Targeted Integration And Stacking Of DNA Through Homologous Recombination

QUE; Qiudeng

Patent Application Summary

U.S. patent application number 13/214002 was filed with the patent office on 2012-01-05 for targeted integration and stacking of dna through homologous recombination. This patent application is currently assigned to Syngenta Participations AG. Invention is credited to Qiudeng QUE.

Application Number20120005789 13/214002
Document ID /
Family ID37395458
Filed Date2012-01-05

United States Patent Application 20120005789
Kind Code A1
QUE; Qiudeng January 5, 2012

Targeted Integration And Stacking Of DNA Through Homologous Recombination

Abstract

The invention provides various methods for the targeted integration and stacking of nucleotide sequences in the genome of a host plant cell using homologous recombination.


Inventors: QUE; Qiudeng; (Research Triangle Park, NC)
Assignee: Syngenta Participations AG
Basel
CH

Family ID: 37395458
Appl. No.: 13/214002
Filed: August 19, 2011

Related U.S. Patent Documents

Application Number Filing Date Patent Number
13030809 Feb 18, 2011
13214002
11001346 Dec 1, 2004 7935862
13030809
60526155 Dec 2, 2003

Current U.S. Class: 800/298 ; 435/419; 435/468
Current CPC Class: C12N 15/8213 20130101
Class at Publication: 800/298 ; 435/468; 435/419
International Class: A01H 5/00 20060101 A01H005/00; C12N 5/10 20060101 C12N005/10; C12N 15/87 20060101 C12N015/87

Claims



1-14. (canceled)

15. A method of preparing a host cell having a genome with a target site for reiterative gene stacking in the host cell genome, the method comprising the steps of: (a) providing a host cell having a genome comprising a target sequence comprising: (i) a truncated functional sequence; and (ii) a host homology sequence; (b) introducing into the host cell a donor sequence comprising: (i) a heterologous sequence of interest or fragment thereof; (ii) a donor homology sequence homologous to the host homology sequence; (iii) a sequence that completes the truncated functional sequence located between the heterologous sequence of interest or fragment thereof and the donor homology sequence; (iv) a first recombinase recognition site located between the donor homology sequence and the sequence that completes the truncated functional sequence; and (v) a second recombinase recognition site located between the sequence that completes the truncated functional sequence and the heterologous sequence of interest or fragment thereof; and further wherein the first and second recombinase recognition sites are oriented relative to one another such that the sequence that completes the truncated functional sequence is excisable in the presence of a recombinase; (c) obtaining in the host cell a recombination product comprising: (i) the heterologous sequence of interest; (ii) a recombined sequence resulting from homologous recombination of the host homology sequence and the donor homology sequence; (iii) a restored functional sequence comprising the truncated functional sequence, the sequence that completes the truncated functional sequence, the recombined sequence located between the truncated functional sequence and the sequence that completes the truncated functional sequence, and the first recombinase recognition site located between the sequence that completes the truncated functional sequence and the recombined sequence; and (iv) the second recombinase recognition site located between the heterologous sequence of interest and the restored functional sequence.

16. The method of claim 15, wherein the restored functional sequence encodes a marker.

17. The method of claim 16, wherein the marker is selected from the group consisting of NPTII, HPT, PAT, BAR, EPSPS, GAT, HPPD, ALS, PPO, PMI, GUS, LUC, GFP, RFP, and CFP.

18. A host cell produced by the method of claim 15.

19. The host cell of claim 18, which is a plant cell.

20. A plant or plant part comprising the plant cell of claim 19.

21. The method of claim 15, wherein the target sequence of step (a) further comprises (iii) a second host homology sequence, and wherein the host homology sequence of (a)(ii) is located between the second host homology sequence and the truncated functional sequence; and wherein the donor sequence of step (b) further comprises (vi) a second donor homology sequence having homology to the second host homology sequence, and wherein the heterologous sequence of interest is located between the second donor homology sequence and the second recombinase recognition site; and wherein the recombination product of step (c) further comprises (v) a second recombined sequence resulting from homologous recombination of the second host homology sequence and the second donor homology sequence, and wherein the heterologous sequence of interest of step (c)(1) is located between the second recombined sequence and the second recombinase recognition site.

22. A host cell produced by the method of claim 21.

23. The host cell of claim 22, which is a plant cell.

24. A plant or plant part comprising the plant cell of claim 23.

25. The method of claim 15, further comprising: (d) introducing into the host cell of step (c) a recombinase or recombinase coding sequence to thereby excise the sequence that completes the truncated functional sequence and yield an excision product comprising: (i) the heterologous sequence of interest; (ii) the truncated functional sequence; (iii) the recombined sequence located between the heterologous sequence of interest and the truncated functional sequence; and (iv) a regenerated target recombinase recognition site located between the heterologous sequence of interest and the recombined sequence.
Description



FIELD OF THE INVENTION

[0001] The present invention relates generally to transgenic plants and, more particularly, to site-specific integration and stacking of nucleotide sequences in the genome of a host cell through homologous recombination.

BACKGROUND

[0002] In recent years, the development of genetic engineering techniques has had dramatic implications in the field of crop improvement. Using these techniques, beneficial traits can be introduced into almost any crop and improved crops can be rapidly obtained. The use of genetic engineering obviates the need for lengthy procedures to introduce the desired trait by conventional breeding methods.

[0003] Present plant transformation methods generally integrate a single transgene into the host genome. Successful integration of each transgene requires repeated confrontation of various issues, such as variability in transgene expression caused by different integration loci, so-called "positions effects," and the risk of creating a mutation in the genome upon integration of the transgene into the host. Consequently, a large number of transformation events must be screened and tested before obtaining a transgenic plant that exhibits the desired level of transgene expression without also exhibiting abnormalities resulting from the inadvertent insertion of the transgene into an important locus in the host genome. Moreover, if an additional transgene is subsequently added to a transgenic plant, the additional transgene likely will be integrated into the genome at a location that is different from the location of the pre-existing transgene, rendering the breeding of elite plant lines with both genes difficult and cumbersome.

[0004] An inherent problem with such single-round integration techniques is that sequence stacking, or the successive integration of multiple nucleotide sequences at a predetermined locus in the host genome, is difficult to accomplish. However, efficient sequence stacking is desirable for a variety of reasons. For example, the ability to achieve targeted insertion of multiple transgenes into a host would facilitate registration of a transgenic plant with government regulatory agencies, since the potential for random alteration of the plant's genetic material would be minimized. Further, in some cases, such as the engineering of traits or metabolic pathways that involve multiple genes, for example, co-location of the transgenes would be highly desirable. Additionally, since only a limited number of selectable and scoreable marker sequences may be available for use in transforming a given crop, the ability to re-use a marker sequence when introducing successive nucleotide sequences into the host genome would also be desirable.

SUMMARY

[0005] The present disclosure provides methods for the targeted integration and stacking of nucleotide sequences in the genome of a host cell using homologous recombination. A target sequence in the genome of a host cell and a donor sequence introduced into the host cell each comprises a homology sequence that permits homologous recombination to occur between the target and donor sequences. In one embodiment, a homology sequence shared by a target sequence and a donor sequence comprises at least one intron sequence that lengthens the region of homology and thereby enhances the frequency of homologous recombination between the target and donor sequences.

BRIEF DESCRIPTION OF THE FIGURES

[0006] FIGS. 1-12B illustrate various exemplary embodiments of the invention.

[0007] FIGS. 13A-13F are schematic representations of a modified nptII gene with multiple introns, target and donor DNA constructs, endonucleases, and a FLP expression construct. FIG. 13A: A schematic representation of a modified nptII gene with four Arabidopsis intron insertions (i.e., the nptII-intron gene sequence). "FRT" indicates a FLP recognition sequence. FIG. 13B: A positive control construct (pNOV2731) containing the full-length nptII-intron. "Phsp80" indicates an HSP80 promoter; "BAR" indicates a Basta.RTM. herbicide resistance gene; "Tnos" indicates a nos terminator; "Pmsmas" indicates a modified SMAS promoter; "Tpal" indicates the Arabidopsis PAL1 terminator. FIG. 13C: A target DNA construct (pNOV2701) containing the modified nptII gene truncated at the 5'-region. FIG. 13D: Donor DNA constructs (pNOV2736, pNOV2737, pNOV2755, pNOV2757) containing the nptII-intron gene truncated at different places in the 3'-coding region. "Hpt" indicates a hygromycin phosphotransferase gene; hpt includes the Arabidopsis ubq3 promoter and terminator. FIG. 13E: A yeast HO endonuclease expression cassette. FIG. 13F: A FLP recombinase expression vector (pNOV2762) with Arabidopsis PPO(dm) as a selectable marker. PPO(dm) is under the control of its native Arabidopsis ptx promoter.

[0008] FIGS. 14A-14C illustrate PCR screening and analysis of targeted events. FIG. 14A: A schematic representation of a target locus derived from pNOV2701, T-DNA of donor pNOV2736, a recombination product, and PCR primers. Striped boxes represent genomic DNA sequences flanking the T-DNA insertion. FIG. 14B: PCR analysis of events targeted to a predetermined location in the genome of tobacco line T2701.06 using PSMASFW2 and NPTR6 primers. Targeted events produce a 3.5 kb fragment. "M" indicates a DNA size marker (i.e., Lambda DNA digested with StyI (19.3, 7.7, 6.2, 3.5, 2.7, 1.9, 1.5, 0.9, 0.4 kb). Lane 1, negative control, untransformed SR1 tobacco; lane 2, positive control T2731.1; lane 3, negative control, target line T2701.6; lane 4, HR-01AB.1; lane 5, HR-01AB.2; lane 6, HR-01AB.3; lane 7, HR-01AC.1; lane 8, HR-01AD.1; lane 9, HR-01AD.4; lane 10, HR-01AE.1; lane 11, HR-01AE.2. FIG. 14C: PCR amplification of targeted events with primers from flanking genomic DNA sequences. "M" is a DNA ladder (10, 8, 6, 5, 4, 3, 2, 1.5, 1.0, 0.5 kb; 3 kb band has the strongest signal, New England Biolab, Beverly Mass.). Lanes 1 to 5 with PDFSP1 and HYGRV1 primers; lane 1, HR-01AB.1; lane 2, HR-03AB.1, lane 3, HR-03AD.2, lane 4, HR-05AA.2; lane 5, HR-02AC.1. Lanes 6 to 9 with PDFSP1 and PALEXONV primers; lane 6, HR-01AB.1; lane 7, HR-01AB.1.times.SR1 kanamycin resistant progeny; lane 8, HR-03AB.1.times.SR1 kanamycin resistant progeny, lane 9, HR-03AD.2.

[0009] FIGS. 15A-15C represent a Southern blot analysis of targeted events. FIG. 15A: A schematic representation of target and donor vectors, restriction sites, and probes. FIG. 15B A blot probed with an HSP80 promoter fragment. "M" is a DNA marker (Lambda DNA digested with StyI). Lanes 1-4, target line T2701.6; lanes 5-8, HR-03AD.2; lanes 9-11, HR-05AA.1; lanes 12-14, HR-05AA.2. Lanes 1, 5, 9, 12 with EcoRV; Lanes 2, 6, 10, 13 with SacI; Lanes 3, 7, 11, 14 with NheI; Lanes 4 and 8 with SpeI. FIG. 15C: The same blot was stripped and re-probed with the nptII exon 5::Pall 3'-UTR fragment.

[0010] FIGS. 16A-16B represent a PCR analysis of recombinant lines that have been re-transformed with a FLP expression vector. FIG. 16A: Tubq3fw and NptR3 primers were used for PCR amplification of lines obtained from HR-03AD.2 progeny re-transformed with pNOV2762. The 1.5 kb band indicates excision of the mSMAS promoter and part of the nptII-intron sequence. FIG. 16B: Tubq3fw and NptR2 primers are used for PCR amplification of progeny of HR-08AA32R2.times.SR1. The 932 bb band indicates excision of the mSMAS promoter and part of the nptII-intron sequence. Lane 1, recombinant HR-03AD.2 control; lanes 2-4, progeny with complete excision of the mSMAS promoter and part of the nptII-intron sequence.

[0011] FIGS. 17A-17B illustrate a PMI-intron gene, a monocot target DNA construct, a donor DNA construct, and a positive control vector. FIG. 17A: A schematic drawing showing a PMI-intron gene sequence, the T-DNA region of monocot target vector pNOV5025, pAdF55, and the positive control vector pNOV5026. "SRRS" indicates a site-specific recombinase recognition sequence. "OsAct1" is a rice actin 1 promoter; "Hpt" is a hygromycin phsosphotransferase gene; "CMPS" is a Cestrum virus promoter; "ZmUbi" indicates a maize ubiquitin promoter; "GUS" is .beta.-glucuronidase gene; "PPO" is a mutant Arabidopsis protoporphyrin oxidase gene. FIG. 17B: Donor vectors pNOV5031, pNOV5045, pNOV5096, and pQD200C6.

[0012] FIG. 18 illustrates an exemplary embodiment of transgene targeting in maize.

[0013] FIGS. 19A-19D represent restriction map and fragment sizes of target locus AW289B1A, T-DNA of donor vector pNOV5045, and putative double crossover recombinant with different probes. The change in size of each restriction fragment is represented in the lower portion with the size in the target locus and recombinant indicated. The short bar under the restriction map represents the location of each probe.

[0014] FIGS. 20A-20D illustrate Southern blot analyses of a maize target plant AW289B1A and a targeted recombination event HR-18FB.1M. The blot was hybridized with the following probes: FIG. 20A: the PMI-intron 3'-region (intron 4-exon 5) that is present in the target T-DNA but not in the donor; FIG. 20B: the rice actin-1 5'-region fragment that is present in the target T-DNA but not in the donor; FIG. 20C: the GUS 3'-probe hybridizes to sequence present only in the donor; and FIG. 20D: the PPO 3'-probe hybridizes to sequences present in both the target and donor. The hybridization probes were spiked with one microliter of labeled Lambda DNA to show the molecular weight marker. Lane M had Lambda DNA digested with StyI. The fragment sizes are: 23578 bps, 19324 bps, 7743 bps, 6225 bps, 4254 bps, 3472 bps, 2690 bps, 1882 bps, 1489 bps, 925 bps, and 421 bps. The 421 by fragment is not shown in the figures. Lanes 1 to 5 include DNA from target plant AW289B1A; lanes 6-10 include DNA from targeted event HR-18FB.1M. The restriction enzymes used to digest DNA in each lane are: lane 1 and 6, Sad; lane 2 and 7, ScaI; lane 3 and 8, KpnI; lane 4 and 9, SpeI; lane 5 and 10, HpaI.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0015] The following definitions are provided to enable a clear and consistent understanding of the specification and the claims. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. The nomenclature for DNA bases as set forth at 37 C.F.R. .sctn.1.822 as well as the standard one- and three-letter nomenclature for amino acid residues are used throughout the disclosure.

[0016] A "coding sequence" is a nucleic acid sequence that can be transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA, within a host cell into which the coding sequence has been introduced. In the case of mRNA, for example, the mRNA then can be translated within the host cell to produce a protein. A "coding region" comprises a coding sequence.

[0017] "Donor," "donor molecule," "donor DNA," and "donor sequence" are used interchangeably to refer to a desired nucleotide sequence that one wishes to recombine into a target DNA sequence using site-directed homologous recombination. The donor sequence can include any desired nucleotide sequence, such as, for example, a gene, an expression cassette, a promoter, a molecular marker, a selectable marker, a visible marker, a portion of any of these, or the like. A "donor construct" or "donor vector" contains a donor sequence.

[0018] "Endogenous," as used herein, means "of the same origin," i.e., derived from a host cell.

[0019] An "excisable sequence" refers to a nucleotide sequence comprising at least a portion of a marker sequence as well as at least one recombinase recognition site. An excisable sequence is contained within a target sequence.

[0020] "Expression" of a gene or other nucleotide sequence of interest refers to the transcription of the nucleotide sequence of interest to produce a corresponding RNA. In the case of an mRNA, the RNA may then be translated to produce a corresponding gene product (i.e., a peptide, a polypeptide, or a protein). Gene expression is controlled or modulated by regulatory elements, including 5' regulatory elements, such as a promoter, for example.

[0021] "Expression cassette," as used herein, includes a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell. An expression cassette typically comprises a promoter operably linked to a nucleotide sequence of interest, which is operably linked to a terminator or termination signal or to sequences containing an RNA polyadenylation signal. The expression cassette may also comprise sequences that permit proper translation of the nucleotide sequence, such as a translation initiation site and a translation termination sequence. Unique endonuclease restriction sites may also be included at the ends of an expression cassette to allow the cassette to be easily inserted or removed when creating a DNA construct. The nucleotide sequence of interest usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA that, in the sense or antisense direction, inhibits expression of a particular gene, e.g., antisense RNA or double-stranded interference RNA. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, that is, the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must be introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of either a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter may also be specific to a particular tissue or organ or stage of development.

[0022] A "foreign" gene or DNA sequence includes a gene or other nucleotide sequence of interest that is not normally found in the host organism but that may be introduced by gene transfer. Foreign genes and DNA that are not integrated into the genome are referred to as "extrachromosomal".

[0023] The term "gene" is used broadly to include any segment of a nucleotide sequence associated with a biological function. Thus, a gene can include a coding sequence either with or without the regulatory sequences needed for their expression. A gene can also include nonexpressed DNA segments, such as 5' and 3' untranslated sequences, recognition sequences for proteins, and/or termination sequences, for example. Further elements that may be present include, for example, introns. Some genes can be transcribed into mRNA and then translated into polypeptides (e.g., structural genes); other genes can be transcribed into RNA (e.g., rRNA and tRNA); and other types of genes function as regulators of expression (i.e., regulatory genes).

[0024] "Gene of interest," "sequence of interest," and "DNA of interest" are used interchangeably and include any nucleotide sequence which, when transferred to a plant, confers upon the plant a desired trait, characteristic, or biological function, such as, for example, virus resistance, insect resistance, resistance to other pests, disease resistance, herbicide tolerance, improved nutritional value, improved performance in an industrial process, or altered reproductive capability, for example. A sequence of interest can be a marker sequence. A sequence of interest can also encode an enzyme involved in a biochemical pathway, the expression of which alters a trait that is important or useful in food, feed, nutraceutical, and/or pharmaceutical production.

[0025] "Genome" refers to the complete genetic material of an organism.

[0026] "Heterologous," as used herein, means "of different natural origin," that is, representing a non-natural state. For example, if a host cell is transformed with a gene derived from another organism, particularly from another species, that gene is heterologous with respect to the host cell and also with respect to descendants of the host cell that carry the gene. Further, "heterologous" may also be used to refer to a nucleotide sequence which is derived from a natural or original cell type and is inserted into that same natural or original cell type, but which is present in a non-natural state, such as, for example, in a different copy number, under the control of different regulatory elements, or the like.

[0027] "Homologous recombination" refers to a reaction between any pair of nucleotide sequences having corresponding sites containing a similar nucleotide sequence (i.e., homologous sequences) through which the two molecules can interact (recombine) to form a new, recombinant DNA sequence. The sites of similar nucleotide sequence are each referred to herein as a "homology sequence". Generally, the frequency of homologous recombination increases as the length of the homology sequence increases. Thus, while homologous recombination can occur between two nucleotide sequences that are less than identical, the recombination frequency (or efficiency) declines as the divergence between the two sequences increases. Recombination may be accomplished using one homology sequence on each of the donor and target molecules, thereby generating a "single-crossover" recombination product. Alternatively, two homology sequences may be placed on each of the target and donor nucleotide sequences. Recombination between two homology sequences on the donor with two homology sequences on the target generates a "double-crossover" recombination product. If the homology sequences on the donor molecule flank a sequence that is to be manipulated (e.g., a sequence of interest), the double-crossover recombination with the target molecule will result in a recombination product wherein the sequence of interest replaces a DNA sequence that was originally between the homology sequences on the target molecule. The exchange of DNA sequence between the target and donor through a double-crossover recombination event is termed "sequence replacement."

[0028] To "identify" a recombination product means that the recombination product is detected and distinguished from the starting target and donor sequences. There are many means of identifying a recombination product. For example, a selectable marker gene can be used, whereby site-specific integration results in the selectable marker gene becoming operatively linked with a promoter only in a recombination product. Alternatively, a visible marker gene can be used, whereby a gain or loss of marker gene expression identifies a recombination product. Alternatively, a negative selectable marker gene can be used, whereby a loss or lack of expression of the marker gene identifies a recombination product. Additionally, molecular markers that are characteristic of the target sequence and/or donor sequence can be used, such that the molecular marker pattern is unique for the recombination product.

[0029] "Integration" refers to the incorporation of a foreign gene or other nucleotide sequence into a host genome through covalent bonding to the host DNA.

[0030] An "isolated" nucleic acid molecule or an isolated protein or toxin is a nucleic acid molecule or protein or toxin that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or protein or toxin may exist in a purified form or may exist in a non-native environment, such as, for example, a recombinant host cell or a transgenic plant.

[0031] A "marker sequence" refers to any nucleotide sequence that can be used to differentiate a transformed cell from a nontransformed cell. Marker sequences include, but are not limited to, selectable markers, scoreable markers, and molecular markers. Exemplary marker sequences include antibiotic resistance genes (such as, e.g., those conferring resistance to tetracycline, ampicillin, kanamycin, neomycin, hygromycin, and spectinomycin), luminescence genes (such as, e.g., genes encoding luciferase, .beta.-galactosidase, green fluorescence protein (GFP), .beta.-lactamase, or choramphenicol acetyl transferase (CAT)), and genes conferring an enhanced capacity, relative to non-transformed cells, to utilize a particular compound as a nutrient, growth factor, or energy source (such as, e.g., a gene encoding phosphomannose isomerase (PMI)).

[0032] "Mega-endonuclease" refers to a rare-cutting endonuclease that is capable of making a site-specific double-strand break in DNA at a particular recognition sequence comprising at least about 12 base pairs. The recognition sequence may be somewhat lengthy and can be as long as about 40 base pairs. One type of mega-endonuclease is referred to as a homing endonuclease, which is an enzyme that is encoded by an intron or an intein (Belfort and Roberts, 1997 Nucl. Acids. Res. 25(17): 3379-3388; see also, Gauthier et al., 1991 Current Genet. 19:43-47). Exemplary mega-endonucleases include, but are not limited to, I-SceI, I-CeuI, I-PpoI, I-CreI, I-DmoI, I-SceII, I-TevI, I-TevII, PI-PfuI, PI-PspI, PI-Scel, and HO, as described herein or otherwise known in the art (see, e.g., Belfort and Roberts (1997).

[0033] "Native" refers to a gene that is present in the genome of an untransformed (e.g., a "wild-type") cell.

[0034] "Naturally occurring" is used to describe an object that can be found in nature, as distinct from being artificially produced by man. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.

[0035] A "nucleic acid molecule," "nucleic acid sequence," or "nucleotide sequence" is a segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.

[0036] "Operably linked" and "operatively linked" refers to a relationship between two or more nucleotide sequences that interact physically or functionally. For example, a promoter or regulatory nucleotide sequence is said to be operably linked to a nucleotide sequence that encodes an RNA or a protein if the two sequences are situated such that the regulatory nucleotide sequence will affect the expression level of the coding or structural nucleotide sequence. A 5' portion of a gene is operatively or operably linked with a 3' portion of a gene if the two portions are situated to form a functional gene.

[0037] The term "plant", as used herein, refers to, without limitation, whole plants, plant organs (e.g., leaves, stems, roots, fruit, etc.), seeds, plant cells and progeny of plant cells, plant tissue, plant cell or tissue cultures, protoplasts, callus, and any groups of plant cells organized into structural and/or functional units. A plant "regenerated" from a plant cell means that all cells of the plant are derived from that plant cell. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Exemplary plants include, without limitation, Acacia, alfalfa, aneth, apple, apricot, artichoke, Arabidopsis, arugula, asparagus, avocado, banana, barley, bean, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, cauliflower, celery, cherry, chicory, clover, cilantro, citrus, clementines, coffee, corn, cotton, cucumber, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, hemp, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, mango, maize, melon, mushroom, nectarine, nut, oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, quince, radicchio, radish, raspberry, rice, rye, safflower, sorghum, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, zucchini, and woody plants such as coniferous and deciduous trees. Once a gene of interest has been transformed into a particular plant species, the gene may be propagated in that species or may be moved into other varieties of the same species, including commercial varieties, using traditional breeding techniques.

[0038] "Plant cell" refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall, and includes, without limitation, seed suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The plant cell may be in the form of an isolated single cell, a cultured cell, or a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0039] "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0040] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0041] A "plant organ" is a distinct and visibly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[0042] "Plant tissue" as used herein means a group of plant cells, including any tissue of a plant either in planta or in culture, organized into a structural and functional unit. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue, as listed above or otherwise embraced by this definition, is not intended to be exclusive of any other type of plant tissue.

[0043] A "promoter" is an untranslated DNA sequence that is located upstream of a coding region, contains a binding site for RNA polymerase II, and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

[0044] A "protoplast" is an isolated plant cell without a cell wall or with only parts of the cell wall.

[0045] "Recognition site" or "recognition sequence" refers to a DNA sequence recognized by an enzymatic protein, such as, for example, a recombinase or an endonuclease. In the case of a recombinase, the recognition site or sequence is the location on the DNA at which the recombinase binds to the DNA and cleavage and strand exchange occur.

[0046] "Recombinase" refers to any enzyme that is capable of performing site-specific recombination of DNA. Recombinase enzymes possess endonuclease and ligase activities. A recombinase may work as a single protein or as a complex of proteins.

[0047] "Regulatory element" includes a nucleotide sequence that is involved in conferring upon a host cell the expression of another nucleotide sequence, such as, for example, a sequence of interest. A regulatory element can comprise a promoter that is operably linked to the nucleotide sequence of interest and to a termination signal. Regulatory elements also typically encompass sequences useful for proper translation of the nucleotide sequence of interest.

[0048] "Selectable marker" or "selectable marker gene" refers to a nucleotide sequence whose expression in a plant cell gives the cell a selective advantage under particular conditions. The selective advantage possessed by the cell transformed with the selectable marker gene can be an improved ability to grow in the presence of a negative selective agent, such as an antibiotic or an herbicide, for example, as compared to the ability of non-transformed cells. Alternatively, the selective advantage possessed by the transformed cells can be an enhanced capacity, relative to non-transformed cells, to utilize a particular compound (such as a particular carbohydrate source like mannose, for example) as a nutrient, growth factor, or energy source, thereby effecting what is termed "positive selection." Alternatively, the selective advantage possessed by the transformed cell can be the loss of a previously possessed trait or characteristic, effecting what is termed "negative selection" or "counter selection." In this last case, the host cell is exposed to or contacted by a compound that is toxic only to cells that have not lost the ability to express a specific trait or characteristic (such as a negative selectable marker gene, for example) that was present in the parent cell, which is typically a transgenic parent cell.

[0049] "Site-directed recombination," as used herein, refers to a recombination of two nucleotide sequences, wherein the recombination occurs between particular recognition sites located on each of the nucleotide sequences.

[0050] "Site-specific" means at a particular nucleotide sequence, which can be in a specific location in the genome of a host cell. The nucleotide sequence can be endogenous to the host cell, either in its natural location in the host genome or at some other location in the genome, or it can be a heterologous nucleotide sequence, which has been previously inserted into the genome of the host cell by any of a variety of known methods.

[0051] "Stably transformed" refers to a host cell that contains a nucleotide sequence of interest that has been integrated into the host cell genome and is capable of being passed to progeny of that host cell.

[0052] "Subcellular organelles" includes intracellular organs of characteristic structure and function. Subcellular organelles include, for example, vacuoles, plastids, mitochondria, the cell nucleus, the endoplasmic reticulum, and the plasma membrane.

[0053] "Substantially identical," as used in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In one embodiment, the substantial identity exists over a region of nucleotide sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the nucleotide sequences are substantially identical over at least about 150 residues. In one embodiment, the nucleotide sequences are substantially identical over the entire length of their coding regions. In another embodiment, the substantial identity exists over a region of protein sequences that is at least about 15 residues in length, more preferably over a region of at least about 30 residues, and most preferably the protein sequences are substantially identical over at least about 50 residues. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function.

[0054] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based upon the designated program parameters.

[0055] Optimal alignment of compared sequences can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0056] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0057] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0058] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0059] "Stringent hybridization conditions" and "stringent hybridization wash conditions," in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations, are sequence dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but to no other sequences.

[0060] The "T.sub.m" is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42.degree. C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72.degree. C. for about 15 minutes. An example of stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30.degree. C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2.times. (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0061] The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

[0062] A further indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

[0063] "Target," "target molecule," "target DNA," and "target sequence" are used interchangeably to refer to a nucleotide sequence that is present naturally in the genome or that has been previously introduced into a chromosome of a host cell and can be inherited stably as part of the genome (i.e., "chromosomally integrated"). The target nucleotide sequence may be a sequence of interest, an expression cassette, a promoter, a molecular marker, a marker sequence, a selectable marker, a portion of any of these, or the like. The target sequence can be stably transformed into a plant cell to create a "target line" comprising the target sequence integrated at a particular chromosomal location in the plant genome. A "target construct" or "target vector" contains a target sequence.

[0064] A "targeted integration event" or "targeted event" is used interchangeably with an "HR-mediated recombination product" to refer to a recombination product formed by target and donor DNA sequences through homologous recombination (i.e., HR).

[0065] "Transformation" is a process for introducing a nucleotide sequence into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of a cell or an organism of interest.

[0066] "Transformed," "transgenic," or "recombinant" refers to a cell, tissue, organ, or organism, such as a bacterium or a plant, into which a particular nucleic acid molecule, such as a recombinant vector, has been introduced. The nucleic acid molecule can be stably integrated into the genome of the recipient cell, tissue, organ, or organism and can also be present as an extra-chromosomal or episomal molecule. Such an extra-chromosomal molecule can be auto-replicating. Transformed or transgenic cells, tissues, organs, or organisms are understood to encompass not only the end product of a transformation process but also the progeny thereof, which includes progeny produced from a breeding program employing a transgenic plant as a parent in a cross and exhibiting an altered genotype resulting from the presence of a heterologous nucleic acid molecule. A "non-transformed," "non-transgenic," or "non-recombinant" host refers to an organism, e.g., a bacterium or plant, which does not contain the particular nucleic acid molecule.

[0067] A "visible marker," "screenable marker," or "scoreable marker" refers to a gene or nucleotide sequence whose expression in a transformed cell may not confer an advantage to that cell but can be made visible or otherwise detectable. Examples of visible markers include, but are not limited to, .beta.-glucuronidase (GUS), luciferase (LUC), and fluorescent proteins (such as green fluorescent protein (GFP) or cyan fluorescent protein (CFP), for example).

[0068] The present disclosure relates to the targeted integration and stacking of nucleotide sequences within the genome of a host cell using homologous recombination. In one embodiment, a homology sequence shared by a target sequence and a donor sequence comprises at least one intron sequence that lengthens the region of homology and thereby enhances the frequency of homologous recombination between the target and donor sequences. In another embodiment, the homology sequence shared by the target and donor sequences comprises two or more intron sequences that lengthen the region of homology shared between the target and donor. In a further embodiment, a site-specific recombination system can be used to mediate the modification of a chromosomally integrated target sequence to prepare the target site for insertion of a subsequent donor sequence. In yet another embodiment, an endonuclease can be used to enhance recombination frequency and to facilitate introduction of the donor sequence into the host cell's genome at the target site. In a further embodiment, the expression level of at least one RecQ gene present in the genome of the host cell is down-regulated to enhance homologous recombination activity in the host cell. In still another embodiment, the expression level of at least one recombination-related gene present in the genome of the host cell is up-regulated to enhance homologous recombination activity in the host cell.

[0069] In one embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising a truncated sequence comprising a homology sequence, the homology sequence comprising at least one intron sequence; (b) introducing into the host cell a donor sequence comprising a sequence of interest and a completion sequence, the completion sequence comprising the homology sequence; and (c) obtaining in the host cell a recombination product comprising the sequence of interest and a functional sequence, the functional sequence comprising the homology sequence (FIG. 1). In another embodiment, the target sequence further comprises a mega-endonuclease recognition sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0070] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence and (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence; (b) introducing into the host cell a donor sequence comprising the first homology sequence, a sequence of interest, and a completion sequence, the completion sequence comprising the second homology sequence; and (c) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, and a functional sequence, the functional sequence comprising the second homology sequence (FIG. 2). Optionally, this embodiment may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, either of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0071] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence and (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising two or more intron sequences; (b) introducing into the host cell a donor sequence comprising the first homology sequence, a sequence of interest, and a completion sequence, the completion sequence comprising the second homology sequence; and (c) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, and a functional sequence, the functional sequence comprising the second homology sequence (FIG. 3). In another embodiment, the target sequence further comprises a mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0072] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence, (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence, and (iii) a mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence; (b) introducing into the host cell a donor sequence comprising the first homology sequence, a sequence of interest, and a completion sequence, the completion sequence comprising the second homology sequence; (c) introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the mega-endonuclease recognition sequence; and (d) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, and a functional sequence, the functional sequence comprising the second homology sequence; wherein (b) and (c) can be performed in any order or simultaneously (FIG. 4). Optionally, this embodiment may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, either of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0073] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence comprising a first sequence of interest and (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence; (b) introducing into the host cell a donor sequence comprising the first homology sequence, a second sequence of interest, and a completion sequence, the completion sequence comprising the second homology sequence; and (c) obtaining in the host cell a recombination product comprising the first homology sequence, the second sequence of interest, and a functional sequence, the functional sequence comprising the second homology sequence (FIG. 5). In another embodiment, the target sequence further comprises a mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0074] In accordance with the methods described herein, a target nucleotide sequence is introduced into a host plant cell. In one embodiment, the target sequence is chromosomally integrated into the plant genome by transformation methods described herein or by methods otherwise known in the art. A plant or plant cell transformed with the target sequence may be used to obtain a target cell line or plant line. Such a target cell line or plant line may comprise a single copy of the target sequence integrated into its genome. Once such a target line has been obtained and identified, it may be further characterized. For example, the location of the target sequence can be precisely determined by genetic methods well known in the art or by using molecular markers, such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), and the like. Additionally, host-plant DNA flanking the site of insertion may be sequenced to ensure that no essential gene has been mutated or otherwise disrupted by the insertion of the target sequence. Once a well-characterized target line is obtained, it may be used as a recipient of one or more subsequently introduced nucleotide sequences. Such additional sequences can be comprised in a donor sequence and can be introduced into the target line by any suitable transformation method, including, but not limited to, Agrobacterium-mediated transformation, biolistic bombardment, electroporation, PEG-mediated transformation, and whiskers technology, as described herein or otherwise known in the art.

[0075] The target sequence comprises a target homology sequence that is used to effect homologous recombination between the target sequence and a donor sequence that comprises a corresponding donor homology sequence. Absolute limits for the length of the homology sequence or the degree of homology are not fixed. Rather, the desired length of the homology sequence and/or the degree of homology depends upon the frequency and/or efficiency that is sought for a particular application. Generally, the longer the homology sequence and the greater the degree of homology, the greater the recombination frequency between the target and donor sequences.

[0076] In one embodiment, the homology sequence contained within each of the target and donor sequences can be any nucleotide sequence that is at least about 200 base pairs in length. The length of the homology sequence can vary and includes unit integral values in the ranges of about 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-700 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, and 5-10 kb or more. These exemplary ranges include both endpoints as well as every integer within the range; for example, the range of 1-2.5 kb includes both 1000 by and 2500 by as well as every integer between those endpoints (i.e., 1000 bp, 1001 bp, 1002 bp, . . . , 2498 bp, 2499 bp, and 2500 bp).

[0077] In another embodiment, the homology sequence includes at least one intron sequence that serves to extend the region of homology shared between the target and donor sequences and thereby enhances targeting efficiency. Any suitable intron sequence can be employed in accordance with the various embodiment of the invention, so long as the intron sequence is capable of being spliced by the host cell from the RNA transcript(s) of a recombination product. As will be appreciated by those of skill in the art, the intron splicing junctions must be properly recognized by the host cell in order to produce an appropriate expression product. Generally, an intron derived from a monocotyledonous plant will tend to be more effectively spliced from an RNA transcript produced by a monocotyledonous host cell. Likewise, an intron derived from a dicotyledonous plant will tend to be more effectively spliced from an RNA transcript produced by a dicotyledonous host cell.

[0078] In one embodiment, each intron sequence is at least about 50 base pairs in length. The length of the intron sequence can vary and includes unit integral values in the ranges of about 40-100 bp, 80-150 bp, 120-200 bp, 160-250 bp, 200-300 bp, 240-350 bp, 280-400 bp, 320-450 bp, 360-500 bp, 400-600 bp, 450-700 bp, 500-800 bp, 550-900 bp, 600-1000 bp, 700-1250 bp, 800-1500 bp, 900-1750 bp, 1-2 kb, and 1.5-3 kb or more. These exemplary ranges include both endpoints as well as every integer within the range; for example, the range of 1.5-3 kb includes both 1500 by and 3000 by as well as every integer between those endpoints (i.e., 1500 bp, 1501 bp, 1502 bp, . . . , 2998 bp, 2999 bp, and 3000 bp).

[0079] In a further embodiment, each homology sequence comprises two or more intron sequences, and each intron sequence is separated from another intron sequence by at least one exon sequence.

[0080] In another embodiment, each homology sequence includes at least one recombinase recognition site (as described in greater detail below).

[0081] In one embodiment, each of the target and donor sequences comprises two homology sequences. In this embodiment, each of the two homology sequences is independently selected. That is, the first and second homology sequences can be the same or they can be different from each other. In one embodiment, at least one of the first and second homology sequences comprises a sequence of interest.

[0082] In one embodiment, the target homology sequence is contained within a truncated, and therefore inactive, nucleotide sequence. The truncated sequence can be, for example, a truncated sequence of interest, a truncated gene, a truncated selectable marker, a truncated visible marker, a truncated negative selectable marker, a truncated promoter sequence, a truncated expression cassette, or the like. In this embodiment, a donor sequence is constructed to include a completion sequence that contains the donor homology sequence. The donor completion sequence completes the truncated nucleotide sequence, in that homologous recombination between the target, which includes the truncated sequence, and the donor, which includes the completion sequence, produces a functional or complete sequence. For example, the truncated sequence can include a 5' portion (or, alternatively, a 3' portion) of a sequence of interest, which optionally may be operably linked to a promoter sequence. The corresponding donor completion sequence then includes the 3' portion (or, alternatively, the 5' portion) of the sequence of interest and optionally may also include a termination sequence. In this manner, homologous recombination between the target and donor sequences ligates or otherwise operably links the 5' portion of the sequence of interest with the 3' portion of the sequence of interest to reconstitute a functional or complete sequence of interest in the recombination product. Only a host cell comprising a desired recombination product has the appropriate expression product (i.e., as derived from a functional sequence of interest).

[0083] In one embodiment, the target's truncated sequence can be a truncated marker sequence. The truncated marker sequence can include either a 5' portion of a marker sequence or a 3' portion of a marker sequence. In one embodiment, the truncated marker sequence includes a 5' portion of a marker sequence, which can be operably linked to a promoter sequence. The corresponding donor completion sequence includes the 3' portion of the marker sequence and can also include a termination sequence. In this manner, homologous recombination between the target and donor sequences ligates or otherwise operably links the 5' portion of the marker sequence with the 3' portion of the marker sequence to reconstitute a functional marker sequence in the recombination product. In another embodiment, the truncated marker sequence includes a 3' portion of a marker sequence and can also include a termination sequence. The corresponding donor completion sequence includes the 5' portion of the marker sequence, which can be operably linked to a promoter sequence. Homologous recombination between the target and donor sequences ligates or otherwise operably links the 5' portion of the marker sequence with the 3' portion of the marker sequence to reconstitute a functional marker sequence in the recombination product.

[0084] In one embodiment, the target sequence comprises a mega-endonuclease recognition sequence. Exemplary mega-endonuclease recognition sequences include those sequences that are recognized and cleaved by various endonucleases, such as, for example, I-SceI (18 by recognition sequence, i.e., 5'-TAGGGATAA CAGGGTAAT-3', where the arrow indicates the cleavage site), I-CeuI (26 by recognition sequence, i.e., 5'-TAACTATAACGGTCCTAA GGTAGCGA-3'), I-PpoI (15 by recognition sequence, i.e., 5'-CTCTCTTAA GGTAGC-3'), PI-PspI (30 by recognition sequence, i.e., 5'-TGGCAAACAGCTATTAT GGGTATTATGGGT-3'), PI-SceI (39 by recognition sequence, i.e., 5'-ATC TAT GTC GGG TGC GGA GAA AGA GGT AAT GAA ATG GCA-3'), and HO (20 by recognition sequence, i.e., 5'-CAG CTT TCC GCA ACA GTA TA-3'). Other mega-endonuclease recognition sequences may also be used, such as any sequence recognized by I-CreI, I-DmoI, I-SceII, I-TevI, I-TevII, PI-PfuI, or any sequence recognized by other mega-endonucleases that are known in the art. See, e.g., Belfort and Roberts, p. 3382, Table 3.

[0085] As will be appreciated by those of skill in the art, mega-endonucleases do not have stringent recognition sequences. The above recognition sequences are but single examples of the recognition sequences that may be used with each of the indicated mega-endonucleases. Other recognition sequences, such as, for example, degenerate variations of the sequences indicated above, may also be used, including recognition sequences having single or multiple base changes. See, e.g., Argast et al. 1998 J. Mol. Biol. 280: 345-353; and Gimble and Wang 1996 J. Mol. Biol. 256: 163-180.

[0086] A mega-endonuclease or a sequence encoding a mega-endonuclease can be introduced into the host plant cell prior to, after, or simultaneously with the introduction of the donor sequence. In one embodiment, a mega-endonuclease is introduced into the host cell as a nucleic acid molecule (DNA and/or RNA) that comprises a coding sequence for the mega-endonuclease. The mega-endonuclease can be introduced as an expression cassette comprising the coding sequence operatively linked to a plant expressible promoter and an appropriate termination sequence. As used herein, "plant expressible" means that the promoter is operable within a plant cell and is therefore capable of driving expression of a nucleotide sequence to which the promoter is operably linked within the plant cell. The promoter may be selected such that expression of the mega-endonuclease can be spatially or temporally regulated in any desired manner. For example, a promoter can be selected such that expression of the mega-endonuclease is constitutive, developmentally regulated, tissue specific, tissue preferred, cell specific, specific to a particular cellular compartment (i.e., organellar-specific), or the like. Additionally, the promoter can be chosen so that expression of the mega-endonuclease can be chemically induced in a plant, resulting in expression of the mega-endonuclease only in response to treatment of the plant cell or tissue with a chemical ligand. By combining promoter elements that confer specific expression with those conferring chemically induced expression, the mega-endonuclease can be expressed or activated within specific cells or tissues of the plant in response to a chemical application. Any of a variety of plant expressible promoters can be used to drive expression of the mega-endonuclease. Several of such promoters are described herein, and other such promoters are known in the art.

[0087] In another embodiment, the mega-endonuclease is introduced into the plant cell by being stably transformed into the genome of the plant cell. For example, the mega-endonuclease can be comprised in an expression cassette comprising the coding sequence of the mega-endonuclease operatively linked to a promoter capable of expression in plant tissues and cells. Suitable methods for stably transforming plant cells are known in the art and are described herein. In one embodiment, a plant cell that is stably transformed with the mega-endonuclease is also stably transformed with a target sequence. In another embodiment, a plant cell that is stably transformed with the mega-endonuclease is also stably transformed with a donor sequence.

[0088] As will be appreciated by one of skill in the art, a whole plant can be regenerated from a plant cell or a group of plant cells that has been stably transformed with a selected nucleotide sequence. This regenerated whole plant is then also referred to as being transformed with the selected nucleotide sequence. Thus, for example, in accordance with the methods disclosed herein, a first plant can be stably transformed with one or more expression cassettes comprising a mega-endonuclease and a donor sequence, and this first plant then can be crossed with a second plant that is stably transformed with a target sequence. Accordingly, expression of the mega-endonuclease in an F1 plant or seed can facilitate recombination between the target and donor sequences such that the HR-mediated recombination product is formed in the F1 plant or seed. The nucleotide sequence encoding the mega-endonuclease and the unrecombined portion(s) of the donor sequence can then be segregated from a nucleotide sequence comprising the recombination product sequence(s) through breeding.

[0089] In another embodiment, the mega-endonuclease can be introduced into a plant cell such that the plant cell transiently expresses the mega-endonuclease. For example, the mega-endonuclease coding sequence can be introduced into a plant cell through any known means for plant transformation, such as, for example, Agrobacterium or microprojectile bombardment. Frequently, the introduced nucleotide sequence is not integrated into the genome but can be transcribed nonetheless into mRNA.

[0090] In another embodiment, the coding sequence of the mega-endonuclease is supplied to the host cell in the form of messenger RNAs (mRNA). In this manner, the mega-endonuclease is provided to the host cell only transiently. The coding sequence for the mega-endonuclease can be inserted into a vector for in-vitro transcription of the RNA using methods described in Lebel et al. 1995 Theor. Appl. Genet. 91:899-906 and U.S. Pat. No. 6,051,409. The RNA then can be transformed into a host cell, such as a cell from a donor line or a target line, for example. In one embodiment, the RNA can be co-transformed into a host cell with a donor sequence. In an exemplary embodiment, the RNA can be transferred to a host cell using microprojectile bombardment, as described in U.S. Pat. No. 6,051,409. In another embodiment, the RNA can be introduced into protoplasts of a host cell by PEG-mediated transformation (see, e.g., Lebel et al. 1995 Theor. Appl. Genet. 91:899-906) or by electroporation. In another embodiment, other transformation techniques, such as microinjection of the RNA, can be used to introduce the RNA into the host cell.

[0091] In a further embodiment, an active mega-endonuclease can be introduced into a host cell as a protein, such as a purified protein, for example. The mega-endonuclease protein can be introduced into the cell by any suitable method known in the art, such as, for example, microinjection or electroporation. In another embodiment, the mega-endonuclease can be introduced into the host cell by microinjection together with a donor DNA sequence (see, e.g., Neuhaus et al. 1993 Cell 73:937-952). In another embodiment, the mega-endonuclease protein is introduced into the host cell through infection with Agrobacterium comprising a VirE2 or VirF fusion protein (see, e.g., Vergunst et al. 2000 Science 290:979-82).

[0092] In one embodiment, the coding sequence of the mega-endonuclease can be optimized for expression in a particular plant host. It is known in the art that the expression of heterologous proteins in plants can be enhanced by optimizing the coding sequences of the proteins according to the codon preference of the host plant. The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. A comparison of the codon usage within a cloned microbial ORF (open reading frame) to the codon usage in plant genes (and, in particular, genes from the selected host plant) enables an identification of the codons within the ORF that can be changed in an effort to optimize the coding sequence for expression in the host plant.

[0093] In one embodiment, the donor sequence comprises at least one sequence of interest. The sequence of interest may be included in an expression cassette, and expression of the sequence of interest may be controlled by any of the promoters described herein or by any other plant expressible promoter known in the art. The promoter that controls or drives expression of the sequence of interest can be included in the expression cassette that comprises the sequence of interest, or the promoter can be otherwise operably linked to the sequence of interest. Exemplary sequences of interest include, but are not limited to, sequences encoding traits related to any of the following desirable characteristics: waxy starch; herbicide tolerance; resistance to bacterial, fungal, or viral disease; insect resistance; abiotic stress resistance; enhanced nutritional quality; improved performance in an industrial process; altered reproductive capability, such as male sterility or male fertility; yield stability; yield enhancement; and the production of commercially valuable enzymes or metabolites in plants.

[0094] In another embodiment, the donor sequence may also include a donor marker sequence, such as a selectable or visible marker gene, for example. The donor marker sequence can be any marker sequence described herein or otherwise known in the art but is typically different from any marker sequence associated with the target homology sequence. In this context, "associated with the target homology sequence" means that the marker sequence, or a truncated form of the marker sequence, is part of the target sequence and includes the target homology sequence, such that the target sequence would be capable of expressing the marker sequence upon recombination with a donor that included the corresponding completion sequence, as described above. In such a case, the donor marker sequence can be selected such that the donor marker sequence is different from the marker sequence associated with the target homology sequence, and recombination of the target and donor results in a recombination product that includes two different marker sequences. As described herein, the donor marker sequence can be operably linked to a suitable promoter and/or a suitable termination sequence.

[0095] In another embodiment, the donor sequence can be stably integrated into a plant genome. A plant or plant cell transformed with the donor sequence can be obtained by any suitable transformation method, as described herein or by methods otherwise known in the art, and is used to form a donor cell line or plant line. Such a donor cell line or plant line may include a single copy of the donor sequence integrated into its genome. Once such a donor line has been obtained and identified, it may be further characterized, as described above with respect to the target line.

[0096] In one embodiment, a target line can be crossed with a donor line by methods of sexual reproduction known in the art, such as, for example, by pollinating the target line with pollen of the donor line and obtaining seed comprising both the target and donor sequences. An HR-mediated recombination product can result from an exchange of nucleotide sequences between a target sequence locus and a donor sequence locus.

[0097] In accordance with another aspect of the methods disclosed herein, a site-specific recombinase can be used to excise a portion of a target sequence that has been introduced into a host cell prior to introducing a donor sequence into that host cell. Exemplary site-specific recombinases (and corresponding recognition sites) include, but are not limited to, FLP (FRT), Cre (Lox), R(RS), Gin (gix), .beta. (six), an integrase from any of bacteriophage-.lamda., HK022, .phi.C31, or R4 (and their corresponding attB/attP or attL/attR sites), as well as any of several other recombinases that are known in the art (see, e.g., Nunes-Duby et al. 1998 Nucleic Acid Research 26:391-406; Smith and Thorpe 2002 Molecular Microbiology 44:299-307).

[0098] In accordance with another aspect of the methods disclosed herein, recombinase recognition sites and a corresponding site-specific recombinase can be used to modify an HR-recombination product in preparation for a successive round of targeted sequence integration and stacking.

[0099] In one embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising a truncated sequence comprising a homology sequence, the homology sequence comprising at least one intron sequence; (b) introducing into the host cell a donor sequence comprising (i) a sequence of interest, (ii) a completion sequence comprising a first recombinase recognition site and the homology sequence, and (iii) a second recombinase recognition site positioned between the sequence of interest and the completion sequence; and (c) obtaining in the host cell a recombination product comprising the sequence of interest, the second recombinase recognition site, and a functional sequence, the functional sequence comprising the first recombinase recognition site and the homology sequence; wherein the first and second recombinase recognition sites can be the same or different (FIG. 6). In another embodiment, the target sequence further comprises a first mega-endonuclease recognition sequence, the donor sequence further comprises a second mega-endonuclease recognition sequence positioned between the sequence of interest and the completion sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0100] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence and (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence; (b) introducing into the host cell a donor sequence comprising (i) the first homology sequence, (ii) a sequence of interest, (iii) a completion sequence comprising a first recombinase recognition site and the second homology sequence, and (iv) a second recombinase recognition site positioned between the sequence of interest and the completion sequence; and (c) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, the second recombinase recognition site, and a functional sequence, the functional sequence comprising the first recombinase recognition site and the second homology sequence; wherein the first and second recombinase recognition sites can be the same or different (FIG. 7A). In another embodiment, the target sequence further comprises a first mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence, the donor sequence further comprises a second mega-endonuclease recognition sequence positioned between the sequence of interest and the completion sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0101] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence and (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising two or more intron sequences; (b) introducing into the host cell a donor sequence comprising (i) the first homology sequence, (ii) a sequence of interest, (iii) a completion sequence comprising a first recombinase recognition site and the second homology sequence, and (iv) a second recombinase recognition site positioned between the sequence of interest and the completion sequence; and (c) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, the second recombinase recognition site, and a functional sequence, the functional sequence comprising the first recombinase recognition site and the second homology sequence; wherein the first and second recombinase recognition sites can be the same or different (FIG. 7B). In another embodiment, the target sequence further comprises a first mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence, the donor sequence further comprises a second mega-endonuclease recognition sequence positioned between the sequence of interest and the completion sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0102] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence comprising a first sequence of interest and (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence; (b) introducing into the host cell a donor sequence comprising (i) the first homology sequence, (ii) a second sequence of interest, (iii) a completion sequence comprising a first recombinase recognition site and the second homology sequence, and (iv) a second recombinase recognition site positioned between the second sequence of interest and the completion sequence; and (c) obtaining in the host cell a recombination product comprising the first homology sequence, the second sequence of interest, the second recombinase recognition site, and a functional sequence, the functional sequence comprising the first recombinase recognition site and the second homology sequence; wherein the first and second recombinase recognition sites can be the same or different (FIG. 8). In another embodiment, the target sequence further comprises a first mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence, the donor sequence further comprises a second mega-endonuclease recognition sequence positioned between the second sequence of interest and the completion sequence, and the method further comprises, prior to obtaining the recombination product, introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0103] In one embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence comprising a first sequence of interest, (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence, and (iii) a first mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence; (b) introducing into the host cell a donor sequence comprising (i) the first homology sequence, (ii) a second sequence of interest, (iii) a completion sequence comprising a first recombinase recognition site and the second homology sequence, (iv) a second mega-endonuclease recognition sequence positioned between the second sequence of interest and the completion sequence, and (v) a second recombinase recognition site positioned between the second mega-endonuclease recognition sequence and the completion sequence; (c) introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence; (d) obtaining in the host cell a recombination product comprising the first homology sequence, the second sequence of interest, the second recombinase recognition site, and a functional sequence, the functional sequence comprising the first recombinase recognition site and the second homology sequence; (e) introducing into the host cell a recombinase or a recombinase coding sequence, the recombinase or an expression product of the recombinase coding sequence being capable of recognizing the first and second recombinase recognition sites; and (f) obtaining in the host cell a recombination product comprising the first homology sequence, the second sequence of interest, the second mega-endonuclease recognition sequence, and a truncated sequence comprising a third recombinase recognition site and the second homology sequence; wherein the first and second recombinase recognition sites can be the same or different; wherein the second and third recombinase recognition sites can be the same or different; and wherein (b) and (c) may be performed in any order or simultaneously (FIG. 9A). Optionally, this embodiment may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, either of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0104] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence comprising a first sequence of interest, (ii) a truncated sequence comprising a second homology sequence, the second homology sequence comprising at least one intron sequence, and (iii) a first mega-endonuclease recognition sequence positioned between the first homology sequence and the truncated sequence; (b) introducing into the host cell a donor sequence comprising (i) the first homology sequence, (ii) a second sequence of interest, (iii) a first completion sequence comprising a first recombinase recognition site and the second homology sequence, (iv) a second mega-endonuclease recognition sequence positioned between the second sequence of interest and the completion sequence, and (v) a second recombinase recognition site positioned between the second mega-endonuclease recognition sequence and the completion sequence; (c) introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence; (d) obtaining in the host cell a recombination product comprising the first homology sequence, the second sequence of interest, the second mega-endonuclease recognition sequence, the second recombinase recognition site, and a functional sequence, the functional sequence comprising the first recombinase recognition site and the second homology sequence; (e) introducing into the host cell a recombinase or a recombinase coding sequence, the recombinase or an expression product of the recombinase coding sequence being capable of recognizing the first and second recombinase recognition sites; (f) obtaining in the host cell a recombination product comprising the first homology sequence, the second sequence of interest, the second mega-endonuclease recognition sequence, and a truncated sequence comprising a third recombinase recognition site and the second homology sequence; (g) introducing into the host cell a second donor sequence comprising (i) a third homology sequence comprising the second sequence of interest, (ii) a third sequence of interest, (iii) a second completion sequence comprising a fourth recombinase recognition site and the second homology sequence, (iv) a third mega-endonuclease recognition sequence positioned between the third sequence of interest and the second completion sequence, and (v) a fifth recombinase recognition site positioned between the third mega-endonuclease recognition sequence and the second completion sequence; (h) introducing into the host cell a second mega-endonuclease or a second mega-endonuclease coding sequence, the second mega-endonuclease or an expression product of the second mega-endonuclease coding sequence being capable of recognizing the second mega-endonuclease recognition sequence; (i) obtaining in the host cell a recombination product comprising the first sequence of interest, the third homology sequence comprising the second sequence of interest, the third sequence of interest, the third mega-endonuclease recognition sequence, the fifth recombinase recognition site, and a functional sequence comprising the fourth recombinase recognition site and the second homology sequence; wherein the first and third mega-endonuclease recognition sequences may be the same or different; wherein the first and second recombinase recognition sites can be the same or different; wherein the second and third recombinase recognition sites can be the same or different; wherein the third and fifth recombinase recognition sites can be the same or different; wherein the fourth and fifth recombinase recognition sites can be the same or different; wherein (b) and (c) may be performed in any order or simultaneously; and wherein (g) and (h) may be performed in any order or simultaneously. As will be readily appreciated by one skilled in the art, steps (e) through (h) may be repeated as desired to obtain a host cell comprising multiple sequences of interest (FIGS. 9A and 9B). Optionally, this embodiment may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, either of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0105] In accordance with another aspect of the methods disclosed herein, a target sequence that has been chromosomally integrated into the host cell genome can include a first recombinase recognition site and a functional sequence, such as a marker sequence, for example. The functional sequence can comprise a target homology sequence that includes a second recombinase recognition site. The portion of the target sequence that is positioned between the first and second recombination recognition sequences is an excisable sequence, which can be removed by a suitable recombinase that is introduced into the cell and is capable of recognizing the first and second recognition sequences. Removal of the exisable sequence by the recombinase transforms the functional sequence into a truncated sequence.

