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Compositions and methods for enhancing oligonucleotide-directed nucleic acid sequence alterationRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidCompositions and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070122822, Compositions and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of priority from United States Application No. 60/326,041, filed Sep. 27, 2001; United States Application No. 60/337,129, filed Dec. 4, 2001 and United States Application No. 60/393,330, filed Jul. 1, 2002, the disclosures of which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION [0002] This invention relates to oligonucleotide-directed repair or alteration of nucleic acid sequence and methods and compositions for enhancing the efficiency of such alteration. [0003] 1. Background of the Invention [0004] A number of different poly- and oligo-nucleotides have been described for use in targeted alteration of nucleic acid sequence including chimeric RNA-DNA oligonucleotides that fold into a double-stranded, double hairpin conformation and single-stranded chemically modified oligonucleotides. These oligonucleotides have been shown to effect targeted alteration of single base pairs as well as frameshift alterations in a variety of host organisms, including bacteria, fungi, plants and animals. [0005] Several cellular pathways and gene groups are believed to be involved in mediating in vivo repair of DNA lesions resulting from radiation or chemical mutagenesis, including the RAD52 epistasis group of proteins, the mismatch repair group of proteins and the nucleotide excision repair group of proteins. The role of these proteins in homologous recombination and maintaining genome integrity has been extensively studied and is reviewed, for example, in Heyer, Experientia 50(3), 223-233 (1994); Thacker, Trends in Genetics 15(5), 166-168 (1999); Paques & Haber, Microbiol. and Molec. Biol. Rev. 63(2), 349-404 (1999); and Thompson & Schild, Mutation Res. 477,131-153 (2001). The specific function of these proteins in oligonucleotide-directed nucleic acid sequence alteration is not well understood. [0006] The utility of oligonucleotide-directed nucleic acid sequence alteration as a means, for example, to generate agricultural products with enhanced traits or to generate animal models or animals with desired traits is affected by its frequency. The utility of oligonucleotide-directed nucleic acid sequence alteration as a therapeutic method would also be enhanced. by increasing its efficiency. A need exists for methods to enhance the efficiency of oligonucleotide-directed. nucleic acid sequence alteration. SUMMARY OF THE INVENTION [0007] The present invention concerns methods and compositions for enhancing oligonucleotide-directed nucleic acid sequence alteration in vivo, ex vivo, and in vitro. Without being limited by theory, it is believed that certain DNA repair proteins are involved in oligonucleotide-directed nucleic acid sequence alteration. However, because these proteins function through multiple complex interactions and because oligonucleotide-directed nucleic acid sequence alteration relies on the activity of molecules that have alternative chemistries as compared to products resulting from radiation or chemical mutagens, whether or how any of these proteins would be involved was unknown and unpredictable prior to this invention. [0008] The invention involves methods of targeted nucleic acid sequence alteration using cells or extracts with altered levels or activity of at least one protein from the RAD52 epistasis group, the mismatch repair group or the nucleotide excision repair group. Generally, in instances where genes are identified herein by gene name, e.g., RAD52, the allele represented is one which is recognized as having normal,. wild-type activity identified using the. yeast (Saccharomyces cerevisiae) nomenclature. Where the. genes have a mutation resulting in reduced or eliminated activity, the gene will be so identified in writing or will be italicized and in lowercase, e.g. rad52. Where a mutation is a deletion of the gene it may also be represented with a Greek letter delta, i.e. .DELTA.. Although these designations use the yeast nomenclature, it is to be understood that homologous proteins, e.g. homologs, orthologs and paralogs, from other organisms, including bacteria, plants, animals and other fungi may be used in the methods of the instant invention. Members of these groups include: RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2 in the RAD52 epistasis group; MSH2, MSH3, MSH6 and PMS1 in the mismatch repair group; and RAD1, RAD2, RAD10, RAD23 and EXO1 in the nucleotide excision repair group. [0009] In another aspect, the invention relates to kits comprising a cell or cell-free extract with increased levels of at least one of the normal allelic RAD10, RAD51, RAD52, RAD54, RAD55, MRE1, PMS1 or XRS2 proteins or with increased activity of one of these proteins. In further embodiments, the kits comprise a cell or cell-free extract with increased levels or activity of at least one of the RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 or XRS2 proteins and decreased levels or activity of at least one protein selected from the group consisting of RAD1, RAD51, RAD52, RAD57 or PMS1. In other embodiments, the kits further comprise at least one oligonucleotide capable of directing nucleic acid sequence alteration. In some embodiments, the kits may include multiple oligonucleotides. [0010] In different embodiments, the invention relates to kits comprising at least one oligonucleotide capable of directing nucleic acid sequence alteration and at least one purified protein selected from the group consisting of the RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 or XRS2 proteins. Other kits may comprise. multiple oligonucleotides. [0011] In still other embodiments, the present invention relates to processes to alter plant genomes by administering to a plant cell or tissue at least one oligonucleotide having a desired nucleic acid sequence alteration