Glu Val Met Arg Glu Leu Ala Pro Glu Leu 195 200 205 Phe Glu Ser Gln Pro Asp Leu Leu His Gln Leu Val Thr Ile Met Asn 210 215 220 Pro Asn Val Leu Met Glu His Gly Val Pro Val Tyr Arg Thr Asn Gln 225 230 235 240 Cys Ala Gly Glu Phe Val Val Thr Phe Pro Arg Ala Tyr His Ser Gly 245 250 255 Phe Asn Gln Gly Tyr Asn Phe Ala Glu Ala Val Asn Phe Cys Thr Ala 260 265 270 Asp Trp Leu Pro Ile Gly Arg Gln Cys Val Asn His Tyr Arg Arg Leu 275 280 285 Arg Arg His Cys Val Phe Ser His Glu Glu Leu Ile Phe 290 295 30010301PRTMus musculus 10 Pro Glu Cys Pro Val Phe Glu Pro Ser Trp Ala Glu Phe Arg Asp Pro 1 5 10 15 Leu Gly Tyr Ile Ala Lys Ile Arg Pro Ile Ala Glu Lys Ser Gly Ile 20 25 30 Cys Lys Ile Arg Pro Pro Gln Ala Thr Arg Glu Tyr Thr Leu Gln Ser 35 40 45 Phe Gly Glu Met Ala Asp Ser Phe Lys Ala Asp Tyr Phe Asn Met Pro 50 55 60 Val His Met Val Pro Thr Glu Leu Val Glu Lys Glu Phe Trp Arg Leu 65 70 75 80 Val Asn Ser Ile Glu Glu Asp Val Thr Val Glu Tyr Gly Ala Asp Ile 85 90 95 His Ser Lys Glu Phe Gly Ser Gly Phe Pro Val Ser Asp Ser Lys Arg 100 105 110 His Leu Thr Pro Glu Glu Glu Glu Tyr Ala Thr Ser Gly Trp Asn Leu 115 120 125 Asn Val Met Pro Val Leu Glu Gln Ser Val Leu Cys His Ile Asn Ala 130 135 140 Asp Ile Ser Gly Met Lys Val Pro Trp Leu Tyr Val Gly Met Val Phe 145 150 155 160 Ser Ala Phe Cys Trp His Ile Glu Asp His Trp Ser Tyr Ser Ile Asn 165 170 175 Tyr Leu His Trp Gly Glu Pro Lys Thr Trp Tyr Gly Val Pro Ser Leu 180 185 190 Ala Ala Glu His Leu Glu Glu Val Met Lys Lys Leu Thr Pro Glu Leu 195 200 205 Phe Asp Ser Gln Pro Asp Leu Leu His Gln Leu Val Thr Leu Met Asn 210 215 220 Pro Asn Thr Leu Met Ser His Gly Val Pro Val Val Arg Thr Asn Gln 225 230 235 240 Cys Ala Gly Glu Phe Val Ile Thr Phe Pro Arg Ala Tyr His Ser Gly 245 250 255 Phe Asn Gln Gly Tyr Asn Phe Ala Glu Ala Val Asn Phe Cys Thr Ala 260 265 270 Asp Trp Leu Pro Ala Gly Arg Gln Cys Ile Glu His Tyr Arg Arg Leu 275 280 285 Arg Arg Tyr Cys Val Phe Ser His Glu Glu Leu Ile Cys 290 295 30011304PRTDrosophila melanogaster 11 Pro Glu Cys Pro Val Phe Arg Pro Thr Thr Glu Glu Phe Lys Asn Pro 1 5 10 15 Leu Ala Tyr Ile Ser Lys Ile Arg Ser Ile Ala Glu Lys Cys Gly Ile 20 25 30 Ala Lys Ile Leu Pro Pro Gln Ala Glu Arg Glu Tyr Thr Leu Gln Gln 35 40 45 Phe Gly Gln Met Ala Asp Gln Phe Lys Gln Glu Tyr Phe Arg Lys Pro 50 55 60 Val His Leu Val Pro Thr Glu Met Val Glu Arg Glu Phe Trp Arg Ile 65 70 75 80 Val Ser Ser Ile Asp Glu Asp Val Thr Val Glu Tyr Gly Ala Asp Leu 85 90 95 His Thr Met Asp His Gly Ser Gly Phe Pro Thr Lys Ser Ser Leu Tyr 100 105 110 Leu Leu Pro Gly Asp Gln Glu Tyr Ala Glu Ser Ser Trp Asn Leu Asn 115 120 125 Asn Leu Pro Leu Leu Glu Asp Ser Ile Leu Gly His Ile Asn Ala Asp 130 135 140 Ile Ser Gly Met Asn Ala Pro Trp Met Tyr Val Gly Met Cys Phe Ala 145 150 155 160 Ala Phe Cys Trp His Asn Glu Asp His Trp Ser Tyr Ser Ile Asn Tyr 165 170 175 Leu His Trp Gly Glu Pro Lys Thr Trp Tyr Gly Val Pro Gly Ser Cys 180 185 190 Ala Glu Gln Phe Glu Glu Thr Met Lys Gln Ala Ala Pro Glu Leu Phe 195 200 205 Ser Ser Gln Pro Asp Leu Leu His Gln Leu Val Thr Ile Met Asn Pro 210 215 220 Asn Ile Leu Met Asn Asn Arg Val Pro Val Phe Arg Thr Asp Gln His 225 230 235 240 Ala Gly Glu Phe Val Ile Thr Phe Pro Arg Ala Tyr His Ala Gly Phe 245 250 255 Asn Gln Gly Tyr Asn Phe Ala Glu Ala Val Asn Phe Ala Pro Ala Asp 260 265 270 Trp Leu Lys Met Gly Arg Glu Cys Val Asn His Tyr Ser Met Leu Arg 275 280 285 Arg Phe Cys Val Phe Ser His Asp Glu Leu Val Cys Lys Met Ala Leu 290 295 30012317PRTSchizosaccharomyces pombe 12 Ile His Gly Leu Pro Val Ala Pro Val Phe Tyr Pro Asp Lys Glu Glu 1 5 10 15 Phe Gln Asp Ser Ile Gly Tyr Ile Asn Lys Ile Ala Pro Ile Gly Glu 20 25 30 Lys Tyr Gly Ile Ile Lys Ile Val Pro Pro Phe Glu Thr Gly Asn Tyr 35 40 45 Tyr Thr Leu Ser Asn Phe Glu Lys Tyr Cys Asp Asn Phe Lys Lys Asn 50 55 60 Tyr Phe Ser Lys Phe Lys Asp Ser Glu Ile Thr Glu Asp Ile Val Glu 65 70 75 80 Lys Glu Tyr Trp Lys Leu Val Lys Asp Asn Asn Thr Ser Leu Glu Val 85 90 95 Glu Tyr Gly Ala Asp Leu Ser Thr Leu Asp Gln Gly Ser Ala Phe Pro 100 105 110 Ser Leu Ala Lys Asn Pro Val Asn Pro Tyr Ser Lys Asp Thr Trp Asn 115 120 125 Leu Asn Val Ile Ala Ser Thr Asn Gly Ser Leu Leu Ser Tyr Ile Asp 130 135 140 Asn Pro Val Ser Gly Ile Thr Cys Pro Trp Leu Tyr Val Gly Met Cys 145 150 155 160 Phe Ser Thr Phe Cys Trp His Val Glu Asp Asn Tyr Thr Tyr Ser Val 165 170 175 Asn Tyr Gln His Tyr Gly Asp Thr Lys Leu Trp Tyr Gly Ile Pro Gly 180 185 190 Asp Gln Ala Glu Arg Phe Glu Arg Ala Ala Leu Asp Ile Ala Pro Asp 195 200 205 Leu Val Lys Lys Gln Lys Asp Leu Leu Tyr Gln Leu Ala Thr Met Ile 210 215 220 Asn Pro Asp Glu Leu Gln Lys Arg Gly Val Asp Val Tyr Phe Ile Asp 225 230 235 240 Gln Gly Pro Asn Glu Phe Val Ile Thr Phe Pro Lys Ser Phe His Ala 245 250 255 Gly Ile Asn His Gly Phe Asn Ile Asn Glu Ala Val Asn Phe Ala Pro 260 265 270 Lys Asp Trp Leu Leu Asn Gly Phe Ser Leu Asn Gly Val Leu Lys Tyr 275 280 285 Gln Ser Leu Leu Lys Pro Pro Val Leu Ser His Asp Met Leu Val Tyr 290 295 300 Asn Leu Ala Thr Asn Pro Ala Ser Glu Ile Ser Val Ser 305 310 31513313PRTArabidopsis thaliana 13 Asn Val Pro Leu Gly Pro Val Tyr Tyr Pro Thr Glu Asp Glu Phe Lys 1 5 10 15 Asp Pro Leu Glu Tyr Ile His Lys Ile Lys Pro Glu Ala Glu Val Tyr 20 25 30 Gly Ile Cys Lys Ile Val Pro Pro Val Ser Arg Thr Gln Ile Glu Lys 35 40 45 Lys Phe Trp Glu Ile Val Glu Gly Ser Gly Gly Glu Val Glu Val Met 50 55 60 Tyr Gly Asn Asp Leu Asp Thr Ser Val Tyr Gly Ser