[0106] In one embodiment, a method for preparing a target sequence for targeted integration and stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a functional sequence comprising a homology sequence, the homology sequence comprising a first recombinase recognition site and at least one intron sequence and (ii) a second recombinase recognition site positioned upstream (i.e., to the 5' side) of the functional sequence; (b) introducing into the host cell a recombinase or a recombinase coding sequence, the recombinase or an expression product of the recombinase coding sequence being capable of recognizing the first and second recombinase recognition sites; and (c) obtaining in the host cell a recombination product comprising a truncated sequence comprising a third recombinase recognition site and the homology sequence; wherein the first and second recombinase recognition sites can be the same or different; and wherein the second and third recombinase recognition sites can be the same or different (FIG. 10). Optionally, the target sequence may further comprise a mega-endonuclease recognition sequence positioned upstream of the second recombinase recognition site.

[0107] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence, (ii) a functional sequence comprising a second homology sequence, the second homology sequence comprising a first recombinase recognition site and at least one intron sequence, and (iii) a second recombinase recognition site positioned between the first homology sequence and the functional sequence; (b) introducing into the host cell a recombinase or a recombinase coding sequence, the recombinase or an expression product of the recombinase coding sequence being capable of recognizing the first and second recombinase recognition sites; (c) obtaining in the host cell a recombination product comprising the first homology sequence and a truncated sequence comprising a third recombinase recognition site and the second homology sequence; (d) introducing into the host cell a donor sequence comprising the first homology sequence, a sequence of interest, and a completion sequence, the completion sequence comprising the second homology sequence; and (e) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, and the functional sequence comprising the second homology sequence; wherein the first and second recombinase recognition sites can be the same or different; and wherein the second and third recombinase recognition sites can be the same or different (FIG. 11). In another embodiment, the target sequence further comprises a mega-endonuclease recognition sequence positioned between the first homology sequence and the functional sequence, and the method further comprises, any time after step (c) and prior to step (e), introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0108] In another embodiment, a method for targeted nucleotide sequence stacking is provided, the method comprising: (a) providing a host cell comprising a chromosomally integrated target sequence, the target sequence comprising (i) a first homology sequence, (ii) a functional sequence comprising a second homology sequence, the second homology sequence comprising a first recombinase recognition site and at least one intron sequence, and (iii) a second recombinase recognition site positioned between the first homology sequence and the functional sequence; (b) introducing into the host cell a recombinase or a recombinase coding sequence, the recombinase or an expression product of the recombinase coding sequence being capable of recognizing the first and second recombinase recognition sites; (c) obtaining in the host cell a recombination product comprising the first homology sequence and a truncated sequence comprising a third recombinase recognition site and the second homology sequence; (d) introducing into the host cell a donor sequence comprising (i) the first homology sequence, (ii) a sequence of interest, (iii) a completion sequence, the completion sequence comprising a fourth recombinase recognition site and the second homology sequence, and (iii) a fifth recombinase recognition site positioned between the sequence of interest and the completion sequence; (e) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, the fifth recombinase recognition site, and a functional sequence comprising the fourth recombinase recognition site and the second homology sequence; (f) introducing into the host cell the recombinase or the recombinase coding sequence; and (g) obtaining in the host cell a recombination product comprising the first homology sequence, the sequence of interest, and a truncated sequence comprising a sixth recombinase recognition site and the second homology sequence; wherein the first and second recombinase recognition sites can be the same or different; wherein the second and third recombinase recognition sites can be the same or different; wherein the third and fifth recombinase recognition sites can be the same or different; wherein the fourth and fifth recombinase recognition sites can be the same or different; and wherein the fifth and sixth recombinase recognition sites can be the same or different. Steps (d) through (f) may be repeated as desired, as detailed in a previously described embodiment, to obtain a host cell comprising multiple sequences of interest (FIGS. 12A-12B). In another embodiment, the target sequence further comprises a first mega-endonuclease recognition sequence positioned between the first homology sequence and the functional sequence, the donor sequence further comprises a second mega-endonuclease recognition sequence positioned between the sequence of interest and the completion sequence, and the method further comprises, any time after step (c) and prior to step (e), introducing into the host cell a mega-endonuclease or a mega-endonuclease coding sequence, the mega-endonuclease or an expression product of the mega-endonuclease coding sequence being capable of recognizing the first mega-endonuclease recognition sequence. Optionally, either of these embodiments may be used in conjunction with a method for down-regulating the expression level of at least one RecQ gene that is present in the genome of the host cell. Optionally, any of these embodiments may be used in conjunction with a method for up-regulating the expression level of at least one recombination-related gene that is present in the genome of the host cell.

[0109] In one embodiment, the recombinase can be introduced into the host cell as one or more nucleic acid molecules (DNA and/or RNA) that comprise the coding sequence for each constituent protein of the recombinase. The recombinase can be introduced as one or more expression cassettes comprising a coding region for each constituent protein, wherein each coding region is operatively linked to a promoter capable of expression in plant cells. Promoters for each expression cassette can be selected such that expression of the recombinase can be spatially or temporally regulated in any desired manner. For example, a promoter can be selected such that expression of the recombinase is constitutive, developmentally regulated, tissue specific, tissue preferred, cell specific, specific to a particular cellular compartment (i.e., organellar-specific), or the like. Additionally, promoters can be chosen so that expression of the recombinase can be chemically induced in a plant, resulting in expression of the recombinase only in response to treatment of the plant cell or tissue with a chemical ligand. By combining promoter elements that confer specific expression with those conferring chemically induced expression, the recombinase can be expressed or activated within specific cells or tissues of the plant in response to a chemical application. Any of a variety of plant expressible promoters can be used to drive expression of the recombinase. Several of such promoters are described herein, and others of such promoters are known in the art.

[0110] In another embodiment, the recombinase can be introduced into the plant cell by being stably transformed into the genome of the plant cell. For example, the recombinase can be comprised in one or more expression cassettes comprising the coding sequences of the recombinase, whereby the coding sequence for each protein component of the recombinase is operatively linked to a promoter capable of expression in plant tissues and cells. Suitable methods for stably transforming plant cells are known in the art and are described herein. In one embodiment, a plant cell that is stably transformed with the recombinase is also stably transformed with a donor sequence.

[0111] In one embodiment, the recombinase can be introduced into a plant cell such that the plant cell transiently expresses the recombinase. For example, one or more nucleotide sequences comprising the recombinase coding sequence can be introduced into a plant cell through Agrobacterium or microprojectile bombardment, for example. Much of the introduced nucleotide sequences are not integrated into the genome but can be transcribed into mRNA.

[0112] In another embodiment, the coding sequence(s) of the recombinase can be supplied to the host cell in the form of messenger RNAs (mRNA). In this manner, the recombinase can be provided to the host cell only transiently. The coding sequence for each of the proteins of the recombinase can be inserted into a vector for in-vitro transcription of the RNA using methods described in Lebel et al. 1995 Theor. Appl. Genet. 91:899-906 and U.S. Pat. No. 6,051,409. The RNA then can be transformed into a host cell, such as a cell from a donor line or a target line, for example. In one embodiment, the RNA is co-transformed into a host cell with a donor sequence. In an exemplary embodiment, the RNA is transferred to a host cell using microprojectile bombardment, as described in U.S. Pat. No. 6,051,409. In another embodiment, the RNA is introduced into protoplasts of a host cell by PEG-mediated transformation (see, e.g., Lebel et al. 1995 Theor. Appl. Genet. 91:899-906) or by electroporation. In another embodiment, other transformation techniques, such as microinjection of the RNA, are used to introduce the RNA into the host cell.

[0113] In a further embodiment, an active recombinase can be introduced into a host cell as one or more proteins, such as one or more purified proteins, for example. The recombinase protein can be introduced into the cell by any suitable method known in the art, such as, for example, microinjection or electroporation. In another embodiment, the recombinase is introduced into the host cell by microinjection together with a donor DNA sequence (see, e.g., Neuhaus et al. 1993 Cell 73:937-952). In another embodiment, the recombinase protein is introduced into the host cell through infection with Agrobacterium comprising a VirE2 or VirF fusion protein (see, e.g., Vergunst et al. 2000 Science 290:979-82).

[0114] In one embodiment, the coding sequence(s) of the recombinase can be optimized for expression in a particular plant host. It is known in the art that the expression of heterologous proteins in plants can be enhanced by optimizing the coding sequences of the proteins according to the codon preference of the host plant. The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. A comparison of the codon usage within a cloned microbial ORF (open reading frame) to the codon usage in plant genes (and, in particular, genes from the selected host plant) enables an identification of the codons within the ORF that can be changed in an effort to optimize the coding sequence for expression in the host plant.

General Methods and Components

[0115] Nucleotide sequences utilized in accordance with various embodiments of the invention can be incorporated into a host cell using conventional recombinant DNA technology. Generally, this involves using standard cloning procedures known in the art to insert a nucleotide sequence into an expression system, such as a vector, for example, with respect to which the nucleotide sequence is heterologous. The vector may contain additional elements that may be used during transcription and/or translation of the inserted coding sequence by the host cell that contains the vector. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses, other modified viruses, and the like. The components of the expression system may also be modified to increase expression levels of the inserted coding sequence. For example, truncated sequences, nucleotide substitutions, or other modifications may be employed. Expression systems known in the art can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells may then be regenerated into whole plants. Methods for transforming dicots and monocots are known to those skilled in the art, as described below.

I. Expression Cassettes

[0116] Coding sequences intended for expression in transgenic plants are first assembled in expression cassettes 3' to a suitable promoter expressible in plants. The expression cassettes can also comprise any further sequences needed or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be transferred to the plant transformation vectors described herein.

[0117] The following is a description of various components of typical expression cassettes.

[0118] A. Promoters

[0119] Selection of the promoter to be used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and selection should reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter can drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non-limiting examples of promoters that can be used in the expression cassettes employed in the present invention.

[0120] 1. Constitutive Promoters

[0121] a. Ubiquitin Promoters

[0122] Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower--Binet et al. 1991 Plant Science 79: 87-94; maize--Christensen et al. 1989 Plant Molec. Biol. 12: 619-632; and Arabidopsis--Norris et al. 1993 Plant Mol. Biol. 21:895-906). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. Taylor et al. (1993 Plant Cell Rep. 12: 491-495) describe a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The Arabidopsis ubiquitin promoter may also be used with the nucleotide sequences of the present invention. The ubiquitin promoter is suitable for gene expression in transgenic plants, including both monocotyledons and dicotyledons. Suitable vectors include derivatives of pAHC25 or any of the transformation vectors described in this application. The vectors can be modified by the introduction of appropriate ubiquitin promoter and/or intron sequences.

[0123] b. The CaMV 35S Promoter

[0124] Construction of the plasmid pCGN1761 is described in published patent application EP 0 392 225 (Example 23). The plasmid contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed which has a modified polylinker, which includes NotI and XhoI sites in addition to the existing EcoRI site. This derivative, designated pCGN1761ENX, is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-coding sequence-tml terminator cassette of such a construction can be excised by HindIII, SphI, SalI, and XbaI sites 5' to the promoter and XbaI, BamHI and BglI sites 3' to the terminator for transfer to transformation vectors such as those described below. Furthermore, the double 35S promoter fragment can be removed by 5' excision with HindIII, SphI, SalI, XbaI, or PstI, and 3' excision with any of the polylinker restriction sites (EcoRI, Nod or XhoI) for replacement with another promoter. If desired, modifications around the cloning sites can be made by the introduction of sequences that can enhance translation. This is particularly useful when over-expression is desired. For example, pCGN1761ENX can be modified by optimization of the translational initiation site as described in Example 37 of U.S. Pat. No. 5,639,949.

[0125] c. The Actin Promoter

[0126] Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is suitable for use as a constitutive promoter. In particular, the promoter from the rice ActI gene has been cloned and characterized (McElroy et al. 1990 Plant Cell 2: 163-171). A 1.3 kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the ActI promoter have been constructed specifically for use in monocotyledons (McElroy et al. 1991 Mol. Gen. Genet. 231: 150-160). These incorporate the ActI-intron 1, AdhI 5' flanking sequence and AdhI-intron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and ActI intron or the ActI 5' flanking sequence and the ActI intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (1991 Mol. Gen. Genet. 231: 150-160)) can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments can be removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice ActI promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. 1993 Plant Cell Rep. 12: 506-509).

[0127] 2. Inducible Expression

[0128] a. PR-1 Promoters

[0129] The double 35S promoter in pCGN1761ENX can be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example, one of the chemically regulatable promoters described in U.S. Pat. No. 5,614,395, such as the tobacco PR-1a promoter, can replace the double 35S promoter. Alternatively, the Arabidopsis PR-1 promoter described in Lebel et al. 1998 Plant J. 16:223-233 can be used. The promoter of choice can be excised from its source by restriction enzymes; alternatively, it can be PCR-amplified using primers that carry appropriate terminal restriction sites. If PCR-amplification is undertaken, then the promoter can be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically/pathogen regulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104) and transferred to plasmid pCGN1761ENX (Uknes et al. 1992 Plant Cell 4: 645-656). The plasmid pCIB1004 is cleaved with NcoI and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with HindIII and the resultant PR-1a promoter-containing fragment is gel purified and cloned into pCGN1761ENX from which the double 35S promoter has been removed. This is done by cleavage with XhoI and blunting with T4 polymerase, followed by cleavage with HindIII and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX derivative with the PR-1a promoter and the tml terminator and an intervening polylinker with unique EcoRI and NotI sites. The selected coding sequence can be inserted into this vector, and the fusion products (i.e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those described infra. Various chemical regulators can be employed to induce expression of the selected coding sequence in plants transformed in accordance with various embodiments of the invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and 5,614,395.

[0130] b. Ethanol-Inducible Promoters

[0131] A promoter inducible by certain alcohols or ketones, such as ethanol, can also be used to confer inducible expression of a coding sequence in accordance with various embodiments of the invention. Such a promoter is, for example, the alcA gene promoter from Aspergillus nidulans (Caddick et al. 1998 Nat. Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer. For the purposes of the present disclosure, the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al. 1998 Nat. Biotechnol 16:177-180) can be replaced by a selected coding sequence to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.

[0132] c. Glucocorticoid-Inducible Promoter

[0133] Induction of expression of a nucleic acid sequence using systems based on steroid hormones is also contemplated. For example, a glucocorticoid-mediated induction system is used (Aoyama and Chua 1997 The Plant Journal 11: 605-612) and gene expression is induced by application of a glucocorticoid, such as a synthetic glucocorticoid (e.g., dexamethasone). In one embodiment, the glutocorticoid is present at a concentration ranging from about 0.1 mM to about 1 mM. In another embodiment, the glutocorticoid is present at a concentration ranging from about 10 mM to 100 mM. For the purposes of the present disclosure, the luciferase gene sequences can be replaced by a sequence of interest to form an expression cassette having a sequence of interest under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods well known in the art. The trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al. 1986 Science 231: 699-704) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg et al. 1988 Genes Devel. 2: 718-729) fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard et al. 1988 Cell 54: 1073-1080). The expression of the fusion protein can be controlled by any promoter suitable for expression in plants, as known in the art or described here. This expression cassette can also comprise a sequence of interest fused to the 6.times.GAL4/minimal promoter. Thus, tissue- or organ-specificity of the fusion protein can be achieved, leading to inducible tissue- or organ-specificity of the expression cassette.

[0134] d. Wound-Inducible Promoters

[0135] Wound-inducible promoters can also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al. 1993 Plant Molec. Biol. 22: 573-588, Logemann et al. 1989 Plant Cell 1: 151-158, Rohrmeier & Lehle 1993 Plant Molec. Biol. 22: 783-792, Firek et al. 1993 Plant Molec. Biol. 22: 129-142, Warner et al. 1993 Plant J. 3: 191-201) and all are suitable for use with various embodiments of the invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pint) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize WipI cDNA, which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to a sequence of interest, for example, and used to express the sequence of interest at sites of plant wounding.

[0136] 3. Tissue-Specific or Tissue-Preferred Expression

[0137] a. Root-Preferred Expression

[0138] Another pattern of gene expression is root expression. A suitable root promoter for use with various embodiments of the invention is the promoter of the maize metallothionein-like (MTL) gene described by de Framond (FEBS 290: 103-106 (1991)) and also in U.S. Pat. No. 5,466,785. This "MTL" promoter is transferred to a suitable vector such as pCGN1761ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.

[0139] b. Pith-Preferred Expression

[0140] Patent Application WO 93/07278 describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells. The gene sequence and promoter extending up to -1726 by from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.

[0141] c. Leaf-Specific Expression

[0142] A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (1989 Plant Molec Biol 12: 579-589). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.

[0143] d. Pollen-Specific Expression

[0144] WO 93/07278 (published Apr. 15, 1993; Ciba Geigy) describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene, which is expressed in pollen cells. The gene sequence and promoter extend up to 1400 by from the start of transcription. Using standard molecular biological techniques, this promoter or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a sequence of interest in a pollen-specific manner.

[0145] B. Transcriptional Terminators

[0146] A variety of transcriptional terminators are available for use in the expression cassettes of the present invention. These are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. Suitable transcriptional terminators are those that are known to function in plants and include, but are not limited to, the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator can be used.

[0147] C. Sequences for the Enhancement or Regulation of Expression

[0148] Numerous sequences have been found to enhance gene expression from within the transcriptional unit, and these sequences can be used in conjunction with various genes to increase their expression in transgenic plants.

[0149] Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize AdhI gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al. 1987 Genes Develop. 1: 1183-1200). In the same experimental system, the intron from the maize bronze1 gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

[0150] A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. 1987 Nucl. Acids Res. 15: 8693-8711; Skuzeski et al. 1990 Plant Molec. Biol. 15: 65-79). Other leader sequences known in the art include but are not limited to: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, Fuerst, and Moss 1989 PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak and Sarnow 1991 Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling and Gehrke 1987 Nature 325:622-625; tobacco mosaic virus leader (TMV), (Gallie et al. 1989 Molecular Biology of RNA, pages 237-256); and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel et al. 1991 Virology 81:382-385). See also, Della-Cioppa et al. 1987 Plant Physiology 84:965-968.

[0151] D. Synthetic Genes

[0152] In various embodiments of the invention, coding sequences for selected proteins, such as a mega-endonuclease or a site-specific recombinase, for example, can be optimized for expression in a particular plant host. It is known in the art that the optimization of protein expression in plants can be enhanced by optimizing the coding regions of genes to the codon preference of the host. Accordingly, the preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the usage of codons within a cloned microbial ORF to usage in plant genes (and in particular genes from the target plant) enables an identification of the codons within the ORF that can be changed. Typically, plant evolution has tended towards a strong preference of the nucleotides C and G in the third base position of monocotyledons, whereas dicotyledons often use the nucleotides A or T at this position. By modifying a gene to incorporate preferred codon usage for a particular target transgenic species, many of the problems described below for GC/AT content and illegitimate splicing will be overcome.

[0153] Plant genes typically have a GC content of more than 35%. ORF sequences which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are believed to cause destabilization of message RNA (mRNA) and are found at the 3' end of many short-lived mRNAs. Secondly, the occurrence of polyadenylation signals, such as AATAAA, at inappropriate positions within the mRNA is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as introns and may identify flanking splice sites (see below).

[0154] Plants differ from microorganisms in that their mRNAs do not possess a defined ribosome-binding site. Rather, it is believed that ribosomes attach to the 5' end of the mRNA and scan for the first available ATG at which to start translation. Nevertheless, it is believed that there is a preference for certain nucleotides adjacent to the ATG and that expression of microbial genes can be achieved by the inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210, incorporated herein by reference) have suggested one sequence as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (1987 NAR 15: 6643-6653) has compared many plant sequences adjacent to the ATG and suggests another consensus sequence. In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these sequences at the initiating ATG may improve translation. In such cases, the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. A survey of 14 maize genes located in the GenBank database provided the following results:

TABLE-US-00001 Position Before the Initiating ATG in 14 Maize Genes: -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 C 3 8 4 6 2 5 6 0 10 7 T 3 0 3 4 3 2 1 1 1 0 A 2 3 1 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5

This analysis can be done for the desired plant species into which the nucleotide sequence is being incorporated, and the sequence adjacent to the ATG modified to incorporate the preferred nucleotides.

[0155] Genes cloned from non-plant sources and not optimized for expression in plants may also contain motifs which may be recognized in plants as 5' or 3' splice sites and may be cleaved, thus generating truncated or deleted mRNAs. These sites can be removed using techniques well known in the art.

[0156] Techniques for modifying coding sequences and adjacent sequences are well known in the art. In cases where the initial expression of a microbial ORF is low and it is deemed appropriate to make alterations to the sequence as described above, then the construction of synthetic genes can be accomplished according to methods well known in the art. See, e.g., EP 0 385 962, EP 0 359 472, and WO 93/07278. In most cases, it is preferable to assay the expression of gene constructions using transient assay protocols (which are well known in the art) prior to their use in generating transgenic plants.

II. Plant Transformation Vectors and Selectable Markers

[0157] Numerous transformation vectors known to those of ordinary skill in the plant transformation arts are available for plant transformation, and the nucleotide sequences pertinent to the invention can be used in conjunction with any such vectors. The selection of a particular vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. 1982 Gene 19: 259-268; Bevan et al. 1983 Nature 304:184-187), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al. 1990 Nucl. Acids Res 18: 1062, Spencer et al. 1990 Theor. Appl. Genet 79: 625-631), the hpt gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al. 1983 EMBO J. 2(7): 1099-1104), the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,835 and 5,188,642), and the mannose-6-phosphate isomerase gene (also referred to herein as the phosphomannose isomerase, or PMI, gene), which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

[0158] A. Vectors Suitable for Agrobacterium Transformation

[0159] Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan Nucl. Acids Res. (1984)). Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.

[0160] 1. pCIB200 and pCIB2001

[0161] The binary vectors pcIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by NarI digestion of pTJS75 (Schmidhauser & Helinski 1985 J. Bacteriol. 164: 446-455) allowing excision of the tetracycline-resistance gene, followed by insertion of an AccI fragment from pUC4K carrying an NPTII (Messing & Vierra 1982 Gene 19: 259-268; Bevan et al. 1983 Nature 304: 184-187; McBride et al. 1990 Plant Molecular Biology 14: 266-276). XhoI linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptII chimeric gene and the pUC polylinker (Rothstein et al. 1987 Gene 53: 153-161), and the Xhol-digested fragment are cloned into Sail-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI, and SalI. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI, Beg XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. pCIB2001, in addition to containing these unique restriction sites, also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

[0162] 2. pCIB10 and Hygromycin Selection Derivatives Thereof

[0163] The binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences. pCIB10 incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (1987 Gene 53: 153-161). Various derivatives of pCIB10 are constructed, which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (1983 Gene 25: 179-188). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

[0164] B. Vectors Suitable for Non-Agrobacterium Transformation

[0165] Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector, and, consequently, vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation), and microinjection. The choice of vector depends largely on the selected transformation method. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.

[0166] 1. pCIB3064

[0167] pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the published PCT application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites SspI and PvuII. The new restriction sites are 96 and 37 by away from the unique SalI site and 101 and 42 by away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with SalI and SacI, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 may be obtained from the John Innes Centre, Norwich and the 400 by SmaI fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the HpaI site of pCIB3060 (Thompson et al. 1987 EMBO J 6: 2519-2523). This generated pCIB3064, which comprises the bar gene (for herbicide selection) under the control of the CaMV 35S promoter and terminator, a gene for ampicillin resistance (for selection in E. coli), and a polylinker with the unique sites SphI, PstI, HindIII, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

[0168] 2. pSOG19 and pSOG35

[0169] The plasmid pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene (-550 bp), and 18 by of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR, and these two PCR fragments are assembled with a SacI-PstI fragment from pB1221 (Clontech), which comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19, which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene, and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites available for the cloning of foreign substances.

[0170] C. Vector Suitable for Chloroplast Transformation

[0171] For expression of a nucleotide sequence in plant plastids, plastid transformation vector pPH143 (WO 97/32011, example 36) can be used. The nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.

III. Transformation Methods

[0172] Target, donor, and other nucleotide sequence cassettes in accordance with the various embodiments of the invention can be introduced into the plant cell in a number of art-recognized ways. Methods for regenerating plants are also well known in the art. For example, Ti plasmid-derived vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells.

[0173] Once a desired DNA sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.

[0174] Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.

[0175] A. Transformation of Dicotyledons

[0176] Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al. 1984 EMBO J 3: 2717-2722, Potrykus et al. 1985 Mol. Gen. Genet. 199: 169-177, Reich et al. 1986 Biotechnology 4: 1001-1004, and Klein et al. 1987 Nature 327: 70-73. In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.

[0177] Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledons because of its high transformation efficiency and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of a binary vector carrying a foreign DNA of interest (e.g., pCIB200 or pCIB2001) to an appropriate Agrobacterium strain, which may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g., strain CIB542 for pCIB200 and pa:132001 (Uknes et al. 1993 Plant Cell 5: 159-169). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, 1988 Nucl. Acids Res. 16: 9877).

[0178] Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on a selection medium containing the compound (e.g., the antibiotic, herbicide, or carbohydrate source) that corresponds to the selectable marker sequence (e.g., antibiotic or herbicide resistance gene or PMI gene) present between the binary plasmid's T-DNA borders.

[0179] Another approach to transforming a plant cell with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all issued to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium, or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.

[0180] B. Transformation of Monocotyledons

[0181] Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG (polyethylene glycol) or electroporation techniques, particle bombardment into callus tissue, and transformation mediated by Agrobacterium. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e., co-transformation), both of which are suitable for use with the methods disclosed herein. Co-transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and either the selectable marker or other sequences, such as those used for improving transformation efficiency, thereby enabling the removal of the selectable marker or other sequences in subsequent generations, should this be regarded as desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. 1986 Biotechnology 4: 1093-1096).

[0182] Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (1990 Plant Cell 2: 603-618) and Fromm et al. (1990 Biotechnology 8: 833-839) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (1993 Biotechnology 11: 194-200) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

[0183] Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al. 1988 Plant Cell Rep 7: 379-384; Shimamoto et al. 1989 Nature 338: 274-277; Datta et al. 1990 Biotechnology 8: 736-740). Both types are also routinely transformable using particle bombardment (Christou et al. 1991 Biotechnology 9: 957-962). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation. Patent Application EP 0 332 581 describes techniques for the generation, transformation, and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat.

[0184] Furthermore, wheat transformation has been described by Vasil et al. (1992 Biotechnology 10: 667-674) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (1993 Biotechnology 11: 1553-1558) and Weeks et al. (1993 Plant Physiol. 102: 1077-1084) using particle bombardment of immature embryos and immature embryo-derived callus.

[0185] One technique for wheat transformation involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any convenient number of embryos (0.75-1 mm in length) can be plated onto MS medium with 3% sucrose (Murashiga & Skoog 1962 Physiologia Plantarum 15: 473-497) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics.RTM. helium device using a burst pressure of 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as "GA7s" which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.

[0186] Transformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616. Rice transformation using Agrobacterium has been described in a number of publications, including Hiei et al. 1994 Plant J. 6:271-282, Dong et al. 1996 Molecular Breeding 2:267-276, and Hiei et al. 1997 Plant Molecular Biol. 35:205-218. Efficient maize transformation using Agrobacterium infection of immature embryos and various selection markers also has been described (Ishida et al. Nature Biotechnology 14:745-750; Negrotto et al. 2000 Plant Cell Reports 19:798-803; and Li et al. 2003 Plant Physiol. 133:736-747).

[0187] C. Transformation of Plastids

[0188] Seeds of Nicotiana tabacum c.v. `Xanthi nc` are germinated seven per plate in a 1 inch circular array on T agar medium and bombarded 12-14 days after sowing with 1 .mu.m tungsten particles (M10, Biorad, Hercules, Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab and Maliga 1993 PNAS 90: 913-917). Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 .mu.mol photons/m.sup.2/s) on plates of RMOP medium (Svab, Hajdukiewicz, and Maliga 1990 PNAS 87: 8526-8530) containing 500 .mu.g/ml spectinomycin dihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned. Complete segregation of transformed plastid genome copies (homoplasmicity) in independent subclones is assessed by standard techniques of Southern blotting (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digested total cellular DNA (Mettler 1987 Plant Mol Biol Reporter 5: 346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with .sup.32P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment from pC8 containing a portion of the rps7/12 plastid targeting sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA medium (McBride et al. 1994 PNAS 91: 7301-7305) and transferred to the greenhouse.

[0189] The foregoing describes various embodiments of the invention and is not intended to limit the scope of the invention as defined in the appended claims. The following Examples are included merely to demonstrate the practice of selected embodiments and should be regarded in an illustrative, rather than a restrictive, manner.

EXAMPLES

[0190] Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Ausubel (ed.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc. (1994); J. Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3d Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (2001); and T. J. Silhavy, M. L. Berman, and L. W. Enquist, EXPERIMENTS WITH GENE FUSIONS, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984).

Example 1

Construction of a Modified Neomycin Phosphotransferase II (nptII) Gene with Four Arabidopsis thaliana Introns

[0191] To introduce four Arabidopsis thaliana introns into the neomycin phosphotransferase II gene (nptII), which confers kanamycin resistance, individual introns and nptII coding sub-regions (artificial exons) were amplified with PCR and then combined by a second round of PCR to form hybrid fragments containing Arabidopsis intron-nptII exon cassettes. Each cassette was cloned individually and combined using standard DNA recombination methods. NptII exon 1 was amplified using primers NPTFA (SEQ ID NO:1: 5'-GAT CTC TAG AAT GAT TGA ACA AGA TGG ATT-3') and NPTRA (SEQ ID NO:2. 5'-TCG CAG CTT GGT ACC TGC AGT TCA TTC AGG GC-3') from pCIB200 (Rothstein et al., Gene 53:153-161, 1987). The PCR product was digested with XbaI/PstI and inserted into XbaI/PstI-digested pNOV2799 to form pNOV2711. pNOV2799 was derived from pNOV205 by replacing the SacII/XbaI polylinker with the SpeI/XbaI polylinker from pLITMUS28 (New England Biolab). pNOV204 is a pBluescript vector containing the Smas promoter (Ni et al. 1996 Plant J. 7: 661-676.) The intron in the untranslated leader of AtBAF60 was amplified from A. thaliana ecotype Columbia DNA with primers IntBAFFW (SEQ ID NO:3: 5'-GCC CTG AAT GAA CTG CAG GTA CCA AGC TGC GA-3') and IntBAFRV (SEQ ID NO:4: 5'-GCC GCG CTG CCT CGT CCT GAA AAA TTC AGA AA-3'). AtBAF60 (CHCl) is a gene that shares homology with the mammalian nucleosome-remodeling factor BAF60 (http://www.chromdb.org/). NptII exon 2 was amplified from pCIB200 using primers NPTF2 (SEQ ID NO:5: 5'-TTT CTG AAT TTT TCA GGA CGA GGC AGC GCG GC-3') and NPTR2 (SEQ ID NO:6: 5'-GAA TAG TAC TAA TAC CTG GCA CTT CGC CCA ATA G-3'). A PAL1 intron was amplified from Arabidopsis thaliana ecotype Landsberg erecta using primers IntPALFW (SEQ ID NO:7: 5'-TTA GTA CTA TTC TTT TGT TCT CTA ATC AGA-3') and IntPALRV (SEQ ID NO:8: 5'-TGA CAG GAG ATC CTG CCC TGT AAC GAA CAA AAA CAT-3'). NptII exon 3 was amplified from pCIB200 using primers NPTFC (SEQ ID NO:9: 5'-ATG TTT TTG TTC GTT ACA GGG CAG GAT CTC CTG TCA-3') and NPTR3 (SEQ ID NO:10: 5'-ATC GAT TCA TAT ATA TAC CTG GTC GAC AAG ACC GGC-3'). A tubulin-1-.beta. intron (760 bps) was amplified from Arabidopsis thaliana ecotype Columbia with primers IntTUBFW (SEQ ID NO:11: 5'-CAG GTA TAT ATA TGA ATC GAT TTC TCC CTT-3') and IntTUBRV (SEQ ID NO:12: 5'-TCG TCC AGA TCA TCC TGT AAT ACA GAA ATG TT-3'). NptII exon 4 was amplified from pCIB200 (Rothstein et al. 1987 Gene 53:153-161) with primers NPTFD (SEQ ID NO:13: 5'-AAC ATT TCT GTA TTA CAG GAT GAT CTG GAC GA) and NPTR4 (SEQ ID NO:14: 5'-GGA AAA GCT TAA TTA CCT CGC CGT CGG GCA TG-3'). A tubulin-1-.alpha. intron (560 bps) was amplified from Arabidopsis thaliana ecotype Columbia with primers IntTUAFW (SEQ ID NO:15: 5'-GTA ATT AAG CTT TTC CAC CTC TCT TGT T-3') and IntTUARV (SEQ. ID NO:16: 5'-GAT CCT GCA GCA ATG GAA AAA TAT TTC AAT AC-3'). NptH exon 5 was amplified from pCIB200 with primers NPTFE (SEQ ID NO:17: 5'-ATT GCT GCA GGA TCT CGT CGT GAC CCA TGG-3') and NPTR5 (SEQ ID NO:18: 5'-CAT TAG GAT CCT CAG AAG AAC TCG TCA A-3'). All of the above PCR products were gel purified and used as templates for a second round of PCR amplification. All PCR reactions were carried out with a mixture of Taq polymerase and Pfu polymerase (30 to 1, unit/unit) in a Perkin-Elmer thermocycler 9600.

[0192] Purified AtBAF60 intron and nptII exon 2 PCR fragments were used as templates for amplification with primers IntBAFFW and NPTR2, and the resulting PCR product was cloned into a pCR2.1-TOPO vector to form pNOV2708. The PAL1 intron and nptII exon 3 were amplified using primers IntPALFW and NPTR3, and the PCR product was cloned into pCR2.1-TOPO to form pNOV2709. The tubulin-1-.beta. intron and nptII exon 4 PCR fragments were amplified using primers IntTUBFW and NPTR4, and the resulting PCR product was cloned into pCR2.1-TOPO to form pNOV2710. The tubulin-1-.alpha. intron and nptII exon 5 fragments were co-amplified using IntTUAFW and NPTR5 primers, and the resulting PCR product was inserted into pCR2.1-TOPO to form pNOV2712. Plasmid pNOV2708 was digested partially with BglII. A linker was formed by annealing two oligonucleotides, FRTBGL2 (SEQ ID NO:19: 5'-GAT CTG AAG TTC CTA TTC TCT AGA AAG TAT AGG AAC TTC G-3') and FRTBAM1 (SEQ ID NO:20: 5'-GAT CCG AAG TTC CTA TAC TTT CTA GAG AAT AGG AAC TTC A-3'). This linker, which contained an FRT site, was inserted into the BglII site in the AtBAF60 intron to form pNOV2715. The PAL1 gene 3'-UTR was amplified from Arabidopsis thaliana ecotype Landsberg erecta using primers TPALBGLII (SEQ ID NO:21: 5'-TGT TAA GAT CTT AGT CCT CTG TTT TTT TCT-3') and TPALSACI (SEQ ID NO:22: 5'-CTT GAG CTC TTC TAT AAC CCT AGA TGG CTA-3'). The PAL1 3'-UTR PCR product was digested with BglII and Sad and then inserted into BglII/SacI-digested pLITMUS28 to form pNOV2707. All the inserts in the above clones were sequenced to ensure that no mutations were introduced into the coding sequence.

[0193] The individual intron-nptII exon cassette was then recombined to form a full-length modified nptII gene. The AtBAF60 intron-nptII exon 2 fragment was removed from pNOV2715 by partial PstI and complete XhoI digestion and inserted into PstI/XbaI-digested pNOV2711 to form pNOV2718. The tubulin-1-.beta. intron-exon 4 fragment was released from pNOV2710 by ClaI/HindIII double-digestion and inserted into ClaI/HindIII-digested pNOV2709 to form pNOV2716, containing PAL1 intron-nptII exon 3-tubulin-1-.beta. intron-nptII exon 4. A BglII/SacI fragment containing the PAL1 3'-UTR was removed from pQD7A1 and inserted into BamHI/SacI-digested pNOV2712 to form pNOV2717. The 1.5 kb XhoI/HindIII and 1.1 kb HindIII/SacI fragments containing intron-exon cassettes were removed from pNOV2716 and pNOV2717, respectively, and inserted into XhoI/SacI-digested pBluescript II KS(+) to form pNOV2719. Plasmid pNOV2719 was digested with Sad and ScaI, and the 2.6 kb SacI/ScaI fragment containing intron-exons and PAL 3'-UTR was isolated into pNOV2718 partially digested with SacI and ScaI to form pNOV2720. Plasmid pNOV2720 then contained the full-length modified nptII gene with four Arabidopsis introns (FIG. 14) under the control of a modified super MAS (mSmas) promoter.

Example 2

Construction of a Control Vector for Dicot Plants

[0194] The 4489 base pair SacI/XhoI fragment containing the mSmas promoter-modified nptII-PAL1 3' end cassette was removed from pNOV2720 and inserted into pNOV2722, which was partially digested with Sad and XhoI to form control construct pNOV2731 (FIG. 16). pNOV2731 was transformed into Agrobacterium LBA4404, and the resulting Agrobacterium strain was used to transform both Arabidopsis and tobacco plants. Phosphinothricin (PPT) resistant transformants produced seeds that were highly resistant to kanamycin. The results demonstrate that the modified nptII gene is fully functional and the introns can be spliced out efficiently.

Example 3

Construction of Target Vectors for Dicot Plants

[0195] The coding region for the bar gene, which provides resistance to the herbicide Basta.RTM., was amplified from pGSFR1 (D'Halluin et al. 1992 Methods Enzymol. 216: 415-26) using two primers, BARCLA (SEQ ID NO:23: 5'-TCA TAT CGA TGA GCC CAG AAC GAC GCC-3') and BARBGL (SEQ ID NO:24: 5'-TTT GAG ATC TTC ATA TCT CGG TGA CGG GCA GG-3'). The gel-purified PCR product was digested with BglII and inserted into SmaI/BamHI-digested pHSPnos to form pNOV2703. pHSPnos is a pSPORT1 base vector (GIBCO BRL, Rockville, Md.) containing the Brassica HSP80 promoter (see U.S. Pat. No. 5,612,472) followed by the nopaline synthase terminator (Bevan et al. 1983 Nucleic Acids Res. 11, 369-385). pNOV2703 was digested with NotI, filled in with a Klenow fragment, and then digested again with XhoI to isolate the 2.4 kb NotI/XhoI fragment containing the Brassica HSP80 promoter-bar-Tnos expression cassette. Binary base vector pHINK078 was digested with ApaI, filled in with a Klenow fragment, and then cut with XhoI. The above-described 2.4 kb NotI/XhoI HSP80 promoter-bar-nos3'UTR fragment was inserted into ApaI/XhoI-digested pHINK078 to form pNOV2797. pNOV2797 was digested with BglII, filled-in with a Klenow fragement, and religated to form pNOV2706. The SacI/NcoI polylinker (88 bps) from pNOV2799 was inserted into SacI/NcoI-digested pNOV2706 to form pNOV2722. pNOV2722 was cut with BglII and then ligated with BglII/BamHI-digested DNA fragments containing a recognition sequence for endonuclease I-SceI, I-CeuI, or HO to form pNOV2723 (I-SceI), pNOV2724 (I-CeuI), and pNOV2725 (HO), respectively. The DNA fragment containing the I-SceI site was synthesized by annealing oligonucleotides ISCEBAM1 (SEQ ID NO:25: 5'-ACT TGG ATC CAT ATT ACC CTG TTA TCC CTA-3') and ISCEBGL2 (SEQ ID NO:26: 5'-TCG AAG ATC TGC TAG GGA TAA CAG GGT AAT-3'), filled-in with a Klenow fragment of E. coli DNA polymerase I, and then digested with BglII and BamHI. A DNA fragment for I-CeuI was synthesized similarly with oligonucleotides ICEUBGL2 (SEQ ID NO:27: 5'-TCG AAG ATC TCT ATA ACG GTC GTA AGG TAG-3') and ICEUBAM1 (SEQ ID NO:28: 5'-ACT TGG ATC CTC GCT ACC TTA GGA CCG TTA-3'). The DNA fragment for the HO site was synthesized with oligonucleotides HOBGL2 (SEQ ID NO:29: 5'-TCG AAG ATC TAG CTT TCC GCA ACA GTA TAA-3') and HOBAM1 (SEQ ID NO:30: 5'-ACT TGG ATC CAT TAT ACT GTT GCG GAA AGC-3'). pNOV2720 was digested with BglII and Sad to isolate a 3054 by BglII/SacI fragment containing truncated modified nptII-PAL1-3'-UTR. This fragment was inserted into BglII/SacI-digested pNOV2723, pNOV2724, and pNOV2725 to form pNOV2700 (with I-SceI site), pNOV2729 (with I-Ceul site), and pNOV2701 (with HO site), respectively (FIG. 13C).

Example 4

Generation of Tobacco Plants Containing a Target Construct

[0196] Agrobacterium tumefaciens strain LBA4404 containing target constructs pNOV2700, pNOV2701, pNOV2729, or control plasmid pNOV2731, respectively, were used to infect leaf explants of Nicotiana tabacum c.v. Petit Havana (SR1). Transgenic plants were obtained from the infected leaf explants using PPT (5 mg/L) as a selection agent. Initially, the tobacco leaves were cut into 1-2 mm wide slices, exposed to the Agrobacterium resuspended in MS3S for 5 minutes, and then moved to sterile paper to blot away excess liquid and placed on co-cultivation medium (MS3S+NAA (0.1 mg/L)+6-BA (1 mg/L)+gelrite agar (2.4 g/L)) for 3 days. The leaf slices were then moved to selection/regeneration medium (MS3S+NAA (0.1 mg/L)+6-BA (1 mg/L)+Carbenicillin (200 mg/L)+gelrite agar (2.4 g/L)+PPT (5 mg/L)). PPT resistant shoots were rooted in selection/rooting medium (MSB+PPT (5 mg/L)+Carbenicillin (200 mg/L)+phytagar (8 g/L) in GA-7 boxes) and then transplanted to soil. As a control, pNOV2731 was placed in rooting medium that included kanamycin (150 mg/L) as well as PPT (5 mg/L) to screen for the expression of the modified nptII gene with four introns. The plants were selfed or outcrossed with pollens from nontransgenic SR1 plants to produce seeds.

Example 5

Molecular Analysis of Transgenic Plants

[0197] The DNA of the transgenic plants was analyzed in accordance with standard molecular biological techniques. DNA was isolated from the leaves of transgenic plants for T-DNA structure analysis using the CTAB protocol (Jorgensen et al. 1996 Plant Mol. Biol. 31:957-973). Samples containing about 5 micrograms of tobacco DNA were digested with a restriction enzyme, such as SacI, NheI, SpeI, KpnI, Seal, HpaI, EcoRI, and EcoRV, separated on an agarose gel, blotted onto Hybond N+ nylon membrane, and then hybridized with a .sup.32P-labeled probe. The probes were prepared from either a PPT fragment or the nptII exon 5/Pal terminator fragment, as appropriate.

Example 6

Construction of Donor Vectors

[0198] pNOV2704 was digested with NotI, blunted with Klenow, cut with XbaI, and ligated with 3.1 kb KpnI/XbaI (blunted) of pNOV2705 containing the UBQ3 5' region (promoter, intron, and leader)-Hyg-TUBG3 to create pNOV2726. pLITMUS28 (New England Biolabs, MA) was digested with BglII and ligated with a fragment containing an FRT site derived from annealed oligonucleotides FRTBGL2 (SEQ ID NO:31: 5'-GAT CTG AAG TTC CTA TTC TCT AGA AAG TAT AGG AAC TTC G-3') and FRTBAM1 (SEQ ID NO:32: 5'-GAT CCG AAG TTC CTA TAC TTT CTA GAG AAT AGG AAC TTC A-3') to create plasmid pNOV2727. pNOV2727 was digested with XhoI, filled-in with a Klenow fragment in the presence of dTTP only, then cut with Sad to isolate a 2.8 kb XhoI/SacI fragment. pNOV2720 was cut with ClaI, filled-in with a Klenow fragment, then digested with Sad to isolate the 4.8 kb SacI/ClaI fragment. The 2.8 kb XhoI/SacI fragment of pNOV2727 was ligated with the 4.8 kb ClaI/SacI fragment of pNOV2720 to create pNOV2732.

[0199] pNOV2700 was digested partially with EcoRV and XhoI to isolate the 10 kb EcoRV/XhoI fragment and then ligated with 3.2 kb Sad (blunted)/XhoI fragment of pNOV2726 to create pNOV2733. pNOV2733 was digested with Sad, blunted with T4 DNA polymerase, and then partially cut with BglII to isolate the 10.4 kb BglII/SacI (blunted) fragment. pNOV2732 was digested with NcoI, filled-in with a Klenow fragment, and then partially cut with BglII to isolate the 3.97 kb BglII/NcoI (blunted) fragment. The donor construct pNOV2736 (FIG. 13D) was created by ligating the 10.4 kb BglII/SacI(blunted) fragment with the 3.97 kb BglII/NcoI fragment.

[0200] pNOV2729 was digested partially with EcoRV and XhoI to isolate the 10 kb EcoRV/XhoI fragment. The fragmant was ligated with a 3.2 kb SacI/XhoI fragment of pNOV2726 (SacI site was blunted by a Klenow treatment) to create pNOV2734.

[0201] pNOV2734 was digested with Sad, blunted with T4 DNA polymerase treatment, then partially cut with BglII to isolate the 10.4 kb SacI/BglII fragment. This fragment was ligated with the 4 kb NcoI/BglII fragment (NcoI was blunted by a Klenow fragment) of pNOV2732 to create donor construct pNOV2737 (FIG. 13D).

[0202] pNOV2701 was digested partially with EcoRV and XhoI to isolate the 10 kb EcoRV/XhoI fragment. This fragment was ligated with the 3.2 kb SacI/XhoI fragement of pNOV2726 (Sad site was blunted by Klenew treatment) to create pNOV2735.

[0203] pNOV2735 was digested with Sad, blunted with T4 DNA polymerase, then partially cut with BglII to isolate the 10.4 kb SacI/BglII fragment. This fragment was ligated with the 4 kb NcoI/BglII fragment (NcoI site was blunted) of pNOV2732 to create donor construct pNOV2738.

[0204] pNOV2734 was digested partially with Ecl136II and BglII to isolate a 10.4 kb Ecl136II/BglII fragment. This fragment was ligated to a 2.5 kb SalI (Blunted)/BglII fragment of pNOV2732 to form donor construct pNOV2755 (FIG. 13D). The pNOV2734 Ecl136II/BglII (10.4 kb) fragment was ligated with a 1.54 kb MscI/BglII fragment of pNOV2732 to form pNOV2756. The pNOV2734 Ecl136II/BglII (10.4 kb) fragment was ligated with 1.42 kb EcoRI (blunted)/BglII fragment of pNOV2732 to form pNOV2757 (FIG. 13D).

[0205] pNOV2733 was digested partially with Ecl136II and BglII to isolate a 10.4 kb Ecl136II/BglII fragment. The fragment was ligated with 3.97 kb NcoI (blunted with Klenow)/BglII fragment of pNOV2732 to form binary donor pNOV2759.

Example 7

Construction of an HO Endonuclease Expression Vector for Dicot Plants

[0206] The coding region of the yeast HO endonuclease gene was amplified from Saccharomyces cereviceae (ATCC48893) using primers HOATG (SEQ ID NO:33: 5'-CTA CTG TCG ACA AAA ATG CTT TCT GAA AAC-3') and HOBAMH (SEQ ID NO:34: 5'-CTA GGA TCC GAC CTG GTC GTC ACA GTA GCT-3'), and the PCR product was cloned into the pCR2.1-TOPO vector to form pNOV2741. pNOV2741 was digested partially with SalI and BamHI, and the SalI/BamHI fragment containing the HO gene was inserted into (SalI)partial/BamHI-digested pNOV2721 to form pNOV2742. The Act2 promoter-HO-act2 terminator cassette was excised from pNOV2742 by KpnI and Sad digestion and was inserted into KpnI/SacI-digested pHINK078 to form binary vector pNOV2747 (FIG. 13E). The HO expression cassette was also excised from pNOV2742 by KpnI and Sad digestion and inserted into KpnI/SacI-cut pCIB100 (Rothstein et al. 1987 Gene 53:153-161) to form pNOV036.

Example 8

Construction of a Synthetic I-CeuI Gene with Maize-Preferred Codons

[0207] The amino acid sequence for the homing endonuclease I-CeuI (Gauthier, Turmel, and Lemieux 1991 Curr. Genet. 19: 43-47) was back-translated into the DNA sequence shown in SEQ ID NO:35 using maize-preferred codons (see U.S. Pat. No. 6,121,014). The unique restriction endonuclease cut site EagI was identified within this DNA sequence, which allowed the DNA to be cloned as two separate segments or sub-fragments of 340 by and 346 bp. Because expression of the I-CeuI endonuclease is toxic to E. coli, an intron was introduced into the 5'-segment before excision and ligation of the segments to form the complete gene. A 189-bp potato ST-LS1 intron sequence (Narasimhulu et al. 1996 Plant Cell 8:873-886) was also inserted into I-CeuI to facilitate cloning in E. coli. Each of the two sub-fragments was constructed from oligonucleotides ranging from 65 to 75 bases in length, with each oligonucleotide overlapping neighboring oligonucleotides by 20 bp.

[0208] Segment 1 of synthetic I-CeuI (SynICeuI) included the first 335 by preceding the EagI site and was constructed from the following oligonucleotides: 1A (SEQ ID NO:36: 5'-GGGGA TCCAT GAGCA ACTTC ATCCT GAAGC CCGGC GAGAA GCTGC CCCGG ACAAG CTGGA GGAGC TGAAG AAGA-3') (GG+BamHI site+top strand bases 1-67), 1B (SEQ ID NO:37: 5'-CGCAG GTCGA TCAGGTACTT GCTGA AGTTC TTGGT CTTCT TCACG GCGTCGTTGA TCTTC TTCAG CTCCT CCAGC-3') (bottom strand bases 48-122), 1C (SEQ ID NO:38: 5'-AAGTA CCTGA TCGAC CTGCG CAAGC TGTTC CAGAT CGACG AGGTG CAGGT GACCA GCGAG AGCAA GCTGT TCCTG-3') (top strand bases 103-177), 1D (SEQ ID NO:39: 5'-TGG CCA GCT TCT TGG TGC TGA TGT TCA GGC TGG CCT CGC CCT CCA GGA AGC CGG CCA GGA ACA GCT TGC TCT CGC-3') (bottom strand bases 158-232), 1E (SEQ ID NO:40: 5'-CAGCA CCAAG AAGCT GGCCA CCAGC AAGTT CGGCC TGGTG GTGGA CCCCG AGTTC AACGT GACCC AGCAC GTGAA-3') (top strand bases 213-287), and 1F (SEQ ID NO:41: 5'-CGCAG GTCGA TCAGG TACTT GCTGA AGTTC TTGGT CTTCT TCACG GCGTCG TTGAT CTTCT TCAGC TCCTC CAGC-3') (bottom strand bases 268-335+5'CCC).

[0209] Segment 1 was constructed in three steps: (1) a Klenow fill-in reaction to form three sets of dimers (AB, CD, and EF); (2) a PCR joining of dimers CD and EF to form a tetramer CDEF; and (3) a second PCR joining of tetramer CDEF to dimer AB, forming hexamer ABCDEF. Three reactions of 50 .mu.l containing 1.times.DNA polymerase salts and 1 .mu.l each of 20 .mu.M solution of 1A and 1B, 1C and 1D, and 1E and 1F, respectively, were heated at 67.degree. C. for 5 minutes and then allowed to cool slowly to 22.degree. C. To each reaction was added 1 .mu.l of a mix of four deoxynucleotide triphosphates (10 mM each), plus 2 .mu.l (10 units) of a Klenow fragment of DNA polymerase (New England Biolabs). The reaction was incubated at 22.degree. C. for 15 minutes, producing AB, CD, and EF precursors of SynICeuI segment 1. Segment CD was joined to overlapping EF by 10 cycles of PCR. A PCR reaction mixture containing 13 .mu.l water, 5 .mu.l each of the CD and EF Klenow reactions, and 1 .mu.l each of the 20 .mu.M solutions of oligo 1C and 1F as primers was added to a Ready-to-Go PCR bead (Amersham Pharmacia Biotech Inc). The PCR reaction conditions were: 95.degree. C. for 5 minutes; (95.degree. C. for 1 min., 56.degree. C. for 30 sec., 72.degree. C. for 1 min.) 10 cycles; 72.degree. C. for 10 min. The yield of tetrameric product was increased by reamplification of the product of this reaction as follows: A new PCR reaction mixture containing 18 .mu.l water, 5 .mu.l of product of the previous PCR reaction, and 1 .mu.l each of the 20 .mu.M solutions of oligo 1C and 1F as primers was added to a Ready-to-Go PCR bead, and the amplification program described above was re-employed. The tetrameric PCR product was excised from an agarose minigel (2% Seaplaque agarose), and the DNA was purified by the QIAquick Gel Extraction Kit (Qiagen, Vanecia, CA91355).