sequence, wherein the plant cell or tissue has altered levels and/or activity of a protein encoded by a plant which is homologous to a RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 or XRS2 gene. The plant cell can be used to generate plants which are a further embodiment of the invention. The invention further relates to methods for genetically altering plants to enhance or generate desirable traits, for example, herbicide or pest resistance. [0012] In a further embodiment, the present invention relates to a process to genetically alter animals, particularly livestock, to enhance expression of desirable traits, comprising administering to a target cell at least one oligonucleotide having a nucleic acid sequence alteration sequence, wherein the cell has altered levels and/or activity of a protein encoded by a gene homologous to the RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 or XRS2 gene from yeast and the animals produced thereby. [0013] In a further embodiment, the present invention relates to an assay to identify activators of RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 and/or XRS2 protein activity and/or one or more activators of RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 or XRS2 gene expression comprising contacting a sample with an oligonucleotide in a system known to provide for nucleic acid sequence alteration and measuring whether the amount of nucleic acid sequence alteration is less, more, or the same as in the absence of sample. In another embodiment this assay may be used to identify mutations in the genes encoding the RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1, or XRS2 protein that increase or decrease their activity. In some embodiments of the invention, the RAD1 0, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1 or XRS2 gene product is increased in amount or activity. In other embodiments of the invention, the RAD10, RAD51, RAD52, RAD54, MRE11, PMS1 or XRS2 gene product is increased in amount or activity by complementation or supplementation and the complemented or supplemented cell contains a knock-out mutation of the RAD52 gene. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIG. 1. Genetic readout system for alteration of a point mutation in plasmid pK.sup.sm4021. A mutant kanamycin gene harbored in plasmid pK.sup.sm4021 is the target for alteration by oligonucleotides. The mutant G is converted to a C by the action of the oligonucleotide. Corrected plasmids confer resistance to kanamycin in E. coli (DH10B) after electroporation leading to the genetic readout and colony counts. The sequence of chimeric, RNA-DNA double-hairpin oligonucleotide KanGG is shown (SEQ ID NO:1). [0015] FIG. 2. Hygromycin-eGFP target plasmids. Diagram of plasmid pAURHYG(x)eGFP. Plasmid pAURHYG(rep)eGFP contains a base substitution mutation introducing a G at nucleotide 137, at codon 46, of the Hygromycin B coding sequence (cds). Plasmid pAURHYG(ins)eGFP contains a single base insertion mutation between nucleotides 136 and 137, at codon 46, of the Hygromycin B coding sequence (cds) which is transcribed from the constitutive ADH1 promoter. Plasmid pAURHYG(.DELTA.)eGFP contains a deletion mutation removing a single nucleotide at codon 46, of the Hygromycin B coding sequence (cds). The target sequence presented below indicates the deletion of an A and the substitution of a C for a T directed by the oligonucleotides to re-establish the resistant phenotype. The target sequence presented below the diagram indicates the amino acid conservative replacement of G with C, restoring gene function. The sequences of the normal hygromycin resistance allele (SEQ ID NO:2) and the desired allele after nucleic acid sequence alteration (SEQ ID NO:3) are shown next to the mutant alleles present in pAURHYG(rep)eGFP (SEQ ID NO: 4), pAURHYG(ins)eGFP (SEQ ID NO:5) and pAURHYG(.DELTA.)eGFP (SEQ ID NO:6). The position of the deletion in the pAURHYG(.DELTA.)eGFP allele is indicated with the symbol .DELTA.. [0016] FIG. 3. Oligonucleotides for alteration of hygromycin resistance gene. The sequence of the oligonucleotides used in experiments to assay alteration of a hygromycin resistance gene are shown. DNA residues are shown in capital letters, RNA residues are shown in lowercase and nucleotides with a phosphorothioate backbone are capitalized and underlined. In FIG. 3, the sequence of HygE3T/25 corresponds to SEQ ID NO: 7, the sequence of HygE3T/74 corresponds to SEQ ID NO:8, the sequence of HygE3T/74a corresponds to SEQ ID NO: 9, the sequence of HygGG/Rev corresponds to SEQ ID NO:10 and the sequence of Kan70T corresponds to SEQ ID NO:11. [0017] FIG. 4. pAURNeo(-)FIAsHplasmid. This figure describes the plasmid structure, target sequence, oligonucleotides, and the basis for detection of the nucleic acid sequence alteration event by fluorescence. In FIG. 9, the sequence of the Neo/kan target mutant corresponds to SEQ ID NO:12 and SEQ ID NO:13, the converted sequence corresponds to SEQ ID NO:14 and SEQ ID NO:15 and the FIAsH peptide sequence corresponds to SEQ ID NO:16. [0018] FIG. 5. Fluorescent microscopy figures of targeting in the FIAsH system. This figure shows confocal microscopy of yeast strains before and after transfection by DNA/RNA CO kanGGrv. Converted yeast cells are indicated by bright green fluorescence. (A) Upper left: wild type, nontargeted. Upper right: .DELTA.rad52, nontargeted. (C) Lower left: wild type, targeted. (D) Lower right: .DELTA.rad52, targeted. [0019] FIG. 6. pYESHyg(x)eGFPplasmid. This plasmid is a construct similar to the pAURHyg(x)eGFP construct shown in FIG. 7, except the promoter is the inducible GALL promoter. This promoter is inducible with galactose, leaky in the presence of raffinose, and repressed in the presence of dextrose. [0020] FIG. 7. pyN132plasmid. This figure shows the plasmid structure including the constitutive promoter from the TPL1 gene, which directs expression of the cDNA cloned downstream. Continue reading about Compositions and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration... 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