Gly Phe Pro Arg 65 70 75 80 Ile Gly Asp Gln Arg Pro Glu Ser Val Glu Ala Asp Ile Trp Asp Glu 85 90 95 Tyr Cys Gly Ser Pro Trp Asn Leu Asn Asn Met Pro Lys Leu Lys Gly 100 105 110 Ser Met Leu Gln Ala Ile Arg His Asn Ile Asn Gly Val Thr Val Pro 115 120 125 Trp Leu Tyr Leu Gly Met Leu Phe Ser Ser Phe Cys Trp His Phe Glu 130 135 140 Asp His Cys Phe Tyr Ser Val Asn Tyr Leu His Trp Gly Glu Ala Lys 145 150 155 160 Cys Trp Tyr Gly Ile Pro Gly Ser Ala Ala Ser Ala Phe Glu Lys Val 165 170 175 Met Arg Lys Thr Leu Pro Asp Leu Phe Asp Ala Gln Pro Asp Leu Leu 180 185 190 Phe Gln Leu Val Thr Met Leu Ser Pro Thr Val Leu Gln Glu Asn Lys 195 200 205 Val Pro Val Tyr Thr Val Leu Gln Glu Pro Gly Asn Phe Val Ile Thr 210 215 220 Phe Pro Lys Ser Phe His Ala Gly Phe Asn Phe Gly Leu Asn Cys Ala 225 230 235 240 Glu Ala Val Asn Phe Ala Thr Ala Asp Trp Leu Pro Tyr Gly Gly Ser 245 250 255 Gly Ala Glu Leu Tyr Arg Leu Tyr Arg Lys Pro Ser Val Ile Ser His 260 265 270 Glu Glu Leu Leu Cys Val Val Ala Lys Gly Asn Cys Cys Asn Asn Glu 275 280 285 Gly Ser Ile His Leu Lys Lys Glu Leu Leu Arg Ile Tyr Ser Lys Glu 290 295 300 Lys Thr Trp Arg Glu Gln Leu Trp Lys 305 31014366PRTArabidopsis thaliana 14 Met Ala Val Ser Glu Gln Ser Gln Asp Val Phe Pro Trp Leu Lys Ser 1 5 10 15 Leu Pro Val Ala Pro Glu Phe Arg Pro Thr Leu Ala Glu Phe Gln Asp 20 25 30 Pro Ile Ala Tyr Ile Leu Lys Ile Glu Glu Glu Ala Ser Arg Tyr Gly 35 40 45 Ile Cys Lys Ile Leu Pro Pro Leu Pro Pro Pro Ser Lys Lys Thr Ser 50 55 60 Ile Ser Asn Leu Asn Arg Ser Leu Ala Ala Arg Ala Ala Ala Arg Val 65 70 75 80 Arg Asp Gly Gly Phe Gly Ala Cys Asp Tyr Asp Gly Gly Pro Thr Phe 85 90 95 Ala Thr Arg Gln Gln Gln Ile Gly Phe Cys Pro Arg Lys Gln Arg Pro 100 105 110 Val Gln Arg Pro Val Trp Gln Ser Gly Glu Glu Tyr Ser Phe Gly Glu 115 120 125 Phe Glu Phe Lys Ala Lys Asn Phe Glu Lys Asn Tyr Leu Lys Lys Cys 130 135 140 Gly Lys Lys Ser Gln Leu Ser Ala Leu Glu Ile Glu Thr Leu Tyr Trp 145 150 155 160 Arg Ala Thr Val Asp Lys Pro Phe Ser Val Glu Tyr Ala Asn Asp Met 165 170 175 Pro Gly Ser Ala Phe Ile Pro Leu Ser Leu Ala Ala Ala Arg Arg Arg 180 185 190 Glu Ser Gly Gly Glu Gly Gly Thr Val Gly Glu Thr Ala Trp Asn Met 195 200 205 Arg Ala Met Ser Arg Ala Glu Gly Ser Leu Leu Lys Phe Met Lys Glu 210 215 220 Glu Ile Pro Gly Val Thr Ser Pro Met Val Tyr Val Ala Met Met Phe 225 230 235 240 Ser Trp Phe Ala Trp His Val Glu Asp His Asp Leu His Ser Leu Asn 245 250 255 Tyr Leu His Met Gly Ala Gly Lys Thr Trp Tyr Gly Val Pro Lys Asp 260 265 270 Ala Ala Leu Ala Phe Glu Glu Val Val Arg Val His Gly Tyr Gly Glu 275 280 285 Glu Leu Asn Pro Leu Val Thr Phe Ser Thr Leu Gly Glu Lys Thr Thr 290 295 300 Val Met Ser Pro Glu Val Phe Val Lys Ala Gly Ile Pro Cys Cys Arg 305 310 315 320 Leu Val Gln Asn Pro Gly Glu Phe Val Val Thr Phe Pro Gly Ala Tyr 325 330 335 His Ser Gly Phe Ser His Gly Phe Asn Phe Gly Glu Ala Ser Asn Ile 340 345 350 Ala Thr Pro Glu Trp Leu Arg Met Ala Lys Asp Ala Ala Ile 355 360 36515336PRTSaccharomyces cerevisiae 15 Pro Val Phe Lys Pro Thr Tyr Glu Gln Phe Glu Asp Phe Tyr Ala Tyr 1 5 10 15 Cys Lys Ala Ile Asn Lys Tyr Gly Met Lys Ser Gly Val Val Lys Val 20 25 30 Ile Pro Pro Lys Glu Trp Lys Asp Lys Leu Asp Leu Pro Tyr Ser Ala 35 40 45 Glu Thr Leu Gln Lys Ile Lys Ile Lys Ser Pro Ile Gln Gln His Ile 50 55 60 Ser Gly Asn Lys Gly Leu Phe Met Val Gln Asn Val Glu Lys Asn Lys 65 70 75 80 Thr Tyr Asn Ile Ile Gln Trp Lys Asp Leu Ser Lys Asp Tyr Val Pro 85 90 95 Pro Glu Asp Pro Lys Ala Arg Arg Asn Ser Arg Lys Gly Ser Val Ser 100 105 110 Lys Ser Thr Lys Leu Lys Leu Lys Asn Phe Glu Ser Ser Phe Asn Ile 115 120 125 Asp Asp Phe Glu Gln Phe Arg Thr Glu Tyr Thr Ile Asp Leu Ser Asp 130 135 140 Phe Gln Asn Thr Glu Arg Leu Lys Phe Leu Glu Glu Tyr Tyr Trp Lys 145 150 155 160 Thr Leu Asn Phe Thr Thr Pro Met Tyr Gly Ala Asp Thr Pro Gly Ser 165 170 175 Ile Phe Pro Glu Gly Leu Asn Val Trp Asn Val Ala Lys Leu Pro Asn 180 185 190 Ile Leu Asp His Met Glu Thr Lys Val Pro Gly Val Asn Asp Ser Tyr 195 200 205 Leu Tyr Ala Gly Leu Trp Lys Ala Ser Phe Ser Trp His Leu Glu Asp 210 215 220 Gln Asp Leu Tyr Ser Ile Asn Tyr Ile His Phe Gly Ala Pro Lys Gln 225 230 235 240 Trp Tyr Ser Ile Pro Gln Glu Asp Arg Phe Lys Phe Tyr Lys Phe Met 245 250 255 Gln Glu Gln Phe Pro Glu Glu Ala Lys Asn Cys Pro Glu Phe Leu Arg 260 265 270 His Lys Met Phe Leu Ala Ser Pro Lys Leu Leu Gln Glu Asn Gly Ile 275 280 285 Arg Cys Asn Glu Ile Val His His Glu Gly Glu Phe Met Ile Thr Tyr 290 295 300 Pro Tyr Gly Tyr His Ala Gly Phe Asn Tyr Gly Tyr Asn Leu Ala Glu 305 310 315 320 Ser Val Asn Phe Ala Leu Glu Glu Trp Leu Pro Ile Gly Lys Lys Ala 325 330 33516362PRTArabidopsis thaliana 16 Ala Pro Val Phe His Pro Thr Ser Glu Glu Phe Glu Asp Thr Leu Ala 1 5 10 15 Tyr Ile Glu Lys Ile Arg Pro Leu Ala Glu Ser Phe Gly Ile Cys Arg 20 25 30 Ile Val Pro Pro Ser Asn Trp Ser Pro Pro Cys Arg Leu Lys Gly Asp 35 40 45 Ser Ile Trp Lys Asn Lys Asn Phe Pro Thr Arg Val Gln Phe Val Asp 50 55 60 Leu Leu Gln Asn Arg Gly Pro Val Lys Lys Lys Thr Pro Lys Gly Arg 65 70 75 80 Lys Arg Lys Arg Gly Lys Tyr Ser Arg Thr Val Ala Pro Lys Lys Arg 85 90 95 Asn Gly Ser Val Ser Lys Ser Val Ser Thr Pro Lys Ala Thr Glu Glu 100 105 110 Glu Asn Phe Gly Phe Glu Ser Gly Pro Glu Phe Thr Leu Glu Lys Phe 115 120 125 Glu Lys Tyr Ala Gln Asp Phe Lys Asp Ser Tyr Phe Glu Arg Lys Asp 130 135 140 Asn Val Gly Asp Pro Ser Val Glu Glu Ile Glu Gly Glu Tyr Trp Arg 145 150 155 160 Ile Ile