[0210] In order to form the hexameric product, the PCR-mediated joining process was repeated using tetramer CDEF plus dimer AB with oligonucleotides 1A and 1F as primers. The resulting hexameric DNA fragment was isolated and purified as described above and then cloned using the TOPO-TA Cloning Kit (InVitrogen, Carlsbad, Calif.). Clones with hexamer-sized inserts were sequenced to identify one of perfect sequence, which is referred to as pCR2.1SynICeuI-1. For assembly of the complete synthetic gene, the fragment was ultimately excised from the TOPO vector with BamHI and EagI, but only after introduction of an intron (see below).

[0211] Segment 2 was constructed from the following oligonucleotides: 2G (SEQ ID NO:42: 5'-CCC CGG CCG CAT CCG CCA CAA GAG CGG CAG CAA CGC CAC CCT GGT GCT GAC CAT CGA CAA CCG CCA GAG CCT GGA-3'), 2H (SEQ ID NO:43: 5'-CTC GGG GCT GCT GAA GGC CAC CAC GTA CTG CTC GTA GAA GGG GAT CAC CTT CTC CTC CAG GCT CTG GCG GTT GTC-3'), 2I (SEQ ID NO:44: 5'-TGG CCT TCA GCA GCC CCG AGA AGG TGA AGC GCG TGG CCA ACT TCA AGG CCC TGC TGG AGC TGT TCA ACA ACG ACG-3'), 2J (SEQ ID NO:45: 5'-ATC TGG TCC CAG ATG GGC AGG ATC TTG TTC ACC AGC TGC TCC AGG TCC TGG TGG GCG TCG TTG TTG AAC AGC TCC-3'), 2K (SEQ ID NO:46: 5'-CTG CCC ATC TGG GAC CAG ATG CGC AAG CAG CAG GGC CAG AGC AAC GAG GGC TTC CCC AAC CTG GAG GCC GCC CAG-3'), and 2L (SEQ ID NO:47: 5'-GGG GAA TTC CTA CTT GAT GCC CTT CTT GTA GTT GCG GGC GAA GTC CTG GGC G,GC CTC CAG GTT GG-3'). In a manner similar to that described above for segment 1, segment 2 was constructed in three steps: (1) a Klenow fill-in reaction to form three sets of dimers (GH, IJ, and KL); (2) a PCR joining of dimers EF and GH to form a tetramer, EFGH; and (3) a second PCR joining of EFGH with IJ to form a hexamer, EFGHIJ.

[0212] The hexamer DNA fragment GHIJKL was cloned into pCR2.1 using the TOPO-TA Cloning Kit (InVitrogen) and was sequenced to identify a clone of perfect sequence, which is referred to as pCR2.11CeuI-2.

Introduction of an Intron into pCR2.1SynICeuI-1

[0213] The potato ST-LS1 intron was PCR-amplified from pBISN1 (Narasimhulu et al. 1996 Plant Cell 8:873-886) using an oligonucleotide primer pair (i.e., SEQ ID NO:48: 5'-GGGTA CGTAA GTTTC TGCTT CTACC TTTG-3' and SEQ ID NO:49: 5'-CCCCAG CTGCA CATCA ACAAA TTTTG GTC-3') to form SnaBl and PvuII sites (shown in bold) at the 5' and 3' ends of the intron, respectively. The PCR product was cloned using the TOPO-TA Cloning Kit (Invitrogen), and a perfect copy, referred to as pInt1, was identified through sequencing. The intron was excised from pInt1 as a SnaB1/PvuII fragment, gel-purified, and then extracted from agarose with the QIAquick gel extraction kit. pCR2.1SynICeuI-1 was cleaved at a unique PmlI site in the insert and, in accordance with methods known in the art, was treated with alkaline phosphatase under appropriately stringent conditions for achieving blunt-end dephosphorylation. The intron fragment was ligated into this vector, and candidate clones were screened by Apol digestion and sequenced to confirm a clone of perfect sequence with the intron in the correct orientation with respect to the coding sequence of ICeuI. The plasmid so-identified is referred to as pCRSynICeuI-1-int.

Assembly of the SynICeuI Gene

[0214] Plasmid pBluescript KS(+) (Stratagene, Inc.) was digested with NotI and EcoRI in the presence of alkaline phosphatase. The 3' end of SynICeuI was excised from pCR2.1IceuI-2 with EagI and EcoRI, gel-purified, and ligated to the bluescript vector, forming pBS-GHIJKL. Because the EagI site of the insert is a half NotI site, the NotI site was reconstituted in the product. This plasmid was next cleaved with NotI in the presence of alkaline phosphatase, and the 5' end of SynICeuI, including the intron excised as an EagI fragment from pCRSynICeuI-1-Int, was ligated into place. Candidate clones were sequenced to identify one with the ABCDEIntF fragment inserted in the correct orientation. The identified clone is referred to as pBS-ICeuI-Int. The sequence of SynICeuI is represented by SEQ ID NO:35, which shows the flanking noncoding DNA between the EcoRI sites in italics.

TABLE-US-00002 SEQ ID NO: 35. I-CeuI endonuclease with maize-preferred codons and potato ST-LS1 intron GAATTCGCCCTTGGGGATCCATGAGCAACTTCATCCTGAAGCCCGGCGAG AAGCTGCCCCAGGACAAGCTGGAGGAGCTGAAGAAGATCAACGACGCCGT GAAGAAGACCAAGAACTTCAGCAAGTACCTGATCGACCTGCGCAAGCTGT TCCAGATCGACGAGGTGCAGGTGACCAGCGAGAGCAAGCTGTTCCTGGCC GGCTTCCTGGAGGGCGAGGCCAGCCTGAACATCAGCACCAAGAAGCTGGC CACCAGCAAGTTCGGCCTGGTGGTGGACCCCGAGTTCAACGTGACCCAGC ACGTAAGTTTCTGCTTCTACCTTTGATATATATATAATAATTATCATTAA TTAGTAGTAATATAATATTTCAAATATTTTTTTCAAAATAAAAGAATGTA GTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTT ATAACTTTTCTAATATATGACCAAAATTTGTTGATGTGCAGGTGAACGGC GTGAAGGTGCTGTACCTGGCCCTGGAGGTGTTCAAGACCGGCCGCATCCG CCACAAGAGCGGCAGCAACGCCACCCTGGTGCTGACCATCGACAACCGCC AGAGCCTGGAGGAGAAGGTGATCCCCTTCTACGAGCAGTACGTGGTGGCC TTCAGCAGCCCCGAGAAGGTGAAGCGCGTGGCCAACTTCAAGGCCCTGCT GGAGCTGTTCAACAACGACGCCCACCAGGACCTGGAGCAGCTGGTGAACA AGATCCTGCCCATCTGGGACCAGATGCGCAAGCAGCAGGGCCAGAGCAAC GAGGGCTTCCCCAACCTGGAGGCCGCCCAGGACTTCGCCCGCAACTACAA GAAGGGCATCAAGTAG GAATTC

Example 9

Construction of a Dicot I-CeuI Endonuclease Expression Vector

[0215] The pBH37 plasmid, an expression vector containing a modified Smas promoter, a Nos terminator, and cloning sites between these two regions, was digested with BglII, and the BglII site was converted to an MfeI site by the introduction of the following site conversion oligonucleotide: (SEQ ID NO:50: 5'-GATCGGCAATTGCC-3'). The resulting plasmid, pBH37M, was digested with MfeI in the presence of alkaline phosphatase. SynICeuI was excised from its bluescript vector as an EcoRI fragment and was ligated into MfeI-cleaved pBH37M. Candidate clones were digested with BstEII/PstI, and a clone having a correctly oriented fragment containing SynICeuI appropriately flanked by the Smas promoter and the Nos terminator was chosen for further cloning into a binary vector. This fragment, referred to as Smas-ICeuI-Int, was excised as a HindIII/EcoRI fragment, ligated into pHINK078, and then digested with HinndIII/EcoRI in the presence of alkaline phosphatase to form pNOV039. Binary vector pNOV100 was digested with HindIII/EcoRI in the presence of alkaline phosphatase, and the HindIII/EcoRI purified fragment of Smas-ICeuI-Int was ligated with it to form pNOVO40.

Example 10

Targeted Integration into a Predetermined Target Loci by Homologous Recombination

[0216] Single copy T-DNA transgenic tobacco target lines (T2701.6 and T2701.27) were selected and infected with Agrobacterium tumefaciens strain LBA4404, which contained a donor vector. Seeds derived from target lines T2701.6 and T2701.27 that had been selfed or backcrossed with untransformed SR1 pollens were germinated on MS3S medium with 5 mg/L PPT. Two different methods for generating targeted events were used. In one method, PPT resistant seedlings were grown in MS3S medium for 3-4 weeks. Leaves of 3 to 6 week old seedlings were used for targeting experiments. The leaves were cut into 1-mm wide slices, exposed for 5 minutes to Agrobacterium resuspended in MS3S, moved to sterile paper to blot away excess liquid, and then placed on co-cultivation medium (MS3S+NAA (0.1 mg/L)+6-BA (1 mg/L)+gelrite agar (2.4 g/L) in standard Petri dishes) for 3 days. The leaf slices were then moved to selection/regeneration medium (MS3S+NAA (0.1 mg/L)+6-BA (1 mg/L)+Carbenicillin (200 mg/L)+gelrite agar (2.4 g/L) with kanamycin (200 mg/L)). Kanamycin-resistant shoots were rooted in selection/rooting medium (MSB+PPO (100 nM)+Carbenicillin (200 mg/L)+phytagar (8 g/L) in GA-7 boxes) and then transplanted to soil. PPT-resistant 9-14 days old seedlings were used for Agrobacterium-mediated transformation using vacuum-infiltration according to the method described in Puchta et al. 1996 Proc. Natl. Acad. Sci. USA 93:5055-5060. Kanamycin-resistant shoots were further verified by PCR analysis.

[0217] Table 1 shows the efficiency of targeted integration in three target lines. Co-delivery of an HO expression vector (pNOV2747 or pNOV036) and an I-CeuI expression vector (pNOV039 or pNOVO40) does not increase targeting efficiency. Overall, up to a 1-2% targeted integration efficiency was obtained. It is believed that the insertion of the 4 Arabidopsis introns in the nptII gene, which extended the region of homology between the target and donor DNA, contributed to the observed targeting efficiency. The enhancing effect on targeting of a longer region of homology is further substantiated by comparing the effect of three different donor vectors (pNOV2736, pNOV2755, pNOV2757) on the targeting efficiency in both line T2701.6 and T2701.27 (Table 1). On average, 1 to 3 targeted events can be obtained with donor pNOV2736, which flanks both sides of the Hyg cassette with 2.4 kb of sequence homology with the target, but no event was obtained with pNOV2757, which flanks one side of the marker with 2.4 kb of sequence homology with the target and the other side of the marker has no homology to the target.

TABLE-US-00003 TABLE 1 Targeting efficiency of two single-copy lines with different vectors Vector(s) Homology Explants Events PCR+ South+ Target line T2701.6 HR-01AB pNOV2737 2.4 & 2.4 kb 237 3 2 2 HR-01AC pNOV2737, 2747 2.4 & 2.4 kb 277 1 1 1 HR-01AD pNOV2755, 2747 2.4 & 1.2 kb 233 4 2 2 HR-02AA pNOV2736, 036 2.4 & 2.4 kb 347 0 0 0 HR-02AC pNOV2755, 036 2.4 & 1.2 kb 303 1 1 1 HR-03AB pNOV2736 2.4 & 2.4 kb 119 2 2 2 HR-03AD pNOV2737 2.4 & 2.4 kb 91 2 1 1 HR-05AA pNOV2736 2.4 & 2.4 kb 247 5 5 3/5* HR-05AC pNOV2755 2.4 & 1.2 kb 194 2 1 1 HR-05AD pNOV2757 2.4 & 0 kb 204 3 0 0 HR-06AA pNOV2736 2.4 & 2.4 kb 183 2 1 1 HR-06AB pNOV2737 2.4 & 2.4 kb 179 1 1 1 HR-11AA.sup.# pNOV2736 2.4 & 2.4 kb .sup. 100.sup.# 3 3 ND Target line T2701.27 HR-01CA pNOV2737 2.4 & 2.4 kb 169 5 1 1 HR-01CC pNOV2737, 2747 2.4 & 2.4 kb 268 4 3 3 HR-02CA pNOV2736, 036 2.4 & 2.4 kb 259 1 0 0 HR-02CC pNOV2755, 036 2.4 & 1.2 kb 211 1 1 1 HR-05CA pNOV2736 2.4 & 2.4 kb 227 8 6 3/3* HR-05CC pNOV2755 2.4 & 1.2 kb 183 8 7 2/2* HR-05CD pNOV2757 2.4 & 0 kb 175 2 0 0 HR-06CB pNOV2737 2.4 & 2.4 kb 193 1 1 ND HR-11CA.sup.# pNOV2736 2.4 & 2.4 kb .sup. 100.sup.# 4 1 ND Target line T2729.26 HR-09CC pNOV2736 2.4 & 2.4 kb 141 1 1 ND HR-09CE pNOV2736 + pNOV040 2.4 & 2.4 kb 183 0 0 ND HR-09CF pNOV2736, pNOV039 2.4 & 2.4 kb 185 1 1 ND HR-12CA pNOV2736 2.4 & 2.4 kb 100 1 1 ND HR-12CB pNOV2736 + pNOV040 2.4 & 2.4 kb 100 2 2 ND HR-12CC pNOV2736, pNOV039 2.4 & 2.4 kb 100 1 1 ND ND: not determined. *Number of events analyzed. .sup.#13 days-old young seedlings instead of leaf explant tissues were used for transformation.

Example 11

Identification of Recombinant Target Lines

[0218] Leaf tissue was collected from these potential recombinants for DNA isolation and PCR analysis (FIG. 14A). In order to identify recombinants, amplification was carried out using the Boehringer Mannheim Expand.TM. High Fidelity PCR system with primers PSMASFW2 (SEQ ID NO:51: 5'-CCG GTG AGT AAT ATT GTA CGG CTA AGA-3') and NPTR6 (SEQ ID NO:52: 5'-AGA TCC TCA GAA GAA CTC GTC AAG AAG-3'). Amplification of recombinant junctions was carried out using Boehringer Mannheim Expand.TM. Long Template PCR system with primers PHSPFWD (SEQ ID NO:53: 5'-AAT ATA GGC GGT ATT CCG GCC ATT ATA ACA-3') and TPalExonV (SEQ ID NO:54: 5'-CTA AGA TCC TCA GAA GAA CTC GTC AAG AAG-3'). FIG. 14B illustrates the identification of targeted events by PCR amplification. FIG. 14C shows recombinants that have successfully integrated a second gene cassette.

[0219] FIGS. 15A, 15B, and 15C illustrate Southern blot analyses of targeted events achieved through homologous recombination. Genomic DNA digested with several enzymes (EcoRV, SacI, NheI, SpeI) was hybridized with two different probes (i.e., the HSP80 promoter and the nptII exonV-PAL1 terminator). The positions of the probes are indicated in FIG. 15A. The HSP80 promoter probe provided information relating to the copy number of the donor sequence (FIG. 15B) and whether recombination occurred at the left end of the target locus (i.e., the end which included the bar gene cassette). Probing with the nptII exonV-PAL1 terminator provided information regarding target sequence copy number and whether there was any rearrangement at the right end of the target locus (i.e., the end which included the nptII gene cassette) (FIG. 15C). If a recombination event derives from recombination at both ends (double crossover recombination), then both probes would be expected to show a shift in the target bands. If a recombination event derives from recombination at a single side (a single crossover recombination), then the target band would be expected to shift with only one probe. If the putatively targeted event is not truly targeted, then none of the bands in the target plant would be expected to shift with either of the probes. FIGS. 15B and 15C illustrate an exemplary analysis of this type.

[0220] Target line T2701.6 (lanes 1 to 4, FIGS. 15B and 15C) gave rise to several restriction fragments that were easily separated by regular gel electrophoresis. FIGS. 15B and 15C (lanes 5-8) show that recombinant HR-03AD.2 had a restriction fragment size-shift, which is consistent with a double-sided recombination event. The other two events (HR-05AA.1 and HR-05AA.2, lanes 9-14) show a band shift that is consistent with recombination at only one side. One of the events (HR-05AA.2, lanes 12-14) shows band shifts with both probes, but the band with NheI digestion (lane 14) is smaller than expected, so there might be some rearrangement or deletion close to the HSP80 promoter. Because there is no restriction polymorphism in the region of homology between the target and donor sequences, it is not possible to distinguish whether the recombinants were derived from a reciprocal crossover or a non-reciprocal gene conversion.

[0221] Target line T2701.27 gave rise to restriction fragments that were not easily separated (larger than 10 kb NheI, Sad, and ScaI fragments with the nptII exon V-PAL 3'-UTR probe), and events derived from this line are analyzed only minimally, due to difficulty in distinguishing small changes for band sizes larger than 15 kb in normal agarose gel electrophoresis.

[0222] Other targeted events were also characterized by Southern blot analysis. The results are summarized as follows: (1) About 70% of the targeted events resulted from single crossover recombination, and about 30% of the recombinants resulted from double crossover recombination. It is not known whether recombinants were the product of a reciprocal crossover or a non-reciprocal gene conversion process using incoming T-DNA as a template. (2) About half of the recombinants had additional copies of the donor sequence inserted elsewhere in the genome. (3) T-DNA is capable of carrying out homologous recombination (by either reciprocal crossover or non-reciprocal gene conversion), and it does not have to be integrated into the host genome first. (4) No unexpected rearrangement of the target locus or ectopic targeting is observed in all of the analyzed events.

[0223] Because a Southern blot analysis will not reveal rearrangements that result in relatively small changes in the size of a band, finer restriction mapping of the recombination breakpoints was done. A primer (PFDSP1, FIG. 14A; SEQ ID NO:55: 5'-ACC CTC CGC TAC TTC TCC GGG AAA AGA CGC-3') was created based upon the flanking plant genomic DNA sequences obtained from I-PCR and used to perform long range PCR amplification in nine recombinant lines derived from T2701.6. PCR amplification was done using two primer pairs. The first pair of primers (PFDSP1 and TPalExonV, FIGS. 14A and 14C; SEQ ID NO:55 and SEQ ID NO:54) produced a 5.5 kb product from a non-targeted copy, a larger than 5.5 kb product from a targeted copy derived from single-sided recombination, and a 10 kb fragment from a targeted copy derived from double-sided recombination. More particularly, using this pair of primers (PFDSP1 and TPalExonV), a ca.10 kb fragment was obtained from one hemizygous recombinant line (HR-03AD.2, see FIG. 14C for examples). A ca. 9 kb fragment was derived from both HR-05AA.2 and HR-01AB.1 and their progeny (FIG. 14C). A ca. 8 kb fragment was amplified from line HR-01AD.1, which is hemizygous for the target transgene locus (not shown). Thus, both HR-01AB.1 and HR-01AD.1 were not the products of a double-crossover recombination. In six other heterozygous lines, only the shorter fragment (5.5 kb) was present, as was predicted from the preferential amplification of the non-targeted copy (result not shown). When the kanamycin resistant progeny of a heterozygous recombinant HR-03AB.1 was subjected to PCR, a 10 kb fragment was produced (FIG. 14C).

[0224] Using another pair of primers (PFDSP1, SEQ ID NO:55 and HygRV1, SEQ ID NO:56: 5'-ACT ATC GGC GAG TAC TTC TAC ACA GCC ATC-3') FIG. 14C, lane 1 to 5), the PCR reaction produced a 5.4 kb product. This indicated that the recombinant was derived from double-sided recombination, because the HygRV1 primer could only bind to the Hyg gene present in the donor vector. The 5.4 kb product was present in both hemizygous and heterozygous recombinants derived from double-crossover recombination. A 5.4 kb fragment was obtained from 5 recombinant lines (HR-01AB.1, HR-02AC.1, HR-03AB.1, HR-03AD.2, and HR-05AA.2). No PCR product was derived from five other targeted events (HR-01AB.3, HR-01AC.1, BR-01AD.1, HR-01AD.4, and HR-03AB.2). In these latter recombinants, it is possible that a DNA rearrangement or a repeat structure was present, such that the PCR reaction was unable to amplify the entire region. Since both HR-01AB.1 and HR-05AA.2 produced a PCR product of only about 9 kb using PFDSP1 and TPalExonV (above) but produced a PCR product of about 5.4 kb with PFDSP1 and HygRV1, it is possible that there was an internal rearrangement (such as a deletion, for example) between the hygromcyin phosphotransferase (HPT) gene and the mSmas promoter during targeting. In summary, Southern blot analyses and PCR results demonstrate that at least three events (HR-02AC.1, HR-03AB.1, and HR-03AD.2) were derived from double-crossover recombination with no additional rearrangement.

Example 12

Progeny Analysis of Targeted Events

[0225] In several recombinants, more than one copy of a donor sequence was integrated into the host cell's genome, as indicated by Southern blot analysis using the HSP80 promoter probe. To study the insertion status of the additional copy(ies) in these lines, plants were pollinated with untransformed SR1. The seeds were plated on PPT, kanamycin, or hygromycin medium. Table 2 shows the number of resistant and sensitive seedlings. In a hemizygous target line, half of the seedlings would be expected to be resistant to PPT, kanamycin, and hygromycin, if all donor copies are integrated into either a single locus or a closely linked locus. Here, all lines had the expected kanamycin resistance segregation ratios, as demonstrated by Southern blot analysis of each plant line. Southern blot analysis indicated that there were several additional copies of the donor sequence present in the HR-01AB.1 genome. The PPT and hygromycin segregation data supported this conclusion.

TABLE-US-00004 TABLE 2 Progeny segregation analysis of targeted events Kan PPT Hyg Crosses R S R S R S Hemizygous target* T2701.6 target locus HR-01AB.1 .times. SR1 56 64 121 10 80 9 HR-03AD.2 .times. SR1 115 111 50 58 38 35 T2701.27 target locus HR-01CB.4 .times. SR1 43 46 37 42 21 21 Homozygous target* T2701.6 target locus HR-01AB.3 .times. SR1 46 57 154 0 NT HR-03AB.1 .times. SR1 28 36 78 0 40 37 T2701.27 target locus HR-01CC.4 .times. SR1 65 80 72 0 58 62 *The target status is extrapolated from Southern blot analysis using npt exonV/PAL 3'-UTR as probe. The plants in bold font are most likely derived from double crossover recombination as indicated by Southern blot analysis. NT: Not tested.

Example 13

Construction of a Site-Specific FLP Recombinase Expression Vector

[0226] A 1.6 kb BamHI fragment containing FLP recombinase was excised from pUCFLP/intron (WO 99/55851) and inserted into pNOV2721 linearized with BamHI to create pNOV2760, thereby placing FLP under the control of the Arabidopsis Act2 promoter. pNOV2760 was digested with Sad and KpnI to isolate the 3.7 kb Act2 promoter/FLP-intron/Act2 terminator cassette. This fragment was then inserted into SacI/KpnI-digested pNOV1511 to create pNOV2762 (FIG. 13E). The PPO gene was isolated from Arabidopsis thaliana, and two mutant amino acids were introduced to obtain PPO(dm) (U.S. Pat. No. 6,308,458), which permitted the selection of transgenic cells with an herbicide (butafenacil, CGA 854,276).

Example 14

Generation of Transgenic Lines Expressing FLP Recombinase

[0227] The FLP recombinase binary vector pNOV2762 was transformed into Agrobacterium strain LBA4404, and the resulting Agrobacterium strain was used to transform tobacco SR1 as described above, with the exception that butafenacil was used as the selection agent. Several FLP-expressing lines were generated. One transgenic line (T2762.2) was selfed to produce progeny lines T2762.2S1 and T2762.2S2.

Example 15

Crossing Recombinant Plant Lines with FLP-Expressing Plant Lines

[0228] To regenerate a truncated nptII (with introns) selectable marker for gene stacking, recombinant line HR-03AD.2, which resulted from double-crossover recombination, was chosen for further studies. HR-03AD.2 was crossed with both T2762.2S1 and T2762.2S2 directly. The seeds from these crosses were plated on medium containing both butafenacil and hygromycin. Double-resistant seedlings were transplanted to soil and grown in a greenhouse. Each seedling was analyzed with a PCR assay to determine whether there was a FLP-mediated excision of the sequence flanked by the two FRT sites. The PCR assay (FIG. 16A) was performed with a forward primer (Tubq3FW; SEQ ID NO:57, 5'-GTG TCT CAT GCA CTT GGG AGG TGA TC-3') located at the Ubq3 terminator and a reverse primer at the nptII exon 3 (NPTR3, SEQ ID NO:10). The wild type target locus produced a 3 kb PCR fragment; the same target locus with the Smas promoter and part of the nptII sequence (i.e., exon 1 and part of intron 1) excised by FLP-mediated site-specific recombination produced a 1.5 kb PCR fragment (see FIG. 16A). 72 progeny seedlings were assayed by PCR. 49 of those seedlings had a detectable 1.5 kb PCR fragment. Several lines with an excised nptII sequence (CFP-A7, CFP-B8, CFP-B11, CFP-C3, CFP-C6, CFP-D1, CFP-D5, CFP-E7, and CFP-E9) were crossed with SR1, and the progeny were selected on hygromycin. Hygromycin-resistant seedlings were then assayed by PCR to recover progeny with FLP-mediated excision.

Example 16

Retransformation of Recombinants with a FLP Expression Vector

[0229] Truncation of the nptII marker sequence can also be achieved by inserting the recombinase expression construct into the target lines and then allowing the recombinase locus to be lost through segregation. Kanamycin-resistant seedlings resulting from crossing HR-03AB.1 with SR1 and HR-03AD.2 with SR1 were re-transformed with Agrobacterium containing pNOV2762 to regenerate a truncated selectable marker gene for gene stacking. In preparation for retransformation with pNOV2762, leaf slices were infected with Agrobacterium (pNOV2762) and then selected on hygromycin and butafenacil. Regenerated shoots were rooted in medium with butafenacil. The rooted shoots were transplanted into soil and assayed by PCR, as described above, to determine whether the mSmas promoter and part of the npaI (with introns) gene were deleted (FIG. 16A). Among 44 independent transformants (HR-08AA's) of HR-03AB.1.times.SR1 kanamycin-resistant seedlings, 19 had a 1.5 kb PCR product. Among 44 transformants (HR-08BA's) of HR-03AD.2.times.SR1 kanamycin-resistant seedlings, 22 had a 1.5 kb PCR product.

[0230] Several lines with an excised nptII sequence (HR-08AA.17, HR-08AA.32, HR-08BA.13 and HR-08BA.20) were crossed with SR1, and the progeny were selected on hygromycin. Hygromycin-resistant seedlings were assayed by PCR to recover progeny with FLP-mediated excision. To facilitate the isolation of lines with complete excision of the nptII sequence, leaves of HR-08AA32 were regenerated. One of the regenerants, HR-08AA32R2, had complete excision of FRT-flanked sequences and was pollinated with pollens from wildtype untransformed SR1. The progeny seedlings were tested for excision of the FRT-flanked mSmas and nptII sequences by PCR. PCR demonstrated that all of the progeny had an mSmas promoter and part of the nptII sequence had been excised (FIG. 16B). Progeny with the regenerated truncated marker gene is then capable of being used to stack additional donor trait cassettes.

[0231] The above Examples describe the production of a recombinant line through site-specific recombination-mediated excision of nucleotide sequences flanked by recombinase recognition sequences. This recombinant line, which includes a partially excised (i.e., truncated) selectable marker gene, can be re-used in further rounds of targeting, thereby permitting the use of a single selectable marker gene in combination with a site-specific recombination system to stack an unlimited number of gene cassettes (i.e., donor sequences) at a single locus in the genome of a host cell (see, e.g., FIGS. 12A and 12B).

Example 17a

Construction of a PMI Gene (PMI-Intron) with Four Arabidopsis Introns

[0232] Four Arabidopsis introns were inserted into the phosphomannose isomerase (PMI) gene to form a PMI-intron sequence (FIG. 17), thereby increasing the length of the PMI gene from 1103 by to 3452 bp. These four introns are from an AtBAF60 homolog, phenylalanine aminolyase (PAL), tubulin-1-alpha, and tubulin-1-beta, as used for constructing the nptII introns in pNOV2720. An R recombinase recognition sequence (RS) was formed by annealing two complementary primers RSFW (SEQ ID NO:58: 5'-GAT CCG CGG TTG ATG AAA GAA TAA CGT ATT CTT TCA TCA A-3') and RSRV (SEQ ID NO:59: 5'-GAT CTT GAT GAA AGA ATA CGT TAT TCT TTC ATC AAC CGC G-3') and inserting them into BglII-digested pNOV2720 to form pNOV2783. PMI intron 1 (488 bps, from AtBAF60 intron) was amplified from pNOV2783 with PMIINTFA (SEQ ID NO:60: 5'-ATG CCG CAG GTA CCA AGC TGC GAA TCT TCG-3') and PMIINTRA (SEQ ID NO:61: 5'-ATC GGG ATA CCT GAA AAA TTC AGA AAC AAA-3'). The other three introns were amplified directly from pNOV2720. PMI intron 2 (from the Arabidopsis PAL1 intron) was amplified from pNOV2720 with PMIINTFB (SEQ ID NO:62: 5'-CGG TCG CAG GTA TTA GTA CTA TTC TTT TGT-3') and PMIINTRB (SEQ ID NO:63: 5'-CGG ATG TGC ACC TGT AAC GAA CAA AAA CAT-3'). PMI intron 3 (from the Arabidopsis tubulin-1-beta intron) was amplified from pNOV2720 with PMIINTFC (SEQ ID NO:64: 5'-ACC TGC AAG GTA TAT ATA TGA ATC GAT TTC-3') and PMIINTRC (SEQ ID NO:65: 5'-GCG CCA CAC CTG TAA TAC AGA AAT GTT AAG-3'). PMI intron 4 (from the Arabidopsis tubulin-1-alpha intron) was amplified from pNOV2720 using PMIINTFD (SEQ ID NO:66: 5'-GTG AAA CAA GGT TAT TAA CGT TTT CCA CCT-3') and PMIINTRD (SEQ ID NO:67: 5'-GTT CTG CAC CTG CAT CAA TGG AAA AAT ATT-3'). PMI exons 1-5 were amplified from pNOV210, which contains the E. coli PMI coding sequence in pBluescript KS(+) (Stratagene, La Jolla, Calif.). PMI exon 1 (346 bps) was amplified from pNOV210 with PMIEXF1 (SEQ ID NO:68: 5'-GTG GAT CCG GCA GCA TGC AAA AAC TCA TTA ACT-3') and PMIEXR1 (SEQ ID NO:69: 5'-TCG CAG CTT GGT ACC TGC GGC ATT TTC TTT GG-3'). PMI exon 2 (140 bps) was amplified from pNOV210 using PMIEXF2 (SEQ ID NO:70: 5'-AAT TTT TCA GGT ATC CCG ATG GAT GCC GCC-3') and PMIEXR2 (SEQ ID NO:71: 5'-TAG TAC TAA TAC CTG CGA CCG GCT GGA GTA-3'). PMI exon 3 (290 bps) was amplified from pNOV210 with PMIEXF3 (SEQ ID NO:72: 5'-GTT CGT TAC AGG TGC ACA TCC GGC GAT TGC-3') and PMIEXR3 (SEQ ID NO:73: 5'-TCA TAT ATA TAC CTT GCA GGT AAG CGT GCG-3'). PMI exon 4 (146 bps) was amplified from pNOV210 with PMIEXF4 (SEQ ID NO:74: 5'-CTG TAT TAC AGG TGT GGC GCT GGA AGT GAT-3') and PMIEXR4 (SEQ ID NO:75: 5'-TGT TAA TAA CCT TGT TTC ACC GGC TGG GTC-3'). PMI exon 5 (283 bps) was amplified from pNOV210 with PMIEXF5 (SEQ ID NO:76: 5'-CGA TTG ATG CAG GTG CAG AAC TGG ACT TCC C-3') and PMIEXR5 (SEQ ID NO:77: 5'-TGC TCG AGT CAT TAG CAA GAG ATG TTA ATT TT-3').

[0233] PMI intron 1 (488 bps) and PMI exon 2 (140 bps) PCR fragments were co-amplified using PMIintFA and PMIEXR2 to form a PMI intron 1::PMI exon 2 PCR fragment (630 bps). PMI exon 1 (346 bps) and PMI intron 1:: PMI exon 2 fragment (630 bps) were co-amplified with PMIEXF1 and PMIEXR2 to form a PMI exon 1::PMI intron 1::PMI exon 2 fragment (976 bps), which was then cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) to form pNOV2784. PMI intron 2 (449 bps) and PMI exon 3 (290 bps) PCR products were co-amplified with PMIINTFB and PMIEXR3 primers to from a PMI intron 2::PMI exon 3 fragment (740 bps). PMI exon 3 (290 bps) and PMI intron 3 (792 bps) were co-amplified with PMIEXF3 and PMIINTRC primers to form a PMI exon 3:: PMI intron 3 fragment (1083 bps). PMI intron 4 (511 bps) and PMI exon 5 (283 bps) PCR products were co-amplified with PMIINTFD and PMIEXR5 to form a PMI intron 4::PMI exon 5 fragment (795 bps). PMI exon 2 (140 bps) and PMI intron 2::PMI exon 3 (740 bps) were co-amplified with PMIEXF2 and PMIEXR3 primers to form a PMI exon 2::PMI intron 2:: PMI exon 3 fragment (881 bps). PMI intron 1 (488 bps) and the PMI exon 2::PMI intron 2::PMI exon 3 fragment (881 bps) were co-amplified with PMIINTFA and PMIEXR3 to form a PMI intron 1::PMI exon 2::PMI intron 2::PMI exon 3 PCR product (1370 bps), which was then cloned into pCR2.1-TOPO to form pNOV2785. A PMI exon 3::PMI intron 3 fragment (1083 bps) and PMI exon 4 (146 bps) were co-amplified using PMIEXF3 and PMIEXR4 to form a PMI exon 3::PMI intron 3::PMI exon 4 fragment (1230 bps), which was inserted into pCR2.1-TOPO to form pNOV2786. The PMI exon 4 fragment (146 bps) and the PMI intron 4::PMI exon 5 fragment (795 bps) were co-amplified with PMIEXF4 and PMIEXR5 primers to form a PMI exon 4::PMI intron 4::PMI exon 5 fragment (942 bps), which was inserted into pCR2.1-TOPO to form pNOV2787. pQD84A1 was partially digested with Sad and Seal to isolate a 4910 bps vector fragment. pQD85B9 was cut with Seal and Sad to isolate the 789 by fragment, which was inserted into the 4910 by pQD84A1 SacI/ScaI vector fragment to form pNOV2788. BstBI/BamHI-digested pQD86A13 was ligated with a BstBI/BamHI fragment (894 bps) of pQD87A19 to form pNOV2789. XhoI/BamHI-digested pBluescript KS(+) (Stratagene, La Jolla, Calif.) was ligated with the BssHI/BamHI fragment (1540 bps) of pQD88A1 and the BssHIIXhoI fragment (1928 bps) of pQD89A7 to form pNOV2790. pNOV2790 contained the full-length PMI sequence with four Arabidopsis introns inserted into pBluescript KS(+).

Example 17b

Construction of a GUS Gene with an AtBAF60 Intron

[0234] To construct a GUS gene with an Arabidopsis intron from the AtBAF60 gene, an AtBAF60 intron (420 bps) was amplified from the Arabidopsis genome using two primers, GUSBAFFW1 (SEQ ID NO:78: 5'-TTG ACT GGC AGG TAC CAA GCT GCG AAT CTT CG-3') and GUSBAFRV1 (SEQ ID NO:79: 5'-ATT GGC CAC CAC CTG AAA AAT TCA GAA ACA AA-3'). AtBAF60 (CHCl) is a gene that shares homology with the mammalian nucleosome-remodeling factor BAF60 (http://www.chromdb.org/). GUS exon 1 (645 bps) was amplified from pBI121 (Clonetech) using two primers, GUSBAMHI (SEQ ID NO:80: 5'-GGA TCC AAC CAT GTT ACG TCC TGT AGA AA-3') and BAFGUSRV1 (SEQ ID NO:81: 5'-CAG CTT GGT ACC TGC CAG TCA ACA GAC GCG AC-3'). GUS exon 2 (1200 bps) was amplified from pBI121 using two primers, BAFGUSFW1 (SEQ ID NO:82 5'-TTG ACT GGC AGG TAC CAA GCT GCG AAT CTT CG-3') and GUSSALI (SEQ ID NO:83: 5'-GTC GAC TCA TTG TTT GCC TCC CTG CTG CGG-3'). The GUS exon 1-AtBAF60 intron fragment (1049 bp) was formed by PCR using gel-purified GUS exon 1 (645 bp) and the AtBAF60 intron (420 bp) fragments as a template and two primers, GUSBAMHI (SEQ ID NO:84: 5'-GGA TCC AAC CAT GTT ACG TCC TGT AGA AA-3') and GUSBAFRV1 (SEQ ID NO:85: 5'-ATT GGC CAC CAC CTG AAA AAT TCA GAA ACA AA-3'). The GUS exon 1::AtBAF60 intron fragment (1049 bp) was cloned into the pCR2.1-TOPO vector to form pNOV5001. The AtBAF60 intron-GUS exon 2 fragment (1620 bp) was formed by PCR using the AtBAF60 intron (420 bp) and GUS exon 2 (1200 bp) fragments as templates and GUSBAFFW1 (SEQ ID NO:86: 5'-TTG ACT GGC AGG TAC CAA GCT GCG AAT CTT CG-3') and GUSSALI (SEQ ID NO:87: 5'-GTC GAC TCA TTG TTT GCC TCC CTG CTG CGG-3') as primers. The AtBAF60 intron::GUS exon 2 fragment (1620 bp) was cloned into pCR2.1-TOPO to form pNOV5002. pNOV5003 was formed through a tripartite ligation of XhoI/BamHI-digested pBluescript KS(+) with two insert fragments, pNOV5001 BamHI/HindIII fragment (961 bp) and pNOV5002 XhoI/HindIII fragment (1312 bps).

Example 18

Construction of a Monocot Expression Vector Including a PMI-Intron Sequence

[0235] Binary backbone vector pNOV2114 was digested with HindIII and Asp718I. The ZmUbi promoter/Nos 3'-UTR fragment was excised from pBH16 as a HindIII/Asp718I fragment and ligated into this vector to form pNOVO44. The pBH16 construct contained the ZmUbi promotor-intron linked to the Nos (nopaline synthase) 3'-UTR by a linker with BamHI and SacI sites. pNOV2790 was digested with BamHI/BglII, and the 3011 by fragment containing the 3'-remainder of the coding region was isolated. This fragment was then ligated into BamHI-digested pNOVO44 to form pNOVO42, which contained the 5'-truncated PMI-intron sequence. pNOV2790 was also digested with AflII, and an oligonucleotide converter was ligated into the site to change it into BamHI: TTAACGGATCCG, producing pQD90C2BamHI. This plasmid was digested with BamHI, and the 2832 by fragment containing the 5'-remainder of the coding region was isolated. This fragment was ligated into the BamHI site of pNOVO44 to form pNOVO43, which contained the 3'-truncated PMI-intron sequence. pNOV2790 was digested with BamHI, and the 3011 by fragment containing the full-length PMI gene was isolated. This PMI fragment was ligated into BamHI-digested p2114UbiNos to form pNOVO41, which contained the full-length PMI-intron sequence.

Example 19

Construction of a PPO-dm Selectable Marker Cassette for Monocots

[0236] The rice actin 1 promoter (McElroy et al. 1990 Plant Cell 1990 2:163-171) was used to drive PPO-dm expression as a selectable marker. PPO-dm is a mutant form of the Arabidopsis PPO gene, which confers tolerance to the herbicide butafenacil. pNOV3010 is a biolistic fragment vector containing a rice actin 1 promoter-PMI expression cassette. pNOV3010 was partially digested with BamHI and filled-in with a Klenow fragment of E. coli DNA polymerase Ito destroy the BamHI site in the intron of the 5'-region of the rice actin 1 gene, thus forming pNOV5004. The 2175 by rice actin promoter sequence was removed from pNOV5004 by BamHI/PstI digestion and was inserted into BamHI/PstI-digested pBluescript KS(+) to form pNOV5012. pNOV5012 was digested with BamHI, filled-in with a Klenow fragment, partially cut with Sad, and then treated with calf intestine phosphatase to isolate the 5.1 kb vector. pNOV1511 (U.S. Pat. No. 6,308,458) was digested with NcoI, filled-in with a Klenow fragment, and then digested with Sad to isolate the 1898 by PPO-dm::35S terminator fragment. This PPO-dm::35S terminator fragment was then inserted into the above pNOV5012 vector (5.1 kb) to form pNOV5013.

Example 20

Construction of a Monocot Target Vector with a PPO Herbicide Resistance Marker Gene

[0237] Two oligonucleotides, ICEUBGL2 (SEQ ID NO:88: 5'-TCG AAG ATC TCT ATA ACG GTC CTA AGG TAG-3') and ICEUBAMH (SEQ ID NO:89: 5'-ACT TGG ATC CTC GCT ACC TTA GGA CCG TTA-3'), were annealed, filled-in with a Klenow fragment, and digested with BglII and BamHI to isolate a fragment containing I-CeuI cleavage site. The isolated I-CeuI site fragment was inserted into BglII-digested pNOV2790 to form pNOV5006. pNOV5013 was digested with PspOMI, filled-in with a Klenow fragment, then partially cut with BamHI to isolate the 4069 by rice Actl promoter::PPO::35S terminator fragment. pNOV5014 was digested with SbfI, blunted with T4 DNA polymerase, and then cut with BglII to isolate the 8972 by fragment. The 4069 by rice Act1 promoter::PPO::35S terminator fragment of pNOV5013 was inserted into the SbfI/BglII vector fragment (8972 bps) of pNOV5014 to form target vector pNOV5025 (FIG. 17A). pNOV5014 was constructed by inserting the BglII/SpeI fragment (3034 bp) of pNOV5006 into BglII/SpeI-digested pNOVO41.

Example 21

Construction of a Monocot Target Vector with the Hygromycin Phosphotransferase (hpt) Gene as an Antibiotic Resistance Marker

[0238] Target binary vector pADF55 was constructed by the following steps and was used to produce target plants through hygromycin selection. The method herein described may be used with any monocot plant and any of a variety of tranformation methods, as described above. In this example, however, rice plants and Agrobacterium-mediated transformation were used (Hiei et al. Plant Journal 6:271-282).

[0239] Step 1: pAdF50 containing a new promoter-gene fusion (the rice Actin 1 promoter fused to the hygromycin gene) was built through a 3-way ligation of (1) pNEB193 cut with SalI and SphI, (2) the 2212 by SaiI-BamHI fragment of pNOV1100 containing the rice Actin 1 promoter, and (3) a 1029 by BamHI-SphI PCR fragment containing the hygromycin gene amplified from pNOV11. Tthe PCR primers for this reaction contained the BamHI and SphI sites: the PCR primer containing the SphI site also contained an additional restriction site, NotI, located between the SphI site and the 3' end of the hygromycin gene, which restriction site was later used to excise the hygromycin gene.

[0240] Step 2: pAdF51 was derived from pAdF50 by adding the CMPS:GIG:Act2 3'-UTR cassette of pQD189A12 and the attP recognition sequence (a phage lambda integrase recognition sequence). pAdF51 was built through a 3-way ligation of (1) pAdF50 cut with Pad and XbaI, (2) the 3224 by KpnI-XbaI fragment of pQD189A12 carrying the CMPS:GIG: Act2 3' gene, and (3) a 260 by PacI-KpnI PCR product carrying the attP recognition site, which was amplified from pQD188A7. The PCR primers used for this reaction contained the Pad and KpnI sites.

[0241] Step 3: pAdF52, a subclone of the EcoO109I-AscI fragment of pNOV5025, which contains a 35S terminator and the truncated PMI-introns::nos 3' gene fragment, was inserted into pNEB193. The construct was made by subcloning the 4864 by EcoO109I/blunt with Klenow-AscI fragment of pNOV5025 into vector pNEB193 cut with EcoO109I/blunt with Klenow and AscI.

[0242] Step 4: pAdF53 was constructed by insterting the phage lambda integrase recognition site attB into pAdF52, downstream of the 35S terminator, using an NcoI site. The attB sequence was added using two annealed oligonucleotides with ends that were compatible with an NcoI site. A BspHI site was also included in the oligo sequence to conveniently assist in identifying particular clones that contained it.

[0243] Step 5: pAdF54 was constructed by adding the Zygosaccharomyces rouxii R recombinase recognition site (RS) to plasmid pAdF53, downstream of the truncated PMI::nos 3' gene fragment, in a KpnI site. The RS sequence was added using two annealed oligos with ends that were compatible with a KpnI site. An Agel site was also included in the oligo sequence to conveniently assist in identifying particular clones that contained it.

[0244] Step 6: The final construct, pAdF55, was built through a 3-way ligation of the 5684 by PacI-AscI vector fragment of pQD199B2 ligated to the 6723 by PacI-NotI fragment of pAdF51 and the 3566 by NotI-AscI fragment of pAdF54.

Example 22

Construction of a Monocot Positive Control Vector

[0245] pNOV5013 was digested with PspOMI, filled-in with a Klenow fragment, and then partially cut with BamHI to isolate the 4069 by rice Act1 promoter::PPO::35S terminator fragment. pNOV5015 was digested with SbfI, blunted with T4 DNA polymerase, and then partially cut with BglII to isolate the 11.5 kb vector fragment. The 4069 by rice Act1 promoter::PPO::35S terminator fragment was inserted into the SbfI/BglII vector fragment (11.5 kb) of pNOV5015 to form pNOV5026, the positive control vector for targeting (FIG. 17A).

Example 23

Construction of Monocot Donor Vectors

[0246] pNOVO41 was digested with AscI, filled-in with a Klenow fragment, and then cut with BamHI to isolate the 7.7 kb AscI/BamHI fragment. pNOV5006 was digested with BstBI, filled-in with a Klenow fragment, cut with BamHI to isolate the 2652 by BstBI/BamHI fragment. pNOV5016 was constructed by ligating the AscI/BamHI fragment (7.7 kb) of pNOVO41 with the BstBI/BamHI fragment (2652 bp) of pNOV5006. pNOV5013 was digested with PspOMI, filled-in with a Klenow fragment, and then partially cut with BamHI to isolate the 4069 by rice Act1 promoter::PPO::35S terminator fragment. pNOV5016 was digested with SbfI, blunted with T4 DNA polymerase, and then partially cut with BglII to isolate the 10.3 kb vector fragment. The 4069 by rice Act1 promoter::PPO::35S terminator fragment was inserted into an SbfI/BglII-digested pNOV5016 vector (10.3 kb) to form pNOV5027. pNOV5027 was partially digested with SrfI and BsrGI to delete part of the rice Act1 promoter, filled-in with a Klenow fragment, and then circularized to form pNOV5030. pNOV5030 was digested with PacI, filled-in with a Klenow fragment, and then partially cut with SalI to isolate a 13,768 by PacI/SalI fragment as a vector. pNOV5019 is a plasmid derived from pBluescript KS(+) containing a rice .alpha.-tubulin promoter::GFPintron::AtAct2 terminator expression cassette. The Ecl136II/XhoI fragment (3286 bp) of pNOV5019 was inserted into the 13,768 by PacI/SalI vector fragment to form a first monocot donor vector, pNOV5031 (FIG. 17B). pNOV5030 was cut with PacI, blunted with a Klenow fragment, partially digested with PspOMI, and then dephosphorylated with CIP to isolate a 13.8 kb PacI/PspOMI vector fragment. pNOV5044 was cut with Ecl136II and PspOMI to isolate a 3525 by Ecl136II/PspOMI insert fragment. pNOV5044 contained a CMPS promoten:GUSbafintron::AtAct2-3'-UTR expression cassette in a pBluescript backbone. The above-described PacI/PspOMI fragment (13.8 kb) of pNOV5030 was ligated with the Ecl136II/PspOMI fragment (3535 bp) of pNOV5044 to form a second monocot donor vector, pNOV5045 (FIG. 17B).

[0247] Donor vectors with attB and attP sites were also constructed. These vectors allowed the use of phage lambda integrase to excise the intervening DNA sequences and thereby regenerate the selectable marker target site to permit gene stacking. To do this, complementary oligos ATTB1 (SEQ ID NO:90: 5'-GAT CCG CTC AAG TTA GTA TAA AAA AGC AGG CTT CAT GA-3') and ATTB2 (SEQ ID NO:91: 5'-GAT CTC ATG AAG CCT GCT TTT TTA TAC TAA CTT GAG CG-3') were annealed and inserted into BglII digested-pNOV2790 to form pQD187A8. The phage lambda attP sequence was amplified by PCR from the phage DNA with two primers, ATTPSPOMI (SEQ ID NO:92: 5'-GGG CCC TCT GTT ACA GGT CAC TAA TAC CAT CTA AG-3') and ATTPSPEI (SEQ ID NO:93: 5'-ACT AGT GAA ATC AAA TAA TGA TTT TAT TTT G-3'), and the PCR product was cloned into the pCR2.1-TOPO vector to form pNOV5088. The attP sequence was removed from pNOV5088 by digestion with ApaI, treatment with a Klenow fragment, and then a second digestion with NotI. pNOV5089 was digested with XbaI, filled-in with a Klenow fragment, and then cut with NotI. The above XbaI/NotI fragment of pNOV5089 was then ligated with the ApaI/NotI fragment of pNOV5088 to form pNOV5094. pNOV5089 was derived from pNOV5044 by replacing the BstBI/SnaBI fragment of the GUSBAFintron with the BstBI/SnaBI fragment of the GUSintron from pNOV3603. The KpnI/SpeI fragment of pNOV5031 was replaced with the KpnI/SpeI fragment from pNOV5087 to form pNOV5095. pNOV5094 was cut with Ecl136II and PspOMI to isolate the 3517 by fragment containing the CMPS promoter::GIG::Tact2::attP site. pNOV5095 was digested with PacI, blunted with a Klenow treatment, and then partially recut with PspOMI to isolate the 13805 by fragment. The Ecl136II/PspOMI fragment of pNOV5094 was inserted into the above 13.8 kb fragment of pQD195A6 to form a third monocot donor vector, pNOV5096 (FIG. 17B). The 3608 by Ecl136II/PspOMI of pNOV5098 was inserted into the above PacUPspOMI-digested pNOV5095 vector to form a fourth monocot donor vector, pQD200C6 (FIG. 17B), which inlcuded RS and FRT sites upstream of the ZmUbi promoter in addition to the attP sequence. A binary vector (pNOV5099) containing a positive control PMI-intron gene with the attB sequence in the first intron was constructed by inserting the 3551 by BamHI fragment of pQD187A8 into BamHI-digested pNOVO41. Another positive control binary vector (pQD203A11) was created by inserting the NcoI(blunt)/PspOMI fragment (3.6 kb) of pNOV5098 into a (PacI)blunt/PspOMI fragment (11535 bps) of pNOV5099.

Example 24

Construction of I-CeuI Expression Vectors for Monocots

[0248] An I-CeuI sequence with maize-preferred codons was released from pSmasICeuIintron as a BamHI/KpnI fragment (1154 bps), and a maize ubiquitin promoter (ZmUbi) was released from pNOV2115 as a BamHI/HindIII (2005 bps) fragment. These two fragments (I-CeuI and ZmUbi) were ligated into KpnI/HindIII-digested pNOV2114 to form pNOV5033. pNOV2114 is a binary backbone vector with a VS1 origin, one copy of the VirG gene, and a spectinomycin resistance gene for selection in bacteria. The BamHUKpnI fragment of I-CeuI and the ZmUbi fragment were also ligated with KpnI/hindIII-digested pNOV2122 to form pNOV5034. pNOV2122 is a binary backbone vector with an RK2 origin of replication, one copy of the VirG gene, and a kanamycin resistance gene for selection in bacteria. In both pNOV5033 and pNOV5034, I-CeuI expression was under the control of a maize ubiquitin promoter.

Example 25

Generation of Target Maize Plants

[0249] Target plants can be generated through Agrobacterium or biolistic-mediated transformation using target vector pNOV5025 and pAdF55 with any of several monocot plants, such as maize, rice, wheat, or barley, for example. Maize examples are provided here to demonstrate the feasibility of gene targeting through homologous recombination in monocot plants. The transformation of immature maize embryos was performed essentially as described in Negrotto et al. (2000 Plant Cell Reports 19: 798-803), which describes the use of PMI as the selectable marker gene and mannose as the selection agent, and Li et al (2003 Plant Physiol. 133:736-747), which describes the use of PPO as the selectable marker gene and butafenacil as the selection agent. For this example, all media constituents are as described in Negrotto et al. and Li et al. supra. However, various media constituents described in the literature may be substituted.

[0250] Target binary vector pNOV5025 contained the mutant protoporphyrin oxidase (protox) (PPO) gene (U.S. Pat. No. 6,308,458), which permitted the selection of transgenic cells with an herbicide-supplemented media (i.e., butafenacil). See, Li et al. 2003 Plant Physiol. 133:736-747. A positive control vector pNOV5026 was also included.

[0251] Agrobacterium strain LBA4404 (pSB1) containing pNOV5025 was grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) solid medium for 2-4 days at 28.degree. C. Approximately 0.8.times.10.sup.9 Agrobacteria (about 0.75 A.sub.660) per ml were resuspended in LS-inf media supplemented with 100 .mu.M As (Negrotto et al. 2000 Plant Cell Rep 19: 798-803). Bacteria were pre-induced in this medium for 30-60 minutes. For this example, immature embryos from A188.times.Hi II were excised from 8-12 day old ears into liquid LS-inf+100 .mu.M As. However, immature embryos derived from various other crosses or selfed A188 or HiII plants can be used as transformation targets. The embryos were rinsed once with fresh infection medium and heat-shocked at 45.degree. C. for 5 minutes. The infection medium was replaced with Agrobacterium solution, and the embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate were transferred to LSDc medium supplemented with ticarcillin (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark for 28.degree. C. for 10 days.