Glu Lys Glu Thr Asn Glu Val Lys Val Leu Tyr Gly Thr Asp 165 170 175 Leu Glu Asn Pro Ile Leu Gly Ser Gly Phe Ser Lys Gly Val Lys Ile 180 185 190 Pro Thr Arg Arg Asn Asp Met Asp Lys Tyr Ile Ser Ser Gly Trp Asn 195 200 205 Leu Asn Asn Leu Ala Arg Leu Gln Gly Ser Leu Leu Ser Phe Glu Asp 210 215 220 Cys Glu Ile Ser Gly Val Gln Val Pro Trp Leu Tyr Val Gly Met Cys 225 230 235 240 Phe Ser Thr Phe Cys Trp His Val Glu Asp Asn His Leu Tyr Ser Leu 245 250 255 Asn Tyr His His Phe Gly Glu Pro Lys Val Trp Tyr Gly Val Pro Gly 260 265 270 Ser His Ala Thr Gly Leu Glu Lys Ala Met Arg Lys His Leu Pro Asp 275 280 285 Leu Phe Asp Glu Gln Pro Asp Leu Leu His Glu Leu Val Thr Gln Phe 290 295 300 Ser Pro Thr Ile Leu Lys Asn Glu Gly Val Pro Val Tyr Arg Ala Val 305 310 315 320 Gln Asn Ala Gly Glu Tyr Val Leu Thr Phe Pro Arg Ala Tyr His Ser 325 330 335 Gly Phe Asn Cys Gly Phe Asn Cys Ala Glu Ala Val Asn Val Ala Pro 340 345 350 Val Asp Trp Leu Ala His Gly Gln Asn Ala 355 36017300PRTDrosophila melanogaster 17 Lys Met Ser Glu Val Pro Arg Ile Lys Val Phe Arg Pro Thr Trp Glu 1 5 10 15 Glu Phe Lys Asp Phe Pro Lys Tyr Val Ala Tyr Met Glu Ser Gln Gly 20 25 30 Ala His Lys Ala Gly Leu Ala Lys Val Val Pro Pro Pro Glu Trp Val 35 40 45 Pro Arg Arg Ser Gly Tyr Ala Asp Leu Asp Ala Leu Asn Val Thr Ile 50 55 60 Pro Ala Pro Ile Cys Gln Val Val Thr Gly Lys Gln Gly Tyr Tyr Gln 65 70 75 80 Gln Ile Asn Ile Gln Lys Lys Pro Leu Thr Val Lys Gln Phe Ser Glu 85 90 95 Leu Ala Ser Thr Glu Arg Tyr Ala Thr Pro Lys His Phe Asp Phe Glu 100 105 110 Asp Leu Glu Arg Lys Tyr Trp Lys Asn Ile Thr Tyr Val Ala Pro Ile 115 120 125 Tyr Gly Ala Asp Val Ser Gly Ser Ile Thr Asp Thr Asp Gln Asp Ser 130 135 140 Trp Asn Ile Asn Arg Leu Gly Thr Ile Leu Asp Tyr Val Asn Lys Asp 145 150 155 160 Tyr Asn Ile Gln Ile Asp Gly Val Asn Thr Ala Tyr Leu Tyr Phe Gly 165 170 175 Met Trp Lys Thr Thr Phe Ala Trp His Thr Glu Asp Met Asp Leu Tyr 180 185 190 Ser Ile Asn Tyr Leu His Phe Gly Ala Pro Lys Thr Trp Tyr Val Val 195 200 205 Pro Pro Glu Cys Gly Arg Lys Leu Glu Lys Val Ala Asn Gln Tyr Phe 210 215 220 Pro Ala Ser Tyr Lys Asn Cys Asn Ala Tyr Leu Arg His Lys Met Thr 225 230 235 240 Leu Ile Ser Pro Gln Ile Leu Lys Gln His Asp Val Pro Val Ser Lys 245 250 255 Ile Thr Gln Glu Ala Gly Glu Ile Met Ile Thr Phe Pro Phe Gly Tyr 260 265 270 His Ala Gly Phe Asn His Gly Phe Asn Cys Ala Glu Ser Thr Asn Phe 275 280 285 Ala Met Glu Arg Trp Ile Glu Tyr Gly Lys Arg Ala 290 295 30018295PRTCaenorhabditis elegans 18 Phe Tyr Pro Thr Met Arg Glu Phe Lys Asn Phe Ser Gln Tyr Ile Lys 1 5 10 15 Lys Ile Glu Gln Asn Gly Gly His Leu Lys Ala Gly Ile Ala Lys Ile 20 25 30 Val Ala Pro Glu Gly Trp Thr Pro Arg Pro Thr Arg Lys Asp Phe Ser 35 40 45 Asp Val Asp Asp Tyr Glu Ile Thr Gln Pro Ala Arg Glu Thr Ile Glu 50 55 60 Ala Thr Glu Lys Pro Gly Ala Tyr Phe Lys Arg Asn Val Thr Cys Arg 65 70 75 80 Arg Lys Met Pro Val Arg Glu Phe Arg Thr Leu Ala Asn Ser Ala Gln 85 90 95 Tyr Arg Asn Pro Arg Pro Asp Leu Lys Gly Ser Glu Ile Glu Lys His 100 105 110 Tyr Phe Asp Asn Ile Leu His Gly Glu Pro Ile Tyr Gly Ala Asp Thr 115 120 125 Glu Gly Ser Phe Tyr Asp Ala Gln Val Glu Glu Trp Asn Met Asn Arg 130 135 140 Leu Gly Thr Ile Leu Glu Asp Thr Asn Tyr Glu Ile Lys Gly Val Asn 145 150 155 160 Thr Val Tyr Leu Tyr Phe Gly Met Tyr Lys Thr Thr Phe Pro Trp His 165 170 175 Ala Glu Asp Met Asp Leu Ser Ile Asn Phe Leu His Phe Gly Ala Pro 180 185 190 Lys Tyr Trp Phe Ala Ile Ser Ser Glu His Ala Asp Arg Phe Glu Arg 195 200 205 Phe Met Ser Gln Gln Phe Ser Tyr Gln Asn Glu Tyr Ala Pro Gln Cys 210 215 220 Lys Ala Phe Leu Arg His Lys Thr Tyr Leu Val Thr Pro Glu Leu Leu 225 230 235 240 Arg Gln Ala Gly Ile Pro Tyr Ala Thr Met Val Gln Arg Pro Asn Glu 245 250 255 Phe Ile Ile Thr Phe Pro Arg Gly Tyr His Met Gly Phe Asn Leu Gly 260 265 270 Tyr Asn Leu Ala Glu Ser Thr Asn Phe Ala Ser Gln Arg Trp Ile Asp 275 280 285 Tyr Gly Lys Asp Ala Val Leu 290 29519306PRTHomo sapiens 19 Met Glu Val Glu Val Glu Ser Pro Leu Asn Pro Ser Cys Lys Ile Met 1 5 10 15 Thr Phe Arg Pro Ser Met Glu Glu Phe Arg Glu Phe Asn Lys Tyr Leu 20 25 30 Ala Tyr Met Glu Ser Lys Gly Ala His Arg Ala Gly Leu Ala Lys Val 35 40 45 Ile Pro Pro Lys Glu Trp Lys Pro Arg Gln Cys Tyr Asp Asp Ile Asp 50 55 60 Asn Leu Leu Ile Pro Ala Pro Ile Gln Gln Met Val Thr Gly Gln Ser 65 70 75 80 Gly Leu Phe Thr Gln Tyr Asn Ile Gln Lys Lys Ala Met Thr Val Lys 85 90 95 Glu Phe Arg Gln Leu Ala Asn Ser Gly Lys Tyr Cys Thr Pro Arg Tyr 100 105 110 Leu Asp Tyr Glu Asp Leu Glu Arg Lys Tyr Trp Lys Asn Leu Thr Phe 115 120 125 Val Ala Pro Ile Tyr Gly Ala Asp Ile Asn Gly Ser Ile Tyr Asp Glu 130 135 140 Gly Val Asp Glu Trp Asn Ile Ala Arg Leu Asn Thr Val Leu Asp Val 145 150 155 160 Val Glu Glu Glu Cys Gly Ile Ser Ile Glu Gly Val Asn Thr Pro Tyr 165 170 175 Leu Tyr Phe Gly Met Trp Lys Thr Thr Phe Ala Trp His Thr Glu Asp 180 185 190 Met Asp Leu Tyr Ser Ile Asn Tyr Leu His Phe Gly Glu Pro Lys Ser 195 200 205 Trp Tyr Ala Ile Pro Pro Glu His Gly Lys Arg Leu Glu Arg Leu Ala 210 215 220 Gln Gly Phe Phe Pro Ser Ser Ser Gln Gly Cys Asp Ala Phe Leu Arg 225 230 235 240 His Lys Met Thr Leu Ile Ser Pro Ser Val Leu Lys Lys Tyr Gly Ile 245 250 255 Pro Phe Asp Lys Ile Thr Gln Glu Ala Gly Glu Phe Met Ile Thr Phe 260 265 270 Pro Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn Cys Ala Glu 275 280 285 Ser Thr Asn Phe Ala Thr Val Arg Trp Ile Asp Tyr Gly Lys Val Ala 290 295 300 Lys Leu 305201359PRTArabidopsis thaliana 20 Met Ala Val Ser Glu Gln Ser Gln Asp Val Phe Pro Trp Leu Lys Ser 1 5 10 15 Leu Pro Val Ala Pro Glu Phe Arg Pro Thr Leu Ala Glu Phe Gln Asp 20 25 30 Pro Ile Ala Tyr Ile Leu Lys Ile Glu Glu Glu Ala Ser Arg Tyr Gly 35 40 45 Ile Cys Lys Ile Leu Pro Pro Leu Pro Pro Pro Ser Lys Lys Thr Ser 50 55 60 Ile Ser Asn Leu Asn Arg Ser Leu Ala Ala Arg Ala Ala Ala Arg Val 65 70 75 80 Arg Asp Gly Gly Phe Gly Ala Cys Asp Tyr Asp Gly Gly Pro Thr Phe 85 90 95 Ala Thr Arg Gln Gln Gln Ile Gly Phe Cys Pro Arg Lys Gln Arg Pro 100 105 110 Val Gln Arg Pro Val Trp Gln Ser Gly Glu Glu Tyr Ser Phe Gly Glu 115 120 125 Phe Glu Phe Lys Ala Lys Asn Phe Glu Lys Asn Tyr Leu Lys Lys Cys 130 135 140 Gly Lys Lys Ser Gln Leu Ser Ala Leu Glu Ile Glu Thr Leu Tyr Trp 145 150 155 160 Arg Ala Thr Val Asp Lys Pro Phe Ser Val Glu Tyr Ala Asn Asp Met 165 170 175 Pro Gly Ser Ala Phe Ile Pro Leu Ser Leu Ala Ala Ala Arg Arg Arg 180 185 190 Glu Ser Gly Gly Glu Gly Gly Thr Val Gly Glu Thr Ala Trp Asn Met 195 200 205 Arg Ala Met Ser Arg Ala Glu Gly Ser Leu Leu Lys Phe Met Lys Glu 210 215 220 Glu Ile Pro Gly Val Thr Ser Pro Met Val Tyr Val Ala Met Met Phe 225 230 235 240 Ser Trp Phe Ala Trp His Val Glu Asp His Asp Leu His Ser Leu Asn 245 250 255 Tyr Leu His Met Gly Ala Gly Lys Thr Trp Tyr Gly Val Pro Lys Asp 260 265 270 Ala Ala Leu Ala Phe Glu Glu Val Val Arg Val His Gly Tyr Gly Glu 275 280 285 Glu Leu Asn Pro Leu Val Thr Phe Ser Thr Leu Gly Glu Lys Thr Thr 290 295 300 Val Met Ser Pro Glu Val Phe Val Lys Ala Gly Ile Pro Cys Cys Arg 305 310 315 320 Leu Val Gln Asn Pro Gly Glu Phe Val Val Thr Phe Pro Gly Ala Tyr 325 330 335 His Ser Gly Phe Ser His Gly Phe Asn Phe Gly Glu Ala Ser Asn Ile 340 345 350 Ala Thr Pro Glu Trp Leu Arg Met Ala Lys Asp Ala Ala Ile Arg Arg 355 360 365 Ala Ala Ile Asn Tyr Pro Pro Met Val Ser His Leu Gln Leu Leu Tyr 370 375 380 Asp Phe Val Leu Ala Leu Gly Ser Arg Val Pro Thr Ser Ile Asn Pro 385 390 395 400 Lys Pro Arg Ser Ser Arg Leu Lys Asp Lys Ala Arg Ser Glu Gly Glu 405 410 415 Arg Leu Thr Lys Lys Leu Phe Val Gln Asn Ile Ile His Asn Asn Glu 420 425 430 Leu Leu Ser Ser Leu Gly Lys Gly Ser Pro Val Ala Leu Leu Pro Gln 435 440 445 Ser Ser Ser Asp Ile Ser Val Cys Ser Asp Leu Arg Ile Gly Ser His 450 455 460 Leu Ile Thr Asn Gln Glu Asn Pro Ile Gln Leu Lys Cys Glu Asp Leu 465 470 475 480 Ser Ser Asp Ser Val Val Val Asp Leu Ser Asn Gly Leu Lys Asp Thr 485 490 495 Val Ser Val Lys Glu Lys Phe Thr Ser Leu Cys Glu Arg Ser Arg Asn 500 505 510 His Leu Ala Ser Thr Glu Lys Asp Thr Gln Glu Thr Leu Ser Asp Ala 515 520 525 Glu Arg Arg Lys Asn Asp Ala Ala Val Ala Leu Ser Asp Gln Arg Leu 530 535 540 Phe Ser Cys Val Thr Cys Gly Val Leu Ser Phe Asp Cys Val Ala Ile 545 550 555 560 Val Gln Pro Lys Glu Ala Ala Ala Arg Tyr Leu Met Ser Ala Asp Cys 565 570 575 Ser Phe Phe Asn Asp Trp Thr Ala Ala Ser Gly Ser Ala Asn Leu Gly 580 585 590 Gln Ala Ala Arg Ser Leu His Pro Gln Ser Lys Glu Lys His Asp Val 595 600 605 Asn Tyr Phe Tyr Asn Val Pro Val Gln Thr Met Asp His Ser Val Lys 610 615 620 Thr Gly Asp Gln Lys Thr Ser Thr Thr Ser Pro Thr Ile Ala His Lys 625 630 635 640 Asp Asn Asp Val Leu Gly Met Leu Ala Ser Ala Tyr Gly Asp Ser Ser 645 650 655 Asp Ser Glu Glu Glu Asp Gln Lys Gly Leu Val Thr Pro Ser Ser Lys 660 665 670 Gly Glu Thr Lys Thr Tyr Asp Gln Glu Gly Ser Asp Gly His Glu Glu 675 680 685 Ala Arg Asp Gly Arg Thr Ser Asp Phe Asn Cys Gln Arg Leu Thr Ser 690 695 700 Glu Gln Asn Gly Leu Ser Lys Gly Gly Lys Ser Ser Leu Leu Glu Ile 705 710 715 720 Ala Leu Pro Phe Ile Pro Arg Ser Asp Asp Asp Ser Cys Arg Leu His 725 730 735 Val Phe Cys Leu Glu His Ala Ala Glu Val Glu Gln Gln Leu Arg Pro 740 745 750 Phe Gly Gly Ile Asn Leu Met Leu Leu Cys His Pro Glu Tyr Pro Arg 755 760 765 Ile Glu Ala Glu Ala Lys Ile Val Ala Glu Glu Leu Val Ile Asn His 770 775 780 Glu Trp Asn Asp Thr Glu Phe Arg Asn Val Thr Arg Glu Asp Glu Glu 785 790 795 800 Thr Ile Gln Ala Ala Leu Asp Asn Val Glu Ala Lys Gly Gly Asn Ser 805 810 815 Asp Trp Thr Val Lys Leu Gly Val Asn Leu Ser Tyr Ser Ala Ile Leu 820 825 830 Ser Arg Ser Pro Leu Tyr Ser Lys Gln Met Pro Tyr Asn Ser Ile Ile 835 840 845 Tyr Lys Ala Phe Gly Arg Ser Ser Pro Val Ala Ser Ser Pro Ser Lys 850 855 860 Pro Lys Val Ser Gly Lys Arg Ser Ser Arg Gln Arg Lys Tyr Val Val 865 870 875 880 Gly Lys Trp Cys Gly Lys Val Trp Met Ser His Gln Val His Pro Phe 885 890 895 Leu Leu Glu Gln Asp Leu Glu Gly Glu Glu Ser Glu Arg Ser Cys His 900 905 910 Leu Arg Val Ala Met Asp Glu Asp Ala Thr Gly Lys Arg Ser Phe Pro 915 920 925 Asn Asn Val Ser Arg Asp Ser Thr Thr Met Phe Gly Arg Lys Tyr Cys 930 935 940 Arg Lys Arg Lys Ile Arg Ala Lys Ala Val Pro Arg Lys Lys Leu Thr 945 950 955 960 Ser Phe Lys Arg Glu Asp Gly Val Ser Asp Asp Thr Ser Glu Asp His 965 970 975 Ser Tyr Lys Gln Gln Trp Arg Ala Ser Gly Asn Glu Glu Glu Ser Tyr 980 985 990 Phe Glu Thr Gly Asn Thr Ala Ser Gly Asp Ser Ser Asn Gln Met Ser 995 1000 1005 Asp Pro His Lys Gly Ile Ile Arg His Lys Gly Tyr Lys Glu Phe Glu 1010 1015 1020 Ser Asp Asp Glu Val Ser Asp Arg Ser Leu Gly Glu Glu Tyr Thr Val 1025 1030 1035 1040 Arg Ala Cys Ala Ala Ser Glu Ser Ser Met Glu Asn Gly Ser Gln His 1045 1050 1055 Ser Met Tyr Asp His Asp Asp Asp Asp Asp Asp Ile Asp Arg Gln Pro 1060 1065 1070 Arg Gly Ile Pro Arg Ser Gln Gln Thr Arg Val Phe Arg Asn Pro Val 1075 1080 1085 Ser Tyr Glu Ser Glu Asp Asn Gly Val Tyr Gln Gln Ser Gly Arg Ile 1090 1095 1100 Ser