[0252] Selection was performed essentially as described in Li et al., supra. Silver nitrate was used in both the initiation and selection media, and sucrose was used at 30 g/L. The protox inhibitory herbicide butafenacil was added to the media at 5 nM for initiation and primary selection, 500 nM for second selection, and 750 nM for the final selection. Regeneration 1 was carried out on media supplemented with 50 nM herbicide with no herbicide selection in subsequent regeneration media. Maize leaf sample were assayed by Taqman analysis for the copy number of PPO and PMI genes. Maize events (for example, AW286B1A to AW289B1C, AW289B1A to AW289B1C, AW289E2D and AW289F2C etc.) with a single copy of both genes were transplanted into soil and grown in the greenhouse.

Example 26

Targeted Integration of a Donor Sequence by Homologous Recombination in Maize

[0253] Once the primary transgenic lines containing the desired T-DNA target (i.e., a target containing single copies of both the PPO and PMI genes) are obtained, various materials derived from these plants and their progeny can be used as target tissue for retransformation to obtain targeted events. These materials can also be used as pollen donors or receptors to produce target tissues for retransformation. For this example, AW289B1A was either selfed or used to pollinate A188 or HiII to produce seeds. Pollen from AW289B1A also was used directly to pollinate untransformed A188 and to generate immature embryos for retransformation with donor vectors pNOV5031, pNOV5045, pNOV5096, and pQD200C6.

[0254] Immature embryos (7-10 days post-pollination) were isolated from immature ears and used for Agrobacterium-mediated transformation, as described by Negrotto et al., supra. In some experiments, an Agrobacterium culture containing an I-CeuI expression vector, pNOV5033, was mixed with an Agrobacterium strain containing the donor vector (in a 1:1 ratio). Targeted events were selected from Agrobacterium-infected immature maize embryos using mannose as a selection agent. Immature embryos producing embryogenic calli were transferred to LSD1M0.5S medium. The cultures were selected on this medium for 3 weeks, transferred to fresh LSD1M0.5S medium, and then incubated for another 3 weeks. Surviving calli were transferred to Reg1 medium supplemented with mannose. Following a culture period of 1 to 2 weeks in the light (16 hour light/8 hour dark regimen), green tissues were then transferred to Reg2 medium without growth regulators and then incubated for 1-2 weeks. Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grown in the light (16 hour light/8 hour dark regimen). After 2-3 weeks, plants were transferred to the greenhouse for planting in soil. Maize lines HR-18FB.1A to HR-18FB.1N are putative mannose resistant targeted lines. These lines were derived from the targeted integration of donor sequence pNOV5045 by homologous recombination in the presence of pNOV5033.

Example 27

Molecular Characterization of the Targeted Event

[0255] Putative mannose-resistant targeted events were confirmed by well-known molecular biological methods, including PCR and Southern blot analysis. For example, a Southern blot was prepared from the DNA of target line AW289B1A and putatively targeted line HR-18FB.1M. DNA samples were digested with various restriction enzymes, including KpnI, ScaI, Sad, SpeI and HpaI, and hybridized with two different target specific probes from 5'-region of the rice actin 1 promoter and 3'-region of the PMlintrons (see FIGS. 20A and 20B for the blot and FIGS. 19A and 19B for the probe location and restriction map). The hybridization patterns were consistent with targeted double crossover recombination of pNOV5045 T-DNA with the target locus, which included T-DNA from pNOV5025.

[0256] The first target locus-specific probe (i.e., the 5'-region of the rice Act1, FIG. 19B) is from the rice actin 1 promoter 5'-upstream region that is not present in the donor vector pNOV5045 and is used to detect recombination at the LB end of the target locus. The second target locus-specific probe (i.e., PMlintrons 3'-region) hybridizes to the region containing the PMlintrons intron 4/exon 5 and is used to detect recombination at the RB end of the target locus (FIG. 19A).

[0257] Southern analysis confirmed that HR-18FB.1M is a truly targeted event derived from AW289B1A (FIGS. 19A to 19D and 20A to 20D). Sad digestion of the DNA samples would be expected to release an internal fragment from the target locus that included most of the introduced T-DNA sequences in both the target locus and the expected recombinant, but the size of the Sad band hybridizing to the PMI 3'-end probe would be expected to shift from 5.4 kb to 11.5 kb. As predicted, the size of the Sad fragment shifted from 5.4 kb of the target locus (AW289B1A) to 11.5 kb of the putative targeted line HR-18FB.1M when the PMI-intron 3'-fragment was used as a probe (FIG. 20B, lane 1a vs 6a).

[0258] KpnI digestion of the DNA samples resulted in a KpnI fragment that was also decreased in size, as predicted, from about 8 kb in the target locus to 3.5 kb in targeted line HR-18FB.1M when the PMI-intron 3' fragment was used as a probe (FIG. 20B, lane 3a vs 8a).

[0259] With ScaI, SpeI, and HpaI digestions, the fragment sizes did not change as predicted when there was targeted integration at the target locus (FIG. 20B, lane 2a vs 7a, lane 4a vs 9a, lane 5a vs 10a). Because ScaI and HpaI digestions hybridized with a PMI-intron 3'-probe detected changes in sequences outside the T-DNA, the results indicated that no DNA rearrangement could be detected on the right border of the T-DNA locus.

[0260] With the rice actin-1 5'-region probe, all five digestions indicated that recombination had occurred on the PPO side of the target locus. With the exception of SpeI, all band shifts in the targeted line HR-18FB.1M as compared with the target line AW289B1A were as expected (FIG. 20B). Because SpeI digestion is sensitive to overlapping cytosine methylation, it is possible that the SpeI site between the PPO and GUS genes was methylated. If this were the case, the size of SpeI fragment would have been expected to increase to 13 kb rather than be reduced to 6 kb. Since the SpeI fragment detected by the rice actin-1 probe was in fact 13 kb, methylation was the likely cause of the band shift (FIG. 20B, lane 4b vs 9b). It is also possible that there was a rearrangement, such as a deletion, that lead to the loss of the SpeI site.

[0261] Overall, the Southern blot data are consistent with the occurrence of targeted integration of the donor T-DNA into the target locus in line HR-18FB.1M by double crossover homologous recombination.

Example 28

Preparation of a Site-Specific R Recombinase Nucleotide Sequence with Maize-Preferred Codons and Construction of an R Recombinase Expression Vector

[0262] A site-specific R recombinase amino acid sequence was back-translated into a DNA nucleotide sequence as shown in SEQ ID NO:94 using maize-preferred codons (U.S. Pat. No. 6,121,014). Sequences flanking the synthetic R recombinase (ZmR) coding region are indicated in lower cases letters.

TABLE-US-00005 SEQ ID NO: 94: R recombinase with maize-preferred codons (ZmR) ctcgagcaaccATGCAGCTGACCAAGGACACCGAGATCAGCACCATCAAC CGCCAGATGAGCGACTTCAGCGAGCTGAGCCAGATCCTGCCCCTGCACCA GATCAGCAAGATCAAGGACATCCTGGAGAACGAGAACCCCCTGCCCAAGG AGAAGCTGGCCAGCCACCTGACCATGATCATCCTGATGGCCAACCTGGCC AGCCAGAAGCGCAAGGACGTGCCCGTGAAGCGCAGCACCTTCCTGAAGTA CCAGCGCAGCATCAGCAAGACCCTGCAGTACGACAGCAGCACCAAGACCG TGAGCTTCGAGTACCACCTGAAGGACCCCAGCAAGCTGATCAAGGGCCTG GAGGACGTGGTGAGCCCCTACCGCTTCGTGGTGGGCGTGCACGAGAAGCC CGACGACGTGATGAGCCACCTGAGCGCCGTGCACATGCGCAAGGAGGCCG GCCGCAAGCGCGACCTGGGCAACAAGATCAACGACGAGATCACCAAGATC GCCGAGACCCAGGAGACCATCTGGGGCTTCGTGGGCAAGACCATGGACCT GATCGAGGCCCGCACCACCCGCCCCACCACCAAGGCCGCCTACAACCTGC TGCTGCAGGCCACCTTCATGAACTGCTGCCGCGCCGACGACCTGAAGAAC ACCGACATCAAGACCTTCGAGGTGATCCCCGACAAGCACCTGGGCCGCAT GCTGCGCGCCTTCGTGCCCGAGACCAAGACCGGCACCCGCTTCGTGTACT TCTTCCCCTGCAAGGGCCGCTGCGACCCCCTGCTGGCCCTGGACAGCTAC CTGCAGTGGACCGACCCCATCCCCAAGACCCGCACCACCGACGAGGACGC CCGCTACGACTACCAGCTGCTGCGCAACAGCCTGCTGGGCAGCTACGACG GCTTCATCAGCAAGCAGAGCGACGAGAGCATCTTCAAGATCCCCAACGGC CCCAAGGCCCACCTGGGCCGCCACGTGACCGCCAGCTACCTGAGCAACAA CGAGATGGACAAGGAGGCCACCCTGTACGGCAACTGGAGCGCCGCCCGCG AGGAGGGCGTGAGCCGCGTGGCCAAGGCCCGCTACATGCACACCATCGAG AAGAGCCCCCCCAGCTACCTGTTCGCCTTCCTGAGCGGCTTCTACAACAT CACCGCCGAGCGCGCCTGCGAGCTGGTGGACCCCAACAGCAACCCCTGCG AGCAGGACAAGAACATCCCCATGATCAGCGACATCGAGACCCTGATGGCC CGCTACGGCAAGAACGCCGAGATCATCCCCATGGACGTGCTGGTGTTCCT GAGCAGCTACGCCCGCTTCAAGAACAACGAGGGCAAGGAGTACAAGCTGC AGGCCCGCAGCAGCCGCGGCGTGCCCGACTTCCCCGACAACGGCCGCACC GCCCTGTACAACGCCCTGACCGCCGCCCACGTGAAGCGCCGCAAGATCAG CATCGTGGTGGGCCGCAGCATCGACACCAGCTGAagctt

[0263] This synthetic R recombinase with maize preferred codons was synthesized and cloned into pUC19 to form pUC19-ZmR by IDT (Coralville, Iowa 52241). A ZmUbi-R expression cassette was inserted into binary vector pNOV2114 for maize transformation. A ZmR HindIII/BamHI fragment (1493 bp) was then removed from pUC19-ZmR by HindIII digetion, filled-in with a Klenow fragment, and then digested with BamHI and inserted into pNOV3603, which then was cut with Sad, blunted with a Klenow fragment, and digested with BamHI to form pQD204B1. pQD204B1 included the maize ubiquitin promoter to drive expression of ZmR, which was followed by a nopaline synthase terminator. The HindIII/KpnI fragment (3784 bp) of pQD204B1 containing the ZmUbi promoter::ZmR::Tnos cassette was inserted into a HindIII/KpnI-digested pNOV2114 binary backbone vector to form pQD205A1. pQD204B1 was also digested with KpnI, blunted by treatment with a Klenow fragment, and then recut with HindIII to isolate the 3780 by KpnI/HindIII fragment containing the ZmUbi promoter::ZmR::Tnos expression cassette. This KpnI/HindIII fragment was inserted into pNOV2819, which was cut with SalI, filled-in with a Klenow fragment, and re-digested with HindIII to form binary vector pQD206B1. pQD206B1 contained a ZmR expression cassette (ZmUbi promoter::ZmR::Tnos) and a selectable marker gene cassette (CMPS promoter::PMI::Tnos). ZmR was also placed under the control of several tissue specific promoters, including OsG, RA-8, P19, and OsMADS13 to avoid any potentially undesirable effects of constitutive expression. These vectors were referred to as pBSC11475 (OsG), pBSC11478 (RA-8), pBSC11479 (P19), and pBSC11480 (OsMADS13), respectively.

Example 29

Construction of Binary Vectors for Expressing Phage Lambda Integrase, an Integrase Mutant, and an Integration Host Factor

[0264] Phage lambda integrase, its double amino acid mutant (IntH218), and host factors with maize preferred codons are described in WO/03083045. Binary vector pNOV2114IntIHFs contained maize-optimized lambda integrase (Int) and IHF .alpha. and .beta. coding sequences under the control of a CMPS promoter followed by a Tnos terminator. The (HindIII)blunt/AscI fragment (4122 bp) containing the Int and IHF expression cassettes were removed from pNOV2114IntIHFs by HindIII digestion, filled-in with a Klenow treatment, recut with HindIII, and ligated with a (BamHI)blunt/AscI fragment (9541 bps) of pWC057 to form pQD208B12. pWC057 is a binary vector containing a ZmUbi promoter::AtPPO(dm)::T35S expression cassette (see U.S. Pat. No. 6,282,837). pQD208B12 is a binary transformation vector containing the CMPS promoter::Int::Tnos, CMPS promoter:IHF.alpha.::Tnos, and CMPS promoter::IHF.beta.::Tnos expression cassettes, as well as the ZmUbi promoter::AtPPOdm::T35S selectable marker cassette. Similarly, binary vector pNOV2114IntH218IHFs contains a maize-optimized lambda integrase mutant (IntH218) and IHF .alpha. and .beta. coding sequences under the control of a CMPS promoter followed by a Tnos terminator. The (HindIII)blunt/AscI fragment (4122 bp) containing the IntH218 and IHF expression cassettes were removed from pNOV2114IntH2181HFs by HindIII digestion, filled-in with a Klenow treatment, recut with HindIII, and ligated with a (BamHI)blunt/AscI fragment (9541 bps) of pWC057 to form pQD209B16. pQD209B16 is a binary transformation vector containing the CMPS promoter::IntH218::Tnos, CMPS promoter::IHFa::Tnos, CMPS promoter::IHF.beta.::Tnos expression cassettes, as well as the ZmUbi promoter::AtPPOdm::T35S selectable marker cassette. Plasmid vector pAdF62A (WO03/083045), containing the synthetic XIS gene with maize optimised codons, was cut with SpeI, filled-in with Klenow, and then re-cut with AscI to isolate the SpeI-AscI fragment containing the CMPS promoter, XIS gene, and nos terminator. This fragment was inserted into AscI/SwaI-digested pQD208B12 and pQD209B16 to form pQD350A7 (aka. pBSC11348) and pQD351A15 (aka. pBSC11349), respectively.

Example 30

Generation of Transgenic Plant Lines Expressing ZmR, IntIHFs, and IntH2181HFs

[0265] Binary vectors pQD206B1, pQD208B12, pQD209B16, pBSC11348, pBSC11349, pBSC11475, pBSC11478, pBSC11479, and pBSC11480 were each transformed, individually, into Agrobacterium strain LAB4404(pSB1). The individual cultures of the Agrobacterium strain were then used for co-cultivation with immature maize embryos. The co-cultivated embryos were placed on a selection medium containing an herbicide (butafenacil) to generate transgenic plants. The transgenic plants were crossed directly to target plants or they were self-pollinated to produce seeds, which were used to generate additional plant material to cross with other plants.

Example 31

Removal of the Promoter and Part of the PMI-Intron Sequence to Regenerate a Truncated PMI-Intron Sequence

[0266] Transgenic maize lines expressing either synthetic R recombinase or phage lambda integrase were obtained by Agrobacterium-mediated transformation using binary pQD208B12, pQD209B16, pBSC11348, pBSC11349, pBSC11475, pBSC11478, pBSC11479, and pBSC11480. R recombinase or integrase-expressing lines can be crossed with desirable targeted recombinants to excise both the promoter sequence and the region of the PMI coding sequence flanked by the RS, attB/attP, or attL/attR sequences to truncate the PMI selectable marker gene. The progeny are screened by PCR for the truncation. Lines with the truncated sequence are backcrossed with a non-transgenic parent line to produce seeds. These seeds are then germinated, and the seedlings are screened by PCR to recover lines with the desired truncated sequence but without the recombinase locus. Lines with a regenerated target site but without the recombinase gene are used for a second round of gene targeting.

[0267] Alternatively, recombinant lines can be re-transformed with either an R recombinase or a Lambda integrase expression vector. Transformed lines are screened by PCR for the desired deletion. Lines with the desired deletion are backcrossed with untransformed plants to obtain seeds. These seeds are then germinated, and the seedlings are screened by PCR to recover lines with the desired deletion but without the R recombinase or Lambda integrase locus. Lines with a regenerated target site but without the R recombinase or integrase gene are used for a second round of gene targeting.

[0268] Recombinase can also be delivered as a virE2/VirF fusion protein expressed by Agrobacterium (Vergunst et al. 2000 Science 290:979-82). Maize tissues, preferably immature embryo or embryogenic callus, are infected with Agrobacterium cells containing vectors expressing R/integrase::virE2/virF fusion proteins. These fusion proteins are transported into plant cells to mediate a site-specific deletion of the sequence flanked by recombinase recognition sequences in suitable orientation allowing excision of the flanked region, such as Lox, FRT, RS, attB/attP or attL/attR sequences. Regenerated plants are screened by PCR for the deletion. With this method, recombinase or integrase expression vector DNA is delivered into the plant cells. Lines with the desired deletion can be used directly for an additional round of gene targeting.

Example 32

Generation of Target Rice Plants

[0269] For this example, the rice (Oryza sativa var. javonica) cultivar "Kaybonnet" was used to generate a target rice plant. However, other rice cultivars also can be used (Hiei et al. (1994) Plant Journal 6:271-282; Dong et al. (1996) Molecular Breeding 2:267-276; Hiei et al. (1997) Plant Molecular Biology 35:205-218). Also, various media constituents described below may be varied or substituted.

[0270] Embryogenic responses were initiated and/or cultures were established from mature embryos by culturing on MS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins (200.times.), 5 ml/liter; Sucrose, 30 g/liter; proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300 mg/liter; 2,4-D (1 mg/ml), 2 ml/liter; adjust pH to 5.8 with 1 N KOH; Phytagel, 3 g/liter). Either mature embryos at the initial stages of culture response or established culture lines were inoculated and co-cultivated with the Agrobacterium strain LBA4404 containing the desired vector construction (i.e., pNOV5025 or pADF55).

[0271] Agrobacterium was cultured from glycerol stocks on solid YP medium (100 mg/L spectinomycin) for 3 days at 28.degree. C., then streaked again and cultured for 1-2 days. Agrobacterium was re-suspended in liquid MS-CIM medium. The Agrobacterium culture was diluted to an OD600 of 0.2-0.3 and acetosyringone was added to a final concentration of 200 uM. Agrobacterium was induced with acetosyringone for at least 30 min before mixing the solution with the rice cultures.

[0272] For inoculation, the cultures were immersed in the bacterial suspension for 30 min. The liquid suspension was removed with a vacuum aspirator, and the inoculated cultures were placed on a Whatman paper filter on co-cultivation medium MS-CIM-As (MS-CIM with 200 uM acetosyringone) and incubated at 22.degree. C. for two days. The cultures were then transferred to MS-CIM medium with ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium. For pNOV5025, a protox inhibitory herbicide (e.g., CGA 856,276 or butafenacil) (U.S. Pat. No. 6,282,837) was used for selection. Cultures are transferred to selection medium containing compound CGA 856,276, MSI/856,276 (MS-CIM with 1000 nM butafenacil, 200 mg/liter timentin) after 14 days and cultured for 28 days in the dark. Resistant colonies were then transferred to regeneration induction medium (MS-CIM with no 2,4-D, 0.5 mg/liter IAA, 1 mg/liter zeatin, 200 mg/liter timentin, and butafenacil) and grown in the dark for 14 days. Proliferating colonies were then transferred to another round of regeneration induction media and moved to the light growth room. Regenerated shoots were transferred to GA7-1 medium (MS without hormones) for 2 weeks and then moved to the greenhouse when they were large enough and had adequate roots. Plants were transplanted to soil in the greenhouse and grown to maturity. For pADF55, a similar protocol was used to generate transgenic plants, except that hygromycin, rather than butafenacil, was used as the selection agent.

Example 33

Targeted Integration of a Donor Construct into Target Rice Lines

[0273] Primary transgenic target rice lines, preferably single copy lines, containing T-DNA from target vector pNOV5025 or pADF55 were self-pollinated to obtain seeds. Seeds from selfed progeny of these lines were also used for establishing embryogenic cultures and suspension cultures for targeting experiments. Immature embryos from young seeds or mature embryos from dry seeds are used to establish embryogenic cultures (Hiei et al. 1994 Plant Journal 6:271-282; Dong et al. 1996 Molecular Breeding 2:267-276; Hiei et al. 1997 Plant Molecular Biology 35:205-218). These cultures or suspension cell clusters are then used for Agrobacterium-mediated transformation.

[0274] Agrobacterium strain LBA4404 containing the targeting donor vector pQD200C6 or pAdF77 was used for generating targeted events from target lines derived from pNOV5025 or pAdF77, respectively. Other targeting vectors using flanking genomic sequences as a region of homology can also be designed and used. In this case, the length of homology could be increased or decreased, as needed, and the selectable marker gene sequences used to introduce the target sequence can be replaced. Targeted events were selected from Agrobacterium-infected rice embryogenic cultures using the selection and regeneration processes described above, with the exception that 2% mannose is used as a selection agent. Two target lines (RITI2001001226A1A and RITI2001001226A5A, referred later to as lines A1A and A5A) containing 2 copies of T-DNAs were used for targeting study with donor vector pQD200C6. Both lines have 2 copies of T-DNA inserted in the genome, mostly likely at unlinked positions. Two lines derived from pAdF55 (AdF55-15A and AdF55-35A) were aslo randomly selected for gene targeting study with donor vector pADF77. Callus or suspension cell cultures were initiated from mature seeds of target plants and were co-cultured with Agrobacterium cells containing donor vector. Co-cultivations were also done with mixture of two Agrobacterium strains, one containing donor vector and another containing pNOV5033, the expression vector of mega-endonuclease 1-CeuI. Co-cultured calli were selected in mannose containing medium to recover targeted events. Mannose resistant callus can be seen within a month after selection. Resistant calli were regenerated into plants. A PCR assay using two primers (PMIExFW1 and PMIExRV5) was used to confirm whether the mannose resistant plants indeed contained a full-length recombinant PMI-intron sequence. Only plants that are derived from recombination between target and donor's truncated PMI-intron gene sequence produce a PCR product of 3.5 Kb. Most of the recovered eventswere tested positive using this assay, suggesting that mannose selection is very effective in recovering targeted events in rice. Co-delivery of I-CeuI expression vector pNOV5033 with donor vector increased the number of targeted events, especially for line A1A. For two target lines derived from pAdF55 (AdF55-15A and AdF55-35A), all targeted events were obtained when the donor vector was co-delivered with the I-CeuI endonuclease expression vector (Table 3).

TABLE-US-00006 TABLE 3 Targeted integration of a donor into target rice lines Donor: No. of I-CeuI Tissue mannose Target Target vector fresh resistant vector line Target tissue Exp. ID. ratio * wt. (g) events pNOV5025 A5A Suspension 664.154 1:0 0.96 2 culture 1:1 1.13 0 cells 1:1/2 1.09 0 from T2 1:1/5 1.08 1 seeds Pos. ctrl 1.13 64 pNOV5025 A5A Suspension 664.165 1:0 1.10 0 culture 1:1 1.15 2 cells 1:1/2 1.58 0 from T2 1:1/5 1.31 1 seeds Pos. ctrl 1.13 55 pNOV5025 A5A, T2 Calli from 664.162 1:0 1.18 0 T2 seeds 1:1 1.26 2 1:1/2 1.31 1 1:1/5 1.34 0 Pos. ctrl 1.15 15 pAdF55 15A, T1 Calli from 664.151 1:0 2.08 0 T1 seeds 1:1 1.98 1 1:1/2 1.98 2 1:1/5 1.98 1 pAdF55 35A, T1 Calli from 664.151 1:0 1.96 0 T1 seeds 1:1 3.1 5 1:1/2 1.9 0 1:1/5 2.07 0 Pos. ctrl 1.93 49 * Note: Donor vectors used were pQD200C6 and pAdF77 for target lines derived from pNOV5025 and pAdF55 respectively. For I-CeuI endonuclease expression, vector pNOV5033 was used; Pos. ctrl: positive control, pNOV2147 was used for estimating overall transformation (random integration) efficiency.

Example 34

Suppression or Down-Regulation of RecQ Homologs to Enhance Gene Targeting Efficiency

[0275] 1. Identification of RecQ Homologs in a Plant Genome

[0276] Plant genomic and cDNA sequence databases can be searched with various bioinformatics programs to identify bacterial, yeast, and animal RecQ homologs. For example, the Arabidopsis genome contains several RecQ homologs (Hartung et al. 2000 Nucleic Acids Res. 21, 4275-4282). To identify RecQ homologs in the rice genome, proprietary Syngenta rice genome (Myriad contigs V8, Nipponbare cultivar) sequences were searched with the TBLASTN program using the E. coli RecQ protein sequence (GenBank accession number: NP.sub.--756603) and the mouse RecQ-like protein (GenBank accession number: BC014735) as queries. Two contigs (CLB1350.2, CLB5120.2) produced a high score (517, E value=e-145). Another three contigs (CL003142.76, CL027228.91, and CLC370) produced lower but significant scores. Gene prediction programs (Fgenesh, Genscan, and Genmark) were used to predict the open reading frame of each hit

[0277] Primers were designed for amplification of the cDNA. OsRecQcfw2 (SEQ ID NO:95: CAC CAT GAA GCA CGG TGT AAT TGA TGA TAA AGA A) and OsRecQcRv1 (SEQ ID NO:96: TCA AGA GGG AAT CTT TAT GCA GTT GTC GCA) amplified a cDNA of 2 kb (OsRecQB) from rice (Oryza sativa, cultivar Kaybonnet) young flowers. OsRecQdFW2 (SEQ ID NO:97: CAC CAT GAT AAA GCC AAG GGT CAA CTG GTC GGA T) and RecQdRV1 (SEQ ID NO:98: CTA GGC TAT TCT GGC GGA CTG CCA CGC AGG) amplified a cDNA of 3.5 kb (OsRecQA) from rice immature flowers. The OsRecQB (2 kb) and OsRecQA (3.5 kb) cDNA PCR products were cloned into pENTR-TOPO vector (Invitrogen) to form pQD356A27 and pQD363C8, respectively. The insert of each clone is sequenced. The OsRecQA cDNA (SEQ ID NO:99) contains an ORF of 3525 bp, which encodes a protein having 1174 amino acid residues (SEQ ID NO:100). The OsRecQB cDNA (SEQ ID NO:101) contains an ORF of 1419 bps, which encodes a protein having 472 amino acid residues (SEQ ID NO:102). When the rice genome open reading frame (ORF) databases (cultivar Nipponbare) were searched, a third homolog, OsRecQC (SEQ ID NO:103), having 4692 bps was also identified. This third homolog encodes a protein having 1563 amino acid residues (SEQ ID NO:104).

TABLE-US-00007 SEQ ID NO: 99: OsRecQA cDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsRecQA cDNA SOURCE Young flower. ORGANISM Oryza sativa Cultivar Kaybonnet REFERENCE 1 (bases 1 to 3525) AUTHORS Qiudeng Que CDS 1 . . . 3525 BASE COUNT 1090 a 736 c 805 g 894 t ORIGIN 1 ATGATAAAGC CAAGGGTCAA CTGGTCGGAT CATGCAAATG CTGTTCAAAG CTCCTGTATC 61 AAAGATGAAT TCCTGAGTTC AAGTTTTTTG TTCTCTTTAC CAACACAAAG GCCTAATCAG 121 GAAGCAGATT GTACGGGAAT GCTTCCTTTA AGGTCTGCTG CTTGCAGAAT TCAAGGCCTA 181 GAGCGTCTTC AAGCTCCATC CATTGAGAAG GCCTGGCGTT CTCTACGCAA CACTCAGGTT 241 GCACGGAAGA ATTATTTAAG ACCTGGTTTA TCTGGAAAAG TGAAAGATTG TGATAGCGAC 301 CATGCTCATA CTTATGGGAC AAGTTCTTCA TATAATGTTA ACAAAGTGGA CAGTGTGTCC 361 AGAAATAGGA ATCCCACCCA GGAAAGTATG CATCAGACGA CTGAAAGTGG TACTATGGAG 421 AAGAACAGTA GCCATCTGCC TGCAGGCACC AAGTCCTGTA CAAGGACTTA CCTGAACAAT 481 CATGTGGTGC AGGCAGATAC CATTACAACA ACAAATCAAA GTCTTGCAAG AACTGGTCCT 541 GAATTATTCA AGACTGCTCC TTTTATTGAC AACATGTGTG ATGATGCTAA ATTAGATGCC 601 ATGGATGAGG ATGAGCTTCT AGCGAGTATT GATGTGGACC GAATAGTCAT GGAACATTAT 661 CAAGCAACAA ATACACCCAG AGGGTCATCC AAATCTCCAT TAGAGAAGTG CAACTTCAAT 721 GGATTTGATG AGAATAATTT ACCACAAGAA CTCTCTATAA TGTGTGACCA CGGTAGCAAG 781 CTAGCTTTTT GCCCAGAGGC GAAGTCTCAT TTGCTTGAAA TGAAGGATAA CTTGCTTGCA 841 ATATCCCATG AGCTTATTGA CGGTCAACTC AGCCCTCAAC AATCTGATGA TCTTCATCAA 901 AAGAGAGCAC TCCTAAAGAA GCAGATTGAG CTGCTTGGGG AGTATACGGC GAGGTTAACC 961 CAAGATGAAG AGCGACAGCA GTCTCATTCT ATGGCCTCCA CAACAGCTCA TCAGGGCCAT 1021 CACCCCACTA GCATCCTAAG TAGCTCTTTT GTAAAGGATA CCAATATATT CCGATCACCG 1081 ATTTACACCA GGAATGAACC TGGGGAGAGT GGTTTATGCT TTTCTTCTGC TCCATATTCC 1141 TATATGGATG GTTTAAGCAT GCCATTACCG TCTGTTCAGA GAGATTACAC TCCAAGGGCT 1201 ATTGATATCA GTTACACTGA AGGTTCTGGT GATAAACAGT GGAGTAGTAC ACACTTTGCA 1261 TGGACTAAGG AACTCGAGGC CAACAACAAA GGAGTATTTG GAAACCGTTC TTTTCGCCCA 1321 AATCAACGAG AAATAACCAA CGCCACAATG AGTGGGAATG ATGTTTTTGT TTTGATGCCA 1381 ACTGGTGGTG GAAAAAGTTT GACATATCAG CTTCCAGCAC TCATTTGTAA TGGCGTTACA 1441 TTGGTAGTTT CTCCTCTCGT ATCGCTCATC CAAGACCAGA TCATGCATTT ATTGCAGGCA 1501 AATATTTCTG CAGCTTACCT TAGCGCCAGC ATGGAGTGGT CAGAACAGCA GGAGATATTA 1561 AGAGAATTAA TGTCTCCTAC ATGCACGTAC AAGTTACTGT ATGTTACGCC TGAAAAGATA 1621 GCCAAGAGTG ATGCTCTGTT GAGACAATTG GAAAATTTAT ATTCGCGAGG CCATCTCTCT 1681 AGAATTGTCA TTGATGAAGC CCACTGTGTT AGCCAGTGGG GTCATGATTT CCGACCTGAT 1741 TACCAGCATC TAGGCATTTT AAAACAGAAG TTCCCGCAGA CGCCGGTCCT GGCCTTGACA 1801 GCAACAGCAA CTGCAAGTGT CAAGGAAGAT GTCGTGCAAG TTCTAGGCCT TGCAAACTGC 1861 ATTATTTTCA GACAAGGTTT TAATCGTCCA AATCTGAGGT ATTTTGTATG GCCCAAGACA 1921 AAGAAGTGCC TCGAGGATAT CCATAACTTT ATACATGCAA ATCATAATAA AGAATGCGGC 1981 ATCATATATT GCCTTTCGAG GATGGATTGT GAGAAAGTGG CTGCTAAATT AAGGGAATAT 2041 GGGCACCAGG CATCACATTA TCATGGTAGC ATGGATCCTG AGGATAGAGC AAATATCCAG 2101 AAACAGTGGA GCAAGGATAG GATCAACATA ATATGTGCTA CAGTTGCATT TGGGATGGGT 2161 ATTAATAAAC CTGATGTCCG TTTTGTTATC CATCATTCCC TGCCCAAATC AATTGAAGGA 2221 TATCATCAGG AGTGTGGACG TGCTGGTCGT GACAGTCAGC TTTCATCTTG TGTCCTGTTC 2281 TACAATTATT CTGATTATAT TCGTCTCAAA CACATGGTTA CCCAAGGATT TGCGGAGCAA 2341 GGAACATCAG CACCACGAGG AGGTTCTTCG CAGGAACAAG CGCTTGAAAC GCATAAGGAA 2401 AATCTCCTGC GAATGGTTAG TTACTGCGAA AATGATGTGG ACTGCAGACG TCTACTACAG 2461 CTGATCCACT TTGGAGAGAT GTTTAATCCT TCATGTTGTG CAAAAACATG TGATAATTGC 2521 TTGAAAGAGT TGAGATGGGT CAAAAAAGAT GTGACCAACA TTGCTAGACA ATTGGTTGAT 2581 CTGGTAATGA TGACAAAGCA AACATATTCA ACTACTCATA TTCTCGAAGT ATACAGAGGT 2641 TCAGTAAACC AAAATGTCAA GAAGCACCGC CATGATACTT TGAGTCTTCA TGGAGCTGGA 2701 AAGCATCTAG CTAAAGGTGA AGCAGCGAGA ATATTGCGCC ATCTAGTAAT TGAGGAAATA 2761 CTCATTGAGG ATGTCAAAAA GAGCGAAAAC TATGGATCTG TATCATCTGT CTTAAAGACT 2821 AATCATAAGA AAAGTGGTGA TCTTCTCTCT GGCAAGCACA ACGTTGTCCT CAAGTTCCCC 2881 ACTCCTGAGA AGGCTCCTAA GATGGGTGTA CTCGATGAAT CGTCAGTTCC ACGAATTAAT 2941 AAGACTAATC AACAGAGTCA AGTGGACGGG AGCCTTGCAG CCGAGCTTTA TGAAGCTTTG 3001 CAATGCCTTA GGACTCAGAT AATGGATGAA AATCCACAAT TATTGGCATA CCACATATTT 3061 AAAAACGAGA CATTGAAGGA AATCAGCAAC CGAATGCCAA GAACGAAAGA GGAACTTGTG 3121 GAGATAAATG GCATCGGCAA GAACAAGCTG AACAAGTACG GGGACCGCGT GCTTGCAACC 3181 ATAGAGGATT TCCTCGCCAG ATATCCAAAT GCGACCAGGA AAACCAGCAG CGGCGGCAGC 3241 AACGAGCACA GCGAGGCGGT CAAGAAGCGA AGAGGCTTCT CCGTCACCAA CACCTCTACC 3301 AACTGTGACG ACTTTGAGGA ACGCACGGTC CAGTCCAAGA AACGCGCTGC AAAGACACGT 3361 ACAAGGCAGG AAATATCTGA TGCTGCCAGC ATCGTCCAGG ACGTCCGCTA CATAGATCTT 3421 GAGCTAGATG GTTGTGAACA AGTCAATGAA GTGCCATACA GTGTACAAAA GCCTGTGGCT 3481 TCTGGTAGGG TTTTACCTGC GTGGCAGTCC GCCAGAATAG CCTAG // SEQ ID NO: 100: Predicted OsRecQA protein sequence DEFINITION Predicted OsRecQA protein sequence, 1174 amino acid residues SOURCE Young flower. ORGANISM Oryza sativa Cultivar Kaybonnet MIKPRVNWSDHANAVQSSCIKDEFLSSSFLFSLPTQRPNQEADCTGMLPLRSAACRIQGL ERLQAPSIEKAWRSLRNTQVARKNYLRPGLSGKVKDCDSDHAHTYGTSSSYNVNKVDSVS RNRNPTQESMHQTTESGTMEKNSSHLPAGTKSCTRTYLNNHVVQADTITTTNQSLARTGP ELFKTAPFIDNMCDDAKLDAMDEDELLASIDVDRIVMEHYQATNTPRGSSKSPLEKCNFN GFDENNLPQELSIMCDHGSKLAFCPEAKSHLLEMKDNLLAISHELIDGQLSPQQSDDLHQ KRALLKKQIELLGEYTARLTQDEERQQSHSMASTTAHQGHHPTSILSSSFVKDTNIFRSP IYTRNEPGESGLCFSSAPYSYMDGLSMPLPSVQRDYTPRAIDISYTEGSGDKQWSSTHFA WTKELEANNKGVFGNRSFRPNQREITNATMSGNDVFVLMPTGGGKSLTYQLPALICNGVT LVVSPLVSLIQDQIMHLLQANISAAYLSASMEWSEQQEILRELMSPTCTYKLLYVTPEKI AKSDALLRQLENLYSRGHLSRIVIDEAHCVSQWGHDFRPDYQHLGILKQKFPQTPVLALT ATATASVKEDVVQVLGLANCIIFRQGFNRPNLRYFVWPKTKKCLEDIHNFIHANHNKECG IIYCLSRMDCEKVAAKLREYGHQASHYHGSMDPEDRANIQKQWSIDRINIICATVAFGMG INKPDVRFVIHHSLPKSIEGYHQECGRAGRDSQLSSCVLEYNYSDYIRLIHMVTQGFAEQ GTSAPRGGSSQEQALETHKENLLRMVSYCENDVDCRRLLQLIHFGEMFNPSCCAKTCDNC LKELRWVKKDVTNIARQLVDLVMMTKQTYSTTHILEVYRGSVNQNVKKHRHDTLSLHGAG KHLAKGEAARILRHLVIEEILIEDVKKSENYGSVSSVLKTNHKKSGDLLSGKHNVVLKFP TPEKAPKMGVLDESSVPRINKTNQQSQVDGSLAAELYEALQCLRTQIMDENPQLLAYHIF KNETLKEISNRMPRTKEELVEINGIGKNKLNKYGDRVLATIEDFLARYPNATRKTSSGGS NEHSEAVKKRRGFSVTNTSTNCDDFEERTVQSKKRAAKTRTRQEISDAASIVQDVRYIDL ELDGCEQVNEVPYSVQKPVASGRVLPAWQSARIA // SEQ ID NO: 101: OsRecQB cDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsRecQB cDNA 1419 bp ORGANISM Oryza sativa Cultivar Kaybonnet SOURCE Young flower REFERENCE 1 (bases 1 to 1419) AUTHORS Qiudeng Que BASE COUNT 427 a 306 c 338 g 348 t ORIGIN 1 ATGAAGCACG GTGTAATTGA TGATAAAGAA GTTGAGGTGA GAACTCCTTT GTTCAGACAG 61 GCAGAATCCT CTGCTCGACA GACTCGCATC AATCTGGACT CCTTCGGGTT CTCCTCAGAT 121 GATGACTTTG AAACGTTGGA GTCCCATTGT GATCGTTCAG TCAGTACCCA GAAGAAGGTG 181 AACAGAGGAA ACAATAGATG TGAGTCATCC ACTTCAACTT CAAACAGAGA AACTCTAAGT 241 TATCAGCAGC TCAACATGGA CACCTTTGTG CTTATGCCAA CAGGTGGTGG GAAGAGCTTG 301 TGTTATCAGC TACCTGCAAC ACTGCACCCA GGTGTTACGG TTGTTGTATG CCCTCTACTG 361 TCACTTATTG AGGATCAAAT TGTGGCATTA AACTTCAAGT TTGCTATACC AGCAGCATTT 421 TTGAACTCTC AGCAGACACC TTCACAGTCA TCTGCAGTAA TCCAAGAGCT TAGAAGTGGT 481 AAACCGTCAT TCAAACTCCT CTACGTCACT CCTGAAAGAA TGGCTGGAAA CAGCTCATTT 541 ATTGGGATCC TCATAGGTTT ACACCAGAGG GGTTTACTGG CGAGATTTGT GATTGATGAA 601 GCCCATTGTG TAAGTCAATG GGGACATGAC TTCCGCCCAG ATTACCGAGG CCTGGGATGC 661 CTCAAACAGA ACTTCCCTCG AGTACCAATT ATGGCTTTAA CAGCTACAGC GACTGCATCT 721 GTCTGCAAGG ACATACTAAG TACCTTGAGG ATCCCTAATG CAACGGTACT CAAGAGGAGC 781 TTTGACAGAA CAAACCTGAA TTATGAGGTG ATTGGCAAGA CAAAAACTCC ACAGAAGCAG 841 CTGGGTGATA TCCTAAAAGA GCGTTTCATG AACATGTCTG GTATCGTGTA CTGTCTGTCC 901 AAAAATGAAT GTGCTGACAC TGCCAAGTTC TTGAGGGAGA AGTACAAGAT AAAATGCGCA 961 CATTACCACG CTGGCTTGGC TGCTCGTCAA CGATCCAATG TACAAGGAAA ATGGCACAGC 1021 GGAGAGGTCA AAGTCATTTG TGCGACCATA GCATTTGGCA TGGGAATAGA CAAACCTGAT 1081 GTGCGCTTTG TTATCCACAA CACCATGTCA AAATCGATAG AAAGCTACTA TCAGGAGTCA 1141 GGGAGAGCAG GAAGAGACAA TCTTCCGGCA CATTGCATTG TGTTATATCA GAAAAAGGAC 1201 CTCGGTCGAA TTGTATGCAT GCTGAGGAAT TCAGGGAACT TCAAGAGTGA GAGCTTCAAG 1261 GTTGCAATGG AGCAAGCAAA GAAAATGCAA ACATATTGCG AGCTGAAGAC AGAATGCCGG 1321 AGGCAAACTC TTCTTGGCCA CTTCGGTGAG CAGTATGACA GGCAAAGGTG CAAACATGGT 1381 TGTAGCCCTT GCGACAACTG CATAAAGATT CCCTCTTGA

// SEQ ID No: 102: Predicted OsREcQB protein sequence DEFINITION OsRecQB protein 472 amino acids ORGANISM Oryza sativa Cultivar Kaybonnet MKHGVIDDKEVEVRTPLFRQAESSARQTRINLDSFGFSSDDDFETLESHCDRSVSTQKKV NRGNNRCESSTSTSNRETLSYQQLNMDTFVLMPTGGGKSLCYQLPATLHPGVTVVVCPLL SLIEDQIVALNFKFAIPAAFLNSQQTPSQSSAVIQELRSGIUSFKLLYVTPERMAGNSSF IGILIGLHQRGLLARFVIDEAHCVSQWGHDFRPDYRGLGCLKQNFPRVPIMALTATATAS VCKDILSTLRIPNATVLKRSFDRTNLNYEVIGKTKTPQKQLGDILKERFMNMSGIVYCLS KNECADTAKFLREKYKIKCAHYHAGLAARQRSNVQGKWHSGEVKVICATIAFGMGIDKPD VRFVIHNTMSKSIESYYQESGRAGRDNLPAHCIVLYQKKDLGRIVCMLRNSGNFKSESFK VAMEQAKKMQTYCELKTECRRQTLLGHFGEQYDRQRCKHGCSPCDNCIKIPS // SEQ ID NO: 103: OsRecQC cDNA from Oryza sativa (cultivar Nipponbare) DEFINITION OsRecQC Open Reading Frame 4692 bp DNA SOURCE Oryza sativa cv Nipponbare REFERENCE 1 (bases 1 to 4692) CDS 1 . . . 4692 BASE COUNT 817 a 1669 c 1511 g 695 t ORIGIN 1 ATGGCTTCCC GTCCCGCGCA CGACCTGCTT CAACGCGTCT TTGGTTACGA CGATTTCCGT 61 GGTCCGCAGC AGGACATCGT GGAGCATGTG GCTGCCGGTC ACGACGCCCT GGTGCTGATG 121 CCCACCGGCG GCGGCAAATC GCTGTGCTAC CAGGTCCCAG CCCTGCTGCG TGACGGTTGC 181 GGCATCGTCA TCTCGCCGCT GATCGCACTG ATGCAGGACC AGGTCGAAGC CCTGCGCCAG 241 CTCGGCGTGC GCGCCGAGTA CCTGAATTCA ACCCTGGACG CCGAGACCGC CGGCCGCGTC 301 GAGCGCGAGC TGCTCGCCGG CGAACTGGAC ATGCTGTATG TCGCCCCTGA GCGGCTGCTG 361 AGCGGGCGTT TCCTGTCGCT GCTGTCGCGC AGCCAGATCG CCCTGTTCGC CATCGACGAA 421 GCACACTGCG TGTCGCAGTG GGGCCATGAC TTCCGCCCCG AATATCGCCA GTTGACCGTG 481 CTGCACGAGC GTTGGCCGCA GATCCCGCGG ATCGCGCTGA CCGCCACCGC CGATCCGCCG 541 ACCCAGCGCG AGATCGCCGA GCGCCTCGAT CTGCAGGAAG CGCGCCATTT TGTCAGTTCC 601 TTCGACCGCC CCAACATCCG CTACACCGTC GTGCAGAAGG ACAACGCCCG CAAGCAGCTG 661 ACCGACTTCC TGCGCGGCCA CCGTGGCGAG GCCGGCATCG TCTACTGCAT GTCGCGGCGC 721 AAGGTCGAGG AGACCGCTGA ATTCCTCTGC GGCCAAGGCG TCAACGCTCT GCCTTACCAC 781 GCCGGCCTGC CGCCGGAAGT GCGCGCCAGC AACCAGCGCC GCTTCCTGCG CGAGGACGGC 841 ATCGTGATGT GTGCCACCAT CGCCTTCGGC ATGGGCATCG ACAAGCCGGA CGTGCGTTTC 901 GTCGCGCATA CCGACCTGCC CAAGTCGATG GAGGGCTACT ACCAGGAAAC CGGACGCGCA 961 GGCCGCGATG GCGAAGCCGC CGAGGCCTGG CTGTGCTACG GCCTGGGTGA TGTGGTACTG 1021 CTCAAGCAGA TGATCGAGCA GTCCGAGGCG GGCGAAGAGC GCAAGCAGCT GGAACGGGCC 1081 AAGCTCGACC ATCTGCTGGG CTACTGCGAA TCGATGCAGT GCCGCCGCCA GGTGCTGCTG 1141 GCCGGCTTCG GCGAAACCTA CCCCCAACCG TGCGGCAACT GCGACAACTG CCTGACGCCA 1201 CCGGCCTCGT GGGACGCGAC CATACCGGCA CAGAAGGCGC TGAGCTGCGT CTACCGCAGC 1261 GGCCAGCGCT TCGGTGTCGG CCACCTGATC GACATCCTGC GCGGCAGCGA GAACGAGAAG 1321 GTGAGGCAGC AGGGCCACGA CAAGCTGAGC ACTTATGCCA TCGGCCGCGA CCTGGATGCA 1381 CGCACCTGGC GCAGCGTGTT CCGCCAGCTG GTCGCGGCCA GCCTGCTGGA AGTGGACAGC 1441 GAGGGCCACG GCGGCCTGCG CCTGACCGAC GCCAGCCGCG ACGTGCTGAC CGGCCGCCGC 1501 CAGATCAGCA TGCGCCGCGA CCCGGCCAGC AGCAGCAGCG GACGCGAGCG CAGTGCGCAG 1561 CGCACCGGCC TGTCGGTACT GCCGCAGGAC CTGGCCCTGT TCAACGCGCT GCGCGGCCTG 1621 CGCGCCGAAC TGGCCCGGGA ACAGAACGTA CCGGCGTTCG TGATCTTCCA CGACAGCACC 1681 CTGCGCAACA TCGCCGAGCG GCGCCCGACC AGCCTGGATG AACTGGCCCG GGTCGGCGGC 1741 ATCGGCGGTA CCAAGCTGAG CCGCTATGGC CCGCGCCTGG TCGAGATCGT GCGCGAAGAA 1801 GGCCTGTTGC TCAACGGGCT GAACGCGGCC ATGGCCCGTG GTCACGAAGA AATGGGGCGG 1861 ATGGCCCACG CCGCAGCCGC TGCTGTTGAT GGCGGCACTG CCGACTGCCA CCACCACGCC 1921 GCCATGCAGG CCGACCCGGC CCCGCAGGCC AAGGCCCCGG CCCACGACGC CCACTGCCAG 1981 ATCAAGGACT GCGTGCGCAG CTGCGCCCAG CACCCGCTGC TGGTGGTGCA GCCGTTGCCG 2041 TTCATGGCCG GACCGGCACT GTCGCTGGCC CCGCAGCCGA TGCCGGCCAC CGGCCGGCCG 2101 GCGCCCCCGT CTGCCGCCGA TCTCACGCCC TCCCATCGGC TGATTCCACA CGCACCGGCC 2161 TGGCCGCCGG TGGCGTGGTT GCCGGCATCG CCGCTGTCGG CGTGCCGCAG CGCGTGCTCG 2221 CCGCCGCCAC TGCCGCCCCA CGCCTGGCCG GCGCCCCCGC CGTGCTCAGC GACACCCGCA 2281 TCGAACTGGC CATCGGCGAA TCGCTGGCCA ACTTTCACTG GCCGCACCCG TCCGGCGATC 2341 ACCGTCAATG GATCGCTGCC GGCACCGATC CTGCGCTGGC GCGAAGGCCA GACCGTGGAC 2401 CTGTTCGTGC GCAACACGCT GGACCGCCAC CCGACCTCGA TCCATTGGCA CGGCATTCTG 2461 CTGCCGGCCA ACATGGACGG CGTGCCCGGC CTGAGCTTCA ATGGCATCGG CCCCGGTGAG 2521 ACCTACCACT ACCACTTCGA ACTGAAGCAG TCGGGTACCT ACTGGTACCA CAGCCACTCG 2581 ATGTTCCAGG AGCAGGCCGG CCTGTACGGA GCGCTGATCA TCGACCCGGC CGAGCCGGCG 2641 CCCTACCAGC ACGACCGCGA GCACGTGATC CTGCTGTCCG ACTGGACCGA CATGGACCCC 2701 GGCGCGCTGT TCCGGCGCAT GAAGAAGCTC GCCGAGCATG ACAACTACTA CAAGCGCACC 2761 CTGCCCGACT TCCTGCGTGA CGTGAAGCGC GACGGTTGGT CGGCCGCGTT GTCCGACCGT 2821 GGCATGTGGG GGCGGATGCG GATGACGCCC ACCGACATCT CCGACATCAA TGCGCACACC 2881 TACACCTACC TGATGAATGG CACCGCGCCG GCCGGCAACT GGACCGGGCT GTTCCGCAGC 2941 GGCGAGAAAG TACTGCTGCG CTTCATCAAC GGCGCCTCGA TGACCTACTT CGACGTGCGC 3001 ATTCCCGGCC TGAAGATGAC CGTGGTCGCC GCCGACGGCC AGTACATCCA TCCGGTCAGC 3061 ATCGACGAGT TCCGCATCGC GCCGGCCGAA ACCTACGACG TGCTGGTGGA ACCGACCGGG 3121 CAGGACGCGT TCACCATCTT CTGCCAGGAC ATGGGCCGCA CCGGTTCCCG CGCGCGACCC 3181 ACGCCCGTTG CTGACGATAG CGACATGGGG CACGACATGG GTAGTGGTGG CCATGGTGGC 3241 CACGACATGG CCGCGATGAA GGGCATGGAA GGCGGCTGCG GCGCCAGCAT GGACCACGGT 3301 GCGCACGGCG GTAGCGATGC CGCCAGCAAG GCACCGAAGC ACCCGGCCAG CGAACGCAAC 3361 AACCCGCTGG TGGACATGCA GAGCTCGGCC ACCGAACCGA AGCTGGACGA TCCCGGCATC 3421 GGCCTGCGCG ACAACGGTCG CCAGGTACTC ACCTACGGCG CGATGCGCAG CCTGTTCGAG 3481 GACCCCGATG GCCGCGAGCC GAGCCGCGAG ATCGAGCTGC ACCTGACCGG CCATATGGAG 3541 AAGTTCTCCT GGTCATTCGA TGGCATTCCG TTCGCCAGCG CCGAGCCGCT GCGGCTGAAC 3601 TACGGCGAGC GCATGCCATC TGATCTGGAG AACGCGCAGG GCGAATTCCA GCTGCGCAAG 3661 CACACCATCG ACATGCCACC CGGCACCCGC CGCAGTTACC GCGTGCGCGC CGATGCGCTC 3721 GGTCGCTGGG CCTACCACTG CCATCTGCTC TACCACATGG AAGCGGGCAT GATGCGCGAA 3781 AACAGCACCG GCCAGGCCTG GGAGGCCACC GGCTGGATCG GTGGCAACAT CAACCGCCTG 3841 TGGTTGCGCA CCGATGGCGA ACGCAGCCGC GGCCGCACGG AATCGTCGTC ACTGGAAGCA 3901 CTGTATGGTC GCAGCGTATC GCCGTGGTGG GACGTGCTGG GCGGCGTGCG CCAGGACTTC 3961 CGGCCGGCCG ACTCGCGCAC CTGGGCGGCC ATCGGCATCC AGGGCCTTGC ACCGTACAAG 4021 TTCGAGAGCT CGGCAACGCT GTACATGGGT TCCGGCGGCC AGGTGCTGGC CAAGGCCGAG 4081 GTCGAGTACG ACGTGCTGCT GACCAACCGC CTGATCCTGC AGCCGCTGCT GGAAGCCACC 4141 ATCGCAGCCA AGGATGAACC GGAGTACGGC ATTGGTCGCG GACTGAACAA GATCCGCCGC 4201 GCCACCCTTG CCGATGTCGA CGCGCTGTCG ACCATCGCCA TCACCACCTA CAACGAAACC 4261 TGGGGCGACT CGTATCCGGC GCAGGAGCTG CAGGATTTCC TGCAGGCGCA CTACAGCAGC 4321 GAACCGCAGC GCGCCGAGTT GTCCGACCCG CGCAGTGCGA TCTGGCTGCT GTTGGACGGC 4381 GACAACGTGG TCGGCTACCT GGCCGCCGGT GCCAACACCC TGCCGCATGC CGAAGCCCGC 4441 GAGGGCGACA TCGAACTGAA GCGCTTCTAC ATCCTGGCCG ACTACCAGAA CGGCGGCCAC 4501 GGCGCGCGCC TGATGGACGC GTTCATGGCC TGGCTGGACC AGCCGCAGCG CCGCACCCTG 4561 TGGGTGGGCG TCTGGGAGGA GAACTTCGGC GCGCAGCGCT TCTACGCGCG CTACGGCTGC 4621 AGCAAGGTCG GCGAGTACGA CTTCATCGTC GGGGATACGC GCGACCGCGA GTTCATCCTG 4681 CGCCGGCTGT GA // SEQ ID NO: 104 Amino Acid Sequence of OsRecQC DEFINITION OsRecQC protein 1563 amino acids ORGANISM Oryza sativa Cultivar Nipponbare MASRPAHDLLQRVFGYDDFRGPQQDIVEHVAAGHDALVLMPTGGGKSLCYQVPALLRDGC GIVISPLIALMQDQVEALRQLGVRAEYLNSTLDAETAGRVERELLAGELDMLYVAPERLL SGRFLSLLSRSQIALFAIDEAHCVSQWGHDFRPEYRQLTVLHERWPQIPRIALTATADPP TQREIAERLDLQEARHFVSSFDRPNIRYTVVQKDNARKQLTDFLRGHRGEAGIVYCMSRR KVEETAEFLCGQGVNALPYHAGLPPEVRASNQRRFLREDGIVMCATIAFGMGIDKPDVRF VAHTDLPKSMEGYYQETGRAGRDGEAAEAWLCYGLGDVVLLKQMIEQSEAGEERKQLERA KLDHLLGYCESMQCRRQVLLAGFGETYPQPCGNCDNCLTPPASWDATIPAQKALSCVYRS GQRFGVGHLIDILRGSENEKVRQQGHDKLSTYAIGRDLDARTWRSVFRQLVAASLLEVDS EGHGGLRLTDASRDVLTGRRQISMRRDPASSSSGRERSAQRTGLSVLPQDLALFNALRGL RAELAREQNVPAFVIFHDSTLRNIAERRPTSLDELARVGGIGGTKLSRYGPRLVEIVREE GLLLNGLNAAMARGHEEMGRMAHAAAAAVDGGTADCHHHAAMQADPAPQAKAPAHDAHCQ IKDCVRSCAQHPLLVVQPLPFMAGPALSLAPQPMPATGRPAPPSAADLTPSHRLIPHAPA WPPVAWLPASPLSACRSACSPPPLPPHAWPAPPPCSATPASNWPSANRWPTFTGRTRPAI TVNGSLPAPILRWREGQTVDLFVRNTLDREPTSIHWHGILLPANMDGVPGLSFNGIGPGE TYHYHFELKQSGTYWYHSHSMFQEQAGLYGALIIDPAEPAPYQHDREHVILLSDWTDMDP GALFRRMKKLAEHDNYYKRTLPDFLRDVKRDGWSAALSDRGMWGRMRMTPTDISDINAHT YTYLMNGTAPAGNWTGLFRSGEKVLLRFINGASMTYFDVRIPGLKMTVVAADGQYIHPVS IDEFRIAPAETYDVLVEPTGQDAFTIFCQDMGRTGSRARPTPVADDSDMGHDMGSGGHGG HDMAAMKGMEGGCGASMDHGAHGGSDAASKAPKHPASERNNPLVDMQSSATEPKLDDPGI GLRDNGRQVLTYGAMRSLFEDPDGREPSREIELHLTGHMEKFSWSFDGIPFASAEPLRLN YGERMPSDLENAQGEFQLRKHTIDMPPGTRRSYRVRADALGRWAYHCHLLYHMEAGMMRE NSTGQAWEATGWIGGNINRLWLRTDGERSRGRTESSSLEALYGRSVSPWWDVLGGVRQDF RPADSRTWAAIGIQGLAPYKFESSATLYMGSGGQVLAKAEVEYDVLLTNRLILQPLLEAT

IAAKDEPEYGIGRGLNKIRRATLADVDALSTIAITTYNETWGDSYPAQELQDFLQAHYSS EPQRAELSDPRSAIWLLLDGDNVVGYLAAGANTLPHAEAREGDIELKRFYILADYQNGGH GARLMDAFMAWLDQPQRRTLWVGVWEENFGAQRFYARYGCSKVGEYDFIVGDTRDREFIL RRL //

[0278] In a similar manner, orthologs of the above rice OsRecQ genes were identified in maize (SEQ ID NO:105 and SEQ ID NO:106). As will be appreciated by those of skill in the art, others of such orthologs can be identified in other monocot or dicot species using the rice OsRecQ amino acid sequence as a query. Standard molecular methods can be used to clone these sequences from other plants.