Ile Ser Asn Arg Gln Ala Asn Arg Met Val Gly Glu Tyr Asp Ser 1105 1110 1115 1120 Ala Glu Asn Ser Leu Glu Glu Arg Gly Phe Cys Ser Thr Gly Lys Arg 1125 1130 1135 Gln Thr Arg Ser Thr Ala Lys Arg Ile Ala Lys Thr Lys Thr Val Gln 1140 1145 1150 Ser Ser Arg Asp Thr Lys Gly Arg Phe Leu Gln Glu Phe Ala Ser Gly 1155 1160 1165 Lys Lys Asn Glu Glu Leu Asp Ser Tyr Met Glu Gly Pro Ser Thr Arg 1170 1175 1180 Leu Arg Val Arg His Gln Lys Pro Ser Arg Gly Ser Leu Glu Thr Lys 1185 1190 1195 1200 Pro Lys Lys Ile Gly Lys Lys Arg Ser Gly Asn Ala Ser Phe Ser Arg 1205 1210 1215 Val Ala Thr Glu Lys Asp Val Glu Glu Lys Glu Glu Glu Glu Glu Glu 1220 1225 1230 Glu Asn Glu Glu Glu Glu Cys Ala Ala Tyr Gln Cys Asn Met Glu Gly 1235 1240 1245 Cys Thr Met Ser Phe Ser Ser Glu Lys Gln Leu Met Leu His Lys Arg 1250 1255 1260 Asn Ile Cys Pro Ile Lys Gly Cys Gly Lys Asn Phe Phe Ser His Lys 1265 1270 1275 1280 Tyr Leu Val Gln His Gln Arg Val His Ser Asp Asp Arg Pro Leu Lys 1285 1290 1295 Cys Pro Trp Lys Gly Cys Lys Met Thr Phe Lys Trp Ala Trp Ser Arg 1300 1305 1310 Thr Glu His Ile Arg Val His Thr Gly Ala Arg Pro Tyr Val Cys Ala 1315 1320 1325 Glu Pro Asp Cys Gly Gln Thr Phe Arg Phe Val Ser Asp Phe Ser Arg 1330 1335 1340 His Lys Arg Lys Thr Gly His Ser Val Lys Lys Thr Asn Lys Arg 1345 1350 135521476PRTArabidopsis thalianaVARSPLIC(1)...(476) 21 Met Ala Val Ser Glu Gln Ser Gln Asp Val Phe Pro Trp Leu Lys Ser 1 5 10 15 Leu Pro Val Ala Pro Glu Phe Arg Pro Thr Leu Ala Glu Phe Gln Asp 20 25 30 Pro Ile Ala Tyr Ile Leu Lys Ile Glu Glu Glu Ala Ser Arg Tyr Gly 35 40 45 Ile Cys Lys Ile Leu Pro Pro Leu Pro Pro Pro Ser Lys Lys Thr Ser 50 55 60 Ile Ser Asn Leu Asn Arg Ser Leu Ala Ala Arg Ala Ala Ala Arg Val 65 70 75 80 Arg Asp Gly Gly Phe Gly Ala Cys Asp Tyr Asp Gly Gly Pro Thr Phe 85 90 95 Ala Thr Arg Gln Gln Gln Ile Gly Phe Cys Pro Arg Lys Gln Arg Pro 100 105 110 Val Gln Arg Pro Val Trp Gln Ser Gly Glu Glu Tyr Ser Phe Gly Glu 115 120 125 Phe Glu Phe Lys Ala Lys Asn Phe Glu Lys Asn Tyr Leu Lys Lys Cys 130 135 140 Gly Lys Lys Ser Gln Leu Ser Ala Leu Glu Ile Glu Thr Leu Tyr Trp 145 150 155 160 Arg Ala Thr Val Asp Lys Pro Phe Ser Val Glu Tyr Ala Asn Asp Met 165 170 175 Pro Gly Ser Ala Phe Ile Pro Leu Ser Leu Ala Ala Ala Arg Arg Arg 180 185 190 Glu Ser Gly Gly Glu Gly Gly Thr Val Gly Glu Thr Ala Trp Asn Met 195 200 205 Arg Ala Met Ser Arg Ala Glu Gly Ser Leu Leu Lys Phe Met Lys Glu 210 215 220 Glu Ile Pro Gly Val Thr Ser Pro Met Val Tyr Val Ala Met Met Phe 225 230 235 240 Ser Trp Phe Ala Trp His Val Glu Asp His Asp Leu His Ser Leu Asn 245 250 255 Tyr Leu His Met Gly Ala Gly Lys Thr Trp Tyr Gly Val Pro Lys Asp 260 265 270 Ala Ala Leu Ala Phe Glu Glu Val Val Arg Val His Gly Tyr Gly Glu 275 280 285 Glu Leu Asn Pro Leu Val Thr Phe Ser Thr Leu Gly Glu Lys Thr Thr 290 295 300 Val Met Ser Pro Glu Val Phe Val Lys Ala Gly Ile Pro Cys Cys Arg 305 310 315 320 Leu Val Gln Asn Pro Gly Glu Phe Val Val Thr Phe Pro Gly Ala Tyr 325 330 335 His Ser Gly Phe Ser His Gly Phe Asn Phe Gly Glu Ala Ser Asn Ile 340 345 350 Ala Thr Pro Glu Trp Leu Arg Met Ala Lys Asp Ala Ala Ile Arg Arg 355 360 365 Ala Ala Ile Asn Tyr Pro Pro Met Val Ser His Leu Gln Leu Leu Tyr 370 375 380 Asp Phe Val Leu Ala Leu Gly Ser Arg Val Pro Thr Ser Ile Asn Pro 385 390 395 400 Lys Pro Arg Ser Ser Arg Leu Lys Asp Lys Ala Arg Ser Glu Gly Glu 405 410 415 Arg Leu Thr Lys Lys Leu Phe Val Gln Asn Ile Ile His Asn Asn Glu 420 425 430 Leu Leu Ser Ser Leu Gly Lys Gly Ser Pro Val Ala Leu Leu Pro Gln 435 440 445 Ser Ser Ser Asp Ile Ser Val Cys Ser Asp Leu Arg Ile Gly Ser His 450 455 460 Leu Ile Thr Asn Gln Glu Asn Pro Ile Gln Leu Lys 465 470 475224575DNAArabidopsis thaliana 22 cagagagacc taagaaacga gatcaacaaa gcccgtcgtt tttttcgagg aaaagtgaat 60 gaccctttaa gaaaataact ctattggccg gagaaatggt ggagactcgt cgattctctt 120 aaagtttgtt tttttttcct ctcttcaaaa tttttgttct gtttatactc tgtaatggcg 180 ttttaatttg atacctagaa gttaaagtga acaaattttg agagaaagtc tctttttttt 240 gttcttccct gtgtgtgtga gagagagaga gatatggcgg tttcagagca gagtcaagat 300 gtgtttccat ggcttaaatc gttaccggtt gctccagagt tcagacctac tctagcagag 360 tttcaagatc cgatcgctta cattttgaag attgaagaag aagcttctag atatggaatc 420 tgtaaaattc tgcctccact gcctcctcct tccaaaaaaa cctcgatttc taatctcaac 480 cgttctctgg cggcaagagc ggcggcgagg gttcgtgacg gcggctttgg cgcgtgtgat 540 tatgacggtg gtcccacatt cgccacgcgc cagcagcaga tcgggttttg ccctaggaaa 600 cagcgtccag tgcagagacc tgtgtggcag agtggagagg agtactcttt tggtgagttc 660 gagtttaaag cgaagaactt tgagaagaat tatctcaaga aatgtggtaa aaagagtcaa 720 ctctctgccc ttgaaattga aacactttac tggagagcca ctgtggataa acccttctct 780 gttgagtatg ccaatgacat gcctggctcg gcttttatcc ctctgagtct ggctgctgcg 840 aggaggagag agtctggtgg tgaaggagga acagttggtg agaccgcttg gaacatgagg 900 gcaatgtcta gagccgaagg atcattgctt aagttcatga aggaagagat ccctggagtt 960 acatcaccaa tggtgtatgt tgctatgatg tttagttggt ttgcttggca tgtggaggac 1020 catgaccttc atagtctcaa ttacttgcat atgggtgctg gtaagacttg gtacggtgtg 1080 ccaaaggatg ctgctctggc ttttgaggag gttgttaggg ttcatggtta cggtgaagag 1140 ctcaatcctc ttgtgacatt ttctactctt ggtgagaaga caactgtgat gtctcctgaa 1200 gtatttgtta aagccggaat accgtgttgc aggttagtgc aaaatcctgg agagtttgtc 1260 gtcacctttc cgggagctta tcattcggga tttagtcatg gatttaattt tggagaagca 1320 tctaacattg ccactcccga atggttgaga atggctaaag atgctgctat ccggcgagct 1380 gctataaatt accctccaat ggtttctcat ctccagctac tttatgactt tgtattggct 1440 ctaggttcta gagtgccaac