TABLE-US-00008 SEQ ID NO: 105 ZmRecQa cDNA from Zea mays LOCUS ZmRecQa 1185 bp ORGANISM Zea mays REFERENCE 1 (bases 1 to 1185) BASE COUNT 347 a 258 c 277 g 303 t ORIGIN 1 GCACGAGCGC AAGGCAAGCT TTCCGCTTCC TATTTCGGAT TGGGATCATC AGCGGCTGTA 61 GCGTGGACCC GACGGGGGTG TCCGGACCAC ATCCCTATTT CATCTTGGTA CCCCGTCCGT 121 CTCCGATTTC AGAAGCACGG CGGGCTCCCC GGCAGCCTCT ACCGAGCAGA AAGCTGAGTT 181 CTACCCCAGA ACCGAGGCAT GGAGGACGAA GAAAACATCG AGGGAGAACT GTTGCTCGTG 241 GAGTCACAAC TCCACGACAT CCAAGGACAA ATTAAAACAT TACTCGATCG CCAAGAGGAG 301 TTGTATGAAC GCCAGGCACA GTTGAAGGCT TTGCTCGAAG CATCTAAATT GACCAGAAAT 361 ACAACAATTA ACACATCTTC AGTTGCTCCG GAAGATTGGT CTGGGAGCTT CCCATGGGAT 421 CTGGAGGCTG ACGATACCAG GTTCAATATA TTTGGCATTT CCTCCTACCG ATCAAATCAA 481 CGAGAAATAA TTAATGCAGT CATGAGTGGA AGAGATGTTC TGGTCATAAT GGCAGCTGGT 541 GGAGGGAAGA GTCTATGTTA CCAGCTCCCA GCTGTACTTC GTGATGGAAT TGCACTGGTT 601 GTCAGTCCTT TACTTTCCCT TATTCAGGAC CAGGTCATGG GACTGTCAGC TTTAGGTATA 661 CCAGCATACA TGCTAACTTC AACTACCAAC AAGGAAGTTG AGAAGTTCAT CTATAAGACA 721 CTTGATAAAG GAGAAGGAGA ACTAAAGATA TTATATGTGA CACCTGAAAA GATCTCAAAA 781 AGTAAAAGGT TCATGTCTAA GCTCGAGAAA TGCCATCATG CCGGTCGTCT TTCTCTGATT 841 GCAATAGATG AGGCTCACTG CTGTAGCCAA TGGGGTCATG ATTTTCGTCC TGACTACAAG 901 AATCTTGGCA TTTTGAAAAT TCAATTTCCC AGTGTTCCAA TGATAGCTTT AACTGCAACT 961 GCAACAAGTA AGGTCCAAAT GGATTTAATG GAGATGCTCC ACATCCCGAG ATGCATCAAG 1021 TTTGTCAGCA CAGTTAACAG GCCCAACCTT TTTTATAAGG TGTCTGAGAA ATCGCCAGTT 1081 GGAAAGGTTG TCATTGATGA GATCACAAAG TTTATAAGTG AATCATACCC AAATAATGAG 1141 TCTGGAATTA TATACTGCTT TTCAAGGAAG GAATGTGAAC AGGTT // SEQ ID NO: 106 ZmRecQb cDNA from Zea mays LOCUS ZmRecQb 870 bp ORGANISM Zea mays REFERENCE 1 (bases 1 to 870) BASE COUNT 239 a 200 c 242 g 189 t ORIGIN 1 CTTGAGGATC CCCAACGCTG TGGTACTGAA GAGGAGCTTC GACAGACTGA ACCTCAACTA 61 CGAGGTAATC GGCAAGACGA AAACTTTCCA GAAGCAGCTG GGCGATCTCC TGAAAGAGCG 121 CTTCATGAAC GAATCTGGTA TCGTGTACTG TCTCTCGAAG AACGAGTGTG CAGACACTGC 181 CAAGTTTTTG AGGAAGAAAT ACAAGATCAA ATGCGCGCAC TACCACGCTA GCCTGGCAGC 241 TCGTCAGCGA ACCAGTGTCC AGGAGAAATG GCACAACGGG GAGGTTAAGG TCATCTGCGC 301 TACCATAGCC TTCGGCATGG GGATCGACAA ACCTGACGTG CGTTTTGTTA TCCACAACAC 361 ATTGTCCAAG TCAATAGAAA GCTACTACCA GGAGTCCGGG AGGGCAGGGC GAGATGAGCT 421 TCCGGCACAC TGTATCGTCT TGTACCAGAA GAAAGACTTC AGCCGTATCG TGTGCATGTT 481 GAGGAACGGT GAGAACTTCA GGAGCGAGAG CTTCAGGGTT GCGATGGAGC AAGCTAAGAA 541 GATGCAGGCA TACTGCGAGC TCAAGACCGA GTGCCGGAGA CAGGCACTTC TGCAGCACTT 601 CGGCGAACAG TACGACAGGC GAAGGTGCCG AGACGGGCCT AGCCCCTGCG ACAACTGCCT 661 CAAGACATAG TTTAGGGTAA TAAACTATGG CGATAAAAAA TGCCATGACG CTTGGTTATG 721 CTCTGAACTT GTGAGGTGTG TGCCACTTCC ACAGTACATT CGTCTGTGTA TATGTAGCAT 781 CCATAGCTCA AACAAGTGGC CGCAACTGCA CTGTGTGTAA CGATGGTCTT TGTTTTCAGT 841 TGGATTGTGA GGTTCGGGGC TTTAAAAAAA //

[0279] 2. Suppression or Down-Regulation of the OsRecQ Gene Expression to Enhance the Efficiency of Targeted Integration through Homologous Recombination: Antisense Suppression, Sense Co-Suppression, dsRNAi, Gene Knockout, and the Use of Dominant Negative Mutants.

[0280] E. coli and yeast cells deficient in RecQ show an elevated level of homologous recombination activity (Nakayama et al. 1985 Mol. Gen. Genet. 200, 266-271; Watt et al. 1995 Genetics 144, 935-945). The above rice and maize RecQ sequence homologs can be used to down-regulate RecQ expression levels and thereby enhance targeting frequency in the previously described target maize and rice lines. Similarly, RecQ homologs from other plants can be used to enhance the frequency and efficiency in those plants of targeted integration through homologous recombination. (See, Bagherieh-Najjar, de Vries, Hille, and Dijkwel, "Increased Homologous Recombination and Altered DNA Damage Response in the Arabidopsis recQ14A Mutant," attached hereto and forming a part hereof).

[0281] Down-regulation can be achieved ectopically by a transgene using methods in the art, including homology dependent gene silencing (antisense suppression, sense suppression, dsRNAi, virus mediated silencing) and dominant-negative mutants of the gene. For homology-dependent silencing, only part of the gene is needed to initiate silencing of the gene. For example, a segment of sense and/or antisense OsRecQ mRNA sequence can be placed under the control of a constitutive or tissue-specific promoter to initiate gene silencing of native genomic OsRecQ genes. Dominant negative mutants are defective variants of a protein, usually deficient in one or more functions that the protein normally has. For example, RecQ has a helicase domain and also interacts with other proteins to carry out its normal biological functions. A dominant negative mutant RecQ may lose its helicase activity but still retain its interactions with other proteins. Sometimes a dominant negative mutant is a truncated protein.

[0282] A particular RecQ gene can also be knocked out totally, and plant lines with a RecQ gene knock-out can be used in gene targeting. In plants, mutagenesis methods such as transposon, T-DNA insertion, UV, gamma rays, X-rays, and chemicals can be used to inactivate these genes. The materials with reduced RecQ expression obtained by the above methods are then used as target tissue when the targeting methods disclosed herein are carried out. For example, rice transgenic target lines with a pNOV5025 or pAdF55 T-DNA insertion locus can be introgressed into lines with the OsRecQ dsRNAi knockout locus, and the resulting lines containing both loci can be re-transformed with the targeting donor vector pQD200C6 or pAdF77, with or without another recombination enhancing vector (e.g., pNOV5033, which expresses the I-CeuI endonuclease to make a dsDNA break at the target locus). Similarly, maize transgenic target lines with a pNOV5025 T-DNA insertion locus can be introgressed into lines with the ZmRecQ dsRNAi knockout locus, and the resulting lines containing both loci can be re-transformed with the targeting donor vector pQD200C6, with or without the recombination enhancing vector pNOV5033, which expresses the I-CeuI endonuclease to make a dsDNA break at the target locus. Down-regulation of the RecQ gene can also be carried out transiently by introducing the interfering protein or RNA or RNA expression cassette during the targeting process, such as, for example, during the Agrobacterium-mediated delivery and transformation of the donor T-DNA into the host cell.

Example 35

Over-Expression or Up-Regulation of OsRad54, OsBRCA1, OsBRCA2, and OsSPO11 to Enhance the Efficiency of Targeted Integration Through Homologous Recombination

[0283] Some genes encode proteins that are involved in the recombination machinery of the cell or that are positive regulators of the recombination process. To clone some of these genes, proprietary Syngenta rice genome (Myriad contigs V8, Nipponbare cultivar) and public rice genome sequence databases were searched with the TBLASTN program using the protein sequences of human BRCA1, BRCA2, RAD54, and yeast SPO11. Primers were designed to amplify predicted cDNAs encoding homologs of these sequences. The following cDNAs were cloned from young rice flowers or mitomycin C-treated callus tissue: OsRad54A (SEQ ID NO:107), OsRad54B (SEQ ID NO:108), OsBRCA1 (SEQ ID NO:109), OsBRCA2 (SEQ ID NO:110), OsSPO11A (SEQ ID NO:111), and OsSPO11B (SEQ ID NO:112). These cDNA sequences are useful for increasing targeting efficiency, since over-expression of these genes can increase the frequence of homologous recombination in plant cells.

[0284] Each of these genes can be put under the control of a regulated promoter, such as a tissue-specific or inducible promoter, for example, so that their expression is tissue-specific or transient. In one embodiment, the recombination enhancing genes is expressed when the donor sequence is delivered to the target cell. In another embodiment, several of the above recombination enhancing genes are co-expressed in the host cell to increase the targeting efficiency. The proteins encoded by these genes (the predicted amino acid sequences of which are shown in SEQ ID NOs:113, 114, 115, 116, 117, and 118) can be introduced into the host cell by any means described herein (such as the methods described above with reference to a mega-endonuclease or a recombinase) or methods that are otherwise known in the art. Such other methods include, for example, introducing the protein (or a fusion protein containing the protein) into the cell through physical or biological means, e.g., electroporation or Agrobacterium. For example, rice target lines with a pNOV5025 or pAdF55 T-DNA insertion locus are introgressed with a line having a transgenic locus for the over-expression of these genes, and the resulting lines containing both loci are re-transformed with a donor vector, such as pQD200C6 or pAdF77, with or without another recombination enhancing vector pNOV5033 which expresses the I-CeuI endonucleases to make a dsDNA break at the target locus. Similarly, maize target lines with a pNOV5025 T-DNA insertion locus can be introgressed into a line with a transgenic locus for the over-expression of these genes, and the resulting lines containing both loci are re-transformed with a donor vector, such as pQD200C6, with or without the recombination enhancing vector pNOV5033 which expresses the I-CeuI endonucleases to make a dsDNA break at the target locus.

TABLE-US-00009 SEQ ID NO: 107: OsRad54A cDNA from Oryza sativ, (cultivar Kaybonnet) LOCUS OsRad54A cDNA 3569 bp ORGANISM Rice, Oryza sativa cv Kaybonnet SOURCE Young flower REFERENCE 1 (bases 1 to 3569) AUTHORS Qiudeng Que CDS 1 . . . 3564 BASE COUNT 1072 a 771 c 865 g 861 t ORIGIN 1 ATGGAGGACG ATGACGATGA CCAACGCTTG CTTCACAGCC TTGGTGTCAC GTCCGCAGAC 61 ATCCACGATA TTGAAAGGAG AATCATATCA CAGGCAACAA CTGATCCTGC CGACTCATCT 121 GGACCAACCA TCAATGGAGG TCATCAGCCT GATGATGCTC TCGCCAAACT GCATCACAAA 181 CTGCGCTCTG TGCAAATTGA AATTGATGCT GTAGCCTCCA CCATCAAAGG AGCTAAGCTT 241 AAGCAACCAT CCGGAAATAA ACCACATGAG CATAAAGGCA AGGACCAGCC AGATCATCAT 301 GGAGCAGGAC ACCTCCAGCA AGCCCTTGCT GCCGACCGTC TTACAAGCCT CAGGAAAGCT 361 AAAGCACAGA TACAGAAAGA GATACTACAG TCACATCTTT CTCCATCTGC CTCCAATCGA 421 AAAGATAAAA TGCTGGCCAT GCTGGTCCAA GACGAGCCGA GGCACAAAAA GCCACCCGTA 481 GGGCCTAAAA ACATCGTGAA ACGCCCGATG AAAACTGTCA CCTATGATGA TGACAACAAC 541 TTCGATGCAG TGCTTGATGG AGCCTCTGCG GGATTTATGG AAACTGAAAG GGAAGAACTG 601 ATCAGGAAGG GTTTGTTGAC ACCATTCCAT AAGTTGAAGG GCTTCGAGAA ACGTGTGGAA 661 CTACCCGAAC CTTCTCATAG ACAAGATGAT TCTGCAGGAC AAACTGAAGA AGCCATGGAA 721 GCTTCCAGGA TTGCTAGAGT TGCTCAGTCG CTAAAGCAGA TTGCACAGAA CCGCCCAGCA 781 ACCAAATTGC TTGATTCAGA GTCTTTACCT AAGCTAGATG CACCTGCTGC CCCATTTCAG 841 AGACTTGGAA AACCCCTAAA GCGTCCTGTC TCTCCCAGTT CAGATGAGCA GGAAAAGAAG 901 AGACCAAGAA ATAAGACCAA AAGACCACTG CCTGGCAAGA AATGGAGGAA AGCAAACTCA 961 ATTAAGGAAT CATCATTGGA TGACAACGAT GTTGGAGAGG CAGCTGTGTC AGTTTCAGAT 1021 GATGATGAAG ATCAGGTTAC AGAAGGCTCT GATGAGTTAA CTGATGTTAC CCTTGAAGGA 1081 GGTTTGAGAA TTCCTGGCAC ACTTTACACG CAACTATTTG ACTACCAGAA AGTGGGAGTG 1141 CAGTGGCTAT GGGAGTTGCA TTGTCAAAGG GCTGGTGGAA TAATTGGAGA TGAAATGGGC 1201 CTGGGAAAGA CTGTGCAGGT CTTGTCATTT CTTGGTTCCT TGCATAACAG TGGGCTCTAC 1261 AAGCCTAGCA TTGTTGTTTG TCCTGTAACC CTTTTGCAAC AGTGGCGAAG GGAGGCCAGT 1321 AGATGGTATC CAAAGTTCAA GGTTGAGATC TTACATGACT CTGCAAACAG TTCATCTAAA 1381 AAGAGCAAGA GGTCTAGTGA TTCTGACAGT GAAGCTTCCT GGGATAGTGA TCAGGAAGAA 1441 GCGGTTACAT GTTCAAAACC CGCAAAGAAG TGGGATGACT TGATTTCACG TGTTGTGAGT 1501 TCAGGATCAG GTTTGCTTCT GACCACATAT GAGCAGTTAA GGATCCTAGG GGAGAAGTTG 1561 CTTGATATAG AATGGGGATA TGCTGTATTG GATGAGGGTC ACCGCATTAG GAATCCTAAT 1621 GCTGAGATTA CTCTTGTGTG CAAGCAATTG CAGACCGTGC ACAGGATAAT TATGACAGGT 1681 GCACCTATTC AAAACAAACT TTCGGAGCTT TGGTCTCTCT TTGATTTTGT GTTCCCTGGA 1741 AAACTAGGTG TCCTGCCTGT GTTTGAGGCT GAGTTTTCTG TTCCAATTAC TGTTGGTGGG 1801 TACGCTAATG CAACACCATT GCAAGTGTCC ACGGCGTATC GATGTGCTGT TGTCCTACGT 1861 GACCTGGTCA TGCCGTACCT TCTTAGAAGA ATGAAAGCTG ATGTCAATGC ACAGCTTCCC 1921 AAGAAAACAG AGCATGTTCT TTTCTGTAGT CTAACTACTG AGCAACGTGC TACTTATCGT 1981 GCATTTCTTG CTAGTTCGGA GGTGGAACAA ATCTTTGATG GTAACAGAAA TTCCCTTTAT 2041 GGGATAGATG TTCTAAGGAA GATATGCAAT CATCCTGATC TACTTGAGAG AGAACATGCT 2101 GCTCAGAATC CTGACTATGG GAATCCAGAA AGAAGTGGAA AGATGAAAGT GGTTGAGCAA 2161 GTTCTTAAAG TATGGAAAGA ACAAGGTCAT CGTGTTCTTC TTTTCACTCA GACACAACAA 2221 ATGCTTGACA TTATGGGGAA CTTCTTGACA GCTTGCGAAT ACCAATACCG AAGAATGGAT 2281 GGACTTACAC CTGCAAAGCA AAGAATGGCA CTTATTGATG AATTCAATAA CACAGATGAA 2341 ATTTTTATTT TCATTCTGAC CACGAAAGTT GGTGGACTGG GTACGAATTT GACTGGTGCA 2401 AACCGGATTA TTATATATGA TCCTGACTGG AATCCTTCAA CTGACATGCA GGCTAGGGAA 2461 CGTGCATGGC GAATTGGGCA AACTAGAGAT GTGACAGTTT ATAGACTGAT CACGCGTGGG 2521 ACAATAGAGG AGAAAGTCTA CCATCGTCAG GTATACAAGC ATTTCCTCAC AAACAAAGTA 2581 CTGAAAGACC CTCAGCAGAG GCGGTTTTTT AAAGCCAGAG ACATGAAGGA TTTGTTTACG 2641 CTGCAAGATG ATGACAATAA TGGCTCAACT GAAACATCAA ATATTTTCAG CCAATTGTCT 2701 GAGGATGTGA ATATCGGAGT TCCGAGTGAC AAGCAACAAG ACCAGCTATA TGCAGCCTCT 2761 GCTACACCGA CAACCTCTGG GACTGAACCG AGCTCATCCA GGCATGGACA GGGTAAAGAA 2821 GACCATTGCC CTGACCAAGC AGATGAAGAA TGCAACATTT TGAAGAGCCT TTTTGATGCT 2881 CAAGGCATTC ATAGTGCGAT CAATCATGAT GCCATAATGA ACGCTAATGA TGACCAGAAG 2941 CTGCGCCTAG AAGCAGAAGC TACACAGGTG GCACAAAGGG CAGCTGAAGC TTTACGCCAA 3001 TCACGGATGC TCAGAAGTCA TGAAAGTTTT TCTGTTCCTA CATGGACTGG AAGAGCTGGT 3061 GCTGCGGGGG CACCATCCTC TGTCCGCAGG AAGTTTGGGT CAACACTCAA TACCCAGTTG 3121 GTTAATTCTT CTCAGCCATC AGAAACTTCA AATGGCAGGG GCCAAAGTCT TCAGGTGGGT 3181 GCTCTAAATG GCAAAGCACT GTCCTCCGCT GAGCTTCTGG CCAGGATACG TGGAACCCGA 3241 GAGGGAGCAG CTTCAGATGC ACTAGAACAT CAACTCAACC TGGGATCAGC TTCCAATCAC 3301 ACATCGAGTT CATCAGGGAA TGGCCGTGCA TCAAGCTCTT CTACTAGGAG CATGATCGTA 3361 CAGCCTGAAG TCCTAATCCG CCAATTGTGC ACCTTCATAC AGCAGCATGG TGGTTCCGCC 3421 AGCTCAACAA GTATAACTGA ACACTTCAAG AACCGGATAC TGTCCAAGGA TATGCTGCTG 3481 TTTAAGAATC TGCTGAAGGA AATAGCTACG TTGCAAAGAG GTGCAAATGG TGCAACGTGG 3541 GTGCTGAAAC CTGACTACCA GTAACTAGT // SEQ ID NO: 108: OsRad54B cDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsRad54B cDNA 3453 ORGANISM Oryza sativa cv Kaybonnet SOURCE Young flower REFERENCE 1 (bases 1 to 3453) AUTHORS Qiudeng Que CDS 1 . . . 3447 BASE COUNT 1134 a 655 c 776 g 888 t ORIGIN 1 ATGCGCACAA GCACCACATC AGATAGCCCA TCCCCATCTC CACAAAACAA AGCCTCTTTT 61 AACACATCAC GTGGTGCTGC ATTTAGGGAT GAAGAACCAG GTGCAAAAGA CAATGAAGTT 121 GAGAAAAGGA AACCATTGAT ATTACATTTG AAGAAGCGTT CAACCAAGGA ACTATCTACA 181 GATACCACAT CATCAAAGTC AGGGTTACTT GGAAAGTCTT CAGAAGAGAA ACAGGAGAAA 241 CACGGAAGTG CTTTGAAAGT GAAGAAACAT CTGCATCCCA TGGAATTATC TCCAAAGAAA 301 TATAAGAACA AGAAGCAACA CAATCACAGA GACAGTAAGA GATCCGAAGC AAAAAAGGTC 361 CAATATTTGG CATCAGATGT GGACAGTGAT TCTTCAATGG AACCATCTAC TTCTCTTGAG 421 CACAGCGAAT CGCCGCCCCC AAAAAGAAAA TCGTTGGATG GAAGAACACC TGCATCAAGT 481 ACCAAGAAAG GAAAAAAGAA AGTGAAATTT ATTGATAAAA AGCACCCTGA GAATGCTGTT 541 CATATAACTG AAAAGGAGCA TGGTGGTGCA GGAGACAAAA TAACAACTCA GGGGGATCTG 601 CAGGTTGATC GCATCCTAGG CTGTCGACTT CAGACAAGCC AAATCATTTC ACCTGCCCAT 661 GCTTCATCAG AGCAGATTGA TATGGCCCCT CCTAGTGCAT CCGGTGCAAC AGAACCTAGT 721 CAAGCCCTTT CAAAAGGACT TCATGAAGAA ATTCAGTCTT CTAATAGTGA TACTAATGTG 781 ACAGAGGATG CATGTGCTGA TGAATTAGCA AACGATGGTG GGGAAAATAA TTTGGATTGT 841 TCTGATGCTC AAAAGGAGAG TAATGTTAGA TCCCATGGAC ACAAGGAATC ACTTAACGCA 901 AAAGAAATCA TGAATACAGC ATCAGCATGT TCCGCTGATC AAATTGTCAC AGTTAAGGAT 961 GCTGGAGCAG TACAGACATA TGTAACGGCT TCAGTAAATG GTGAATATGA GACAGTAACT 1021 GATATTCCAG AAGAAAAGAA TGACACCAAA CATCCAGTTT CCAAAGCTGA CACAGAAGTC 1081 CACACTAAAC AAGAACATAC ACCTGATAGT AAATTGCATG GGAAACTAGA AAACTACAAA 1141 GCAAAGTACG GAACAGGTTT GATAAACATC TGCAAAGAAC AATGGTGCCA ACCGCAACGA 1201 GTTATTGCTC TGCGCACTTC TTTAGATGAA ATAGAAGAGG CTTTGATCAA ATGGTGTGCC 1261 CTTCCATATG ACGAATGCAC GTGGGAAAGA TTAGATGAAC CTACAATGGT GAAGTATGCA 1321 CATTTGGTCA CTCAGTTCAA AAAATTTGAA TCCCAGGCTT TGGATAAGGA TAAGGGAGGT 1381 AGCCATGCAA AGCCAAGGGA ACACCAAGAG TTTAATATGC TGGTTGAGCA GCCAAAAGAA 1441 CTCCAGGGAG GCATGCTCTT CCCTCATCAA CTGGAAGCAT TGAACTGGCT ACGCAAATGC 1501 TGGTACAAGT CAAAAAATGT TATCCTTGCT GATGAGATGG GTCTTGGAAA GACTGTGTCT 1561 GCCTGTGCTT TTCTATCATC CCTATGTTGT GAATATAAGA TTAACTTGCC ATGTCTTGTC 1621 TTGGTTCCTC TTTCTACTAT GCCCAACTGG ATGGCTGAAT TTGCATCATG GGCACCTCAT 1681 TTAAATGTTG TGGAGTATCA TGGTTCTGCA CGGGCAAGAT CTATTATTCG TCAATATGAG 1741 TGGCATGAGG GTGATGCAAG CCAGATGGGT AAAATCAAGA AATCTCATAA GTTCAATGTA 1801 TTGCTCACTA CTTATGAAAT GGTGCTTGTT GATGCTGCAT ATCTTCGGTC TGTGTCATGG 1861 GAGGTTCTTA TAGTCGATGA GGGTCATCGT CTGAAGAATT CTAGCAGCAA ACTTTTCAGT 1921 TTACTCAATA CATTATCATT TCAGCATAGA GTTTTGCTGA CTGGAACTCC GTTACAGAAT 1981 AACATTGGTG AAATGTATAA CTTATTGAAC TTCTTACAAC CTGCTTCTTT CCCTTCTCTA 2041 GCTTCATTTG AGGAGAAATT CAATGACCTT ACAACAACAG AGAAAGTGGA GGAGCTGAAG 2101 AACCTTGTAG CTCCACATAT GCTTCGAAGA CTGAAAAAGG ATGCAATGCA AAATATCCCT 2161 CCAAAGACTG AACGAATGGT GCCTGTTGAA TTGACATCAA TCCAGGCTGA ATACTACCGT 2221 GCTATGCTTA CAAAGAACTA CCAAGTATTG CGCAATATTG GGAAAGGTGG TGCTCACCAG 2281 TCATTGTTGA ACATAGTAAT GCAACTTCGG AAAGTCTGCA ATCATCCGTA TCTTATTCCT 2341 GGAACTGAAC CTGAATCAGG ATCACCAGAG TTCTTGCATG AAATGCGAAT AAAGGCCTCA 2401 GCAAAGTTAA CTTTGTTGCA CTCTATGCTT AAAATCCTAC ACAAGGATGG TCATCGAGTT 2461 CTTATTTTTT CTCAGATGAC AAAGCTTCTT GACATCCTTG AAGATTACCT GACCTGGGAG 2521 TTTGGTCCGA AAACATTTGA AAGAGTGGAT GGTTCAGTAT CTGTGGCAGA ACGCCAGGCA 2581 GCAATTGCTC GTTTTAATCA GGACAAGAGT CGTTTTGTAT TCCTGCTATC TACGCGGTCA 2641 TGTGGGCTTG GAATTAATTT GGCAACTGCA GATACTGTTA TCATATATGA TTCTGATTTC 2701 AATCCACATG CTGATATACA GGCAATGAAC AGAGCACACA GAATTGGACA GTCAAACAGA 2761 CTTTTAGTTT ACAGGCTTGT CGTGCGTGCT AGTGTTGAGG AGCGTATCTT GCACCTTGCG

2821 AAGAAAAAAT TGATGCTTGA TCAACTTTTT GTTAACAAAT CAGAATCACA GAAGGAAGTG 2881 GAAGATATCA TTCGCTGGGG AACAGAGGAA CTCTTCAGGA ATAGCGATGT TGCAGTTAAA 2941 GATAATAATG AAGCTTCTGG TGCTAAAAAT GATGTAGCAG AGGTTGAGTT TAAGCATAAA 3001 AGAAAAACTG GTGGACTAGG CGATGTTTAT GAAGACAGAT GTGCTGATGG TTCTGCTAAA 3061 TTTAATTGGG ATGAAAATGC TATCACAAAG CTTCTTGACA GATCCAACGT TCCATCAACA 3121 GTAGCTGAAA GCACTGATGG GGACTTGGAC AATGATATGC TTGGCACTGT AAAGTCAATA 3181 GATTGGAACG ATGAGCTGAA TGATGACCCT GGTGCCACCG AGGACATCCC AAATATTGAT 3241 AATGATGGTT GCGAGCAGGC ATCTGAAGCA AAGCAGGATG CAGCTAATCG TGTTGAAGAA 3301 AATGAATGGG ATAAACTCTT ACGTGTCAGA TGGGAGCAGT ATCAAACTGA GGAGGAAGCA 3361 TCTCTTGGTC GAGGTAAGCG TTTAAGGAAG GCTGTTTCTT ACAGGGAAAC ATTTGCAACC 3421 ATTCCTAATG AAGCTTTAAG CGAGTAGAAC TAG // SEQ ID NO: 109: OsBRCA1 cDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsBRCA1 cDNA 2964 bp ORGANISM Oryza sativa cv Kaybonnet SOURCE Young flower AUTHORS Qiudeng Que CDS 1 . . . 2964 BASE COUNT 957 a 623 c 694 g 690 t ORIGIN 1 ATGGCGGACA CGGGGAGCCT GGAGAAGATG GGGCGAGAGC TCAAGTGCCC CATCTGCCTG 61 AGCCTTCTCA GTTCGGCGGT ATCCATCTCC TGCAACCACG TCTTCTGCAA TGATTGCCTC 121 ACGGAATCGA TGAAATCCAC GTCGAGCTGC CCCGTGTGCA AGGTCCCGTT CCGACGACGA 181 GAAATGCGAC CAGCACCTCA CATGGACAAT CTGGTCAGCA TTTTCAAAAG CATGGAGGCT 241 GCAGCAGGTA CCAATGTTGT CTCAACACAG GAGGCTCCTG TGGTAAAACT TGCAGATGGA 301 TCAGATTGTG TCAACAGCGG GAAAAATTCC AAAAGGTCAC AAAAATCATT GACACGAAAA 361 AGGAAGGTAA CATCCGAGAT GGAAAAAAAT ACAGCAAAGG ATGCTACAGC TTCTGCATCC 421 CAACCTACTA CAAAGCCTTC CTTCTCTACT AACAAAAGAA TACAAGTGAA ACCATTCCCT 481 GAATCTGAGA CACCAATAAG AGCTGAGAAG ATTATGAAGC CTGAAGAGCC AAAAAATAAT 541 CTGAATAATG ATGTTGAAGG AAAGAATAAA GCAGTGGCAT CGGGTCAACC TGGAAGTCCT 601 TCATTGTCAC CCTTTTTTTG GCTAAGGGAA CAAGAAGAAC AAGAAGGCTG TACCGCTGAG 661 ACGTTAAGTG AAACGCAATC TTTAGACACA CCCTTGCGTC ATAATGCACC CTCTTTTAGC 721 GATATTAAAG ATTCTGATGA CGAAATCCCT TTAAATACAA CTCCAAATAG CAAAGCTGCG 781 GCTACAGAAC TCTTTGACAG TGAAATATTT GAATGGACCC AGAGACCATG CTCTCCTGAA 841 TTGTATTCCA CTCCATTGAA AAAGCAGAGT AAAGCTAAGA GTAAACTAGA TCAAATTGAA 901 GAGAAGGGTG ATGAAGAAGA TGTGCATATT GGTGGTTCAT TTGATAAGCT GGGCAGTGCA 961 AGTAATGCAG CTCAGCTTGT CAATACAAAA GCAACAAAGC AGAAGAGAAA GAAAACAAGT 1021 CCCAGTAACA AAAACAGTGC AAAATTGTCC AATCGTGCTG AGCCCTGCAT AAAAAAGTCT 1081 GATGCCAATC AACAAGGTTC AAATAGACGT AAAAGTGCTG CCCTAAAATC TTGTCAGAAA 1141 AGCAGCAGTG CTGTAGGGAG GAATACTTCA GGTAGAAGAA ACAAGGCCTC TAGCAACAGC 1201 AAGCCAATTC ATGGCTCTAG TGATAACTCC CCAGAGTCAT ATCTTCCTAA GGAGGGTTTG 1261 GATGTTGAAG CACCTGACAA ACCCCTTTCT GAAAGGATCC AAAACTTGGA GAAAACTAGT 1321 CGACGAAAGG GAAGTGCAAG GAAGCTGGAA ATGGCAGGGA AAACTATTTC AGATACTACA 1381 GAGAAGAATA GTGAGCCAAG AAGTAAGAGA GTCAGAAGAA TGTCTGACCA CGCTATAGCT 1441 AAACCGGTTG AAGTTCCTTC AGGATCTGGA AATGAAACAG AAATACCACA GCTTCACACC 1501 CTCACAAAAG GCAGCATTCA ACGCAAATCC TCCAACGCTA GAAGACATAG CAAAGTTTGT 1561 GGAGAACAGG AAGGTAAGAA TAAACTTGAG AACACGACAA TGACACCTAT TATTTTACAT 1621 GGGAAATGCC AAAATAAAGA GGCAGTATGT ACAGCTCCTT CAGTAAGGAC TGCATCTGTT 1681 AAGTACAAGC AAGCAAAATT TAGCGAACAA CCAGATTGTT TTGGAACGGA GAACTTTGGA 1741 AACCTTCAAG CATGCCCTGC ACGTAATGTT TTACTGAAGA AGTGTGAGGT ATCTACTTTG 1801 AAGGTTTCCT GTGCTTTCTG CCAGACCGAT GTCATCACAG AGGAGTCTGG AGAGATGGTT 1861 CATTATCAAA ATGGGAAGCA AGTCCCTGCA GAGTTCAATG GAGGAGCCAA TGTGGTGCAC 1921 TCTCACAAGA ACTGCCTTGA GTGGGCTCCT GATGTCTACT TCGAAGATGA TTCTGCCTTT 1981 AATCTTACAA CTGAATTGGC GAGAAGCAGA CGGATCAAAT GTGCTTGCTG TGGAATTAAA 2041 GGAGCTGCAC TTGGATGCTT TGAGATGAGT TGTCGGAGAA GTTTCCACTT CACCTGTGCT 2101 AAACTAATCC CAGAATGCAG ATGGGATAAT GAAAATTTTG TGATGTTATG CCCTCTACAT 2161 CGGTCTACAA AGTTACCCAA TGAAAATTCT GAACAGCAAA AGCAACCTAA AAGGAAAACA 2221 ACACTCAAAG GGTCATCTCA AATAGGATCC AATCAAGATT GTGGTAATAA CTGGAAATGG 2281 CCATCTGGAT CACCACAGAA GTGGGTTCTC TGCTGCTCAT CACTTTCTAG TTCTGAGAAG 2341 GGACTTGTAT CAGAATTTGC AAAGTTAGCT GGCGTGCCTA TTTCGGCAAC TTGGAGTCCA 2401 AATGTTACCC ATGTTATTGC ATCAACTGAT CTCTCTGGTG CTTGCAAACG GACGCTGAAG 2461 TTTCTCATGG CAATCTTGAA TGGCAGATGG ATTGTCTCCA TAGATTGGGT TAAAACTTGC 2521 ATGGAGTGCA TGGAACCAAT TGATGAGCAC AAATTTGAAG TCGCTACTGA TGTTCATGGG 2581 ATCACTGATG GTCCTAGGTT AGGAAGATGC AGGGTTATTG ACAGGCAACC TAAGCTGTTC 2641 GACAGCATGA GGTTCTACCT CCATGGGGAC TACACAAAAT CCTACAGAGG CTACCTGCAA 2701 GATCTCGTGG TTGCAGCAGG TGGAATAGTT CTTCAGAGGA AGCCCGTATC AAGAGACCAG 2761 CAAAAGCTTC TTGATGACAG CTCTGACCTC CTCATCGTTT ACAGCTTCGA GAATCAAGAT 2821 AGGGCAAAAT CCAAGGCCGA AACCAAGGCT GCTGATCGCA GGCAGGCTGA TGCTCAGGCT 2881 CTTGCTTGCG CTTCTGGAGG CAGAGTTGTG AGCAGTGCAT GGGTGATTGA CTCAATTGCA 2941 GCCTGCAATC TGCAACCTCT TTGA // SEQ ID NO: 110: OsBRCA2 cDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsBRCA2 cDNA 4500 bp ORGANISM Oryza sativa cv Kaybonnet SOURCE Oryza sativa cv. Kaybonnet Mitomycin-C treated calli REFERENCE 1 (bases 1 to 4500) BASE COUNT 1379 a 856 c 1102 g 1163 t ORIGIN 1 ATGGCTGACC TCTTCAACCA AGCTTTGGAT AAGCTGGTTG CTGCTGATGG AATGGCCGAA 61 GCGATCGAGG ATTCAGGGAA AGGTGCGGTG TTCTGCACTG GGTTGGGGGG ATCAGTTGCC 121 GTCAGCGAGA GGGCTGTAGA GAGGGCCAAG GCATTGGTTG GGGAGGTCGC GGAGGAGATA 181 AGTAATGAGA GGAGGCAACC ATTTGGTGAT GGTTCTAATT TGGAGTGCGG ATTGGGAGAA 241 AGTAATGTTT CATTTAAAGG TGGTGTACAT AAAGATAGTT TGTCTCCGAT GTTCCAAACC 301 GGATCGGGTA AAATGGTTTC GCTGAGCAAG GGCTCAATTC AGAAGGCTAG AGCTGTTTTA 361 GAAGGAAATG CCGAGAATTC TTCTGTCATT GCTGTACAGT CTATGTTCCA TACTGGATTG 421 GTTAGGCCAG ACCCAGTCAG CAGGAGCTCC ACTGATAATG CAATGACTGT TTTGGAGGGA 481 CAAACAAATC CAAAACAAGG AGATGTGGCA GATGTGTATG ACAAGGAAAA TTTTCCATTG 541 TTCCAAACTG GTTCAGGTAA AGCTGTATCG GTCAGTGTAG CATCTATCCA GAAAGCTAAG 601 GCTGTCCTGG AGCAAAATAA TACAGAAAAC ACGGAAGATT TTGGTAGGCC TGACCAATCT 661 CTGATTTTCC AAACTGGTTC GCGAAGACCA GTCTTGATCA GTGAAAGATC TAGCTCTGTG 721 GTGAAGGATG GAGGTGCTGA AAATATTGTG TTCCAAACGG GGTTAGGGAG GCCTGTTGTG 781 GTGAGCCAGA CCTCAATTCA AAAGGCAAGG ACAGTATTAG ATCAAGAATG TGCCAAAAGA 841 AGTGGACATG GAGATACTAA TGTCTCCACC ACTACTTTTC AAACTGAAAC ACCAACGCCT 901 GTTCTGATGA GTGGTGGCCT GACTATGAAT GATAGATCTG TTACACCTGA GGGGGGTGTT 961 TCAATGCAAG GAAATTTTTT GGAGGCTGAT GGTCACTTGC CATTATTTCA AACTGGGTTA 1021 GGGAGGTCCA TTTCAGTAAG TAAAGGCTCA ATTAAGAGAG CAAGTGCACT TCTGGAGCCA 1081 AGGAACATTA CAAAAGAACT GGAAGATGAA GCTCACTCAG ATGATGGCTG TGCCACTCCA 1141 ATGTTCAAAA CTGGATCAGG AAGGTCTATC ACAGCAAGTG AAAATTCTAG AAAGAAAGCC 1201 CACGTTGTCT TAGAGGGCGA GGAACCAGTA AAAAATGTAA ATAATGACAC TGGAGAAGCC 1261 ATTGCTCCAA TGCTCCATGC TGGAATGCAG AAGTTTGCAC CCCAAAATAG AAACTCAAGT 1321 CATAAGGCGA TCACCCTCAT GGAGCAAGGG AGCTCTATGG AAGAAGACCG TGGAAACGAA 1381 CCACCAATGT TTCGAACTGG ATCTGGGAAG TCAGTCTTGA TTAGTCACAG CTCCGTGCAG 1441 AAGGCAAGGG CGGTTCTGGA GGAAGAAGGC AATATGAAGA AAGAAAATCA CAAACAACTT 1501 AGCAATGTGG ACAAATATAT TCCGATCTTT ACTTCACCTC TCAAGACAAG CTATGCAAGG 1561 ACTGTACATA TATCTTCAGT TGGTGTTTCT CGAGCTGCAA CTTTGTTGGG TTTGGAGGAG 1621 AATACCCTTT CAACACAACT TTTAGGACAT GTGGGTGATA AGCTAGGTAC AAAGATAACT 1681 GTTGAGAGGG AAAATTCAGA GCACCAGTTT GGTGTAGCAT CAGTCAGTGG AATTTCTGGT 1741 GGCTGCCCTA TAAGCTCTGG CCCAGCTGAA AACCAAGTAC TTATGGATCC ACATCAGCAT 1801 TTTGCATTTT CTAAAACAAC GTTCTCTGAT TCCAGTGAGC AAGCTATCAG GTTCAGCACT 1861 GCTGGCGGCA GAACAATGGC TATTCCTAGT GATGCACTTC AGCGTGCGAA AAATCTTCTG 1921 GGTGAATCGG ATTTAGAGGT TTCACCAAAT AATTTATTAG GCCACTCTTC AGCATCTGCT 1981 TGTAAAGAGA ATATACAAAA TTCAACTGGT CTGCGAAAAG AAGGTGAACC TGATTTATTG 2041 AAAAGTAGGG GGAACAGCAA AACTGAGCCA GCACAATTTT CCATTCCAGC AAAACCTGAT 2101 AGGAAGCACA CAGATTCCTT GGAATATGCT GTACCTGATG CCACTTTGGC TAACGGAAAC 2161 TCCGTCAGGC TTCATGCGGC AAGAGATTTT CATCCTATCA ATGAAATTCC AAAGATATCC 2221 AAGCCTTCTT CCAGATGTTC ATTTGGAACT GAAAATGCAA GTGACACTAA AGATAAGGCT 2281 CGAAGACTCC AAATGCCATC TGGACCATTG ATTGACATCA CTAATTACAT CGATACACAT 2341 TCTGTTAATA CTGACTACCT GGCCGGTGAG AAGAGAAGAT TTGGGGGAAG AAACTCCATA 2401 TCTCCCTTTA AACGTCCTCG TTCTTCCAGG TTCATCGCAC CTATCAACAT CAATAATCCA 2461 TCCCCTTCTG GAGTATCCAA ACTACCTATT CAGATTAATC CCTGTCGAAC AAAGCTATCT 2521 TCATGCTATC CTTTTCAACA TCAAAGAAAA TCGTGTGAAG AGTATTTTGG TGGTCCCCCA 2581 TGCTTCAAAT ATTTGACAGA AGATGTAACA GATGAAGTGA AGCTCATGGA TGCAAAAAAG 2641 GCTGAGAAGT ACAAGTTTAA AACAGATACT GGTGCAGAAG AATTTCAGAA GATGCTTCTT 2701 GCCTGTGGTG CTTCATTGAC ATACACAACT AAAGAATGGG TCAGCAACCA CTACAAATGG 2761 ATTGTTTGGA AGCTTGCTTC ATTGGAGAGA TGCTATCCAA CTAGAGCTGC TGGCAAATTC 2821 TTAAAAGTTG GTAATGTTTT GGAAGAGCTG AAGTACAGGT ATGACAGAGA AGTGAACAAT

2881 GGCCACCGCT CAGCCATAAA GAAAATTTTG GAAGGGAATG CTTCACCATC TTTGATGATG 2941 GTGCTGTGCA TTTCTGCTAT TTACTCTTGT CCTGACCTAA ACAACAGTAA GCCAGAGGAT 3001 GATAGGGCAC ATACAGACGA CGACAACAGT GAGAATAAAA GCTTGAGACC TGCTAAAAGG 3061 AACATGTCTA CAAAGATTGA ACTAACTGAT GGATGGTATT CTCTAGATGC GTCATTAGAT 3121 CTGGCACTTT TGGAGCAACT AGAGAAAAGA AAACTTTTTA TAGGACAGAA GCTTCGGATA 3181 TGGGGAGCTT CACTATGTGG GTGGGCTGGG CCTGTGTCAT TTCATGAGGC ATCGGGTACC 3241 GTCAAATTAA TGATCCACAT AAATGGCACC TATCGTGCAA GATGGGATGA GACTTTGGGG 3301 TTATGCAAGC ATGCTGGAGT CCCACTGGCA TTCAAGTGCA TAAAAGCTTC AGGTGGCAGA 3361 GTTCCTAGGA CACTGGTTGG AGTTACAAGG ATTTATCCTG TTATGTACAG GGAGAGGTTT 3421 TCTGACGGTC GTTTTGTGGT GAGGTCTGAA AGGATGGAAA GAAAAGCACT ACAGCTGTAT 3481 CACCAGAGAG TGTCTAAGAT TGCAGAAGAC ATTCAGTCAG AACATGGAGA ACACTGCGAC 3541 AACACTGATG ATAACGATGA AGGGGCAAAA ATATGCAAAA TGCTAGAGAG GGCAGCTGAG 3601 CCTGAAATTC TTATGTCCAG CATGAGTTCA GAGCAGCTGC TGTCTTTCTC ATATTATCAA 3661 GAAAAGCAAA AGATTGTCAG GCAAAATGAA GTAGCTAAGA AGGTTGAAAA TGCTCTTAAA 3721 GTTGCTGGGC TTAGTTCAAG AGATGTTACA CCATTTTTGA AAGTGAGGGT GACGGGCCTT 3781 ATCAGCAAAC ACTCCGCCAC AAAATCTGGC TGCAGGGAAG GGTTAATAAC AATTTGGAAC 3841 CCTACCGAGA AGCAAAAATC CGACCTGGTG GAGGGACAAA TTTATTCTGT CACAGGACTG 3901 TTGGCTTCAA GCTACTTTAC AGAAGTATCC TACTTGAGTG GTAGAGGATC ATCTACAGCA 3961 TGGACGCCTT TAGCAACCGC ACAGACTACA AATTTTGAAC CATTTTTCAC CCCTCGTAAA 4021 GCAGTTGAAT TGTCACATTT TGGTGAAGTG CCACTTACAA GCGAATTTGA CATTGCAGGT 4081 GTTATTTTGT ATGTTGGGAA TGTTTATTTA TTGAACAACC AGAATAGGCA GTGGCTCTTT 4141 TTGACAGATG GATCTAAATT TATCTCTGGA GAAAAGTATG AAGAGCAAGA TGACTGTCTT 4201 CTGGCAGTTA GCTTTTCTTC CAAAACCACT GGCGAGGATT CTGCATTCTT CAATTATGCC 4261 CTTTCTGGAC ATATAGTTGG TTTTAGTAAT CTGGTCAAGC GAGATAAAGA CCAGATGAGG 4321 CACGTGTGGG TAGCTGAGGC GACAGAGAGC TCCACCTATA GTCTCTCCCA CGAGATACCT 4381 AAAAAATCAC ATCTCAAAGA GGCTGCCACT TCTGCTGAAA AATGGGCTTC AAATTCTCAT 4441 CCTATGATTC AGCATCTGAA GGAAAGAGTT CTGCAAATAG TTGGTGACAG TGGTGGCTGA // SEQ ID NO: 111: OsSPO11A cDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsSPO11A cDNA 1329 bp ORGANISM Oryza sativa cv Kaybonnet SOURCE Young flower AUTHORS Qiudeng Que CDS 1 . . . 1329 BASE COUNT 225 a 460 c 405 g 239 t ORIGIN 1 ATGTCGGAGA AGAAGCGCCG CGGCGGGGCA GGCGCGGGGG CCGCGTCGGG CTCCGCCTCC 61 AAGAAGCCGC GGGTCTCCAC GGCGGCGTCG TACGCCGAGT CGCTCCGCTC GAAGCTCCGC 121 CCCGACGCCT CCATCCTCGC CACCCTCCGC TCCCTGGCCT CCGCCTGCTC CAAACCCAAG 181 CCCGCGGGGT CGTCGTCGTC GTCGTCGTCC GCCTCGAAGG CGCTCGCAGC CGAGGACGAC 241 CCGGCCGCCA GCTACATCGT GGTGGCCGAC CAGGACTCCG CCTCCGTCAC CTCCCGCATC 301 AACCGCCTCG TGCTCGCCGC GGCGCGCAGC ATCCTGTCCG GCCGGGGCTT CTCCTTCGCG 361 GTGCCCTCCC GCGCCGCCTC CAACCAGGTC TACCTCCCGG ACCTCGACCG CATCGTGCTC 421 GTCCGCCGCG AGTCCGCCAG GCCCTTCGCC AACGTCGCCA CCGCGCGGAA GGCCACCATC 481 ACCGCGCGCG TCCTCTCCTT GGTCCACGCC GTCCTCCGCA GGGGGATCCA CGTCACCAAG 541 CGTGACCTCT TCTACACCGA CGTCAAGCTC TTCGGCGACC AGGCGCAGTC CGACGCCGTC 601 CTCGACGACG TCTCCTGTAT GCTCGGCTGC ACCCGCTCCT CCCTCCACGT CGTCGCGTCC 661 GAGAAGGGCG TCGTCGTCGG GCGCCTCACC TTCGCCGACG ACGGCGACCG GATCGACTGC 721 ACGCGCATGG GCGTCGGCGG GAAGGCCATC CCGCCCAACA TCGACAGGGT CTCAGGCATC 781 GAGAGCGACG CTCTCTTCAT CTTGCTGGTG GAGAAGGACG CCGCGTTCAT GCGTCTCGCC 841 GAGGACCGGT TCTACAACCG CTTCCCGTGC ATCATCTTGA CGGCGAAGGG GCAGCCGGAT 901 GTCGCCACAC GGCTGTTCTT GCGGCGGCTT AAGGTGGAGC TGAAGCTGCC AGTGCTGGCA 961 TTGGTGGACT CCGACCCATA TGGGCTGAAG ATCTTGTCAG TGTACATGTG TGGTTCCAAG 1021 AACATGTCAT ATGACAGTGC CAACCTGACA ACACCGGATA TCAAGTGGCT CGGAGTGCGG 1081 CCAAGCGATC TGGACAAGTA TCGGGTGCCG GAGCAGTGCC GGCTTCCGAT GACTGATCAC 1141 GATATCAAGG TGGGGAAGGA GCTGCTTGAG GAGGACTTTG TGAAGCAGAA TGAAGGATGG 1201 GTGAAGGAGC TGGAGACGAT GTTGCGGACG AGGCAGAAGG CTGAGATACA GGCTCTCAGT 1261 TCATTTGGTT TCCAGTATCT CACTGAGGTC TATCTACCTC TCAAGCTGCA GCAACAGGAC 1321 TGGATTTGA // SEQ ID NO: 112: OsSPO11B gDNA from Oryza sativa (cultivar Kaybonnet) DEFINITION OsSpollB gDNA 1456 bp ORGANISM Oryza sativa cv Kaybonnet SOURCE Oryza sativa cv. Kaybonnet calli REFERENCE 1 (bases 1 to 1456) CDS 1 . . . 1444 BASE COUNT 452 a 268 c 326 g 410 t ORIGIN 1 AGCAACCATG GATGATTCAA CGGATGACGA TTCGTATCAT CCAAGAAAAC ACTATGCTTA 61 TGATCGTCAG GTTTCTTCAA GCAGATGGCG TACCAGCCGC GAGTATATCA GAGGTCCCGG 121 CCCCGAAACT CATACTACTG AGAGTGCTCA AGATGGACAG GATCCACCTG CTGGAGTATA 181 TTCCTATGGT TATTTTTCTG GCAGTGGTAA TGATCCTCAA GTTCAAGGAC ACTTTGTTCC 241 GGAGATTCAA AAGTACAACC CTTACGTGAT TTTCAAAGGT GAACAACTCC CGGTTCCTAT 301 ATGGGAACTG CCAGAGGAGA AGGTCCAAGA TTTTCATGAT AGGTACTTTA TTGCAAAAGA 361 CAAGAGTCGA GTTGAAGCCA GGAAGACTCT GAATAGGTTG TTAGAGGGGA ACATCAATAC 421 AATTGAAAGG GGACATGGAT ATAAATTCAA TATTCCAAAA TATACAGATA ACATGGAGTT 481 TAATGAGGAA GTCAAGGTTT CTCTAGCAAA AGCAGGCAAG ACCATAAGCC GTTCCTTTTG 541 CAATGCGAAT CAGCGGGAAG TTGCATCTAG GACTGGCTAT ACCATTGATC TAATAGAACG 601 GACACTTGGG GCTGGATTGA ACATCTCGAA GAGAACTGTC TTATACACAA ACAAGGATCT 661 GTTTGGGGAT CAAAGTAAAT CAGATCAAGC GATCAATGAC ATCTGCGCTT TGACAAATAT 721 CAGAAGGGGC TCTTTGGGTA TAATAGCAGC TGAAAAAGGA ATTGTAGTTG GAAACATTTT 781 CCTGGAATTG ACAAATGGCA AATCGATTAG TTGTTCTATT GGAGTGCAGA TACCACACAG 841 GCTTGACCAG ATCAAAGATG TTTGTGTTGA AATAGGTTCA CGCAACATAG AGTATATTCT 901 TGTTGTGGAA AAGCATACAA TGTTGAATTA TCTACTAGAG ATGGACTATC ACACCAATAA 961 CAACTGTATA ATTCTGACAG GATGTGGCAT GCCAACCCTC CAAACAAGGG ATTTCCTCAG 1021 ATTCTTGAAA CAACGCACTG GACTACCTGT CTTTGGACTT TGTGATCCAG ATCCTGAAGG 1081 TATAAGTATT CTTGCTACGT ATGCTAGAGG GTCTTGCAAT TCAGCATATG ACAATTTCAA 1141 TATTTCCGTG CCATCTATTT GTTGGGTTGG ATTGTCATCC TCAGACATGA TAAAGTTGAA 1201 TTTGTCTGAG ACCAACTACT CACGTTTGTC TCGCGAGGAC AAAACTATGT TGAAGAACCT 1261 TTGGCAGGAC GATTTGTCCG ATGTATGGAA ACGCAGAATC GAAGAAATGA TAAGTTTTGA 1321 CAAGAAGGCC TCTTTTGAAG CTATTCATAG TTTGGGGTTT GATTATTTTG CAACCAATTT 1381 GCTTCCGGAT ATGATTAACA AAGTACGAGA AGGCTATGTT CAGGTATATT TCTCACTCCT 1441 ATAGCAACTT GTATTT // SEQ ID NO: 113: OsRad54A protein sequence LOCUS OsRad54A protein 1187 amino acid residues ORGANISM Rice, Oryza sativa cv Kaybonnet MEDDDDDQRLLHSLGVTSADIHDIERRIISQATTDPADSSGPTINGGHQPDDALAKLHHK LRSVQIEIDAVASTIKGAKLKQPSGNKPHEHKGKDQPDHHGAGHLQQALAADRLTSLRKA KAQIQKEILQSHLSPSASNRKDKMLAMLVQDEPRHKKPPVGPKNIVKRPMKTVTYDDDNN FDAVLDGASAGFMETEREELIRKGLLTPFHKLKGFEKRVELPEPSHRQDDSAGQTEEAME ASRIARVAQSLKQIAQNRPATKLLDSESLPKLDAPAAPFQRLGKPLKRPVSPSSDEQEKK RPRNKTKRPLPGKKWRKANSIKESSLDDNDVGEAAVSVSDDDEDQVTEGSDELTDVTLEG GLRIPGTLYTQLFDYQKVGVQWLWELHCQRAGGIIGDEMGLGKTVQVLSFLGSLHNSGLY KPSIVVCPVTLLQQWRREASRWYPKFKVEILHDSANSSSKKSKRSSDSDSEASWDSDQEE AVTCSKPAKKWDDLISRVVSSGSGLLLTTYEQLRILGEKLLDIEWGYAVLDEGHRIRNPN AEITLVCKQLQTVHRIIMTGAPIQNKLSELWSLFDFVFPGKLGVLPVFEAEFSVPITVGG YANATPLQVSTAYRCAVVLRDLVMPYLLRRMKADVNAQLPKKTEHVLFCSLTTEQRATYR AFLASSEVEQIFDGNRNSLYGIDVLRKICNHPDLLEREHAAQNPDYGNPERSGKMKVVEQ VLKVWKEQGHRVLLFTQTQQMLDIMGNFLTACEYQYRRMDGLTPAKQRMALIDEFNNTDE IFIFILTTKVGGLGINLTGANRIIIYDPDWNPSTDMQARERAWRIGQTRDVTVYRLITRG TIEEKVYHRQVYKHFLTNKVLKDPQQRRFFKARDMKDLFTLQDDDNNGSTETSNIFSQLS EDVNIGVPSDKQQDQLYAASATPTTSGTEPSSSRHGQGKEDHCPDQADEECNILKSLFDA QGIHSAINHDAIMNANDDQKLRLEAEATQVAQRAAEALRQSRMLRSHESFSVPTWTGRAG AAGAPSSVRRKFGSTLNTQLVNSSQPSETSNGRGQSLQVGALNGKALSSAELLARIRGTR EGAASDALEHQLNLGSASNHTSSSSGNGRASSSSTRSMIVQPEVLIRQLCTFIQQHGGSA SSTSITEHFKNRILSKDMLLFKNLLKEIATLQRGANGATWVLKPDYQ // SEQ ID NO: 114: OsRad54B Protein sequence DEFINITION OsRad54B protein 1148 amino acid residues ORGANISM Oryza sativa cv Kaybonnet MRTSTTSDSPSPSPQNKASFNTSRGAAFRDEEPGAKDNEVEKRKPLILHLKKRSTKELST DTTSSKSGLLGKSSEEKQEKHGSALKVKKHLHPMELSPKKYKNKKQHNHRDSKRSEAKKV QYLASDVDSDSSMEPSTSLEHSESPPPKRKSLDGRTPASSTKKGKKKVKFIDKKHPENAV HITEKEHGGAGDKITTQGDLQVDRILGCRLQTSQIISPAHASSEQIDMAPPSASGATEPS QALSKGLHEEIQSSNSDTNVTEDACADELANDGGENNLDCSDAQKESNVRSHGHKESLNA KEIMNTASACSADQIVTVKDAGAVQTYVTASVNGEYETVTDIPEEKNDTKHPVSKADTEV

HTKQEHTPDSKLHGKLENYKAKYGTGLINICKEQWCQPQRVIALRTSLDEIEEALIKWCA LPYDECTWERLDEPTMVKYAHLVTQFKKFESQALDKDKGGSHAKPREHQEFNMLVEQPKE LQGGMLFPHQLEALNWLRKCWYKSKNVILADEMGLGKTVSACAFLSSLCCEYKINLPCLV LVPLSTMPNWMAEFASWAPHLNVVEYHGSARARSIIRQYEWHEGDASQMGKIKKSHKFNV LLTTYEMVLVDAAYLRSVSWEVLIVDEGHRLKNSSSKLFSLLNTLSFQHRVLLTGTPLQN NIGEMYNLLNFLQPASFPSLASFEEKFNDLTTTEKVEELKNLVAPHMLRRLKKDAMQNIP PKTERMVPVELTSIQAEYYRAMLTKNYQVLRNIGKGGAHQSLLNIVMQLRKVCNHPYLIP GTEPESGSPEFLHEMRIKASAKLTLLHSMLKILHKDGHRVLIFSQMTKLLDILEDYLTWE FGPKTFERVDGSVSVAERQAAIARFNQDKSRFVFLLSTRSCGLGINLATADTVIIYDSDF NPHADIQAMNRAHRIGQSNRLLVYRLVVRASVEERILHLAKKKLMLDQLFVNKSESQKEV EDIIRWGTEELFRNSDVAVKDNNEASGAKNDVAEVEFKHKRKTGGLGDVYEDRCADGSAK FNWDENAITKLLDRSNVPSTVAESTDGDLDNDMLGTVKSIDWNDELNDDPGATEDIPNID NDGCEQASEAKQDAANRVEENEWDKLLRVRWEQYQTEEEASLGRGKRLRKAVSYRETFAT IPNEALSE // SEQ ID NO: 115: OsBRCA1 protein sequence DEFINITION OsBRCA1 protein 987 amino acid residues ORGANISM Oryza sativa cv Kaybonnet MADTGSLEKMGRELKCPICLSLLSSAVSISCNHVFCNDCLTESMKSTSSCPVCKVPFRRR EMRPAPHMDNLVSIFKSMEAAAGTNVVSTQEAPVVKLADGSDCVNSGKNSKRSQKSLTRK RKVTSEMEKNTAKDATASASQPTTKPSFSTNKRIQVKPFPESETPIRAEKIMKPEEPKNN LNNDVEGKNKAVASGQPGSPSLSPFFWLREQEEQEGCTAETLSETQSLDTPLRHNAPSFS DIKDSDDEIPLNTTPNSKAAATELFDSEIFEWTQRPCSPELYSTPLKKQSKAKSKLDQIE EKGDEEDVHIGGSFDKLGSASNAAQLVNTKATKQKRKKTSPSNKNSAKLSNRAEPCIKKS DANQQGSNRRKSAALKSCQKSSSAVGRNTSGRRNKASSNSKPIHGSSDNSPESYLPKEGL DVEAPDKPLSERIQNLEKTSRRKGSARKLEMAGKTISDTTEKNSEPRSKRVRRMSDHAIA KPVEVPSGSGNETEIPQLHTLTKGSIQRKSSNARRHSKVCGEQEGKNKLENTTMTPIILH GKCQNKEAVCTAPSVRTASVKYKQAKFSEQPDCFGTENFGNLQACPARNVLLKKCEVSTL KVSCAFCQTDVITEESGEMVHYQNGKQVPAEFNGGANVVHSHKNCLEWAPDVYFEDDSAF NLTTELARSRRIKCACCGIKGAALGCFEMSCRRSFHFTCAKLIPECRWDNENFVMLCPLH RSTKLPNENSEQQKQPKRKTTLKGSSQIGSNQDCGNNWKWPSGSPQKWVLCCSSLSSSEK GLVSEFAKLAGVPISATWSPNVTHVIASTDLSGACKRTLKFLMAILNGRWIVSIDWVKTC MECMEPIDEHKFEVATDVHGITDGPRLGRCRVIDRQPKLFDSMRFYLHGDYTKSYRGYLQ DLVVAAGGIVLQRKPVSRDQQKLLDDSSDLLIVYSFENQDRAKSKAETKAADRRQADAQA LACASGGRVVSSAWVIDSIAACNLQPL // SEQ ID NO: 116: OsBRCA2 Protein sequence DEFINITION OsBRCA2 protein 1499 amino acid resisues ORGANISM Oryza sativa cv Kaybonnet MADLFNQALDKLVAADGMAEAIEDSGKGAVFCTGLGGSVAVSERAVERAKALVGEVAEEI SNERRQPFGDGSNLECGLGESNVSFKGGVHKDSLSPMFQTGSGKMVSLSKGSIQKARAVL EGNAENSSVIAVQSMFHTGLVRPDPVSRSSTDNAMTVLEGQTNPKQGDVADVYDKENFPL FQTGSGKAVSVSVASIQKAKAVLEQNNTENTEDFGRPDQSLIFQTGSRRPVLISERSSSV VKDGGAENIVFQTGLGRPVVVSQTSIQKARTVLDQECAKRSGHGDTNVSTTTFQTETPTP VLMSGGLTMNDRSVTPEGGVSMQGNFLEADGHLPLFQTGLGRSISVSKGSIKRASALLEP RNITKELEDEAHSDDGCATPMFKTGSGRSITASENSRKKAHVVLEGEEPVKNVNNDTGEA IAPMLHAGMQKFAPQNRNSSHKAITLMEQGSSMEEDRGNEPPMFRTGSGKSVLISHSSVQ KARAVLEEEGNMKKENHKQLSNVDKYIPIFTSPLKTSYARTVHISSVGVSRAATLLGLEE NTLSTQLLGHVGDKLGTKITVERENSEHQFGVASVSGISGGCPISSGPAENQVLMDPHQH FAFSKTTFSDSSEQAIRFSTAGGRTMAIPSDALQRAKNLLGESDLEVSPNNLLGHSSASA CKENIQNSTGLRKEGEPDLLKSRGNSKTEPAQFSIPAKPDRKHTDSLEYAVPDATLANGN SVRLHAARDFHPINEIPKISKPSSRCSFGTENASDTKDKARRLQMPSGPLIDITNYIDTH SVNTDYLAGEKRRFGGRNSISPFKRPRSSRFIAPININNPSPSGVSKLPIQINPCRTKLS SCYPFQHQRKSCEEYFGGPPCFKYLTEDVTDEVKLMDAKKAEKYKFKTDTGAEEFQKMLL ACGASLTYTTKEWVSNHYKWIVWKLASLERCYPTRAAGKFLKVGNVLEELKYRYDREVNN GHRSAIKKILEGNASPSLMMVLCISAIYSCPDLNNSKPEDDRAHTDDDNSENKSLRPAKR NMSTKIELTDGWYSLDASLDLALLEQLEKRKLFIGQKLRIWGASLCGWAGPVSFHEASGT VKLMIHINGTYRARWDETLGLCKHAGVPLAFKCIKASGGRVPRTLVGVTRIYPVMYRERF SDGRFVVRSERMERKALQLYHQRVSKIAEDIQSEHGEHCDNTDDNDEGAKICKMLERAAE PEILMSSMSSEQLLSFSYYQEKQKIVRQNEVAKKVENALKVAGLSSRDVTPFLKVRVTGL ISKHSATKSGCREGLITIWNPTEKQKSDLVEGQIYSVTGLLASSYFTEVSYLSGRGSSTA WTPLATAQTTNEEPFFTPRKAVELSHFGEVPLTSEFDIAGVILYVGNVYLLNNQNRQWLF LTDGSKFISGEKYEEQDDCLLAVSFSSKTTGEDSAFFNYALSGHIVGESNLVKRDKDQMR HVWVAEATESSTYSLSHEIPKKSHLKEAATSAEKWASNSHPMIQHLKERVLQIVGDSGG // SEQ ID NO: 117: OsSPO11A protein DEFINITION OsSPO1lA protein, 442 amino acids ORGANISM Oryza sativa cv Kaybonnet MSEKKRRGGAGAGAASGSASKKPRVSTAASYAESLRSKLRPDASILATLRSLASACSLPK PAGSSSSSSSASLALAAEDDPAASYIVVADQDSASVTSRINRLVLAAARSILSGRGFSFA VPSRAASNQVYLPDLDRIVLVRRESARPFANVATARLATITARVLSLVHAVLRRGIHVTK RDLFYTDVKLFGDQAQSDAVLDDVSCMLGCTRSSLHVVASEKGVVVGRLTFADDGDRIDC TRMGVGGKAIPPNIDRVSGIESDALFILLVEKDAAFMRLAEDRFYNRFPCIILTAKGQPD VATRLFLRRLKVELKLPVLALVDSDPYGLKILSVYMCGSKNMSYDSANLTTPDIKWLGVR PSDLDKYRVPEQCRLPMTDHDIKVGKELLEEDFVKQNEGWVKELETMLRTRQKAEIQALS SFGFQYLTEVYLPLKLQQQDWI // SEQ ID NO: 118: OsSPO11B DEFINITION OsSpo11B protein, 478 amino acid residues ORGANISM Oryza sativa cv Kaybonnet MDDSTDDDSYHPRKHYAYDRQVSSSRWRTSREYIRGPGPETHTTESAQDGQDPPAGVYSY GYFSGSGNDPQVQGHFVPEIQKYNPYVIFKGEQLPVPIWELPEEKVQDFHDRYFIAKDKS RVEARKTLNRLLEGNINTIERGHGYKFNIPKYTDNMEFNEEVKVSLAKAGKTISRSFCNA NQREVASRTGYTIDLIERTLGAGLNISKRTVLYTNKDLFGDQSKSDQAINDICALTNIRR GSLGIIAAEKGIVVGNIFLELTNGKSISCSIGVQIPHRLDQIKDVCVEIGSRNIEYILVV EKHTMLNYLLEMDYHTNNNCIILTGCGMPTLQTRDFLRFLKQRTGLPVFGLCDPDPEGIS ILATYARGSCNSAYDNFNISVPSICWVGLSSSDMIKLNLSETNYSRLSREDKTMLKNLWQ DDLSDVWKRRIEEMISFDKKASFEAIHSLGFDYFATNLLPDMINKVREGYVQVYFSLL //

[0285] All publications, published patent documents, and patent applications cited in this specification are indicative of the level of skill in the art(s) to which the invention pertains. All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

[0286] The foregoing describes the invention with reference to various embodiments and examples. No particular embodiment, example, or element of a particular embodiment or example is to be construed as a critical, required, or essential element or feature of any or all of the claims. As used herein, the terms "comprises," "comprising," "includes," "including," "contains," "containing," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter. Further, no element described herein is required for the practice of the invention unless expressly described as "essential" or "critical."

[0287] It will be appreciated that various modifications and substitutions can be made to the disclosed embodiments without departing from the scope of the invention as set forth in the claims below. The specification, including the drawings and examples, is to be regarded in an illustrative manner, rather than a restrictive one, and all such modifications and substitutions are intended to be included within the scope of the invention. Accordingly, the scope of the invention should be determined by the appended claims and their legal equivalents, rather then by the examples given above. For example, the steps recited in any of the method or process claims may be executed in any feasible order and are not limited to an order presented in any of claims.

Sequence CWU 1

1

118130DNAArtificial SequenceNPTFA primer 1gatctctaga atgattgaac aagatggatt 30232DNAArtificial SequenceNPTRA primer 2tcgcagcttg gtacctgcag ttcattcagg gc 32332DNAArtificial SequenceIntBAFFW primer 3gccctgaatg aactgcaggt accaagctgc ga 32432DNAArtificial SequenceIntBAFRV primer 4gccgcgctgc ctcgtcctga aaaattcaga aa 32532DNAArtificial sequenceNPTF2 primer 5tttctgaatt tttcaggacg aggcagcgcg gc 32634DNAArtificial sequenceNPTR2 primer 6gaatagtact aatacctggc acttcgccca atag 34730DNAArtificial sequenceIntPALFW primer 7ttagtactat tcttttgttc tctaatcaga 30836DNAArtificial sequenceIntPALRV primer 8tgacaggaga tcctgccctg taacgaacaa aaacat 36936DNAArtificial sequenceNPTFC primer 9atgtttttgt tcgttacagg gcaggatctc ctgtca 361036DNAArtificial sequenceNPTR3 primer 10atcgattcat atatatacct ggtcgacaag accggc 361130DNAArtificial sequenceIntTUBFW primer 11caggtatata tatgaatcga tttctccctt 301232DNAArtificial sequenceIntTUBRV primer 12tcgtccagat catcctgtaa tacagaaatg tt 321332DNAArtificial sequenceNPTFD primer 13aacatttctg tattacagga tgatctggac ga 321432DNAArtificial sequenceNPTR4 primer 14ggaaaagctt aattacctcg ccgtcgggca tg 321528DNAArtificial sequenceIntTUAFW primer 15gtaattaagc ttttccacct ctcttgtt 281632DNAArtificial sequenceIntTUARV primer 16gatcctgcag caatggaaaa atatttcaat ac 321730DNAArtificial sequenceNPTFE primer 17attgctgcag gatctcgtcg tgacccatgg 301828DNAArtificial sequenceNPTR5 primer 18cattaggatc ctcagaagaa ctcgtcaa 281940DNAArtificial sequenceFRTBGL2 primer 19gatctgaagt tcctattctc tagaaagtat aggaacttcg 402040DNAArtificial sequenceFRTBAM1 primer 20gatccgaagt tcctatactt tctagagaat aggaacttca 402130DNAArtificial sequenceTPALBGLI primer 21tgttaagatc ttagtcctct gtttttttct 302230DNAArtificial sequenceTPALSAC primer 22cttgagctct tctataaccc tagatggcta 302327DNAArtificial sequenceBARCLA primer 23tcatatcgat gagcccagaa cgacgcc 272432DNAArtificial sequenceBARBGL primer 24tttgagatct tcatatctcg gtgacgggca gg 322530DNAArtificial sequenceISCEBAM1 primer 25acttggatcc atattaccct gttatcccta 302630DNAArtificial sequenceISCEBGL2 primer 26tcgaagatct gctagggata acagggtaat 302730DNAArtificial sequenceICEUBGL2 primer 27tcgaagatct ctataacggt cgtaaggtag 302830DNAArtificial sequenceICEUBAM1 primer 28acttggatcc tcgctacctt aggaccgtta 302930DNAArtificial sequenceHOBGL2 primer 29tcgaagatct agctttccgc aacagtataa 303030DNAArtificial sequenceHOBAM1 primer 30acttggatcc attatactgt tgcggaaagc 303140DNAArtificial sequenceFRTBGL2 31gatctgaagt tcctattctc tagaaagtat aggaacttcg 403240DNAArtificial sequenceFRTBAM1 32gatccgaagt tcctatactt tctagagaat aggaacttca 403330DNAArtificial sequenceHOATG primer 33ctactgtcga caaaaatgct ttctgaaaac 303430DNAArtificial sequenceHOBAMH primer 34ctaggatccg acctggtcgt cacagtagct 3035872DNAArtificial sequenceI-CeuI endonuclease with maize-preferred codons and potato ST-LS1 intron 35gaattcgccc ttggggatcc atgagcaact tcatcctgaa gcccggcgag aagctgcccc 60aggacaagct ggaggagctg aagaagatca acgacgccgt gaagaagacc aagaacttca 120gcaagtacct gatcgacctg cgcaagctgt tccagatcga cgaggtgcag gtgaccagcg 180agagcaagct gttcctggcc ggcttcctgg agggcgaggc cagcctgaac atcagcacca 240agaagctggc caccagcaag ttcggcctgg tggtggaccc cgagttcaac gtgacccagc 300acgtaagttt ctgcttctac ctttgatata tatataataa ttatcattaa ttagtagtaa 360tataatattt caaatatttt tttcaaaata aaagaatgta gtatatagca attgcttttc 420tgtagtttat aagtgtgtat attttaattt ataacttttc taatatatga ccaaaatttg 480ttgatgtgca ggtgaacggc gtgaaggtgc tgtacctggc cctggaggtg ttcaagaccg 540gccgcatccg ccacaagagc ggcagcaacg ccaccctggt gctgaccatc gacaaccgcc 600agagcctgga ggagaaggtg atccccttct acgagcagta cgtggtggcc ttcagcagcc 660ccgagaaggt gaagcgcgtg gccaacttca aggccctgct ggagctgttc aacaacgacg 720cccaccagga cctggagcag ctggtgaaca agatcctgcc catctgggac cagatgcgca 780agcagcaggg ccagagcaac gagggcttcc ccaacctgga ggccgcccag gacttcgccc 840gcaactacaa gaagggcatc aagtaggaat tc 8723674DNAArtificial sequence1A primer 36ggggatccat gagcaacttc atcctgaagc ccggcgagaa gctgccccgg acaagctgga 60ggagctgaag aaga 743775DNAArtificial sequence1B primer 37cgcaggtcga tcaggtactt gctgaagttc ttggtcttct tcacggcgtc gttgatcttc 60ttcagctcct ccagc 753875DNAArtificial sequence1C primer 38aagtacctga tcgacctgcg caagctgttc cagatcgacg aggtgcaggt gaccagcgag 60agcaagctgt tcctg 753975DNAArtificial sequence1Dprimer 39tggccagctt cttggtgctg atgttcaggc tggcctcgcc ctccaggaag ccggccagga 60acagcttgct ctcgc 754075DNAArtificial sequence1E primer 40cagcaccaag aagctggcca ccagcaagtt cggcctggtg gtggaccccg agttcaacgt 60gacccagcac gtgaa 754175DNAArtificial sequence1F primer 41cgcaggtcga tcaggtactt gctgaagttc ttggtcttct tcacggcgtc gttgatcttc 60ttcagctcct ccagc 754275DNAArtificial sequence2G primer 42ccccggccgc atccgccaca agagcggcag caacgccacc ctggtgctga ccatcgacaa 60ccgccagagc ctgga 754375DNAArtificial sequence2H primer 43ctcggggctg ctgaaggcca ccacgtactg ctcgtagaag gggatcacct tctcctccag 60gctctggcgg ttgtc 754475DNAArtificial sequence2I primer 44tggccttcag cagccccgag aaggtgaagc gcgtggccaa cttcaaggcc ctgctggagc 60tgttcaacaa cgacg 754575DNAArtificial sequence2J primer 45atctggtccc agatgggcag gatcttgttc accagctgct ccaggtcctg gtgggcgtcg 60ttgttgaaca gctcc 754675DNAArtificial sequence2K primer 46ctgcccatct gggaccagat gcgcaagcag cagggccaga gcaacgaggg cttccccaac 60ctggaggccg cccag 754765DNAArtificial sequence2L primer 47ggggaattcc tacttgatgc ccttcttgta gttgcgggcg aagtcctggg cggcctccag 60gttgg 654829DNAArtificial sequenceprimer 48gggtacgtaa gtttctgctt ctacctttg 294929DNAArtificial sequenceprimer 49ccccagctgc acatcaacaa attttggtc 295014DNAArtificial sequenceprimer 50gatcggcaat tgcc 145127DNAArtificial sequenceprimer 51ccggtgagta atattgtacg gctaaga 275227DNAArtificial sequenceprimer 52agatcctcag aagaactcgt caagaag 275330DNAArtificial sequenceprimer 53aatataggcg gtattccggc cattataaca 305430DNAArtificial sequenceprimer 54ctaagatcct cagaagaact cgtcaagaag 305530DNAArtificial sequenceprimer 55accctccgct acttctccgg gaaaagacgc 305630DNAArtificial sequenceprimer 56actatcggcg agtacttcta cacagccatc 305726DNAArtificial sequenceprimer 57gtgtctcatg cacttgggag gtgatc 265840DNAArtificial sequenceprimer 58gatccgcggt tgatgaaaga ataacgtatt ctttcatcaa 405940DNAArtificial sequenceprimer 59gatcttgatg aaagaatacg ttattctttc atcaaccgcg 406030DNAArtificial sequenceprimer 60atgccgcagg taccaagctg cgaatcttcg 306130DNAArtificial sequencePMIINTRA primer 61atcgggatac ctgaaaaatt cagaaacaaa 306230DNAArtificial sequencePMIINTFB primer 62cggtcgcagg tattagtact attcttttgt 306330DNAArtificial sequencePMIINTRB primer 63cggatgtgca cctgtaacga acaaaaacat 306430DNAArtificial sequencePMIINTFC primer 64acctgcaagg tatatatatg aatcgatttc 306530DNAArtificial sequencePMIINTRC primer 65gcgccacacc tgtaatacag aaatgttaag 306630DNAArtificial sequencePMIINTFD primer 66gtgaaacaag gttattaacg ttttccacct 306730DNAArtificial sequencePMIINTRD primer 67gttctgcacc tgcatcaatg gaaaaatatt 306833DNAArtificial sequencePMIEXF1 primer 68gtggatccgg cagcatgcaa aaactcatta act 336932DNAArtificial sequencePMIEXR1 primer 69tcgcagcttg gtacctgcgg cattttcttt gg 327030DNAArtificial sequencePMIEXF2 primer 70aatttttcag gtatcccgat ggatgccgcc 307130DNAArtificial sequencePMIEXR2 primer 71tagtactaat acctgcgacc ggctggagta 307230DNAArtificial sequencePMIEXF3 primer 72gttcgttaca ggtgcacatc cggcgattgc 307330DNAArtificial sequencePMIEXR3 primer 73tcatatatat accttgcagg taagcgtgcg 307430DNAArtificial sequencePMIEXF4 primer 74ctgtattaca ggtgtggcgc tggaagtgat 307530DNAArtificial sequencePMIEXR4 primer 75tgttaataac cttgtttcac cggctgggtc 307631DNAArtificial sequencePMIEXF5 primer 76cgattgatgc aggtgcagaa ctggacttcc c 317732DNAArtificial sequencePMIEXR5 primer 77tgctcgagtc attagcaaga gatgttaatt tt 327832DNAArtificial sequenceGUSBAFFW1 primer 78ttgactggca ggtaccaagc tgcgaatctt cg 327929DNAArtificial sequenceGUSBAFRV1 primer 79ggccaccacc tgaaaaattc agaaacaaa 298026DNAArtificial sequenceGUSBAMHI primer 80tccaaccatg ttacgtcctg tagaaa 268132DNAArtificial sequenceBAFGUSRV1 primer 81cagcttggta cctgccagtc aacagacgcg ac 328232DNAArtificial sequenceBAFGUSFW primer 82ttgactggca ggtaccaagc tgcgaatctt cg 328330DNAArtificial sequenceGUSSALI primer 83gtcgactcat tgtttgcctc cctgctgcgg 308429DNAArtificial sequenceGUSBAMHI 84ggatccaacc atgttacgtc ctgtagaaa 298532DNAArtificial sequenceGUSBAFRV1 primer 85attggccacc acctgaaaaa ttcagaaaca aa 328632DNAArtificial sequenceGUSBAFFW1 86ttgactggca ggtaccaagc tgcgaatctt cg 328730DNAArtificial sequenceGUSSALI 87gtcgactcat tgtttgcctc cctgctgcgg 308830DNAArtificial sequenceICEUBGL2 88tcgaagatct ctataacggt cctaaggtag 308930DNAArtificial sequenceICEUBAMH primer 89acttggatcc tcgctacctt aggaccgtta 309038DNAArtificial sequenceATTB1 primer 90gatccgctca agttagtata aaaaagcagg cttcatga 389138DNAArtificial sequenceATTB2 primer 91gatctcatga agcctgcttt tttatactaa cttgagcg 389235DNAArtificial sequenceATTPSPOMI primer 92gggccctctg ttacaggtca ctaataccat ctaag 359331DNAArtificial sequenceATTPSPEI primer 93actagtgaaa tcaaataatg attttatttt g 31941489DNADNA 94ctcgagcaac catgcagctg accaaggaca ccgagatcag caccatcaac cgccagatga 60gcgacttcag cgagctgagc cagatcctgc ccctgcacca gatcagcaag atcaaggaca 120tcctggagaa cgagaacccc ctgcccaagg agaagctggc cagccacctg accatgatca 180tcctgatggc caacctggcc agccagaagc gcaaggacgt gcccgtgaag cgcagcacct 240tcctgaagta ccagcgcagc atcagcaaga ccctgcagta cgacagcagc accaagaccg 300tgagcttcga gtaccacctg aaggacccca gcaagctgat caagggcctg gaggacgtgg 360tgagccccta ccgcttcgtg gtgggcgtgc acgagaagcc cgacgacgtg atgagccacc 420tgagcgccgt gcacatgcgc aaggaggccg gccgcaagcg cgacctgggc aacaagatca 480acgacgagat caccaagatc gccgagaccc aggagaccat ctggggcttc gtgggcaaga 540ccatggacct gatcgaggcc cgcaccaccc gccccaccac caaggccgcc tacaacctgc 600tgctgcaggc caccttcatg aactgctgcc gcgccgacga cctgaagaac accgacatca 660agaccttcga ggtgatcccc gacaagcacc tgggccgcat gctgcgcgcc ttcgtgcccg 720agaccaagac cggcacccgc ttcgtgtact tcttcccctg caagggccgc tgcgaccccc 780tgctggccct ggacagctac ctgcagtgga ccgaccccat ccccaagacc cgcaccaccg 840acgaggacgc ccgctacgac taccagctgc tgcgcaacag cctgctgggc agctacgacg 900gcttcatcag caagcagagc gacgagagca tcttcaagat ccccaacggc cccaaggccc 960acctgggccg ccacgtgacc gccagctacc tgagcaacaa cgagatggac aaggaggcca 1020ccctgtacgg caactggagc gccgcccgcg aggagggcgt gagccgcgtg gccaaggccc 1080gctacatgca caccatcgag aagagccccc ccagctacct gttcgccttc ctgagcggct 1140tctacaacat caccgccgag cgcgcctgcg agctggtgga ccccaacagc aacccctgcg 1200agcaggacaa gaacatcccc atgatcagcg acatcgagac cctgatggcc cgctacggca 1260agaacgccga gatcatcccc atggacgtgc tggtgttcct gagcagctac gcccgcttca 1320agaacaacga gggcaaggag tacaagctgc aggcccgcag cagccgcggc gtgcccgact 1380tccccgacaa cggccgcacc gccctgtaca acgccctgac cgccgcccac gtgaagcgcc 1440gcaagatcag catcgtggtg ggccgcagca tcgacaccag ctgaagctt 14899534DNAArtificial sequenceOsRecQcfw2 primer 95caccatgaag cacggtgtaa ttgatgataa agaa 349630DNAArtificial sequenceOsRecQcRv1 primer 96tcaagaggga atctttatgc agttgtcgca 309734DNAArtificial sequenceOsRecQdFW2 primer 97caccatgata aagccaaggg tcaactggtc ggat 349830DNAArtificial sequenceRecQdRV1 primer 98ctaggctatt ctggcggact gccacgcagg 30993525DNADNA 99atgataaagc caagggtcaa ctggtcggat catgcaaatg ctgttcaaag ctcctgtatc 60aaagatgaat tcctgagttc aagttttttg ttctctttac caacacaaag gcctaatcag 120gaagcagatt gtacgggaat gcttccttta aggtctgctg cttgcagaat tcaaggccta 180gagcgtcttc aagctccatc cattgagaag gcctggcgtt ctctacgcaa cactcaggtt 240gcacggaaga attatttaag acctggttta tctggaaaag tgaaagattg tgatagcgac 300catgctcata cttatgggac aagttcttca tataatgtta acaaagtgga cagtgtgtcc 360agaaatagga atcccaccca ggaaagtatg catcagacga ctgaaagtgg tactatggag 420aagaacagta gccatctgcc tgcaggcacc aagtcctgta caaggactta cctgaacaat 480catgtggtgc aggcagatac cattacaaca acaaatcaaa gtcttgcaag aactggtcct 540gaattattca agactgctcc ttttattgac aacatgtgtg atgatgctaa attagatgcc 600atggatgagg atgagcttct agcgagtatt gatgtggacc gaatagtcat ggaacattat 660caagcaacaa atacacccag agggtcatcc aaatctccat tagagaagtg caacttcaat 720ggatttgatg agaataattt accacaagaa ctctctataa tgtgtgacca cggtagcaag 780ctagcttttt gcccagaggc gaagtctcat ttgcttgaaa tgaaggataa cttgcttgca 840atatcccatg agcttattga cggtcaactc agccctcaac aatctgatga tcttcatcaa 900aagagagcac tcctaaagaa gcagattgag ctgcttgggg agtatacggc gaggttaacc 960caagatgaag agcgacagca gtctcattct atggcctcca caacagctca tcagggccat 1020caccccacta gcatcctaag tagctctttt gtaaaggata ccaatatatt ccgatcaccg 1080atttacacca ggaatgaacc tggggagagt ggtttatgct tttcttctgc tccatattcc 1140tatatggatg gtttaagcat gccattaccg tctgttcaga gagattacac tccaagggct 1200attgatatca gttacactga aggttctggt gataaacagt ggagtagtac acactttgca 1260tggactaagg aactcgaggc caacaacaaa ggagtatttg gaaaccgttc ttttcgccca 1320aatcaacgag aaataaccaa cgccacaatg agtgggaatg atgtttttgt tttgatgcca 1380actggtggtg gaaaaagttt gacatatcag cttccagcac tcatttgtaa tggcgttaca 1440ttggtagttt ctcctctcgt atcgctcatc caagaccaga tcatgcattt attgcaggca 1500aatatttctg cagcttacct tagcgccagc atggagtggt cagaacagca ggagatatta 1560agagaattaa tgtctcctac atgcacgtac aagttactgt atgttacgcc tgaaaagata 1620gccaagagtg atgctctgtt gagacaattg gaaaatttat attcgcgagg ccatctctct 1680agaattgtca ttgatgaagc ccactgtgtt agccagtggg gtcatgattt ccgacctgat 1740taccagcatc taggcatttt aaaacagaag ttcccgcaga cgccggtcct ggccttgaca 1800gcaacagcaa ctgcaagtgt caaggaagat gtcgtgcaag ttctaggcct tgcaaactgc 1860attattttca gacaaggttt taatcgtcca aatctgaggt attttgtatg gcccaagaca 1920aagaagtgcc tcgaggatat ccataacttt atacatgcaa atcataataa agaatgcggc 1980atcatatatt gcctttcgag gatggattgt gagaaagtgg ctgctaaatt aagggaatat 2040gggcaccagg catcacatta tcatggtagc atggatcctg aggatagagc aaatatccag 2100aaacagtgga gcaaggatag gatcaacata atatgtgcta cagttgcatt tgggatgggt 2160attaataaac ctgatgtccg ttttgttatc catcattccc tgcccaaatc aattgaagga 2220tatcatcagg agtgtggacg tgctggtcgt gacagtcagc tttcatcttg tgtcctgttc 2280tacaattatt ctgattatat tcgtctcaaa cacatggtta cccaaggatt tgcggagcaa 2340ggaacatcag caccacgagg aggttcttcg caggaacaag cgcttgaaac gcataaggaa 2400aatctcctgc gaatggttag ttactgcgaa aatgatgtgg actgcagacg tctactacag 2460ctgatccact ttggagagat gtttaatcct tcatgttgtg caaaaacatg tgataattgc

2520ttgaaagagt tgagatgggt caaaaaagat gtgaccaaca ttgctagaca attggttgat 2580ctggtaatga tgacaaagca aacatattca actactcata ttctcgaagt atacagaggt 2640tcagtaaacc aaaatgtcaa gaagcaccgc catgatactt tgagtcttca tggagctgga 2700aagcatctag ctaaaggtga agcagcgaga atattgcgcc atctagtaat tgaggaaata 2760ctcattgagg atgtcaaaaa gagcgaaaac tatggatctg tatcatctgt cttaaagact 2820aatcataaga aaagtggtga tcttctctct ggcaagcaca acgttgtcct caagttcccc 2880actcctgaga aggctcctaa gatgggtgta ctcgatgaat cgtcagttcc acgaattaat 2940aagactaatc aacagagtca agtggacggg agccttgcag ccgagcttta tgaagctttg 3000caatgcctta ggactcagat aatggatgaa aatccacaat tattggcata ccacatattt 3060aaaaacgaga cattgaagga aatcagcaac cgaatgccaa gaacgaaaga ggaacttgtg 3120gagataaatg gcatcggcaa gaacaagctg aacaagtacg gggaccgcgt gcttgcaacc 3180atagaggatt tcctcgccag atatccaaat gcgaccagga aaaccagcag cggcggcagc 3240aacgagcaca gcgaggcggt caagaagcga agaggcttct ccgtcaccaa cacctctacc 3300aactgtgacg actttgagga acgcacggtc cagtccaaga aacgcgctgc aaagacacgt 3360acaaggcagg aaatatctga tgctgccagc atcgtccagg acgtccgcta catagatctt 3420gagctagatg gttgtgaaca agtcaatgaa gtgccataca gtgtacaaaa gcctgtggct 3480tctggtaggg ttttacctgc gtggcagtcc gccagaatag cctag 35251001174PRTProtein 100Met Ile Lys Pro Arg Val Asn Trp Ser Asp His Ala Asn Ala Val Gln1 5 10 15Ser Ser Cys Ile Lys Asp Glu Phe Leu Ser Ser Ser Phe Leu Phe Ser 20 25 30Leu Pro Thr Gln Arg Pro Asn Gln Glu Ala Asp Cys Thr Gly Met Leu 35 40 45Pro Leu Arg Ser Ala Ala Cys Arg Ile Gln Gly Leu Glu Arg Leu Gln 50 55 60Ala Pro Ser Ile Glu Lys Ala Trp Arg Ser Leu Arg Asn Thr Gln Val65 70 75 80Ala Arg Lys Asn Tyr Leu Arg Pro Gly Leu Ser Gly Lys Val Lys Asp 85 90 95Cys Asp Ser Asp His Ala His Thr Tyr Gly Thr Ser Ser Ser Tyr Asn 100 105 110Val Asn Lys Val Asp Ser Val Ser Arg Asn Arg Asn Pro Thr Gln Glu 115 120 125Ser Met His Gln Thr Thr Glu Ser Gly Thr Met Glu Lys Asn Ser Ser 130 135 140His Leu Pro Ala Gly Thr Lys Ser Cys Thr Arg Thr Tyr Leu Asn Asn145 150 155 160His Val Val Gln Ala Asp Thr Ile Thr Thr Thr Asn Gln Ser Leu Ala 165 170 175Arg Thr Gly Pro Glu Leu Phe Lys Thr Ala Pro Phe Ile Asp Asn Met 180 185 190Cys Asp Asp Ala Lys Leu Asp Ala Met Asp Glu Asp Glu Leu Leu Ala 195 200 205Ser Ile Asp Val Asp Arg Ile Val Met Glu His Tyr Gln Ala Thr Asn 210 215 220Thr Pro Arg Gly Ser Ser Lys Ser Pro Leu Glu Lys Cys Asn Phe Asn225 230 235 240Gly Phe Asp Glu Asn Asn Leu Pro Gln Glu Leu Ser Ile Met Cys Asp 245 250 255His Gly Ser Lys Leu Ala Phe Cys Pro Glu Ala Lys Ser His Leu Leu 260 265 270Glu Met Lys Asp Asn Leu Leu Ala Ile Ser His Glu Leu Ile Asp Gly 275 280 285Gln Leu Ser Pro Gln Gln Ser Asp Asp Leu His Gln Lys Arg Ala Leu 290 295 300Leu Lys Lys Gln Ile Glu Leu Leu Gly Glu Tyr Thr Ala Arg Leu Thr305 310 315 320Gln Asp Glu Glu Arg Gln Gln Ser His Ser Met Ala Ser Thr Thr Ala 325 330 335His Gln Gly His His Pro Thr Ser Ile Leu Ser Ser Ser Phe Val Lys 340 345 350Asp Thr Asn Ile Phe Arg Ser Pro Ile Tyr Thr Arg Asn Glu Pro Gly 355 360 365Glu Ser Gly Leu Cys Phe Ser Ser Ala Pro Tyr Ser Tyr Met Asp Gly 370 375 380Leu Ser Met Pro Leu Pro Ser Val Gln Arg Asp Tyr Thr Pro Arg Ala385 390 395 400Ile Asp Ile Ser Tyr Thr Glu Gly Ser Gly Asp Lys Gln Trp Ser Ser 405 410 415Thr His Phe Ala Trp Thr Lys Glu Leu Glu Ala Asn Asn Lys Gly Val 420 425 430Phe Gly Asn Arg Ser Phe Arg Pro Asn Gln Arg Glu Ile Thr Asn Ala 435 440 445Thr Met Ser Gly Asn Asp Val Phe Val Leu Met Pro Thr Gly Gly Gly 450 455 460Lys Ser Leu Thr Tyr Gln Leu Pro Ala Leu Ile Cys Asn Gly Val Thr465 470 475 480Leu Val Val Ser Pro Leu Val Ser Leu Ile Gln Asp Gln Ile Met His 485 490 495Leu Leu Gln Ala Asn Ile Ser Ala Ala Tyr Leu Ser Ala Ser Met Glu 500 505 510Trp Ser Glu Gln Gln Glu Ile Leu Arg Glu Leu Met Ser Pro Thr Cys 515 520 525Thr Tyr Lys Leu Leu Tyr Val Thr Pro Glu Lys Ile Ala Lys Ser Asp 530 535 540Ala Leu Leu Arg Gln Leu Glu Asn Leu Tyr Ser Arg Gly His Leu Ser545 550 555 560Arg Ile Val Ile Asp Glu Ala His Cys Val Ser Gln Trp Gly His Asp 565 570 575Phe Arg Pro Asp Tyr Gln His Leu Gly Ile Leu Lys Gln Lys Phe Pro 580 585 590Gln Thr Pro Val Leu Ala Leu Thr Ala Thr Ala Thr Ala Ser Val Lys 595 600 605Glu Asp Val Val Gln Val Leu Gly Leu Ala Asn Cys Ile Ile Phe Arg 610 615 620Gln Gly Phe Asn Arg Pro Asn Leu Arg Tyr Phe Val Trp Pro Lys Thr625 630 635 640Lys Lys Cys Leu Glu Asp Ile His Asn Phe Ile His Ala Asn His Asn 645 650 655Lys Glu Cys Gly Ile Ile Tyr Cys Leu Ser Arg Met Asp Cys Glu Lys 660 665 670Val Ala Ala Lys Leu Arg Glu Tyr Gly His Gln Ala Ser His Tyr His 675 680 685Gly Ser Met Asp Pro Glu Asp Arg Ala Asn Ile Gln Lys Gln Trp Ser 690 695 700Lys Asp Arg Ile Asn Ile Ile Cys Ala Thr Val Ala Phe Gly Met Gly705 710 715 720Ile Asn Lys Pro Asp Val Arg Phe Val Ile His His Ser Leu Pro Lys 725 730 735Ser Ile Glu Gly Tyr His Gln Glu Cys Gly Arg Ala Gly Arg Asp Ser 740 745 750Gln Leu Ser Ser Cys Val Leu Phe Tyr Asn Tyr Ser Asp Tyr Ile Arg 755 760 765Leu Lys His Met Val Thr Gln Gly Phe Ala Glu Gln Gly Thr Ser Ala 770 775 780Pro Arg Gly Gly Ser Ser Gln Glu Gln Ala Leu Glu Thr His Lys Glu785 790 795 800Asn Leu Leu Arg Met Val Ser Tyr Cys Glu Asn Asp Val Asp Cys Arg 805 810 815Arg Leu Leu Gln Leu Ile His Phe Gly Glu Met Phe Asn Pro Ser Cys 820 825 830Cys Ala Lys Thr Cys Asp Asn Cys Leu Lys Glu Leu Arg Trp Val Lys 835 840 845Lys Asp Val Thr Asn Ile Ala Arg Gln Leu Val Asp Leu Val Met Met 850 855 860Thr Lys Gln Thr Tyr Ser Thr Thr His Ile Leu Glu Val Tyr Arg Gly865 870 875 880Ser Val Asn Gln Asn Val Lys Lys His Arg His Asp Thr Leu Ser Leu 885 890 895His Gly Ala Gly Lys His Leu Ala Lys Gly Glu Ala Ala Arg Ile Leu 900 905 910Arg His Leu Val Ile Glu Glu Ile Leu Ile Glu Asp Val Lys Lys Ser 915 920 925Glu Asn Tyr Gly Ser Val Ser Ser Val Leu Lys Thr Asn His Lys Lys 930 935 940Ser Gly Asp Leu Leu Ser Gly Lys His Asn Val Val Leu Lys Phe Pro945 950 955 960Thr Pro Glu Lys Ala Pro Lys Met Gly Val Leu Asp Glu Ser Ser Val 965 970 975Pro Arg Ile Asn Lys Thr Asn Gln Gln Ser Gln Val Asp Gly Ser Leu 980 985 990Ala Ala Glu Leu Tyr Glu Ala Leu Gln Cys Leu Arg Thr Gln Ile Met 995 1000 1005Asp Glu Asn Pro Gln Leu Leu Ala Tyr His Ile Phe Lys Asn Glu 1010 1015 1020Thr Leu Lys Glu Ile Ser Asn Arg Met Pro Arg Thr Lys Glu Glu 1025 1030 1035Leu Val Glu Ile Asn Gly Ile Gly Lys Asn Lys Leu Asn Lys Tyr 1040 1045 1050Gly Asp Arg Val Leu Ala Thr Ile Glu Asp Phe Leu Ala Arg Tyr 1055 1060 1065Pro Asn Ala Thr Arg Lys Thr Ser Ser Gly Gly Ser Asn Glu His 1070 1075 1080Ser Glu Ala Val Lys Lys Arg Arg Gly Phe Ser Val Thr Asn Thr 1085 1090 1095Ser Thr Asn Cys Asp Asp Phe Glu Glu Arg Thr Val Gln Ser Lys 1100 1105 1110Lys Arg Ala Ala Lys Thr Arg Thr Arg Gln Glu Ile Ser Asp Ala 1115 1120 1125Ala Ser Ile Val Gln Asp Val Arg Tyr Ile Asp Leu Glu Leu Asp 1130 1135 1140Gly Cys Glu Gln Val Asn Glu Val Pro Tyr Ser Val Gln Lys Pro 1145 1150 1155Val Ala Ser Gly Arg Val Leu Pro Ala Trp Gln Ser Ala Arg Ile 1160 1165 1170Ala1011419PRTProtein 101Ala Thr Gly Ala Ala Gly Cys Ala Cys Gly Gly Thr Gly Thr Ala Ala1 5 10 15Thr Thr Gly Ala Thr Gly Ala Thr Ala Ala Ala Gly Ala Ala Gly Thr 20 25 30Thr Gly Ala Gly Gly Thr Gly Ala Gly Ala Ala Cys Thr Cys Cys Thr 35 40 45Thr Thr Gly Thr Thr Cys Ala Gly Ala Cys Ala Gly Gly Cys Ala Gly 50 55 60Ala Ala Thr Cys Cys Thr Cys Thr Gly Cys Thr Cys Gly Ala Cys Ala65 70 75 80Gly Ala Cys Thr Cys Gly Cys Ala Thr Cys Ala Ala Thr Cys Thr Gly 85 90 95Gly Ala Cys Thr Cys Cys Thr Thr Cys Gly Gly Gly Thr Thr Cys Thr 100 105 110Cys Cys Thr Cys Ala Gly Ala Thr Gly Ala Thr Gly Ala Cys Thr Thr 115 120 125Thr Gly Ala Ala Ala Cys Gly Thr Thr Gly Gly Ala Gly Thr Cys Cys 130 135 140Cys Ala Thr Thr Gly Thr Gly Ala Thr Cys Gly Thr Thr Cys Ala Gly145 150 155 160Thr Cys Ala Gly Thr Ala Cys Cys Cys Ala Gly Ala Ala Gly Ala Ala 165 170 175Gly Gly Thr Gly Ala Ala Cys Ala Gly Ala Gly Gly Ala Ala Ala Cys 180 185 190Ala Ala Thr Ala Gly Ala Thr Gly Thr Gly Ala Gly Thr Cys Ala Thr 195 200 205Cys Cys Ala Cys Thr Thr Cys Ala Ala Cys Thr Thr Cys Ala Ala Ala 210 215 220Cys Ala Gly Ala Gly Ala Ala Ala Cys Thr Cys Thr Ala Ala Gly Thr225 230 235 240Thr Ala Thr Cys Ala Gly Cys Ala Gly Cys Thr Cys Ala Ala Cys Ala 245 250 255Thr Gly Gly Ala Cys Ala Cys Cys Thr Thr Thr Gly Thr Gly Cys Thr 260 265 270Thr Ala Thr Gly Cys Cys Ala Ala Cys Ala Gly Gly Thr Gly Gly Thr 275 280 285Gly Gly Gly Ala Ala Gly Ala Gly Cys Thr Thr Gly Thr Gly Thr Thr 290 295 300Ala Thr Cys Ala Gly Cys Thr Ala Cys Cys Thr Gly Cys Ala Ala Cys305 310 315 320Ala Cys Thr Gly Cys Ala Cys Cys Cys Ala Gly Gly Thr Gly Thr Thr 325 330 335Ala Cys Gly Gly Thr Thr Gly Thr Thr Gly Thr Ala Thr Gly Cys Cys 340 345 350Cys Thr Cys Thr Ala Cys Thr Gly Thr Cys Ala Cys Thr Thr Ala Thr 355 360 365Thr Gly Ala Gly Gly Ala Thr Cys Ala Ala Ala Thr Thr Gly Thr Gly 370 375 380Gly Cys Ala Thr Thr Ala Ala Ala Cys Thr Thr Cys Ala Ala Gly Thr385 390 395 400Thr Thr Gly Cys Thr Ala Thr Ala Cys Cys Ala Gly Cys Ala Gly Cys 405 410 415Ala Thr Thr Thr Thr Thr Gly Ala Ala Cys Thr Cys Thr Cys Ala Gly 420 425 430Cys Ala Gly Ala Cys Ala Cys Cys Thr Thr Cys Ala Cys Ala Gly Thr 435 440 445Cys Ala Thr Cys Thr Gly Cys Ala Gly Thr Ala Ala Thr Cys Cys Ala 450 455 460Ala Gly Ala Gly Cys Thr Thr Ala Gly Ala Ala Gly Thr Gly Gly Thr465 470 475 480Ala Ala Ala Cys Cys Gly Thr Cys Ala Thr Thr Cys Ala Ala Ala Cys 485 490 495Thr Cys Cys Thr Cys Thr Ala Cys Gly Thr Cys Ala Cys Thr Cys Cys 500 505 510Thr Gly Ala Ala Ala Gly Ala Ala Thr Gly Gly Cys Thr Gly Gly Ala 515 520 525Ala Ala Cys Ala Gly Cys Thr Cys Ala Thr Thr Thr Ala Thr Thr Gly 530 535 540Gly Gly Ala Thr Cys Cys Thr Cys Ala Thr Ala Gly Gly Thr Thr Thr545 550 555 560Ala Cys Ala Cys Cys Ala Gly Ala Gly Gly Gly Gly Thr Thr Thr Ala 565 570 575Cys Thr Gly Gly Cys Gly Ala Gly Ala Thr Thr Thr Gly Thr Gly Ala 580 585 590Thr Thr Gly Ala Thr Gly Ala Ala Gly Cys Cys Cys Ala Thr Thr Gly 595 600 605Thr Gly Thr Ala Ala Gly Thr Cys Ala Ala Thr Gly Gly Gly Gly Ala 610 615 620Cys Ala Thr Gly Ala Cys Thr Thr Cys Cys Gly Cys Cys Cys Ala Gly625 630 635 640Ala Thr Thr Ala Cys Cys Gly Ala Gly Gly Cys Cys Thr Gly Gly Gly 645 650 655Ala Thr Gly Cys Cys Thr Cys Ala Ala Ala Cys Ala Gly Ala Ala Cys 660 665 670Thr Thr Cys Cys Cys Thr Cys Gly Ala Gly Thr Ala Cys Cys Ala Ala 675 680 685Thr Thr Ala Thr Gly Gly Cys Thr Thr Thr Ala Ala Cys Ala Gly Cys 690 695 700Thr Ala Cys Ala Gly Cys Gly Ala Cys Thr Gly Cys Ala Thr Cys Thr705 710 715 720Gly Thr Cys Thr Gly Cys Ala Ala Gly Gly Ala Cys Ala Thr Ala Cys 725 730 735Thr Ala Ala Gly Thr Ala Cys Cys Thr Thr Gly Ala Gly Gly Ala Thr 740 745 750Cys Cys Cys Thr Ala Ala Thr Gly Cys Ala Ala Cys Gly Gly Thr Ala 755 760 765Cys Thr Cys Ala Ala Gly Ala Gly Gly Ala Gly Cys Thr Thr Thr Gly 770 775 780Ala Cys Ala Gly Ala Ala Cys Ala Ala Ala Cys Cys Thr Gly Ala Ala785 790 795 800Thr Thr Ala Thr Gly Ala Gly Gly Thr Gly Ala Thr Thr Gly Gly Cys 805 810 815Ala Ala Gly Ala Cys Ala Ala Ala Ala Ala Cys Thr Cys Cys Ala Cys 820 825 830Ala Gly Ala Ala Gly Cys Ala Gly Cys Thr Gly Gly Gly Thr Gly Ala 835 840 845Thr Ala Thr Cys Cys Thr Ala Ala Ala Ala Gly Ala Gly Cys Gly Thr 850 855 860Thr Thr Cys Ala Thr Gly Ala Ala Cys Ala Thr Gly Thr Cys Thr Gly865 870 875 880Gly Thr Ala Thr Cys Gly Thr Gly Thr Ala Cys Thr Gly Thr Cys Thr 885 890 895Gly Thr Cys Cys Ala Ala Ala Ala Ala Thr Gly Ala Ala Thr Gly Thr 900 905 910Gly Cys Thr Gly Ala Cys Ala Cys Thr Gly Cys Cys Ala Ala Gly Thr 915 920 925Thr Cys Thr Thr Gly Ala Gly Gly Gly Ala Gly Ala Ala Gly Thr Ala 930 935 940Cys Ala Ala Gly Ala Thr Ala Ala Ala Ala Thr Gly Cys Gly Cys Ala945 950 955 960Cys Ala Thr Thr Ala Cys Cys Ala Cys Gly Cys Thr Gly Gly Cys Thr 965 970 975Thr Gly Gly Cys Thr Gly Cys Thr Cys Gly Thr Cys Ala Ala Cys Gly 980 985 990Ala Thr Cys Cys Ala Ala Thr Gly Thr Ala Cys Ala Ala Gly Gly Ala 995 1000 1005Ala Ala Ala Thr Gly Gly Cys Ala Cys Ala Gly Cys Gly Gly Ala 1010 1015 1020Gly Ala Gly Gly Thr Cys Ala Ala Ala Gly Thr Cys Ala Thr Thr 1025 1030 1035Thr Gly Thr Gly Cys Gly Ala Cys Cys Ala Thr Ala Gly Cys Ala 1040 1045 1050Thr Thr Thr Gly Gly Cys Ala Thr Gly Gly Gly Ala Ala Thr Ala 1055 1060 1065Gly Ala Cys Ala Ala Ala Cys Cys Thr Gly Ala Thr Gly Thr Gly 1070 1075 1080Cys Gly Cys Thr Thr Thr Gly Thr Thr Ala Thr Cys Cys Ala Cys 1085 1090 1095Ala Ala Cys Ala Cys Cys Ala Thr Gly Thr Cys Ala Ala Ala Ala 1100 1105 1110Thr Cys Gly Ala Thr Ala Gly Ala Ala Ala Gly Cys Thr Ala Cys