aagcatcaat cccaaaccac ggagttctag attaaaagat 1500 aaggcaagaa gcgaaggaga aagattgacc aaaaagctat ttgtgcaaaa cattatccac 1560 aacaacgaat tgctttcttc tctcggaaaa ggatccccag tggcccttct cccacagagt 1620 tcctcagata tatcagtttg ttctgacctg cgaattggat cccatttgat aaccaaccag 1680 gaaaacccaa tccagttaaa gtgtgaggac ttaagttctg atagtgttgt ggttgatctc 1740 agtaacggtt taaaggatac agtttcagtg aaagaaaaat ttacatcttt atgtgaaagg 1800 agcagaaatc acctagcaag cacggagaag gacactcaag aaactctgtc tgatgctgaa 1860 aggaggaaga atgatgcagc tgttgcgctt tcggatcaaa ggcttttctc ttgtgttaca 1920 tgtggagtct taagctttga ttgtgtagct atcgttcaac ctaaagaagc agctgctaga 1980 tatctcatgt ctgcagattg tagcttcttc aatgattgga cagctgcttc tggatctgca 2040 aatcttggtc aggctgcaag atcacttcat cctcaaagca aagagaagca tgatgtaaat 2100 tacttctaca atgttcctgt tcaaactatg gatcattcag tgaagactgg cgatcaaaaa 2160 acttcaacaa cttccccgac aatagcgcat aaagataatg atgttcttgg gatgttagct 2220 tcagcatatg gagactcttc tgattccgag gaagaagatc aaaaaggctt agttacccct 2280 agttccaaag gggaaacaaa aacgtatgat caagaaggtt cagatggcca tgaggaggct 2340 agagatggta gaacttctga ttttaactgc cagagactaa ccagcgaaca gaatgggtta 2400 agcaaaggcg gaaaatcatc acttctggaa atagctttac catttattcc aagatctgat 2460 gacgattcat gtcggttgca cgtgttttgt cttgagcatg ctgcggaagt ggaacagcaa 2520 cttcgtcctt ttggggggat taacttaatg ttactgtgcc atccagagta ccccaggata 2580 gaggctgaag caaagatagt tgccgaagag ctcgtcatca atcacgaatg gaatgatact 2640 gaattcagga atgtgacccg agaggatgag gaaacgattc aggcagcgtt ggataatgtt 2700 gaagctaagg gtgggaacag tgattggacc gtaaaattgg gtgttaacct ttcttacagc 2760 gctattctca gtcgctctcc tctgtacagt aagcagatgc cgtataactc catcatatac 2820 aaggcgttcg gtcgcagctc tccagtagcg agctcaccct cgaaacccaa agtctctggt 2880 aaaagatcgt ccagacagag gaaatatgtt gttggaaaat ggtgtggtaa ggtttggatg 2940 tcacatcagg tgcatccctt tttgctggag caggacttag agggggaaga atctgaaaga 3000 agttgtcatc ttcgagttgc tatggatgag gatgccactg gaaagagatc gtttcctaat 3060 aatgtttcca gggattcgac aacaatgttt ggaagaaagt attgtaggaa gagaaagata 3120 agagcaaagg cggtgccacg caagaagctt acttctttta agagggaaga tggagtttct 3180 gatgacacat cagaagatca ttcttataag cagcaatgga gggcttccgg gaatgaggaa 3240 gagtcttatt ttgagacagg gaacacagct tctggtgatt catcaaatca aatgtctgat 3300 ccgcacaagg gaattatcag acataaaggt tataaagaat ttgagtcaga tgatgaggtt 3360 tcagaccgtt cacttgggga agagtatact gtacgggcat gtgcagcttc agagagctca 3420 atggagaatg gctctcagca ttcaatgtat gaccatgatg atgatgatga tgatatcgac 3480 aggcagccta gggggattcc aaggagccaa cagacaagag tttttaggaa tccagtttca 3540 tatgagtcag aagataatgg cgtttatcag caaagcggaa gaatatccat aagtaatagg 3600 caagctaatc gaatggttgg tgaatatgat tcagcagaga attctttgga ggaacgaggc 3660 ttttgcagta cagggaaaag gcaaaccagg tcaacagcca aacgaatagc aaaaaccaag 3720 acagttcaga gttcgagaga cacaaaaggt cgctttttgc aagaatttgc atctggaaag 3780 aagaatgaag aattggattc atacatggag ggacctagca cacggcttag ggtgagacat 3840 cagaagccgt cgagagggtc tttagaaaca aaaccaaaga agattggtaa gaagagaagt 3900 ggtaatgctt ccttctccag agttgcaact gaaaaagatg tggaggaaaa agaagaagaa 3960 gaagaagaag agaatgagga agaggaatgt gcagcatacc aatgtaacat ggagggttgc 4020 acgatgagtt tcagttcgga aaaacagttg atgttacaca aaagaaacat atgcccaatt 4080 aaaggctgtg gtaaaaactt cttctcacac aagtatttgg ttcaacacca gcgtgttcac 4140 tcagacgacc gtcctctgaa atgtccatgg aagggatgta agatgacgtt caagtgggct 4200 tggtctagaa ccgagcacat aagggttcac acaggcgcta ggccttatgt ttgcgctgaa 4260 ccggattgtg gtcaaacatt caggtttgtc tctgacttca gccggcataa aaggaagacc 4320 ggtcattcgg ttaagaagac caacaaaagg tgatttaggc tctaacatgg aatctatcat 4380 gaagaagatt actccattga tctattagaa gaaaattgga ttgtacgaat attaggggtt 4440 ttctaagtgt gtatgtctct ctggtcctct gtcaattcgg gattataaag tcaaagtctt 4500 aacatttgga atagccattg tttgttgcag acgttttcac tttcagacag attctctctt 4560 tgttgtattc tctat 4575235841DNAArabidopsis thalianavariation(1)...(5841)ein6 mutant gene 23 gtgcaaacgg atttacagag aggacaacaa caaatgtgtg ttagaaatgg cagaatcgta 60 aataataaga aaatcatacc cttttaaaaa ccatttctct ctctaactaa gtgttcttta 120 cttgttttaa tacagagata tcacaatctc ttgctctgcc caaacccatc ccaaccccct 180 aaaaaaaata caattttttc ctctgtttct ttaatccaat aaaaacagag agacctaaga 240 aacgagatca acaaagcccg tcgttttttt cgaggaaaag tgaatgaccc tttaagaaaa 300 taactctatt ggccggagaa atggtggaga ctcgtcgatt ctcttaaagt ttgttttttt 360 ttcctctctt caaaattttt gttctgttta tactctgtaa tggcgtttta atttgatacc 420 tagaagttaa agtgaacaaa ttttgagaga aagtctcttt tttttgttct tccctgtgtg 480 tgtgagagag agagagagat atggcggttt cagagcagag tcaagatgtg tttccatggc 540 ttaaatcgtt accggttgct ccagagttca gacctactct agcagagttt caagatccga 600 tcgcttacat tttgaagatt gaagaagaag cttctagata tggaatctgt aaaattctgc 660 ctccactgcc tcctccttcc aaaaaaacct cgatttctaa tctcaaccgt tctctggcgg 720 caagagcggc ggcgagggtt cgtgacggcg gctttggcgc gtgtgattat gacggtggtc 780 ccacattcgc cacgcgccag cagcagatcg ggttttgccc taggaaacag cgtccagtgc 840 agagacctgt gtggcagagt ggagaggagt actcttttgg tgagttcgag tttaaagcta 900 agaactttga gaagaattat ctcaagaaat gtggtaaaaa gagtcaactc tctgcccttg 960 aaattgaaac actttactgg agagccactg tggataaacc cttctctgtt gagtatgcca 1020 atgacatgcc tggctcggct tttatccctc tgagtctggc tgctgcgagg aggagagagt 1080 ctggtggtga aggaggaaca gttggtgaga ccgcttggaa catgagggca atgtctagag 1140 ccgaaggatc attgcttaag ttcatgaagg aagagatccc tggagttaca tcaccaatgg 1200 tgtatgttgc tatgatgttt agttggtttg cttggcatgt ggaggaccat gaccttcata 1260 gtctcaatta cttgcatatg ggtgctggta agacttggta cggtgtgcca aaggatgctg 1320 ctctggcttt tgaggaggtt