1115 1120 1125Thr Ala Thr Cys Ala Gly Gly Ala Gly Thr Cys Ala Gly Gly Gly 1130 1135 1140Ala Gly Ala Gly Cys Ala Gly Gly Ala Ala Gly Ala Gly Ala Cys 1145 1150 1155Ala Ala Thr Cys Thr Thr Cys Cys Gly Gly Cys Ala Cys Ala Thr 1160 1165 1170Thr Gly Cys Ala Thr Thr Gly Thr Gly Thr Thr Ala Thr Ala Thr 1175 1180 1185Cys Ala Gly Ala Ala Ala Ala Ala Gly Gly Ala Cys Cys Thr Cys 1190 1195 1200Gly Gly Thr Cys Gly Ala Ala Thr Thr Gly Thr Ala Thr Gly Cys 1205 1210 1215Ala Thr Gly Cys Thr Gly Ala Gly Gly Ala Ala Thr Thr Cys Ala 1220 1225 1230Gly Gly Gly Ala Ala Cys Thr Thr Cys Ala Ala Gly Ala Gly Thr 1235 1240 1245Gly Ala Gly Ala Gly Cys Thr Thr Cys Ala Ala Gly Gly Thr Thr 1250 1255 1260Gly Cys Ala Ala Thr Gly Gly Ala Gly Cys Ala Ala Gly Cys Ala 1265 1270 1275Ala Ala Gly Ala Ala Ala Ala Thr Gly Cys Ala Ala Ala Cys Ala 1280 1285 1290Thr Ala Thr Thr Gly Cys Gly Ala Gly Cys Thr Gly Ala Ala Gly 1295 1300 1305Ala Cys Ala Gly Ala Ala Thr Gly Cys Cys Gly Gly Ala Gly Gly 1310 1315 1320Cys Ala Ala Ala Cys Thr Cys Thr Thr Cys Thr Thr Gly Gly Cys 1325 1330 1335Cys Ala Cys Thr Thr Cys Gly Gly Thr Gly Ala Gly Cys Ala Gly 1340 1345 1350Thr Ala Thr Gly Ala Cys Ala Gly Gly Cys Ala Ala Ala Gly Gly 1355 1360 1365Thr Gly Cys Ala Ala Ala Cys Ala Thr Gly Gly Thr Thr Gly Thr 1370 1375 1380Ala Gly Cys Cys Cys Thr Thr Gly Cys Gly Ala Cys Ala Ala Cys 1385 1390 1395Thr Gly Cys Ala Thr Ala Ala Ala Gly Ala Thr Thr Cys Cys Cys 1400 1405 1410Thr Cys Thr Thr Gly Ala 1415102472PRTProtein 102Met Lys His Gly Val Ile Asp Asp Lys Glu Val Glu Val Arg Thr Pro1 5 10 15Leu Phe Arg Gln Ala Glu Ser Ser Ala Arg Gln Thr Arg Ile Asn Leu 20 25 30Asp Ser Phe Gly Phe Ser Ser Asp Asp Asp Phe Glu Thr Leu Glu Ser 35 40 45His Cys Asp Arg Ser Val Ser Thr Gln Lys Lys Val Asn Arg Gly Asn 50 55 60Asn Arg Cys Glu Ser Ser Thr Ser Thr Ser Asn Arg Glu Thr Leu Ser65 70 75 80Tyr Gln Gln Leu Asn Met Asp Thr Phe Val Leu Met Pro Thr Gly Gly 85 90 95Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Thr Leu His Pro Gly Val 100 105 110Thr Val Val Val Cys Pro Leu Leu Ser Leu Ile Glu Asp Gln Ile Val 115 120 125Ala Leu Asn Phe Lys Phe Ala Ile Pro Ala Ala Phe Leu Asn Ser Gln 130 135 140Gln Thr Pro Ser Gln Ser Ser Ala Val Ile Gln Glu Leu Arg Ser Gly145 150 155 160Lys Pro Ser Phe Lys Leu Leu Tyr Val Thr Pro Glu Arg Met Ala Gly 165 170 175Asn Ser Ser Phe Ile Gly Ile Leu Ile Gly Leu His Gln Arg Gly Leu 180 185 190Leu Ala Arg Phe Val Ile Asp Glu Ala His Cys Val Ser Gln Trp Gly 195 200 205His Asp Phe Arg Pro Asp Tyr Arg Gly Leu Gly Cys Leu Lys Gln Asn 210 215 220Phe Pro Arg Val Pro Ile Met Ala Leu Thr Ala Thr Ala Thr Ala Ser225 230 235 240Val Cys Lys Asp Ile Leu Ser Thr Leu Arg Ile Pro Asn Ala Thr Val 245 250 255Leu Lys Arg Ser Phe Asp Arg Thr Asn Leu Asn Tyr Glu Val Ile Gly 260 265 270Lys Thr Lys Thr Pro Gln Lys Gln Leu Gly Asp Ile Leu Lys Glu Arg 275 280 285Phe Met Asn Met Ser Gly Ile Val Tyr Cys Leu Ser Lys Asn Glu Cys 290 295 300Ala Asp Thr Ala Lys Phe Leu Arg Glu Lys Tyr Lys Ile Lys Cys Ala305 310 315 320His Tyr His Ala Gly Leu Ala Ala Arg Gln Arg Ser Asn Val Gln Gly 325 330 335Lys Trp His Ser Gly Glu Val Lys Val Ile Cys Ala Thr Ile Ala Phe 340 345 350Gly Met Gly Ile Asp Lys Pro Asp Val Arg Phe Val Ile His Asn Thr 355 360 365Met Ser Lys Ser Ile Glu Ser Tyr Tyr Gln Glu Ser Gly Arg Ala Gly 370 375 380Arg Asp Asn Leu Pro Ala His Cys Ile Val Leu Tyr Gln Lys Lys Asp385 390 395 400Leu Gly Arg Ile Val Cys Met Leu Arg Asn Ser Gly Asn Phe Lys Ser 405 410 415Glu Ser Phe Lys Val Ala Met Glu Gln Ala Lys Lys Met Gln Thr Tyr 420 425 430Cys Glu Leu Lys Thr Glu Cys Arg Arg Gln Thr Leu Leu Gly His Phe 435 440 445Gly Glu Gln Tyr Asp Arg Gln Arg Cys Lys His Gly Cys Ser Pro Cys 450 455 460Asp Asn Cys Ile Lys Ile Pro Ser465 4701034692DNADNA 103atggcttccc gtcccgcgca cgacctgctt caacgcgtct ttggttacga cgatttccgt 60ggtccgcagc aggacatcgt ggagcatgtg gctgccggtc acgacgccct ggtgctgatg 120cccaccggcg gcggcaaatc gctgtgctac caggtcccag ccctgctgcg tgacggttgc 180ggcatcgtca tctcgccgct gatcgcactg atgcaggacc aggtcgaagc cctgcgccag 240ctcggcgtgc gcgccgagta cctgaattca accctggacg ccgagaccgc cggccgcgtc 300gagcgcgagc tgctcgccgg cgaactggac atgctgtatg tcgcccctga gcggctgctg 360agcgggcgtt tcctgtcgct gctgtcgcgc agccagatcg ccctgttcgc catcgacgaa 420gcacactgcg tgtcgcagtg gggccatgac ttccgccccg aatatcgcca gttgaccgtg 480ctgcacgagc gttggccgca gatcccgcgg atcgcgctga ccgccaccgc cgatccgccg 540acccagcgcg agatcgccga gcgcctcgat ctgcaggaag cgcgccattt tgtcagttcc 600ttcgaccgcc ccaacatccg ctacaccgtc gtgcagaagg acaacgcccg caagcagctg 660accgacttcc tgcgcggcca ccgtggcgag gccggcatcg tctactgcat gtcgcggcgc 720aaggtcgagg agaccgctga attcctctgc ggccaaggcg tcaacgctct gccttaccac 780gccggcctgc cgccggaagt gcgcgccagc aaccagcgcc gcttcctgcg cgaggacggc 840atcgtgatgt gtgccaccat cgccttcggc atgggcatcg acaagccgga cgtgcgtttc 900gtcgcgcata ccgacctgcc caagtcgatg gagggctact accaggaaac cggacgcgca 960ggccgcgatg gcgaagccgc cgaggcctgg ctgtgctacg gcctgggtga tgtggtactg 1020ctcaagcaga tgatcgagca gtccgaggcg ggcgaagagc gcaagcagct ggaacgggcc 1080aagctcgacc atctgctggg ctactgcgaa tcgatgcagt gccgccgcca ggtgctgctg 1140gccggcttcg gcgaaaccta cccccaaccg tgcggcaact gcgacaactg cctgacgcca 1200ccggcctcgt gggacgcgac cataccggca cagaaggcgc tgagctgcgt ctaccgcagc 1260ggccagcgct tcggtgtcgg ccacctgatc gacatcctgc gcggcagcga gaacgagaag 1320gtgaggcagc agggccacga caagctgagc acttatgcca tcggccgcga cctggatgca 1380cgcacctggc gcagcgtgtt ccgccagctg gtcgcggcca gcctgctgga agtggacagc 1440gagggccacg gcggcctgcg cctgaccgac gccagccgcg acgtgctgac cggccgccgc 1500cagatcagca tgcgccgcga cccggccagc agcagcagcg gacgcgagcg cagtgcgcag 1560cgcaccggcc tgtcggtact gccgcaggac ctggccctgt tcaacgcgct gcgcggcctg 1620cgcgccgaac tggcccggga acagaacgta ccggcgttcg tgatcttcca cgacagcacc 1680ctgcgcaaca tcgccgagcg gcgcccgacc agcctggatg aactggcccg ggtcggcggc 1740atcggcggta ccaagctgag ccgctatggc ccgcgcctgg tcgagatcgt gcgcgaagaa 1800ggcctgttgc tcaacgggct gaacgcggcc atggcccgtg gtcacgaaga aatggggcgg 1860atggcccacg ccgcagccgc tgctgttgat ggcggcactg ccgactgcca ccaccacgcc 1920gccatgcagg ccgacccggc cccgcaggcc aaggccccgg cccacgacgc ccactgccag 1980atcaaggact gcgtgcgcag ctgcgcccag cacccgctgc tggtggtgca gccgttgccg 2040ttcatggccg gaccggcact gtcgctggcc ccgcagccga tgccggccac cggccggccg 2100gcgcccccgt ctgccgccga tctcacgccc tcccatcggc tgattccaca cgcaccggcc 2160tggccgccgg tggcgtggtt gccggcatcg ccgctgtcgg cgtgccgcag cgcgtgctcg 2220ccgccgccac tgccgcccca cgcctggccg gcgcccccgc cgtgctcagc gacacccgca 2280tcgaactggc catcggcgaa tcgctggcca actttcactg gccgcacccg tccggcgatc 2340accgtcaatg gatcgctgcc ggcaccgatc ctgcgctggc gcgaaggcca gaccgtggac 2400ctgttcgtgc gcaacacgct ggaccgccac ccgacctcga tccattggca cggcattctg 2460ctgccggcca acatggacgg cgtgcccggc ctgagcttca atggcatcgg ccccggtgag 2520acctaccact accacttcga actgaagcag tcgggtacct actggtacca cagccactcg 2580atgttccagg agcaggccgg cctgtacgga gcgctgatca tcgacccggc cgagccggcg 2640ccctaccagc acgaccgcga gcacgtgatc ctgctgtccg actggaccga catggacccc 2700ggcgcgctgt tccggcgcat gaagaagctc gccgagcatg acaactacta caagcgcacc 2760ctgcccgact tcctgcgtga cgtgaagcgc gacggttggt cggccgcgtt gtccgaccgt 2820ggcatgtggg ggcggatgcg gatgacgccc accgacatct ccgacatcaa tgcgcacacc 2880tacacctacc tgatgaatgg caccgcgccg gccggcaact ggaccgggct gttccgcagc 2940ggcgagaaag tactgctgcg cttcatcaac ggcgcctcga tgacctactt cgacgtgcgc 3000attcccggcc tgaagatgac cgtggtcgcc gccgacggcc agtacatcca tccggtcagc 3060atcgacgagt tccgcatcgc gccggccgaa acctacgacg tgctggtgga accgaccggg 3120caggacgcgt tcaccatctt ctgccaggac atgggccgca ccggttcccg cgcgcgaccc 3180acgcccgttg ctgacgatag cgacatgggg cacgacatgg gtagtggtgg ccatggtggc 3240cacgacatgg ccgcgatgaa gggcatggaa ggcggctgcg gcgccagcat ggaccacggt 3300gcgcacggcg gtagcgatgc cgccagcaag gcaccgaagc acccggccag cgaacgcaac 3360aacccgctgg tggacatgca gagctcggcc accgaaccga agctggacga tcccggcatc 3420ggcctgcgcg acaacggtcg ccaggtactc acctacggcg cgatgcgcag cctgttcgag 3480gaccccgatg gccgcgagcc gagccgcgag atcgagctgc acctgaccgg ccatatggag 3540aagttctcct ggtcattcga tggcattccg ttcgccagcg ccgagccgct gcggctgaac 3600tacggcgagc gcatgccatc tgatctggag aacgcgcagg gcgaattcca gctgcgcaag 3660cacaccatcg acatgccacc cggcacccgc cgcagttacc gcgtgcgcgc cgatgcgctc 3720ggtcgctggg cctaccactg ccatctgctc taccacatgg aagcgggcat gatgcgcgaa 3780aacagcaccg gccaggcctg ggaggccacc ggctggatcg gtggcaacat caaccgcctg 3840tggttgcgca ccgatggcga acgcagccgc ggccgcacgg aatcgtcgtc actggaagca 3900ctgtatggtc gcagcgtatc gccgtggtgg gacgtgctgg gcggcgtgcg ccaggacttc 3960cggccggccg actcgcgcac ctgggcggcc atcggcatcc agggccttgc accgtacaag 4020ttcgagagct cggcaacgct gtacatgggt tccggcggcc aggtgctggc caaggccgag 4080gtcgagtacg acgtgctgct gaccaaccgc ctgatcctgc agccgctgct ggaagccacc 4140atcgcagcca aggatgaacc ggagtacggc attggtcgcg gactgaacaa gatccgccgc 4200gccacccttg ccgatgtcga cgcgctgtcg accatcgcca tcaccaccta caacgaaacc 4260tggggcgact cgtatccggc gcaggagctg caggatttcc tgcaggcgca ctacagcagc 4320gaaccgcagc gcgccgagtt gtccgacccg cgcagtgcga tctggctgct gttggacggc 4380gacaacgtgg tcggctacct ggccgccggt gccaacaccc tgccgcatgc cgaagcccgc 4440gagggcgaca tcgaactgaa gcgcttctac atcctggccg actaccagaa cggcggccac 4500ggcgcgcgcc tgatggacgc gttcatggcc tggctggacc agccgcagcg ccgcaccctg 4560tgggtgggcg tctgggagga gaacttcggc gcgcagcgct tctacgcgcg ctacggctgc 4620agcaaggtcg gcgagtacga cttcatcgtc ggggatacgc gcgaccgcga gttcatcctg 4680cgccggctgt ga 46921041563PRTProtein 104Met Ala Ser Arg Pro Ala His Asp Leu Leu Gln Arg Val Phe Gly Tyr1 5 10 15Asp Asp Phe Arg Gly Pro Gln Gln Asp Ile Val Glu His Val Ala Ala 20 25 30Gly His Asp Ala Leu Val Leu Met Pro Thr Gly Gly Gly Lys Ser Leu 35 40 45Cys Tyr Gln Val Pro Ala Leu Leu Arg Asp Gly Cys Gly Ile Val Ile 50 55 60Ser Pro Leu Ile Ala Leu Met Gln Asp Gln Val Glu Ala Leu Arg Gln65 70 75 80Leu Gly Val Arg Ala Glu Tyr Leu Asn Ser Thr Leu Asp Ala Glu Thr 85 90 95Ala Gly Arg Val Glu Arg Glu Leu Leu Ala Gly Glu Leu Asp Met Leu 100 105 110Tyr Val Ala Pro Glu Arg Leu Leu Ser Gly Arg Phe Leu Ser Leu Leu 115 120 125Ser Arg Ser Gln Ile Ala Leu Phe Ala Ile Asp Glu Ala His Cys Val 130 135 140Ser Gln Trp Gly His Asp Phe Arg Pro Glu Tyr Arg Gln Leu Thr Val145 150 155 160Leu His Glu Arg Trp Pro Gln Ile Pro Arg Ile Ala Leu Thr Ala Thr 165 170 175Ala Asp Pro Pro Thr Gln Arg Glu Ile Ala Glu Arg Leu Asp Leu Gln 180 185 190Glu Ala Arg His Phe Val Ser Ser Phe Asp Arg Pro Asn Ile Arg Tyr 195 200 205Thr Val Val Gln Lys Asp Asn Ala Arg Lys Gln Leu Thr Asp Phe Leu 210 215 220Arg Gly His Arg Gly Glu Ala Gly Ile Val Tyr Cys Met Ser Arg Arg225 230 235 240Lys Val Glu Glu Thr Ala Glu Phe Leu Cys Gly Gln Gly Val Asn Ala 245 250 255Leu Pro Tyr His Ala Gly Leu Pro Pro Glu Val Arg Ala Ser Asn Gln 260 265 270Arg Arg Phe Leu Arg Glu Asp Gly Ile Val Met Cys Ala Thr Ile Ala 275 280 285Phe Gly Met Gly Ile Asp Lys Pro Asp Val Arg Phe Val Ala His Thr 290 295 300Asp Leu Pro Lys Ser Met Glu Gly Tyr Tyr Gln Glu Thr Gly Arg Ala305 310 315 320Gly Arg Asp Gly Glu Ala Ala Glu Ala Trp Leu Cys Tyr Gly Leu Gly 325 330 335Asp Val Val Leu Leu Lys Gln Met Ile Glu Gln Ser Glu Ala Gly Glu 340 345 350Glu Arg Lys Gln Leu Glu Arg Ala Lys Leu Asp His Leu Leu Gly Tyr 355 360 365Cys Glu Ser Met Gln Cys Arg Arg Gln Val Leu Leu Ala Gly Phe Gly 370 375 380Glu Thr Tyr Pro Gln Pro Cys Gly Asn Cys Asp Asn Cys Leu Thr Pro385 390 395 400Pro Ala Ser Trp Asp Ala Thr Ile Pro Ala Gln Lys Ala Leu Ser Cys 405 410 415Val Tyr Arg Ser Gly Gln Arg Phe Gly Val Gly His Leu Ile Asp Ile 420 425 430Leu Arg Gly Ser Glu Asn Glu Lys Val Arg Gln Gln Gly His Asp Lys 435 440 445Leu Ser Thr Tyr Ala Ile Gly Arg Asp Leu Asp Ala Arg Thr Trp Arg 450 455 460Ser Val Phe Arg Gln Leu Val Ala Ala Ser Leu Leu Glu Val Asp Ser465 470 475 480Glu Gly His Gly Gly Leu Arg Leu Thr Asp Ala Ser Arg Asp Val Leu 485 490 495Thr Gly Arg Arg Gln Ile Ser Met Arg Arg Asp Pro Ala Ser Ser Ser 500 505 510Ser Gly Arg Glu Arg Ser Ala Gln Arg Thr Gly Leu Ser Val Leu Pro 515 520 525Gln Asp Leu Ala Leu Phe Asn Ala Leu Arg Gly Leu Arg Ala Glu Leu 530 535 540Ala Arg Glu Gln Asn Val Pro Ala Phe Val Ile Phe His Asp Ser Thr545 550 555 560Leu Arg Asn Ile Ala Glu Arg Arg Pro Thr Ser Leu Asp Glu Leu Ala 565 570 575Arg Val Gly Gly Ile Gly Gly Thr Lys Leu Ser Arg Tyr Gly Pro Arg 580 585 590Leu Val Glu Ile Val Arg Glu Glu Gly Leu Leu Leu Asn Gly Leu Asn 595 600 605Ala Ala Met Ala Arg Gly His Glu Glu Met Gly Arg Met Ala His Ala 610 615 620Ala Ala Ala Ala Val Asp Gly Gly Thr Ala Asp Cys His His His Ala625 630 635 640Ala Met Gln Ala Asp Pro Ala Pro Gln Ala Lys Ala Pro Ala His Asp 645 650 655Ala His Cys Gln Ile Lys Asp Cys Val Arg Ser Cys Ala Gln His Pro 660 665 670Leu Leu Val Val Gln Pro Leu Pro Phe Met Ala Gly Pro Ala Leu Ser 675 680 685Leu Ala Pro Gln Pro Met Pro Ala Thr Gly Arg Pro Ala Pro Pro Ser 690 695 700Ala Ala Asp Leu Thr Pro Ser His Arg Leu Ile Pro His Ala Pro Ala705 710 715 720Trp Pro Pro Val Ala Trp Leu Pro Ala Ser Pro Leu Ser Ala Cys Arg 725 730 735Ser Ala Cys Ser Pro Pro Pro Leu Pro Pro His Ala Trp Pro Ala Pro 740 745 750Pro Pro Cys Ser Ala Thr Pro Ala Ser Asn Trp Pro Ser Ala Asn Arg 755 760 765Trp Pro Thr Phe Thr Gly Arg Thr Arg Pro Ala Ile Thr Val Asn Gly 770 775 780Ser Leu Pro Ala Pro Ile Leu Arg Trp Arg Glu Gly Gln Thr Val Asp785 790 795 800Leu Phe Val Arg Asn Thr Leu Asp Arg His Pro Thr Ser Ile His Trp 805 810 815His Gly Ile Leu Leu Pro Ala Asn Met Asp Gly Val Pro Gly Leu Ser 820 825 830Phe Asn Gly Ile Gly Pro Gly Glu Thr Tyr His Tyr His Phe Glu Leu 835 840 845Lys Gln Ser Gly Thr Tyr Trp Tyr His Ser His Ser Met Phe Gln Glu 850 855 860Gln Ala Gly Leu Tyr Gly Ala Leu Ile Ile Asp Pro Ala Glu Pro Ala865 870 875 880Pro Tyr Gln His Asp Arg Glu His Val Ile Leu Leu Ser Asp Trp Thr 885 890 895Asp Met Asp Pro Gly Ala Leu Phe Arg Arg Met Lys Lys Leu Ala Glu 900 905 910His Asp Asn Tyr Tyr Lys Arg Thr Leu Pro Asp Phe

Leu Arg Asp Val 915 920 925Lys Arg Asp Gly Trp Ser Ala Ala Leu Ser Asp Arg Gly Met Trp Gly 930 935 940Arg Met Arg Met Thr Pro Thr Asp Ile Ser Asp Ile Asn Ala His Thr945 950 955 960Tyr Thr Tyr Leu Met Asn Gly Thr Ala Pro Ala Gly Asn Trp Thr Gly 965 970 975Leu Phe Arg Ser Gly Glu Lys Val Leu Leu Arg Phe Ile Asn Gly Ala 980 985 990Ser Met Thr Tyr Phe Asp Val Arg Ile Pro Gly Leu Lys Met Thr Val 995 1000 1005Val Ala Ala Asp Gly Gln Tyr Ile His Pro Val Ser Ile Asp Glu 1010 1015 1020Phe Arg Ile Ala Pro Ala Glu Thr Tyr Asp Val Leu Val Glu Pro 1025 1030 1035Thr Gly Gln Asp Ala Phe Thr Ile Phe Cys Gln Asp Met Gly Arg 1040 1045 1050Thr Gly Ser Arg Ala Arg Pro Thr Pro Val Ala Asp Asp Ser Asp 1055 1060 1065Met Gly His Asp Met Gly Ser Gly Gly His Gly Gly His Asp Met 1070 1075 1080Ala Ala Met Lys Gly Met Glu Gly Gly Cys Gly Ala Ser Met Asp 1085 1090 1095His Gly Ala His Gly Gly Ser Asp Ala Ala Ser Lys Ala Pro Lys 1100 1105 1110His Pro Ala Ser Glu Arg Asn Asn Pro Leu Val Asp Met Gln Ser 1115 1120 1125Ser Ala Thr Glu Pro Lys Leu Asp Asp Pro Gly Ile Gly Leu Arg 1130 1135 1140Asp Asn Gly Arg Gln Val Leu Thr Tyr Gly Ala Met Arg Ser Leu 1145 1150 1155Phe Glu Asp Pro Asp Gly Arg Glu Pro Ser Arg Glu Ile Glu Leu 1160 1165 1170His Leu Thr Gly His Met Glu Lys Phe Ser Trp Ser Phe Asp Gly 1175 1180 1185Ile Pro Phe Ala Ser Ala Glu Pro Leu Arg Leu Asn Tyr Gly Glu 1190 1195 1200Arg Met Pro Ser Asp Leu Glu Asn Ala Gln Gly Glu Phe Gln Leu 1205 1210 1215Arg Lys His Thr Ile Asp Met Pro Pro Gly Thr Arg Arg Ser Tyr 1220 1225 1230Arg Val Arg Ala Asp Ala Leu Gly Arg Trp Ala Tyr His Cys His 1235 1240 1245Leu Leu Tyr His Met Glu Ala Gly Met Met Arg Glu Asn Ser Thr 1250 1255 1260Gly Gln Ala Trp Glu Ala Thr Gly Trp Ile Gly Gly Asn Ile Asn 1265 1270 1275Arg Leu Trp Leu Arg Thr Asp Gly Glu Arg Ser Arg Gly Arg Thr 1280 1285 1290Glu Ser Ser Ser Leu Glu Ala Leu Tyr Gly Arg Ser Val Ser Pro 1295 1300 1305Trp Trp Asp Val Leu Gly Gly Val Arg Gln Asp Phe Arg Pro Ala 1310 1315 1320Asp Ser Arg Thr Trp Ala Ala Ile Gly Ile Gln Gly Leu Ala Pro 1325 1330 1335Tyr Lys Phe Glu Ser Ser Ala Thr Leu Tyr Met Gly Ser Gly Gly 1340 1345 1350Gln Val Leu Ala Lys Ala Glu Val Glu Tyr Asp Val Leu Leu Thr 1355 1360 1365Asn Arg Leu Ile Leu Gln Pro Leu Leu Glu Ala Thr Ile Ala Ala 1370 1375 1380Lys Asp Glu Pro Glu Tyr Gly Ile Gly Arg Gly Leu Asn Lys Ile 1385 1390 1395Arg Arg Ala Thr Leu Ala Asp Val Asp Ala Leu Ser Thr Ile Ala 1400 1405 1410Ile Thr Thr Tyr Asn Glu Thr Trp Gly Asp Ser Tyr Pro Ala Gln 1415 1420 1425Glu Leu Gln Asp Phe Leu Gln Ala His Tyr Ser Ser Glu Pro Gln 1430 1435 1440Arg Ala Glu Leu Ser Asp Pro Arg Ser Ala Ile Trp Leu Leu Leu 1445 1450 1455Asp Gly Asp Asn Val Val Gly Tyr Leu Ala Ala Gly Ala Asn Thr 1460 1465 1470Leu Pro His Ala Glu Ala Arg Glu Gly Asp Ile Glu Leu Lys Arg 1475 1480 1485Phe Tyr Ile Leu Ala Asp Tyr Gln Asn Gly Gly His Gly Ala Arg 1490 1495 1500Leu Met Asp Ala Phe Met Ala Trp Leu Asp Gln Pro Gln Arg Arg 1505 1510 1515Thr Leu Trp Val Gly Val Trp Glu Glu Asn Phe Gly Ala Gln Arg 1520 1525 1530Phe Tyr Ala Arg Tyr Gly Cys Ser Lys Val Gly Glu Tyr Asp Phe 1535 1540 1545Ile Val Gly Asp Thr Arg Asp Arg Glu Phe Ile Leu Arg Arg Leu 1550 1555 15601051185DNADNA 105gcacgagcgc aaggcaagct ttccgcttcc tatttcggat tgggatcatc agcggctgta 60gcgtggaccc gacgggggtg tccggaccac atccctattt catcttggta ccccgtccgt 120ctccgatttc agaagcacgg cgggctcccc ggcagcctct accgagcaga aagctgagtt 180ctaccccaga accgaggcat ggaggacgaa gaaaacatcg agggagaact gttgctcgtg 240gagtcacaac tccacgacat ccaaggacaa attaaaacat tactcgatcg ccaagaggag 300ttgtatgaac gccaggcaca gttgaaggct ttgctcgaag catctaaatt gaccagaaat 360acaacaatta acacatcttc agttgctccg gaagattggt ctgggagctt cccatgggat 420ctggaggctg acgataccag gttcaatata tttggcattt cctcctaccg atcaaatcaa 480cgagaaataa ttaatgcagt catgagtgga agagatgttc tggtcataat ggcagctggt 540ggagggaaga gtctatgtta ccagctccca gctgtacttc gtgatggaat tgcactggtt 600gtcagtcctt tactttccct tattcaggac caggtcatgg gactgtcagc tttaggtata 660ccagcataca tgctaacttc aactaccaac aaggaagttg agaagttcat ctataagaca 720cttgataaag gagaaggaga actaaagata ttatatgtga cacctgaaaa gatctcaaaa 780agtaaaaggt tcatgtctaa gctcgagaaa tgccatcatg ccggtcgtct ttctctgatt 840gcaatagatg aggctcactg ctgtagccaa tggggtcatg attttcgtcc tgactacaag 900aatcttggca ttttgaaaat tcaatttccc agtgttccaa tgatagcttt aactgcaact 960gcaacaagta aggtccaaat ggatttaatg gagatgctcc acatcccgag atgcatcaag 1020tttgtcagca cagttaacag gcccaacctt ttttataagg tgtctgagaa atcgccagtt 1080ggaaaggttg tcattgatga gatcacaaag tttataagtg aatcataccc aaataatgag 1140tctggaatta tatactgctt ttcaaggaag gaatgtgaac aggtt 1185106870DNADNA 106cttgaggatc cccaacgctg tggtactgaa gaggagcttc gacagactga acctcaacta 60cgaggtaatc ggcaagacga aaactttcca gaagcagctg ggcgatctcc tgaaagagcg 120cttcatgaac gaatctggta tcgtgtactg tctctcgaag aacgagtgtg cagacactgc 180caagtttttg aggaagaaat acaagatcaa atgcgcgcac taccacgcta gcctggcagc 240tcgtcagcga accagtgtcc aggagaaatg gcacaacggg gaggttaagg tcatctgcgc 300taccatagcc ttcggcatgg ggatcgacaa acctgacgtg cgttttgtta tccacaacac 360attgtccaag tcaatagaaa gctactacca ggagtccggg agggcagggc gagatgagct 420tccggcacac tgtatcgtct tgtaccagaa gaaagacttc agccgtatcg tgtgcatgtt 480gaggaacggt gagaacttca ggagcgagag cttcagggtt gcgatggagc aagctaagaa 540gatgcaggca tactgcgagc tcaagaccga gtgccggaga caggcacttc tgcagcactt 600cggcgaacag tacgacaggc gaaggtgccg agacgggcct agcccctgcg acaactgcct 660caagacatag tttagggtaa taaactatgg cgataaaaaa tgccatgacg cttggttatg 720ctctgaactt gtgaggtgtg tgccacttcc acagtacatt cgtctgtgta tatgtagcat 780ccatagctca aacaagtggc cgcaactgca ctgtgtgtaa cgatggtctt tgttttcagt 840tggattgtga ggttcggggc tttaaaaaaa 8701073569DNADNA 107atggaggacg atgacgatga ccaacgcttg cttcacagcc ttggtgtcac gtccgcagac 60atccacgata ttgaaaggag aatcatatca caggcaacaa ctgatcctgc cgactcatct 120ggaccaacca tcaatggagg tcatcagcct gatgatgctc tcgccaaact gcatcacaaa 180ctgcgctctg tgcaaattga aattgatgct gtagcctcca ccatcaaagg agctaagctt 240aagcaaccat ccggaaataa accacatgag cataaaggca aggaccagcc agatcatcat 300ggagcaggac acctccagca agcccttgct gccgaccgtc ttacaagcct caggaaagct 360aaagcacaga tacagaaaga gatactacag tcacatcttt ctccatctgc ctccaatcga 420aaagataaaa tgctggccat gctggtccaa gacgagccga ggcacaaaaa gccacccgta 480gggcctaaaa acatcgtgaa acgcccgatg aaaactgtca cctatgatga tgacaacaac 540ttcgatgcag tgcttgatgg agcctctgcg ggatttatgg aaactgaaag ggaagaactg 600atcaggaagg gtttgttgac accattccat aagttgaagg gcttcgagaa acgtgtggaa 660ctacccgaac cttctcatag acaagatgat tctgcaggac aaactgaaga agccatggaa 720gcttccagga ttgctagagt tgctcagtcg ctaaagcaga ttgcacagaa ccgcccagca 780accaaattgc ttgattcaga gtctttacct aagctagatg cacctgctgc cccatttcag 840agacttggaa aacccctaaa gcgtcctgtc tctcccagtt cagatgagca ggaaaagaag 900agaccaagaa ataagaccaa aagaccactg cctggcaaga aatggaggaa agcaaactca 960attaaggaat catcattgga tgacaacgat gttggagagg cagctgtgtc agtttcagat 1020gatgatgaag atcaggttac agaaggctct gatgagttaa ctgatgttac ccttgaagga 1080ggtttgagaa ttcctggcac actttacacg caactatttg actaccagaa agtgggagtg 1140cagtggctat gggagttgca ttgtcaaagg gctggtggaa taattggaga tgaaatgggc 1200ctgggaaaga ctgtgcaggt cttgtcattt cttggttcct tgcataacag tgggctctac 1260aagcctagca ttgttgtttg tcctgtaacc cttttgcaac agtggcgaag ggaggccagt 1320agatggtatc caaagttcaa ggttgagatc ttacatgact ctgcaaacag ttcatctaaa 1380aagagcaaga ggtctagtga ttctgacagt gaagcttcct gggatagtga tcaggaagaa 1440gcggttacat gttcaaaacc cgcaaagaag tgggatgact tgatttcacg tgttgtgagt 1500tcaggatcag gtttgcttct gaccacatat gagcagttaa ggatcctagg ggagaagttg 1560cttgatatag aatggggata tgctgtattg gatgagggtc accgcattag gaatcctaat 1620gctgagatta ctcttgtgtg caagcaattg cagaccgtgc acaggataat tatgacaggt 1680gcacctattc aaaacaaact ttcggagctt tggtctctct ttgattttgt gttccctgga 1740aaactaggtg tcctgcctgt gtttgaggct gagttttctg ttccaattac tgttggtggg 1800tacgctaatg caacaccatt gcaagtgtcc acggcgtatc gatgtgctgt tgtcctacgt 1860gacctggtca tgccgtacct tcttagaaga atgaaagctg atgtcaatgc acagcttccc 1920aagaaaacag agcatgttct tttctgtagt ctaactactg agcaacgtgc tacttatcgt 1980gcatttcttg ctagttcgga ggtggaacaa atctttgatg gtaacagaaa ttccctttat 2040gggatagatg ttctaaggaa gatatgcaat catcctgatc tacttgagag agaacatgct 2100gctcagaatc ctgactatgg gaatccagaa agaagtggaa agatgaaagt ggttgagcaa 2160gttcttaaag tatggaaaga acaaggtcat cgtgttcttc ttttcactca gacacaacaa 2220atgcttgaca ttatggggaa cttcttgaca gcttgcgaat accaataccg aagaatggat 2280ggacttacac ctgcaaagca aagaatggca cttattgatg aattcaataa cacagatgaa 2340atttttattt tcattctgac cacgaaagtt ggtggactgg gtacgaattt gactggtgca 2400aaccggatta ttatatatga tcctgactgg aatccttcaa ctgacatgca ggctagggaa 2460cgtgcatggc gaattgggca aactagagat gtgacagttt atagactgat cacgcgtggg 2520acaatagagg agaaagtcta ccatcgtcag gtatacaagc atttcctcac aaacaaagta 2580ctgaaagacc ctcagcagag gcggtttttt aaagccagag acatgaagga tttgtttacg 2640ctgcaagatg atgacaataa tggctcaact gaaacatcaa atattttcag ccaattgtct 2700gaggatgtga atatcggagt tccgagtgac aagcaacaag accagctata tgcagcctct 2760gctacaccga caacctctgg gactgaaccg agctcatcca ggcatggaca gggtaaagaa 2820gaccattgcc ctgaccaagc agatgaagaa tgcaacattt tgaagagcct ttttgatgct 2880caaggcattc atagtgcgat caatcatgat gccataatga acgctaatga tgaccagaag 2940ctgcgcctag aagcagaagc tacacaggtg gcacaaaggg cagctgaagc tttacgccaa 3000tcacggatgc tcagaagtca tgaaagtttt tctgttccta catggactgg aagagctggt 3060gctgcggggg caccatcctc tgtccgcagg aagtttgggt caacactcaa tacccagttg 3120gttaattctt ctcagccatc agaaacttca aatggcaggg gccaaagtct tcaggtgggt 3180gctctaaatg gcaaagcact gtcctccgct gagcttctgg ccaggatacg tggaacccga 3240gagggagcag cttcagatgc actagaacat caactcaacc tgggatcagc ttccaatcac 3300acatcgagtt catcagggaa tggccgtgca tcaagctctt ctactaggag catgatcgta 3360cagcctgaag tcctaatccg ccaattgtgc accttcatac agcagcatgg tggttccgcc 3420agctcaacaa gtataactga acacttcaag aaccggatac tgtccaagga tatgctgctg 3480tttaagaatc tgctgaagga aatagctacg ttgcaaagag gtgcaaatgg tgcaacgtgg 3540gtgctgaaac ctgactacca gtaactagt 35691083453DNADNA 108atgcgcacaa gcaccacatc agatagccca tccccatctc cacaaaacaa agcctctttt 60aacacatcac gtggtgctgc atttagggat gaagaaccag gtgcaaaaga caatgaagtt 120gagaaaagga aaccattgat attacatttg aagaagcgtt caaccaagga actatctaca 180gataccacat catcaaagtc agggttactt ggaaagtctt cagaagagaa acaggagaaa 240cacggaagtg ctttgaaagt gaagaaacat ctgcatccca tggaattatc tccaaagaaa 300tataagaaca agaagcaaca caatcacaga gacagtaaga gatccgaagc aaaaaaggtc 360caatatttgg catcagatgt ggacagtgat tcttcaatgg aaccatctac ttctcttgag 420cacagcgaat cgccgccccc aaaaagaaaa tcgttggatg gaagaacacc tgcatcaagt 480accaagaaag gaaaaaagaa agtgaaattt attgataaaa agcaccctga gaatgctgtt 540catataactg aaaaggagca tggtggtgca ggagacaaaa taacaactca gggggatctg 600caggttgatc gcatcctagg ctgtcgactt cagacaagcc aaatcatttc acctgcccat 660gcttcatcag agcagattga tatggcccct cctagtgcat ccggtgcaac agaacctagt 720caagcccttt caaaaggact tcatgaagaa attcagtctt ctaatagtga tactaatgtg 780acagaggatg catgtgctga tgaattagca aacgatggtg gggaaaataa tttggattgt 840tctgatgctc aaaaggagag taatgttaga tcccatggac acaaggaatc acttaacgca 900aaagaaatca tgaatacagc atcagcatgt tccgctgatc aaattgtcac agttaaggat 960gctggagcag tacagacata tgtaacggct tcagtaaatg gtgaatatga gacagtaact 1020gatattccag aagaaaagaa tgacaccaaa catccagttt ccaaagctga cacagaagtc 1080cacactaaac aagaacatac acctgatagt aaattgcatg ggaaactaga aaactacaaa 1140gcaaagtacg gaacaggttt gataaacatc tgcaaagaac aatggtgcca accgcaacga 1200gttattgctc tgcgcacttc tttagatgaa atagaagagg ctttgatcaa atggtgtgcc 1260cttccatatg acgaatgcac gtgggaaaga ttagatgaac ctacaatggt gaagtatgca 1320catttggtca ctcagttcaa aaaatttgaa tcccaggctt tggataagga taagggaggt 1380agccatgcaa agccaaggga acaccaagag tttaatatgc tggttgagca gccaaaagaa 1440ctccagggag gcatgctctt ccctcatcaa ctggaagcat tgaactggct acgcaaatgc 1500tggtacaagt caaaaaatgt tatccttgct gatgagatgg gtcttggaaa gactgtgtct 1560gcctgtgctt ttctatcatc cctatgttgt gaatataaga ttaacttgcc atgtcttgtc 1620ttggttcctc tttctactat gcccaactgg atggctgaat ttgcatcatg ggcacctcat 1680ttaaatgttg tggagtatca tggttctgca cgggcaagat ctattattcg tcaatatgag 1740tggcatgagg gtgatgcaag ccagatgggt aaaatcaaga aatctcataa gttcaatgta 1800ttgctcacta cttatgaaat ggtgcttgtt gatgctgcat atcttcggtc tgtgtcatgg 1860gaggttctta tagtcgatga gggtcatcgt ctgaagaatt ctagcagcaa acttttcagt 1920ttactcaata cattatcatt tcagcataga gttttgctga ctggaactcc gttacagaat 1980aacattggtg aaatgtataa cttattgaac ttcttacaac ctgcttcttt cccttctcta 2040gcttcatttg aggagaaatt caatgacctt acaacaacag agaaagtgga ggagctgaag 2100aaccttgtag ctccacatat gcttcgaaga ctgaaaaagg atgcaatgca aaatatccct 2160ccaaagactg aacgaatggt gcctgttgaa ttgacatcaa tccaggctga atactaccgt 2220gctatgctta caaagaacta ccaagtattg cgcaatattg ggaaaggtgg tgctcaccag 2280tcattgttga acatagtaat gcaacttcgg aaagtctgca atcatccgta tcttattcct 2340ggaactgaac ctgaatcagg atcaccagag ttcttgcatg aaatgcgaat aaaggcctca 2400gcaaagttaa ctttgttgca ctctatgctt aaaatcctac acaaggatgg tcatcgagtt 2460cttatttttt ctcagatgac aaagcttctt gacatccttg aagattacct gacctgggag 2520tttggtccga aaacatttga aagagtggat ggttcagtat ctgtggcaga acgccaggca 2580gcaattgctc gttttaatca ggacaagagt cgttttgtat tcctgctatc tacgcggtca 2640tgtgggcttg gaattaattt ggcaactgca gatactgtta tcatatatga ttctgatttc 2700aatccacatg ctgatataca ggcaatgaac agagcacaca gaattggaca gtcaaacaga 2760cttttagttt acaggcttgt cgtgcgtgct agtgttgagg agcgtatctt gcaccttgcg 2820aagaaaaaat tgatgcttga tcaacttttt gttaacaaat cagaatcaca gaaggaagtg 2880gaagatatca ttcgctgggg aacagaggaa ctcttcagga atagcgatgt tgcagttaaa 2940gataataatg aagcttctgg tgctaaaaat gatgtagcag aggttgagtt taagcataaa 3000agaaaaactg gtggactagg cgatgtttat gaagacagat gtgctgatgg ttctgctaaa 3060tttaattggg atgaaaatgc tatcacaaag cttcttgaca gatccaacgt tccatcaaca 3120gtagctgaaa gcactgatgg ggacttggac aatgatatgc ttggcactgt aaagtcaata 3180gattggaacg atgagctgaa tgatgaccct ggtgccaccg aggacatccc aaatattgat 3240aatgatggtt gcgagcaggc atctgaagca aagcaggatg cagctaatcg tgttgaagaa 3300aatgaatggg ataaactctt acgtgtcaga tgggagcagt atcaaactga ggaggaagca 3360tctcttggtc gaggtaagcg tttaaggaag gctgtttctt acagggaaac atttgcaacc 3420attcctaatg aagctttaag cgagtagaac tag 34531092964DNADNA 109atggcggaca cggggagcct ggagaagatg gggcgagagc tcaagtgccc catctgcctg 60agccttctca gttcggcggt atccatctcc tgcaaccacg tcttctgcaa tgattgcctc 120acggaatcga tgaaatccac gtcgagctgc cccgtgtgca aggtcccgtt ccgacgacga 180gaaatgcgac cagcacctca catggacaat ctggtcagca ttttcaaaag catggaggct 240gcagcaggta ccaatgttgt ctcaacacag gaggctcctg tggtaaaact tgcagatgga 300tcagattgtg tcaacagcgg gaaaaattcc aaaaggtcac aaaaatcatt gacacgaaaa 360aggaaggtaa catccgagat ggaaaaaaat acagcaaagg atgctacagc ttctgcatcc 420caacctacta caaagccttc cttctctact aacaaaagaa tacaagtgaa accattccct 480gaatctgaga caccaataag agctgagaag attatgaagc ctgaagagcc aaaaaataat 540ctgaataatg atgttgaagg aaagaataaa gcagtggcat cgggtcaacc tggaagtcct 600tcattgtcac cctttttttg gctaagggaa caagaagaac aagaaggctg taccgctgag 660acgttaagtg aaacgcaatc tttagacaca cccttgcgtc ataatgcacc ctcttttagc 720gatattaaag attctgatga cgaaatccct ttaaatacaa ctccaaatag caaagctgcg 780gctacagaac tctttgacag tgaaatattt gaatggaccc agagaccatg ctctcctgaa 840ttgtattcca ctccattgaa aaagcagagt aaagctaaga gtaaactaga tcaaattgaa 900gagaagggtg atgaagaaga tgtgcatatt ggtggttcat ttgataagct gggcagtgca 960agtaatgcag ctcagcttgt caatacaaaa gcaacaaagc agaagagaaa gaaaacaagt 1020cccagtaaca aaaacagtgc aaaattgtcc aatcgtgctg agccctgcat aaaaaagtct 1080gatgccaatc aacaaggttc aaatagacgt aaaagtgctg ccctaaaatc ttgtcagaaa 1140agcagcagtg ctgtagggag gaatacttca ggtagaagaa acaaggcctc tagcaacagc 1200aagccaattc atggctctag tgataactcc ccagagtcat atcttcctaa ggagggtttg 1260gatgttgaag cacctgacaa acccctttct gaaaggatcc aaaacttgga gaaaactagt 1320cgacgaaagg gaagtgcaag gaagctggaa atggcaggga aaactatttc agatactaca 1380gagaagaata gtgagccaag aagtaagaga gtcagaagaa tgtctgacca cgctatagct 1440aaaccggttg aagttccttc aggatctgga aatgaaacag aaataccaca gcttcacacc 1500ctcacaaaag gcagcattca acgcaaatcc tccaacgcta gaagacatag caaagtttgt 1560ggagaacagg aaggtaagaa taaacttgag aacacgacaa tgacacctat tattttacat 1620gggaaatgcc aaaataaaga ggcagtatgt acagctcctt cagtaaggac tgcatctgtt 1680aagtacaagc aagcaaaatt tagcgaacaa ccagattgtt ttggaacgga gaactttgga 1740aaccttcaag catgccctgc acgtaatgtt ttactgaaga agtgtgaggt atctactttg