gttagggttc atggttacgg tgaagagctc aatcctcttg 1380 gtgagtatga taagtgttat gtgttagtga ctcatagtta ctcaatgtat caattcattg 1440 ttctgattgt tggctttgtt gtgtctctgt tctttttgtt gcttttaaat gcttttcttt 1500 ccctccaaaa gttgtcaggg gtagaagctg tatgcaagca aactaattgg ttatgcctct 1560 tacgttcttg tcttattagt gacattttct actcttggtg agaagacaac tgtgatgtct 1620 cctgaagtat ttgttaaagc cggaataccg tgttgcaggt aatggttttt tatgtttgtg 1680 ctctgtttat ctcatcatct tgttgtttgc atgtctcttc tttggtctct tataattggc 1740 acactttttc ttcttctggt ttttcacgca agtggttttt ttgtatcagg ttagtgcaaa 1800 atcctggaga gtttgtcgtc acctttccgg gagcttatca ttcgggattt agtcatggtg 1860 agtaagcgac ctatatttag tatctttaag tcaaccattc aaaggcctag tatgttgaaa 1920 taacttatat tatccataaa aacaatcgtg ggggttgtac aggatttaat tttggagaag 1980 catctaacat tgccactccc gaatggttga gaatggctaa agatgctgct atccggcgag 2040 ctgctataaa ttaccctcca atggtttctc atctccagct actttatgac tttgtattgg 2100 ctctaggttc taggtactct ttcttttcta tagacacact caaacttttt aagctgcttg 2160 aagggctcat ataaggtatg tttcactcat tgcagagtgc caacaagcat caatcccaaa 2220 ccacggagtt ctagattaaa agataaggca agaagcgaag gagaaagatt gaccaaaaag 2280 ctatttgtgc aaaacattat ccacaacaac gaattgcttt cttctctcgg aaaaggatcc 2340 ccagtggccc acagagttcc tcagatatat cagtttgttc tgacctgcga attggatccc 2400 atttgataac caaccaggaa aacccaatcc agttaaagtg tgaggactta agttctgata 2460 gtgttgtggt tgatctcagt aacggtttaa aggatacagt ttcagtgaaa gaaaaattta 2520 catctttatg tgaaaggagc agaaatcacc tagcaagcac ggagaaggac actcaagaaa 2580 ctctgtctga tgctgaaagg aggaagaatg atgcagctgt tgcgctttcg gatcaaaggc 2640 ttttctcttg tgttacatgt ggagtcttaa gctttgattg tgtagctatc gttcaaccta 2700 aagaagcagc tgctagatat ctcatgtctg cagattgtag cttcttcaat gattggacag 2760 ctgcttctgg atctgcaaat cttggtcagg ctgcaagatc acttcatcct cgtaagttac 2820 aggtcgcact ttcggttatt ggctgttatt tagcatttac taactttttt tcatcatgca 2880 gaaagcaaag agaagcatga tgtaaattac ttctacaatg ttcctgttca aactatggat 2940 cattcagtga agactggcga tcaaaaaact tcaacaactt ccccgacaat agcgcataaa 3000 gataatgatg ttcttgggat gttagcttca gcatatggag actcttctga ttccgaggaa 3060 gaagatcaaa aaggcttagt tacccctagt tccaaagggg aaacaaaaac gtatgatcaa 3120 gaaggttcag atggccatga ggaggctaga gatggtagaa cttctgattt taactgccag 3180 agactaacca gcgaacagaa tgggttaagc aaaggcggaa aatcatcact tctggaaata 3240 gctttaccat ttattccaag atctgatgac gattcatgtc ggttgcacgt gttttgtctt 3300 gagcatgctg cggaagtgga acagcaactt cgtccttttg gggggattaa cttaatgtta 3360 ctgtgccatc caggtaacat caaaactaca agattcattt tattagtatt tctgtatcag 3420 ttatgcaatt ctttagtctt tttcatcatt ttgacgcaca acatttgcag agtaccccag 3480 gatagaggct gaagcaaaga tagttgccga agagctcgtc atcaatcacg aatggaatga 3540 tactgaattc aggaatgtga cccgagagga tgaggaaacg attcaggcag cgttggataa 3600 tgttgaagct aagggtggga acagtgattg gaccgtaaaa ttgggtgtta acctttctta 3660 cagcgctatt ctcagtcgct ctcctctgta cagtaagcag atgccgtata actccatcat 3720 atacaaggcg ttcggtcgca gctctccagt agcgagctca ccctcgaaac ccaaagtctc 3780 tggtaaaaga tcgtccagac agaggaaata tgttgttgga aaatggtgtg gtaaggtttg 3840 gatgtcacat caggtactat tgtctaacct gaatccatac taattcagac attagtacac 3900 ttctgtttgg ttcaatttgc ttttctttct tcaggtgcat ccctttttgc tggagcagga 3960 cttagagggg gaagaatctg aaagaagttg tcatcttcga gttgctatgg atgaggatgc 4020 cactggaaag agatcgtttc ctaataatgt ttccagggat tcgacaacaa tgtttggaag 4080 aaagtattgt aggaagagaa agataagagc aaaggcggtg ccacgcaaga agcttacttc 4140 ttttaagagg gaagatggag tttctgatga cacatcagaa gatcattctt ataagcagca 4200 atggagggct tccgggaatg aggaagagtc ttattttgag acagggaaca cagcttctgg 4260 tgattcatca aatcaaatgt ctgatccgca caagggaatt atcagacata aaggttataa 4320 agaatttgag tcagatgatg aggtttcaga ccgttcactt ggggaagagt atactgtacg 4380 ggcatgtgca gcttcagaga gctcaatgga gaatggctct cagcattcaa tgtatgacca 4440 tgatgatgat gatgatgata tcgacaggca gcctaggggg attccaagga gccaacagac 4500 aagagttttt aggaatccag tttcatatga gtcagaagat aatggcgttt atcagcaaag 4560 cggaagaata tccataagta ataggcaagc taatcgaatg gttggtgaat atgattcagc 4620 agagaattct ttggaggaac gaggcttttg cagtacaggg aaaaggcaaa ccaggtcaac 4680 agccaaacga atagcaaaaa ccaagacagt tcagagttcg agagacacaa aaggtcgctt 4740 tttgcaagaa tttgcatctg gaaagaagaa tgaagaattg gattcataca tggagggacc 4800 tagcacacgg cttagggtga gacatcagaa gccgtcgaga gggtctttag aaacaaaacc 4860 aaagaagatt ggtaagaaga gaagtggtaa tgcttccttc tccagagttg caactgaaaa 4920 agatgtggag gaaaaagaag aagaagaaga agaagaagag aatgaggaag aggaatgtgc 4980 agcataccaa tgtaacatgg agggttgcac gatgagtttc agttcggaaa aacagttgat 5040 gttacacaaa agaaacatat gcccaattaa aggctgtggt aaaaacttct tctcacacaa 5100 gtatttggtt caacaccagc gtgttcactc agacgaccgt cctctgaaat gtccatggaa 5160 gggatgtaag atgacgttca agtgggcttg gtctagaacc gagcacataa gggttcacac 5220 aggcgctagg ccttatgttt gcgctgaacc ggattgtggt caaacattca ggtttgtctc 5280 tgacttcagc cggcataaaa ggaagaccgg tcattcggtt aagaagacca acaaaaggtg 5340 atttaggctc taacatggaa tctatcatga agaagattac tccattgatc tattagaaga 5400 aaattggatt gtacgaatat taggggtttt ctaagtgtgt atgtctctct ggtcctctgt 5460 caattcggga ttataaagtc aaagtcttaa catttggaat agccattgtt tgttgcagac 5520 gttttcactt tcagacagat tctctctttg ttgtattctc tatttttgca gttgaatgaa 5580 tctgtaacaa caacaaatcc tggtgtagga aaagcattgt gttactcaag tatttgacat 5640 atatggttgt atacgtggct tatcctggtt ggttgacagt cagagttcat cgccgtggac 5700 ttcatttgac tttgataaag actcaaacct accaaattgc taatgaatga cattacctgt 5760 gtcaactagg atgtaaaact ctctgacttc agcacacggc acaaacgaaa gaccaaatga 5820 cttaacattt ggaattgcca t 5841