1800aaggtttcct gtgctttctg ccagaccgat gtcatcacag aggagtctgg agagatggtt 1860cattatcaaa atgggaagca agtccctgca gagttcaatg gaggagccaa tgtggtgcac 1920tctcacaaga actgccttga gtgggctcct gatgtctact tcgaagatga ttctgccttt 1980aatcttacaa ctgaattggc gagaagcaga cggatcaaat gtgcttgctg tggaattaaa 2040ggagctgcac ttggatgctt tgagatgagt tgtcggagaa gtttccactt cacctgtgct 2100aaactaatcc cagaatgcag atgggataat gaaaattttg tgatgttatg ccctctacat 2160cggtctacaa agttacccaa tgaaaattct gaacagcaaa agcaacctaa aaggaaaaca 2220acactcaaag ggtcatctca aataggatcc aatcaagatt gtggtaataa ctggaaatgg 2280ccatctggat caccacagaa gtgggttctc tgctgctcat cactttctag ttctgagaag 2340ggacttgtat cagaatttgc aaagttagct ggcgtgccta tttcggcaac ttggagtcca 2400aatgttaccc atgttattgc atcaactgat ctctctggtg cttgcaaacg gacgctgaag 2460tttctcatgg caatcttgaa tggcagatgg attgtctcca tagattgggt taaaacttgc 2520atggagtgca tggaaccaat tgatgagcac aaatttgaag tcgctactga tgttcatggg 2580atcactgatg gtcctaggtt aggaagatgc agggttattg acaggcaacc taagctgttc 2640gacagcatga ggttctacct ccatggggac tacacaaaat cctacagagg ctacctgcaa 2700gatctcgtgg ttgcagcagg tggaatagtt cttcagagga agcccgtatc aagagaccag 2760caaaagcttc ttgatgacag ctctgacctc ctcatcgttt acagcttcga gaatcaagat 2820agggcaaaat ccaaggccga aaccaaggct gctgatcgca ggcaggctga tgctcaggct 2880cttgcttgcg cttctggagg cagagttgtg agcagtgcat gggtgattga ctcaattgca 2940gcctgcaatc tgcaacctct ttga 29641104500DNADNA 110atggctgacc tcttcaacca agctttggat aagctggttg ctgctgatgg aatggccgaa 60gcgatcgagg attcagggaa aggtgcggtg ttctgcactg ggttgggggg atcagttgcc 120gtcagcgaga gggctgtaga gagggccaag gcattggttg gggaggtcgc ggaggagata 180agtaatgaga ggaggcaacc atttggtgat ggttctaatt tggagtgcgg attgggagaa 240agtaatgttt catttaaagg tggtgtacat aaagatagtt tgtctccgat gttccaaacc 300ggatcgggta aaatggtttc gctgagcaag ggctcaattc agaaggctag agctgtttta 360gaaggaaatg ccgagaattc ttctgtcatt gctgtacagt ctatgttcca tactggattg 420gttaggccag acccagtcag caggagctcc actgataatg caatgactgt tttggaggga 480caaacaaatc caaaacaagg agatgtggca gatgtgtatg acaaggaaaa ttttccattg 540ttccaaactg gttcaggtaa agctgtatcg gtcagtgtag catctatcca gaaagctaag 600gctgtcctgg agcaaaataa tacagaaaac acggaagatt ttggtaggcc tgaccaatct 660ctgattttcc aaactggttc gcgaagacca gtcttgatca gtgaaagatc tagctctgtg 720gtgaaggatg gaggtgctga aaatattgtg ttccaaacgg ggttagggag gcctgttgtg 780gtgagccaga cctcaattca aaaggcaagg acagtattag atcaagaatg tgccaaaaga 840agtggacatg gagatactaa tgtctccacc actacttttc aaactgaaac accaacgcct 900gttctgatga gtggtggcct gactatgaat gatagatctg ttacacctga ggggggtgtt 960tcaatgcaag gaaatttttt ggaggctgat ggtcacttgc cattatttca aactgggtta 1020gggaggtcca tttcagtaag taaaggctca attaagagag caagtgcact tctggagcca 1080aggaacatta caaaagaact ggaagatgaa gctcactcag atgatggctg tgccactcca 1140atgttcaaaa ctggatcagg aaggtctatc acagcaagtg aaaattctag aaagaaagcc 1200cacgttgtct tagagggcga ggaaccagta aaaaatgtaa ataatgacac tggagaagcc 1260attgctccaa tgctccatgc tggaatgcag aagtttgcac cccaaaatag aaactcaagt 1320cataaggcga tcaccctcat ggagcaaggg agctctatgg aagaagaccg tggaaacgaa 1380ccaccaatgt ttcgaactgg atctgggaag tcagtcttga ttagtcacag ctccgtgcag 1440aaggcaaggg cggttctgga ggaagaaggc aatatgaaga aagaaaatca caaacaactt 1500agcaatgtgg acaaatatat tccgatcttt acttcacctc tcaagacaag ctatgcaagg 1560actgtacata tatcttcagt tggtgtttct cgagctgcaa ctttgttggg tttggaggag 1620aatacccttt caacacaact tttaggacat gtgggtgata agctaggtac aaagataact 1680gttgagaggg aaaattcaga gcaccagttt ggtgtagcat cagtcagtgg aatttctggt 1740ggctgcccta taagctctgg cccagctgaa aaccaagtac ttatggatcc acatcagcat 1800tttgcatttt ctaaaacaac gttctctgat tccagtgagc aagctatcag gttcagcact 1860gctggcggca gaacaatggc tattcctagt gatgcacttc agcgtgcgaa aaatcttctg 1920ggtgaatcgg atttagaggt ttcaccaaat aatttattag gccactcttc agcatctgct 1980tgtaaagaga atatacaaaa ttcaactggt ctgcgaaaag aaggtgaacc tgatttattg 2040aaaagtaggg ggaacagcaa aactgagcca gcacaatttt ccattccagc aaaacctgat 2100aggaagcaca cagattcctt ggaatatgct gtacctgatg ccactttggc taacggaaac 2160tccgtcaggc ttcatgcggc aagagatttt catcctatca atgaaattcc aaagatatcc 2220aagccttctt ccagatgttc atttggaact gaaaatgcaa gtgacactaa agataaggct 2280cgaagactcc aaatgccatc tggaccattg attgacatca ctaattacat cgatacacat 2340tctgttaata ctgactacct ggccggtgag aagagaagat ttgggggaag aaactccata 2400tctcccttta aacgtcctcg ttcttccagg ttcatcgcac ctatcaacat caataatcca 2460tccccttctg gagtatccaa actacctatt cagattaatc cctgtcgaac aaagctatct 2520tcatgctatc cttttcaaca tcaaagaaaa tcgtgtgaag agtattttgg tggtccccca 2580tgcttcaaat atttgacaga agatgtaaca gatgaagtga agctcatgga tgcaaaaaag 2640gctgagaagt acaagtttaa aacagatact ggtgcagaag aatttcagaa gatgcttctt 2700gcctgtggtg cttcattgac atacacaact aaagaatggg tcagcaacca ctacaaatgg 2760attgtttgga agcttgcttc attggagaga tgctatccaa ctagagctgc tggcaaattc 2820ttaaaagttg gtaatgtttt ggaagagctg aagtacaggt atgacagaga agtgaacaat 2880ggccaccgct cagccataaa gaaaattttg gaagggaatg cttcaccatc tttgatgatg 2940gtgctgtgca tttctgctat ttactcttgt cctgacctaa acaacagtaa gccagaggat 3000gatagggcac atacagacga cgacaacagt gagaataaaa gcttgagacc tgctaaaagg 3060aacatgtcta caaagattga actaactgat ggatggtatt ctctagatgc gtcattagat 3120ctggcacttt tggagcaact agagaaaaga aaacttttta taggacagaa gcttcggata 3180tggggagctt cactatgtgg gtgggctggg cctgtgtcat ttcatgaggc atcgggtacc 3240gtcaaattaa tgatccacat aaatggcacc tatcgtgcaa gatgggatga gactttgggg 3300ttatgcaagc atgctggagt cccactggca ttcaagtgca taaaagcttc aggtggcaga 3360gttcctagga cactggttgg agttacaagg atttatcctg ttatgtacag ggagaggttt 3420tctgacggtc gttttgtggt gaggtctgaa aggatggaaa gaaaagcact acagctgtat 3480caccagagag tgtctaagat tgcagaagac attcagtcag aacatggaga acactgcgac 3540aacactgatg ataacgatga aggggcaaaa atatgcaaaa tgctagagag ggcagctgag 3600cctgaaattc ttatgtccag catgagttca gagcagctgc tgtctttctc atattatcaa 3660gaaaagcaaa agattgtcag gcaaaatgaa gtagctaaga aggttgaaaa tgctcttaaa 3720gttgctgggc ttagttcaag agatgttaca ccatttttga aagtgagggt gacgggcctt 3780atcagcaaac actccgccac aaaatctggc tgcagggaag ggttaataac aatttggaac 3840cctaccgaga agcaaaaatc cgacctggtg gagggacaaa tttattctgt cacaggactg 3900ttggcttcaa gctactttac agaagtatcc tacttgagtg gtagaggatc atctacagca 3960tggacgcctt tagcaaccgc acagactaca aattttgaac catttttcac ccctcgtaaa 4020gcagttgaat tgtcacattt tggtgaagtg ccacttacaa gcgaatttga cattgcaggt 4080gttattttgt atgttgggaa tgtttattta ttgaacaacc agaataggca gtggctcttt 4140ttgacagatg gatctaaatt tatctctgga gaaaagtatg aagagcaaga tgactgtctt 4200ctggcagtta gcttttcttc caaaaccact ggcgaggatt ctgcattctt caattatgcc 4260ctttctggac atatagttgg ttttagtaat ctggtcaagc gagataaaga ccagatgagg 4320cacgtgtggg tagctgaggc gacagagagc tccacctata gtctctccca cgagatacct 4380aaaaaatcac atctcaaaga ggctgccact tctgctgaaa aatgggcttc aaattctcat 4440cctatgattc agcatctgaa ggaaagagtt ctgcaaatag ttggtgacag tggtggctga 45001111329DNADNA 111atgtcggaga agaagcgccg cggcggggca ggcgcggggg ccgcgtcggg ctccgcctcc 60aagaagccgc gggtctccac ggcggcgtcg tacgccgagt cgctccgctc gaagctccgc 120cccgacgcct ccatcctcgc caccctccgc tccctggcct ccgcctgctc caaacccaag 180cccgcggggt cgtcgtcgtc gtcgtcgtcc gcctcgaagg cgctcgcagc cgaggacgac 240ccggccgcca gctacatcgt ggtggccgac caggactccg cctccgtcac ctcccgcatc 300aaccgcctcg tgctcgccgc ggcgcgcagc atcctgtccg gccggggctt ctccttcgcg 360gtgccctccc gcgccgcctc caaccaggtc tacctcccgg acctcgaccg catcgtgctc 420gtccgccgcg agtccgccag gcccttcgcc aacgtcgcca ccgcgcggaa ggccaccatc 480accgcgcgcg tcctctcctt ggtccacgcc gtcctccgca gggggatcca cgtcaccaag 540cgtgacctct tctacaccga cgtcaagctc ttcggcgacc aggcgcagtc cgacgccgtc 600ctcgacgacg tctcctgtat gctcggctgc acccgctcct ccctccacgt cgtcgcgtcc 660gagaagggcg tcgtcgtcgg gcgcctcacc ttcgccgacg acggcgaccg gatcgactgc 720acgcgcatgg gcgtcggcgg gaaggccatc ccgcccaaca tcgacagggt ctcaggcatc 780gagagcgacg ctctcttcat cttgctggtg gagaaggacg ccgcgttcat gcgtctcgcc 840gaggaccggt tctacaaccg cttcccgtgc atcatcttga cggcgaaggg gcagccggat 900gtcgccacac ggctgttctt gcggcggctt aaggtggagc tgaagctgcc agtgctggca 960ttggtggact ccgacccata tgggctgaag atcttgtcag tgtacatgtg tggttccaag 1020aacatgtcat atgacagtgc caacctgaca acaccggata tcaagtggct cggagtgcgg 1080ccaagcgatc tggacaagta tcgggtgccg gagcagtgcc ggcttccgat gactgatcac 1140gatatcaagg tggggaagga gctgcttgag gaggactttg tgaagcagaa tgaaggatgg 1200gtgaaggagc tggagacgat gttgcggacg aggcagaagg ctgagataca ggctctcagt 1260tcatttggtt tccagtatct cactgaggtc tatctacctc tcaagctgca gcaacaggac 1320tggatttga 13291121456DNADNA 112agcaaccatg gatgattcaa cggatgacga ttcgtatcat ccaagaaaac actatgctta 60tgatcgtcag gtttcttcaa gcagatggcg taccagccgc gagtatatca gaggtcccgg 120ccccgaaact catactactg agagtgctca agatggacag gatccacctg ctggagtata 180ttcctatggt tatttttctg gcagtggtaa tgatcctcaa gttcaaggac actttgttcc 240ggagattcaa aagtacaacc cttacgtgat tttcaaaggt gaacaactcc cggttcctat 300atgggaactg ccagaggaga aggtccaaga ttttcatgat aggtacttta ttgcaaaaga 360caagagtcga gttgaagcca ggaagactct gaataggttg ttagagggga acatcaatac 420aattgaaagg ggacatggat ataaattcaa tattccaaaa tatacagata acatggagtt 480taatgaggaa gtcaaggttt ctctagcaaa agcaggcaag accataagcc gttccttttg 540caatgcgaat cagcgggaag ttgcatctag gactggctat accattgatc taatagaacg 600gacacttggg gctggattga acatctcgaa gagaactgtc ttatacacaa acaaggatct 660gtttggggat caaagtaaat cagatcaagc gatcaatgac atctgcgctt tgacaaatat 720cagaaggggc tctttgggta taatagcagc tgaaaaagga attgtagttg gaaacatttt 780cctggaattg acaaatggca aatcgattag ttgttctatt ggagtgcaga taccacacag 840gcttgaccag atcaaagatg tttgtgttga aataggttca cgcaacatag agtatattct 900tgttgtggaa aagcatacaa tgttgaatta tctactagag atggactatc acaccaataa 960caactgtata attctgacag gatgtggcat gccaaccctc caaacaaggg atttcctcag 1020attcttgaaa caacgcactg gactacctgt ctttggactt tgtgatccag atcctgaagg 1080tataagtatt cttgctacgt atgctagagg gtcttgcaat tcagcatatg acaatttcaa 1140tatttccgtg ccatctattt gttgggttgg attgtcatcc tcagacatga taaagttgaa 1200tttgtctgag accaactact cacgtttgtc tcgcgaggac aaaactatgt tgaagaacct 1260ttggcaggac gatttgtccg atgtatggaa acgcagaatc gaagaaatga taagttttga 1320caagaaggcc tcttttgaag ctattcatag tttggggttt gattattttg caaccaattt 1380gcttccggat atgattaaca aagtacgaga aggctatgtt caggtatatt tctcactcct 1440atagcaactt gtattt 14561131187PRTProtein 113Met Glu Asp Asp Asp Asp Asp Gln Arg Leu Leu His Ser Leu Gly Val1 5 10 15Thr Ser Ala Asp Ile His Asp Ile Glu Arg Arg Ile Ile Ser Gln Ala 20 25 30Thr Thr Asp Pro Ala Asp Ser Ser Gly Pro Thr Ile Asn Gly Gly His 35 40 45Gln Pro Asp Asp Ala Leu Ala Lys Leu His His Lys Leu Arg Ser Val 50 55 60Gln Ile Glu Ile Asp Ala Val Ala Ser Thr Ile Lys Gly Ala Lys Leu65 70 75 80Lys Gln Pro Ser Gly Asn Lys Pro His Glu His Lys Gly Lys Asp Gln 85 90 95Pro Asp His His Gly Ala Gly His Leu Gln Gln Ala Leu Ala Ala Asp 100 105 110Arg Leu Thr Ser Leu Arg Lys Ala Lys Ala Gln Ile Gln Lys Glu Ile 115 120 125Leu Gln Ser His Leu Ser Pro Ser Ala Ser Asn Arg Lys Asp Lys Met 130 135 140Leu Ala Met Leu Val Gln Asp Glu Pro Arg His Lys Lys Pro Pro Val145 150 155 160Gly Pro Lys Asn Ile Val Lys Arg Pro Met Lys Thr Val Thr Tyr Asp 165 170 175Asp Asp Asn Asn Phe Asp Ala Val Leu Asp Gly Ala Ser Ala Gly Phe 180 185 190Met Glu Thr Glu Arg Glu Glu Leu Ile Arg Lys Gly Leu Leu Thr Pro 195 200 205Phe His Lys Leu Lys Gly Phe Glu Lys Arg Val Glu Leu Pro Glu Pro 210 215 220Ser His Arg Gln Asp Asp Ser Ala Gly Gln Thr Glu Glu Ala Met Glu225 230 235 240Ala Ser Arg Ile Ala Arg Val Ala Gln Ser Leu Lys Gln Ile Ala Gln 245 250 255Asn Arg Pro Ala Thr Lys Leu Leu Asp Ser Glu Ser Leu Pro Lys Leu 260 265 270Asp Ala Pro Ala Ala Pro Phe Gln Arg Leu Gly Lys Pro Leu Lys Arg 275 280 285Pro Val Ser Pro Ser Ser Asp Glu Gln Glu Lys Lys Arg Pro Arg Asn 290 295 300Lys Thr Lys Arg Pro Leu Pro Gly Lys Lys Trp Arg Lys Ala Asn Ser305 310 315 320Ile Lys Glu Ser Ser Leu Asp Asp Asn Asp Val Gly Glu Ala Ala Val 325 330 335Ser Val Ser Asp Asp Asp Glu Asp Gln Val Thr Glu Gly Ser Asp Glu 340 345 350Leu Thr Asp Val Thr Leu Glu Gly Gly Leu Arg Ile Pro Gly Thr Leu 355 360 365Tyr Thr Gln Leu Phe Asp Tyr Gln Lys Val Gly Val Gln Trp Leu Trp 370 375 380Glu Leu His Cys Gln Arg Ala Gly Gly Ile Ile Gly Asp Glu Met Gly385 390 395 400Leu Gly Lys Thr Val Gln Val Leu Ser Phe Leu Gly Ser Leu His Asn 405 410 415Ser Gly Leu Tyr Lys Pro Ser Ile Val Val Cys Pro Val Thr Leu Leu 420 425 430Gln Gln Trp Arg Arg Glu Ala Ser Arg Trp Tyr Pro Lys Phe Lys Val 435 440 445Glu Ile Leu His Asp Ser Ala Asn Ser Ser Ser Lys Lys Ser Lys Arg 450 455 460Ser Ser Asp Ser Asp Ser Glu Ala Ser Trp Asp Ser Asp Gln Glu Glu465 470 475 480Ala Val Thr Cys Ser Lys Pro Ala Lys Lys Trp Asp Asp Leu Ile Ser 485 490 495Arg Val Val Ser Ser Gly Ser Gly Leu Leu Leu Thr Thr Tyr Glu Gln 500 505 510Leu Arg Ile Leu Gly Glu Lys Leu Leu Asp Ile Glu Trp Gly Tyr Ala 515 520 525Val Leu Asp Glu Gly His Arg Ile Arg Asn Pro Asn Ala Glu Ile Thr 530 535 540Leu Val Cys Lys Gln Leu Gln Thr Val His Arg Ile Ile Met Thr Gly545 550 555 560Ala Pro Ile Gln Asn Lys Leu Ser Glu Leu Trp Ser Leu Phe Asp Phe 565 570 575Val Phe Pro Gly Lys Leu Gly Val Leu Pro Val Phe Glu Ala Glu Phe 580 585 590Ser Val Pro Ile Thr Val Gly Gly Tyr Ala Asn Ala Thr Pro Leu Gln 595 600 605Val Ser Thr Ala Tyr Arg Cys Ala Val Val Leu Arg Asp Leu Val Met 610 615 620Pro Tyr Leu Leu Arg Arg Met Lys Ala Asp Val Asn Ala Gln Leu Pro625 630 635 640Lys Lys Thr Glu His Val Leu Phe Cys Ser Leu Thr Thr Glu Gln Arg 645 650 655Ala Thr Tyr Arg Ala Phe Leu Ala Ser Ser Glu Val Glu Gln Ile Phe 660 665 670Asp Gly Asn Arg Asn Ser Leu Tyr Gly Ile Asp Val Leu Arg Lys Ile 675 680 685Cys Asn His Pro Asp Leu Leu Glu Arg Glu His Ala Ala Gln Asn Pro 690 695 700Asp Tyr Gly Asn Pro Glu Arg Ser Gly Lys Met Lys Val Val Glu Gln705 710 715 720Val Leu Lys Val Trp Lys Glu Gln Gly His Arg Val Leu Leu Phe Thr 725 730 735Gln Thr Gln Gln Met Leu Asp Ile Met Gly Asn Phe Leu Thr Ala Cys 740 745 750Glu Tyr Gln Tyr Arg Arg Met Asp Gly Leu Thr Pro Ala Lys Gln Arg 755 760 765Met Ala Leu Ile Asp Glu Phe Asn Asn Thr Asp Glu Ile Phe Ile Phe 770 775 780Ile Leu Thr Thr Lys Val Gly Gly Leu Gly Thr Asn Leu Thr Gly Ala785 790 795 800Asn Arg Ile Ile Ile Tyr Asp Pro Asp Trp Asn Pro Ser Thr Asp Met 805 810 815Gln Ala Arg Glu Arg Ala Trp Arg Ile Gly Gln Thr Arg Asp Val Thr 820 825 830Val Tyr Arg Leu Ile Thr Arg Gly Thr Ile Glu Glu Lys Val Tyr His 835 840 845Arg Gln Val Tyr Lys His Phe Leu Thr Asn Lys Val Leu Lys Asp Pro 850 855 860Gln Gln Arg Arg Phe Phe Lys Ala Arg Asp Met Lys Asp Leu Phe Thr865 870 875 880Leu Gln Asp Asp Asp Asn Asn Gly Ser Thr Glu Thr Ser Asn Ile Phe 885 890 895Ser Gln Leu Ser Glu Asp Val Asn Ile Gly Val Pro Ser Asp Lys Gln 900 905 910Gln Asp Gln Leu Tyr Ala Ala Ser Ala Thr Pro Thr Thr Ser Gly Thr 915 920 925Glu Pro Ser Ser Ser Arg His Gly Gln Gly Lys Glu Asp His Cys Pro 930 935 940Asp Gln Ala Asp Glu Glu Cys Asn Ile Leu Lys Ser Leu Phe Asp Ala945 950 955 960Gln Gly Ile His Ser Ala Ile Asn His Asp Ala Ile Met Asn Ala Asn 965 970 975Asp Asp Gln Lys Leu Arg Leu Glu Ala Glu Ala Thr Gln Val Ala Gln 980 985 990Arg Ala Ala Glu Ala Leu Arg Gln Ser Arg Met Leu Arg Ser His Glu 995 1000 1005Ser Phe Ser Val Pro Thr Trp Thr Gly Arg Ala Gly Ala Ala Gly 1010 1015 1020Ala Pro Ser Ser Val Arg Arg Lys Phe Gly Ser Thr Leu Asn Thr 1025 1030 1035Gln Leu Val Asn Ser Ser Gln Pro Ser Glu Thr Ser Asn Gly Arg 1040 1045

1050Gly Gln Ser Leu Gln Val Gly Ala Leu Asn Gly Lys Ala Leu Ser 1055 1060 1065Ser Ala Glu Leu Leu Ala Arg Ile Arg Gly Thr Arg Glu Gly Ala 1070 1075 1080Ala Ser Asp Ala Leu Glu His Gln Leu Asn Leu Gly Ser Ala Ser 1085 1090 1095Asn His Thr Ser Ser Ser Ser Gly Asn Gly Arg Ala Ser Ser Ser 1100 1105 1110Ser Thr Arg Ser Met Ile Val Gln Pro Glu Val Leu Ile Arg Gln 1115 1120 1125Leu Cys Thr Phe Ile Gln Gln His Gly Gly Ser Ala Ser Ser Thr 1130 1135 1140Ser Ile Thr Glu His Phe Lys Asn Arg Ile Leu Ser Lys Asp Met 1145 1150 1155Leu Leu Phe Lys Asn Leu Leu Lys Glu Ile Ala Thr Leu Gln Arg 1160 1165 1170Gly Ala Asn Gly Ala Thr Trp Val Leu Lys Pro Asp Tyr Gln 1175 1180 11851141148PRTProtein 114Met Arg Thr Ser Thr Thr Ser Asp Ser Pro Ser Pro Ser Pro Gln Asn1 5 10 15Lys Ala Ser Phe Asn Thr Ser Arg Gly Ala Ala Phe Arg Asp Glu Glu 20 25 30Pro Gly Ala Lys Asp Asn Glu Val Glu Lys Arg Lys Pro Leu Ile Leu 35 40 45His Leu Lys Lys Arg Ser Thr Lys Glu Leu Ser Thr Asp Thr Thr Ser 50 55 60Ser Lys Ser Gly Leu Leu Gly Lys Ser Ser Glu Glu Lys Gln Glu Lys65 70 75 80His Gly Ser Ala Leu Lys Val Lys Lys His Leu His Pro Met Glu Leu 85 90 95Ser Pro Lys Lys Tyr Lys Asn Lys Lys Gln His Asn His Arg Asp Ser 100 105 110Lys Arg Ser Glu Ala Lys Lys Val Gln Tyr Leu Ala Ser Asp Val Asp 115 120 125Ser Asp Ser Ser Met Glu Pro Ser Thr Ser Leu Glu His Ser Glu Ser 130 135 140Pro Pro Pro Lys Arg Lys Ser Leu Asp Gly Arg Thr Pro Ala Ser Ser145 150 155 160Thr Lys Lys Gly Lys Lys Lys Val Lys Phe Ile Asp Lys Lys His Pro 165 170 175Glu Asn Ala Val His Ile Thr Glu Lys Glu His Gly Gly Ala Gly Asp 180 185 190Lys Ile Thr Thr Gln Gly Asp Leu Gln Val Asp Arg Ile Leu Gly Cys 195 200 205Arg Leu Gln Thr Ser Gln Ile Ile Ser Pro Ala His Ala Ser Ser Glu 210 215 220Gln Ile Asp Met Ala Pro Pro Ser Ala Ser Gly Ala Thr Glu Pro Ser225 230 235 240Gln Ala Leu Ser Lys Gly Leu His Glu Glu Ile Gln Ser Ser Asn Ser 245 250 255Asp Thr Asn Val Thr Glu Asp Ala Cys Ala Asp Glu Leu Ala Asn Asp 260 265 270Gly Gly Glu Asn Asn Leu Asp Cys Ser Asp Ala Gln Lys Glu Ser Asn 275 280 285Val Arg Ser His Gly His Lys Glu Ser Leu Asn Ala Lys Glu Ile Met 290 295 300Asn Thr Ala Ser Ala Cys Ser Ala Asp Gln Ile Val Thr Val Lys Asp305 310 315 320Ala Gly Ala Val Gln Thr Tyr Val Thr Ala Ser Val Asn Gly Glu Tyr 325 330 335Glu Thr Val Thr Asp Ile Pro Glu Glu Lys Asn Asp Thr Lys His Pro 340 345 350Val Ser Lys Ala Asp Thr Glu Val His Thr Lys Gln Glu His Thr Pro 355 360 365Asp Ser Lys Leu His Gly Lys Leu Glu Asn Tyr Lys Ala Lys Tyr Gly 370 375 380Thr Gly Leu Ile Asn Ile Cys Lys Glu Gln Trp Cys Gln Pro Gln Arg385 390 395 400Val Ile Ala Leu Arg Thr Ser Leu Asp Glu Ile Glu Glu Ala Leu Ile 405 410 415Lys Trp Cys Ala Leu Pro Tyr Asp Glu Cys Thr Trp Glu Arg Leu Asp 420 425 430Glu Pro Thr Met Val Lys Tyr Ala His Leu Val Thr Gln Phe Lys Lys 435 440 445Phe Glu Ser Gln Ala Leu Asp Lys Asp Lys Gly Gly Ser His Ala Lys 450 455 460Pro Arg Glu His Gln Glu Phe Asn Met Leu Val Glu Gln Pro Lys Glu465 470 475 480Leu Gln Gly Gly Met Leu Phe Pro His Gln Leu Glu Ala Leu Asn Trp 485 490 495Leu Arg Lys Cys Trp Tyr Lys Ser Lys Asn Val Ile Leu Ala Asp Glu 500 505 510Met Gly Leu Gly Lys Thr Val Ser Ala Cys Ala Phe Leu Ser Ser Leu 515 520 525Cys Cys Glu Tyr Lys Ile Asn Leu Pro Cys Leu Val Leu Val Pro Leu 530 535 540Ser Thr Met Pro Asn Trp Met Ala Glu Phe Ala Ser Trp Ala Pro His545 550 555 560Leu Asn Val Val Glu Tyr His Gly Ser Ala Arg Ala Arg Ser Ile Ile 565 570 575Arg Gln Tyr Glu Trp His Glu Gly Asp Ala Ser Gln Met Gly Lys Ile 580 585 590Lys Lys Ser His Lys Phe Asn Val Leu Leu Thr Thr Tyr Glu Met Val 595 600 605Leu Val Asp Ala Ala Tyr Leu Arg Ser Val Ser Trp Glu Val Leu Ile 610 615 620Val Asp Glu Gly His Arg Leu Lys Asn Ser Ser Ser Lys Leu Phe Ser625 630 635 640Leu Leu Asn Thr Leu Ser Phe Gln His Arg Val Leu Leu Thr Gly Thr 645 650 655Pro Leu Gln Asn Asn Ile Gly Glu Met Tyr Asn Leu Leu Asn Phe Leu 660 665 670Gln Pro Ala Ser Phe Pro Ser Leu Ala Ser Phe Glu Glu Lys Phe Asn 675 680 685Asp Leu Thr Thr Thr Glu Lys Val Glu Glu Leu Lys Asn Leu Val Ala 690 695 700Pro His Met Leu Arg Arg Leu Lys Lys Asp Ala Met Gln Asn Ile Pro705 710 715 720Pro Lys Thr Glu Arg Met Val Pro Val Glu Leu Thr Ser Ile Gln Ala 725 730 735Glu Tyr Tyr Arg Ala Met Leu Thr Lys Asn Tyr Gln Val Leu Arg Asn 740 745 750Ile Gly Lys Gly Gly Ala His Gln Ser Leu Leu Asn Ile Val Met Gln 755 760 765Leu Arg Lys Val Cys Asn His Pro Tyr Leu Ile Pro Gly Thr Glu Pro 770 775 780Glu Ser Gly Ser Pro Glu Phe Leu His Glu Met Arg Ile Lys Ala Ser785 790 795 800Ala Lys Leu Thr Leu Leu His Ser Met Leu Lys Ile Leu His Lys Asp 805 810 815Gly His Arg Val Leu Ile Phe Ser Gln Met Thr Lys Leu Leu Asp Ile 820 825 830Leu Glu Asp Tyr Leu Thr Trp Glu Phe Gly Pro Lys Thr Phe Glu Arg 835 840 845Val Asp Gly Ser Val Ser Val Ala Glu Arg Gln Ala Ala Ile Ala Arg 850 855 860Phe Asn Gln Asp Lys Ser Arg Phe Val Phe Leu Leu Ser Thr Arg Ser865 870 875 880Cys Gly Leu Gly Ile Asn Leu Ala Thr Ala Asp Thr Val Ile Ile Tyr 885 890 895Asp Ser Asp Phe Asn Pro His Ala Asp Ile Gln Ala Met Asn Arg Ala 900 905 910His Arg Ile Gly Gln Ser Asn Arg Leu Leu Val Tyr Arg Leu Val Val 915 920 925Arg Ala Ser Val Glu Glu Arg Ile Leu His Leu Ala Lys Lys Lys Leu 930 935 940Met Leu Asp Gln Leu Phe Val Asn Lys Ser Glu Ser Gln Lys Glu Val945 950 955 960Glu Asp Ile Ile Arg Trp Gly Thr Glu Glu Leu Phe Arg Asn Ser Asp 965 970 975Val Ala Val Lys Asp Asn Asn Glu Ala Ser Gly Ala Lys Asn Asp Val 980 985 990Ala Glu Val Glu Phe Lys His Lys Arg Lys Thr Gly Gly Leu Gly Asp 995 1000 1005Val Tyr Glu Asp Arg Cys Ala Asp Gly Ser Ala Lys Phe Asn Trp 1010 1015 1020Asp Glu Asn Ala Ile Thr Lys Leu Leu Asp Arg Ser Asn Val Pro 1025 1030 1035Ser Thr Val Ala Glu Ser Thr Asp Gly Asp Leu Asp Asn Asp Met 1040 1045 1050Leu Gly Thr Val Lys Ser Ile Asp Trp Asn Asp Glu Leu Asn Asp 1055 1060 1065Asp Pro Gly Ala Thr Glu Asp Ile Pro Asn Ile Asp Asn Asp Gly 1070 1075 1080Cys Glu Gln Ala Ser Glu Ala Lys Gln Asp Ala Ala Asn Arg Val 1085 1090 1095Glu Glu Asn Glu Trp Asp Lys Leu Leu Arg Val Arg Trp Glu Gln 1100 1105 1110Tyr Gln Thr Glu Glu Glu Ala Ser Leu Gly Arg Gly Lys Arg Leu 1115 1120 1125Arg Lys Ala Val Ser Tyr Arg Glu Thr Phe Ala Thr Ile Pro Asn 1130 1135 1140Glu Ala Leu Ser Glu 1145115987PRTProtein 115Met Ala Asp Thr Gly Ser Leu Glu Lys Met Gly Arg Glu Leu Lys Cys1 5 10 15Pro Ile Cys Leu Ser Leu Leu Ser Ser Ala Val Ser Ile Ser Cys Asn 20 25 30His Val Phe Cys Asn Asp Cys Leu Thr Glu Ser Met Lys Ser Thr Ser 35 40 45Ser Cys Pro Val Cys Lys Val Pro Phe Arg Arg Arg Glu Met Arg Pro 50 55 60Ala Pro His Met Asp Asn Leu Val Ser Ile Phe Lys Ser Met Glu Ala65 70 75 80Ala Ala Gly Thr Asn Val Val Ser Thr Gln Glu Ala Pro Val Val Lys 85 90 95Leu Ala Asp Gly Ser Asp Cys Val Asn Ser Gly Lys Asn Ser Lys Arg 100 105 110Ser Gln Lys Ser Leu Thr Arg Lys Arg Lys Val Thr Ser Glu Met Glu 115 120 125Lys Asn Thr Ala Lys Asp Ala Thr Ala Ser Ala Ser Gln Pro Thr Thr 130 135 140Lys Pro Ser Phe Ser Thr Asn Lys Arg Ile Gln Val Lys Pro Phe Pro145 150 155 160Glu Ser Glu Thr Pro Ile Arg Ala Glu Lys Ile Met Lys Pro Glu Glu 165 170 175Pro Lys Asn Asn Leu Asn Asn Asp Val Glu Gly Lys Asn Lys Ala Val 180 185 190Ala Ser Gly Gln Pro Gly Ser Pro Ser Leu Ser Pro Phe Phe Trp Leu 195 200 205Arg Glu Gln Glu Glu Gln Glu Gly Cys Thr Ala Glu Thr Leu Ser Glu 210 215 220Thr Gln Ser Leu Asp Thr Pro Leu Arg His Asn Ala Pro Ser Phe Ser225 230 235 240Asp Ile Lys Asp Ser Asp Asp Glu Ile Pro Leu Asn Thr Thr Pro Asn 245 250 255Ser Lys Ala Ala Ala Thr Glu Leu Phe Asp Ser Glu Ile Phe Glu Trp 260 265 270Thr Gln Arg Pro Cys Ser Pro Glu Leu Tyr Ser Thr Pro Leu Lys Lys 275 280 285Gln Ser Lys Ala Lys Ser Lys Leu Asp Gln Ile Glu Glu Lys Gly Asp 290 295 300Glu Glu Asp Val His Ile Gly Gly Ser Phe Asp Lys Leu Gly Ser Ala305 310 315 320Ser Asn Ala Ala Gln Leu Val Asn Thr Lys Ala Thr Lys Gln Lys Arg 325 330 335Lys Lys Thr Ser Pro Ser Asn Lys Asn Ser Ala Lys Leu Ser Asn Arg 340 345 350Ala Glu Pro Cys Ile Lys Lys Ser Asp Ala Asn Gln Gln Gly Ser Asn 355 360 365Arg Arg Lys Ser Ala Ala Leu Lys Ser Cys Gln Lys Ser Ser Ser Ala 370 375 380Val Gly Arg Asn Thr Ser Gly Arg Arg Asn Lys Ala Ser Ser Asn Ser385 390 395 400Lys Pro Ile His Gly Ser Ser Asp Asn Ser Pro Glu Ser Tyr Leu Pro 405 410 415Lys Glu Gly Leu Asp Val Glu Ala Pro Asp Lys Pro Leu Ser Glu Arg 420 425 430Ile Gln Asn Leu Glu Lys Thr Ser Arg Arg Lys Gly Ser Ala Arg Lys 435 440 445Leu Glu Met Ala Gly Lys Thr Ile Ser Asp Thr Thr Glu Lys Asn Ser 450 455 460Glu Pro Arg Ser Lys Arg Val Arg Arg Met Ser Asp His Ala Ile Ala465 470 475 480Lys Pro Val Glu Val Pro Ser Gly Ser Gly Asn Glu Thr Glu Ile Pro 485 490 495Gln Leu His Thr Leu Thr Lys Gly Ser Ile Gln Arg Lys Ser Ser Asn 500 505 510Ala Arg Arg His Ser Lys Val Cys Gly Glu Gln Glu Gly Lys Asn Lys 515 520 525Leu Glu Asn Thr Thr Met Thr Pro Ile Ile Leu His Gly Lys Cys Gln 530 535 540Asn Lys Glu Ala Val Cys Thr Ala Pro Ser Val Arg Thr Ala Ser Val545 550 555 560Lys Tyr Lys Gln Ala Lys Phe Ser Glu Gln Pro Asp Cys Phe Gly Thr 565 570 575Glu Asn Phe Gly Asn Leu Gln Ala Cys Pro Ala Arg Asn Val Leu Leu 580 585 590Lys Lys Cys Glu Val Ser Thr Leu Lys Val Ser Cys Ala Phe Cys Gln 595 600 605Thr Asp Val Ile Thr Glu Glu Ser Gly Glu Met Val His Tyr Gln Asn 610 615 620Gly Lys Gln Val Pro Ala Glu Phe Asn Gly Gly Ala Asn Val Val His625 630 635 640Ser His Lys Asn Cys Leu Glu Trp Ala Pro Asp Val Tyr Phe Glu Asp 645 650 655Asp Ser Ala Phe Asn Leu Thr Thr Glu Leu Ala Arg Ser Arg Arg Ile 660 665 670Lys Cys Ala Cys Cys Gly Ile Lys Gly Ala Ala Leu Gly Cys Phe Glu 675 680 685Met Ser Cys Arg Arg Ser Phe His Phe Thr Cys Ala Lys Leu Ile Pro 690 695 700Glu Cys Arg Trp Asp Asn Glu Asn Phe Val Met Leu Cys Pro Leu His705 710 715 720Arg Ser Thr Lys Leu Pro Asn Glu Asn Ser Glu Gln Gln Lys Gln Pro 725 730 735Lys Arg Lys Thr Thr Leu Lys Gly Ser Ser Gln Ile Gly Ser Asn Gln 740 745 750Asp Cys Gly Asn Asn Trp Lys Trp Pro Ser Gly Ser Pro Gln Lys Trp 755 760 765Val Leu Cys Cys Ser Ser Leu Ser Ser Ser Glu Lys Gly Leu Val Ser 770 775 780Glu Phe Ala Lys Leu Ala Gly Val Pro Ile Ser Ala Thr Trp Ser Pro785 790 795 800Asn Val Thr His Val Ile Ala Ser Thr Asp Leu Ser Gly Ala Cys Lys 805 810 815Arg Thr Leu Lys Phe Leu Met Ala Ile Leu Asn Gly Arg Trp Ile Val 820 825 830Ser Ile Asp Trp Val Lys Thr Cys Met Glu Cys Met Glu Pro Ile Asp 835 840 845Glu His Lys Phe Glu Val Ala Thr Asp Val His Gly Ile Thr Asp Gly 850 855 860Pro Arg Leu Gly Arg Cys Arg Val Ile Asp Arg Gln Pro Lys Leu Phe865 870 875 880Asp Ser Met Arg Phe Tyr Leu His Gly Asp Tyr Thr Lys Ser Tyr Arg 885 890 895Gly Tyr Leu Gln Asp Leu Val Val Ala Ala Gly Gly Ile Val Leu Gln 900 905 910Arg Lys Pro Val Ser Arg Asp Gln Gln Lys Leu Leu Asp Asp Ser Ser 915 920 925Asp Leu Leu Ile Val Tyr Ser Phe Glu Asn Gln Asp Arg Ala Lys Ser 930 935 940Lys Ala Glu Thr Lys Ala Ala Asp Arg Arg Gln Ala Asp Ala Gln Ala945 950 955 960Leu Ala Cys Ala Ser Gly Gly Arg Val Val Ser Ser Ala Trp Val Ile 965 970 975Asp Ser Ile Ala Ala Cys Asn Leu Gln Pro Leu 980 9851161499PRTProtein 116Met Ala Asp Leu Phe Asn Gln Ala Leu Asp Lys Leu Val Ala Ala Asp1 5 10 15Gly Met Ala Glu Ala Ile Glu Asp Ser Gly Lys Gly Ala Val Phe Cys 20 25 30Thr Gly Leu Gly Gly Ser Val Ala Val Ser Glu Arg Ala Val Glu Arg 35 40 45Ala Lys Ala Leu Val Gly Glu Val Ala Glu Glu Ile Ser Asn Glu Arg 50 55 60Arg Gln Pro Phe Gly Asp Gly Ser Asn Leu Glu Cys Gly Leu Gly Glu65 70 75 80Ser Asn Val Ser Phe Lys Gly Gly Val His Lys Asp Ser Leu Ser Pro 85 90 95Met Phe Gln Thr Gly Ser Gly Lys Met Val Ser Leu Ser Lys Gly Ser 100 105 110Ile Gln Lys Ala Arg Ala Val Leu Glu Gly Asn Ala Glu Asn Ser Ser 115 120 125Val Ile Ala Val Gln Ser Met Phe His Thr Gly Leu Val Arg Pro Asp 130 135 140Pro Val Ser Arg Ser Ser Thr Asp Asn Ala Met Thr Val Leu Glu Gly145 150 155 160Gln Thr Asn Pro Lys Gln Gly Asp Val Ala Asp Val Tyr Asp Lys Glu 165 170 175Asn Phe Pro Leu Phe Gln Thr Gly Ser Gly Lys Ala Val Ser Val Ser 180 185

190Val Ala Ser Ile Gln Lys Ala Lys Ala Val Leu Glu Gln Asn Asn Thr 195 200 205Glu Asn Thr Glu Asp Phe Gly Arg Pro Asp Gln Ser Leu Ile Phe Gln 210 215 220Thr Gly Ser Arg Arg Pro Val Leu Ile Ser Glu Arg Ser Ser Ser Val225 230 235 240Val Lys Asp Gly Gly Ala Glu Asn Ile Val Phe Gln Thr Gly Leu Gly 245 250 255Arg Pro Val Val Val Ser Gln Thr Ser Ile Gln Lys Ala Arg Thr Val 260 265 270Leu Asp Gln Glu Cys Ala Lys Arg Ser Gly His Gly Asp Thr Asn Val 275 280 285Ser Thr Thr Thr Phe Gln Thr Glu Thr Pro Thr Pro Val Leu Met Ser 290 295 300Gly Gly Leu Thr Met Asn Asp Arg Ser Val Thr Pro Glu Gly Gly Val305 310 315 320Ser Met Gln Gly Asn Phe Leu Glu Ala Asp Gly His Leu Pro Leu Phe 325 330 335Gln Thr Gly Leu Gly Arg Ser Ile Ser Val Ser Lys Gly Ser Ile Lys 340 345 350Arg Ala Ser Ala Leu Leu Glu Pro Arg Asn Ile Thr Lys Glu Leu Glu 355 360 365Asp Glu Ala His Ser Asp Asp Gly Cys Ala Thr Pro Met Phe Lys Thr 370 375 380Gly Ser Gly Arg Ser Ile Thr Ala Ser Glu Asn Ser Arg Lys Lys Ala385 390 395 400His Val Val Leu Glu Gly Glu Glu Pro Val Lys Asn Val Asn Asn Asp 405 410 415Thr Gly Glu Ala Ile Ala Pro Met Leu His Ala Gly Met Gln Lys Phe 420 425 430Ala Pro Gln Asn Arg Asn Ser Ser His Lys Ala Ile Thr Leu Met Glu 435 440 445Gln Gly Ser Ser Met Glu Glu Asp Arg Gly Asn Glu Pro Pro Met Phe 450 455 460Arg Thr Gly Ser Gly Lys Ser Val Leu Ile Ser His Ser Ser Val Gln465 470 475 480Lys Ala Arg Ala Val Leu Glu Glu Glu Gly Asn Met Lys Lys Glu Asn 485 490 495His Lys Gln Leu Ser Asn Val Asp Lys Tyr Ile Pro Ile Phe Thr Ser 500 505 510Pro Leu Lys Thr Ser Tyr Ala Arg Thr Val His Ile Ser Ser Val Gly 515 520 525Val Ser Arg Ala Ala Thr Leu Leu Gly Leu Glu Glu Asn Thr Leu Ser 530 535 540Thr Gln Leu Leu Gly His Val Gly Asp Lys Leu Gly Thr Lys Ile Thr545 550 555 560Val Glu Arg Glu Asn Ser Glu His Gln Phe Gly Val Ala Ser Val Ser 565 570 575Gly Ile Ser Gly Gly Cys Pro Ile Ser Ser Gly Pro Ala Glu Asn Gln 580 585 590Val Leu Met Asp Pro His Gln His Phe Ala Phe Ser Lys Thr Thr Phe 595 600 605Ser Asp Ser Ser Glu Gln Ala Ile Arg Phe Ser Thr Ala Gly Gly Arg 610 615 620Thr Met Ala Ile Pro Ser Asp Ala Leu Gln Arg Ala Lys Asn Leu Leu625 630 635 640Gly Glu Ser Asp Leu Glu Val Ser Pro Asn Asn Leu Leu Gly His Ser 645 650 655Ser Ala Ser Ala Cys Lys Glu Asn Ile Gln Asn Ser Thr Gly Leu Arg 660 665 670Lys Glu Gly Glu Pro Asp Leu Leu Lys Ser Arg Gly Asn Ser Lys Thr 675 680 685Glu Pro Ala Gln Phe Ser Ile Pro Ala Lys Pro Asp Arg Lys His Thr 690 695 700Asp Ser Leu Glu Tyr Ala Val Pro Asp Ala Thr Leu Ala Asn Gly Asn705 710 715 720Ser Val Arg Leu His Ala Ala Arg Asp Phe His Pro Ile Asn Glu Ile 725 730 735Pro Lys Ile Ser Lys Pro Ser Ser Arg Cys Ser Phe Gly Thr Glu Asn 740 745 750Ala Ser Asp Thr Lys Asp Lys Ala Arg Arg Leu Gln Met Pro Ser Gly 755 760 765Pro Leu Ile Asp Ile Thr Asn Tyr Ile Asp Thr His Ser Val Asn Thr 770 775 780Asp Tyr Leu Ala Gly Glu Lys Arg Arg Phe Gly Gly Arg Asn Ser Ile785 790 795 800Ser Pro Phe Lys Arg Pro Arg Ser Ser Arg Phe Ile Ala Pro Ile Asn 805 810 815Ile Asn Asn Pro Ser Pro Ser Gly Val Ser Lys Leu Pro Ile Gln Ile 820 825 830Asn Pro Cys Arg Thr Lys Leu Ser Ser Cys Tyr Pro Phe Gln His Gln 835 840 845Arg Lys Ser Cys Glu Glu Tyr Phe Gly Gly Pro Pro Cys Phe Lys Tyr 850 855 860Leu Thr Glu Asp Val Thr Asp Glu Val Lys Leu Met Asp Ala Lys Lys865 870 875 880Ala Glu Lys Tyr Lys Phe Lys Thr Asp Thr Gly Ala Glu Glu Phe Gln 885 890 895Lys Met Leu Leu Ala Cys Gly Ala Ser Leu Thr Tyr Thr Thr Lys Glu 900 905 910Trp Val Ser Asn His Tyr Lys Trp Ile Val Trp Lys Leu Ala Ser Leu 915 920 925Glu Arg Cys Tyr Pro Thr Arg Ala Ala Gly Lys Phe Leu Lys Val Gly 930 935 940Asn Val Leu Glu Glu Leu Lys Tyr Arg Tyr Asp Arg Glu Val Asn Asn945 950 955 960Gly His Arg Ser Ala Ile Lys Lys Ile Leu Glu Gly Asn Ala Ser Pro 965 970 975Ser Leu Met Met Val Leu Cys Ile Ser Ala Ile Tyr Ser Cys Pro Asp 980 985 990Leu Asn Asn Ser Lys Pro Glu Asp Asp Arg Ala His Thr Asp Asp Asp 995 1000 1005Asn Ser Glu Asn Lys Ser Leu Arg Pro Ala Lys Arg Asn Met Ser 1010 1015 1020Thr Lys Ile Glu Leu Thr Asp Gly Trp Tyr Ser Leu Asp Ala Ser 1025 1030 1035Leu Asp Leu Ala Leu Leu Glu Gln Leu Glu Lys Arg Lys Leu Phe 1040 1045 1050Ile Gly Gln Lys Leu Arg Ile Trp Gly Ala Ser Leu Cys Gly Trp 1055 1060 1065Ala Gly Pro Val Ser Phe His Glu Ala Ser Gly Thr Val Lys Leu 1070 1075 1080Met Ile His Ile Asn Gly Thr Tyr Arg Ala Arg Trp Asp Glu Thr 1085 1090 1095Leu Gly Leu Cys Lys His Ala Gly Val Pro Leu Ala Phe Lys Cys 1100 1105 1110Ile Lys Ala Ser Gly Gly Arg Val Pro Arg Thr Leu Val Gly Val 1115 1120 1125Thr Arg Ile Tyr Pro Val Met Tyr Arg Glu Arg Phe Ser Asp Gly 1130 1135 1140Arg Phe Val Val Arg Ser Glu Arg Met Glu Arg Lys Ala Leu Gln 1145 1150 1155Leu Tyr His Gln Arg Val Ser Lys Ile Ala Glu Asp Ile Gln Ser 1160 1165 1170Glu His Gly Glu His Cys Asp Asn Thr Asp Asp Asn Asp Glu Gly 1175 1180 1185Ala Lys Ile Cys Lys Met Leu Glu Arg Ala Ala Glu Pro Glu Ile 1190 1195 1200Leu Met Ser Ser Met Ser Ser Glu Gln Leu Leu Ser Phe Ser Tyr 1205 1210 1215Tyr Gln Glu Lys Gln Lys Ile Val Arg Gln Asn Glu Val Ala Lys 1220 1225 1230Lys Val Glu Asn Ala Leu Lys Val Ala Gly Leu Ser Ser Arg Asp 1235 1240 1245Val Thr Pro Phe Leu Lys Val Arg Val Thr Gly Leu Ile Ser Lys 1250 1255 1260His Ser Ala Thr Lys Ser Gly Cys Arg Glu Gly Leu Ile Thr Ile 1265 1270 1275Trp Asn Pro Thr Glu Lys Gln Lys Ser Asp Leu Val Glu Gly Gln 1280 1285 1290Ile Tyr Ser Val Thr Gly Leu Leu Ala Ser Ser Tyr Phe Thr Glu 1295 1300 1305Val Ser Tyr Leu Ser Gly Arg Gly Ser Ser Thr Ala Trp Thr Pro 1310 1315 1320Leu Ala Thr Ala Gln Thr Thr Asn Phe Glu Pro Phe Phe Thr Pro 1325 1330 1335Arg Lys Ala Val Glu Leu Ser His Phe Gly Glu Val Pro Leu Thr 1340 1345 1350Ser Glu Phe Asp Ile Ala Gly Val Ile Leu Tyr Val Gly Asn Val 1355 1360 1365Tyr Leu Leu Asn Asn Gln Asn Arg Gln Trp Leu Phe Leu Thr Asp 1370 1375 1380Gly Ser Lys Phe Ile Ser Gly Glu Lys Tyr Glu Glu Gln Asp Asp 1385 1390 1395Cys Leu Leu Ala Val Ser Phe Ser Ser Lys Thr Thr Gly Glu Asp 1400 1405 1410Ser Ala Phe Phe Asn Tyr Ala Leu Ser Gly His Ile Val Gly Phe 1415 1420 1425Ser Asn Leu Val Lys Arg Asp Lys Asp Gln Met Arg His Val Trp 1430 1435 1440Val Ala Glu Ala Thr Glu Ser Ser Thr Tyr Ser Leu Ser His Glu 1445 1450 1455Ile Pro Lys Lys Ser His Leu Lys Glu Ala Ala Thr Ser Ala Glu 1460 1465 1470Lys Trp Ala Ser Asn Ser His Pro Met Ile Gln His Leu Lys Glu 1475 1480 1485Arg Val Leu Gln Ile Val Gly Asp Ser Gly Gly 1490 1495117442PRTProtein 117Met Ser Glu Lys Lys Arg Arg Gly Gly Ala Gly Ala Gly Ala Ala Ser1 5 10 15Gly Ser Ala Ser Lys Lys Pro Arg Val Ser Thr Ala Ala Ser Tyr Ala 20 25 30Glu Ser Leu Arg Ser Lys Leu Arg Pro Asp Ala Ser Ile Leu Ala Thr 35 40 45Leu Arg Ser Leu Ala Ser Ala Cys Ser Lys Pro Lys Pro Ala Gly Ser 50 55 60Ser Ser Ser Ser Ser Ser Ala Ser Lys Ala Leu Ala Ala Glu Asp Asp65 70 75 80Pro Ala Ala Ser Tyr Ile Val Val Ala Asp Gln Asp Ser Ala Ser Val 85 90 95Thr Ser Arg Ile Asn Arg Leu Val Leu Ala Ala Ala Arg Ser Ile Leu 100 105 110Ser Gly Arg Gly Phe Ser Phe Ala Val Pro Ser Arg Ala Ala Ser Asn 115 120 125Gln Val Tyr Leu Pro Asp Leu Asp Arg Ile Val Leu Val Arg Arg Glu 130 135 140Ser Ala Arg Pro Phe Ala Asn Val Ala Thr Ala Arg Lys Ala Thr Ile145 150 155 160Thr Ala Arg Val Leu Ser Leu Val His Ala Val Leu Arg Arg Gly Ile 165 170 175His Val Thr Lys Arg Asp Leu Phe Tyr Thr Asp Val Lys Leu Phe Gly 180 185 190Asp Gln Ala Gln Ser Asp Ala Val Leu Asp Asp Val Ser Cys Met Leu 195 200 205Gly Cys Thr Arg Ser Ser Leu His Val Val Ala Ser Glu Lys Gly Val 210 215 220Val Val Gly Arg Leu Thr Phe Ala Asp Asp Gly Asp Arg Ile Asp Cys225 230 235 240Thr Arg Met Gly Val Gly Gly Lys Ala Ile Pro Pro Asn Ile Asp Arg 245 250 255Val Ser Gly Ile Glu Ser Asp Ala Leu Phe Ile Leu Leu Val Glu Lys 260 265 270Asp Ala Ala Phe Met Arg Leu Ala Glu Asp Arg Phe Tyr Asn Arg Phe 275 280 285Pro Cys Ile Ile Leu Thr Ala Lys Gly Gln Pro Asp Val Ala Thr Arg 290 295 300Leu Phe Leu Arg Arg Leu Lys Val Glu Leu Lys Leu Pro Val Leu Ala305 310 315 320Leu Val Asp Ser Asp Pro Tyr Gly Leu Lys Ile Leu Ser Val Tyr Met 325 330 335Cys Gly Ser Lys Asn Met Ser Tyr Asp Ser Ala Asn Leu Thr Thr Pro 340 345 350Asp Ile Lys Trp Leu Gly Val Arg Pro Ser Asp Leu Asp Lys Tyr Arg 355 360 365Val Pro Glu Gln Cys Arg Leu Pro Met Thr Asp His Asp Ile Lys Val 370 375 380Gly Lys Glu Leu Leu Glu Glu Asp Phe Val Lys Gln Asn Glu Gly Trp385 390 395 400Val Lys Glu Leu Glu Thr Met Leu Arg Thr Arg Gln Lys Ala Glu Ile 405 410 415Gln Ala Leu Ser Ser Phe Gly Phe Gln Tyr Leu Thr Glu Val Tyr Leu 420 425 430Pro Leu Lys Leu Gln Gln Gln Asp Trp Ile 435 440118478PRTProtein 118Met Asp Asp Ser Thr Asp Asp Asp Ser Tyr His Pro Arg Lys His Tyr1 5 10 15Ala Tyr Asp Arg Gln Val Ser Ser Ser Arg Trp Arg Thr Ser Arg Glu 20 25 30Tyr Ile Arg Gly Pro Gly Pro Glu Thr His Thr Thr Glu Ser Ala Gln 35 40 45Asp Gly Gln Asp Pro Pro Ala Gly Val Tyr Ser Tyr Gly Tyr Phe Ser 50 55 60Gly Ser Gly Asn Asp Pro Gln Val Gln Gly His Phe Val Pro Glu Ile65 70 75 80Gln Lys Tyr Asn Pro Tyr Val Ile Phe Lys Gly Glu Gln Leu Pro Val 85 90 95Pro Ile Trp Glu Leu Pro Glu Glu Lys Val Gln Asp Phe His Asp Arg 100 105 110Tyr Phe Ile Ala Lys Asp Lys Ser Arg Val Glu Ala Arg Lys Thr Leu 115 120 125Asn Arg Leu Leu Glu Gly Asn Ile Asn Thr Ile Glu Arg Gly His Gly 130 135 140Tyr Lys Phe Asn Ile Pro Lys Tyr Thr Asp Asn Met Glu Phe Asn Glu145 150 155 160Glu Val Lys Val Ser Leu Ala Lys Ala Gly Lys Thr Ile Ser Arg Ser 165 170 175Phe Cys Asn Ala Asn Gln Arg Glu Val Ala Ser Arg Thr Gly Tyr Thr 180 185 190Ile Asp Leu Ile Glu Arg Thr Leu Gly Ala Gly Leu Asn Ile Ser Lys 195 200 205Arg Thr Val Leu Tyr Thr Asn Lys Asp Leu Phe Gly Asp Gln Ser Lys 210 215 220Ser Asp Gln Ala Ile Asn Asp Ile Cys Ala Leu Thr Asn Ile Arg Arg225 230 235 240Gly Ser Leu Gly Ile Ile Ala Ala Glu Lys Gly Ile Val Val Gly Asn 245 250 255Ile Phe Leu Glu Leu Thr Asn Gly Lys Ser Ile Ser Cys Ser Ile Gly 260 265 270Val Gln Ile Pro His Arg Leu Asp Gln Ile Lys Asp Val Cys Val Glu 275 280 285Ile Gly Ser Arg Asn Ile Glu Tyr Ile Leu Val Val Glu Lys His Thr 290 295 300Met Leu Asn Tyr Leu Leu Glu Met Asp Tyr His Thr Asn Asn Asn Cys305 310 315 320Ile Ile Leu Thr Gly Cys Gly Met Pro Thr Leu Gln Thr Arg Asp Phe 325 330 335Leu Arg Phe Leu Lys Gln Arg Thr Gly Leu Pro Val Phe Gly Leu Cys 340 345 350Asp Pro Asp Pro Glu Gly Ile Ser Ile Leu Ala Thr Tyr Ala Arg Gly 355 360 365Ser Cys Asn Ser Ala Tyr Asp Asn Phe Asn Ile Ser Val Pro Ser Ile 370 375 380Cys Trp Val Gly Leu Ser Ser Ser Asp Met Ile Lys Leu Asn Leu Ser385 390 395 400Glu Thr Asn Tyr Ser Arg Leu Ser Arg Glu Asp Lys Thr Met Leu Lys 405 410 415Asn Leu Trp Gln Asp Asp Leu Ser Asp Val Trp Lys Arg Arg Ile Glu 420 425 430Glu Met Ile Ser Phe Asp Lys Lys Ala Ser Phe Glu Ala Ile His Ser 435 440 445Leu Gly Phe Asp Tyr Phe Ala Thr Asn Leu Leu Pro Asp Met Ile Asn 450 455 460Lys Val Arg Glu Gly Tyr Val Gln Val Tyr Phe Ser Leu Leu465 470 475

* * * * *

References


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