Claims (22)

What is claimed is:

1. An isolated polynucleotide comprising a nucleic acid sequence that hybridizes to the sequence of SEQ ID NO: 22 under high stringency and encodes an EIN6 protein.

2. The isolated polynucleotide ofclaim 1, wherein said polynucleotide comprises the sequence shown in SEQ ID NO: 23.

3. The isolated polynucleotide ofclaim 1, wherein said polynucleotide comprises the sequence shown in SEQ ID NO: 22.

4. The isolated polynucleotide ofclaim 1, wherein said EIN6 protein comprises the amino acid sequence shown in SEQ ID NO: 20.

5. An isolated polynucleotide comprising a nucleotide sequence having at least 80% homology to SEQ ID NO: 22.

6. The polynucleotide ofclaim 5, wherein the polynucleotide has at least 85% homology to SEQ ID NO: 22.

7. The polynucleotide ofclaim 5, wherein the polynucleotide has at least 90% homology to SEQ ID NO: 22.

8. The polynucleotide ofclaim 5, wherein the polynucleotide has at least 95% homology to SEQ ID NO: 22.

9. An isolated polypeptide having at least 80% homology to SEQ ID NO: 20.

10. The polypeptide ofclaim 9, wherein the polypeptide has at least 85% homology to SEQ ID NO: 20.

11. The polypeptide ofclaim 9, wherein the polypeptide has at least 90% homology to SEQ ID NO: 20.

12. The polypeptide ofclaim 9, wherein the polypeptide has at least 95% homology to SEQ ID NO: 20.

13. The polypeptide ofclaim 9, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

14. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 21.

15. A method for making a plant insensitive to ethylene, comprising:

contacting plant cells with a vector that disrupts the EIN6 gene, wherein the EIN6 gene comprises the nucleic acid sequence of SEQ ID NO: 22;

producing a plant from the plant cells; and

selecting plants that a found to be insensitive to ethylene.

16. The method ofclaim 15, wherein said plant is a dicotyledonous plant.

17. The method ofclaim 15, wherein the plant is a monocotyledonous plant.

18. The method ofclaim 15, wherein said vector comprises the nucleic acid sequence shown in SEQ ID NO: 22.

19. The method ofclaim 15, comprising contacting the plant cells with a vector that disrupts the een gene.

20. The method ofclaim 15, wherein the contacting is by physical means.

21. The method ofclaim 15, wherein the contacting is by chemical means.

22. The method ofclaim 15, wherein the plant cell is selected from the group consisting of protoplasts, gamete producing cells, and cells which regenerate into a whole plant.

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