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`UNITED StaTreS PATENT AND TRADEMARK OFFICE
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`
`APPLICATION
`FILINGor
`GRP AR’
`
`NUMBER
`371(¢) DATE
`UNI
`FIL FEE REC'D
`ATTY.DOCKET.NO
`[TOT CLAIMS§IND CLAIMS
`61/559,618
`11/14/2011
`560
`B248 1400.P1
`
`59392
`WOMBLE CARLYLE SANDRIDGE & RIGE, LLP
`
`Attn: IP Docketing
`
`P.O. BOX 7037
`ATLANTA, GA 30357-0037
`
`CONFIRMATIONNO. 3269
`
`FILING RECEIPT
`
`IACAA
`
`3479
`
`Date Mailed: 12/12/2011
`
`It will not be examined for patentability and will
`Receipt is acknowledged of this provisional patent application.
`become abandoned notlater than twelve monthsafter its filing date. Any correspondence concerning the application
`mustinclude the following identification information: the U.S. APPLICATION NUMBER, FILING DATE, NAME OF
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`changesnotedthereon. If you received a "Notice to File Missing Parts" for this application, please submit
`any corrections to this Filing Receipt with your reply to the Notice. When the USPTO processesthe reply
`to the Notice, the USPTO will generate another Filing Receipt incorporating the requested corrections
`
`Applicant(s)
`
`Leon Neuteboom, Youngsville, NC;
`Sherry R. Whitt, Raleigh, NC;
`John McElver, Durham, NC;
`Jill Stevenson Paulik, Cary, NC;
`Scots L. Mankin, Raleigh, NC;
`Powerof Attorney:
`Lawrence Carroll Il--40940
`
`If Required, Foreign Filing License Granted: 12/07/2011
`The country code and number of your priority application, to be used for filing abroad under the Paris Convention,
`is US 61/559,618
`Projected Publication Date: None, application is not eligible for pre-grant publication
`Non-Publication Request: No
`Early Publication Request: No
`Title
`
`METHODS AND COMPOSITIONS FOR ISOLATING, IDENTIFYING AND CHARACTERIZING
`MONOCOTPLASTIDIG AGGASE HERBICIDE TOLERANT MUTATIONS USING A MODEL
`SYSTEM
`
`PROTECTING YOUR INVENTION OUTSIDE THE UNITED STATES
`
`Since the rights granted by a U.S. patent extend only throughout the territory of the United States and have no
`effect in a foreign country, an inventor who wishes patent protection in another country must apply for a patent
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`LICENSE FOR FOREIGN FILING UNDER
`
`Title 35, United States Code, Section 184
`
`Title 37, Code of Federal Regulations, 5.11 & 5.15
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`This license is to be retained by the licensee and may be usedat any time onor after the effective date thereof unless
`itis revoked. This license is automatically transferred to any related applications(s)filed under 37 CFR 1.53(d). This
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`
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`The grantof a license does not in any way lessen the responsibility of a licensee for the security of the subject matter
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`SelectUSA
`
`The United States represents the largest, most dynamic marketplace in the world and is an unparalleled location
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`PTO/SB/16 (12-08)
`Approved for use through 06/30/2010. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERGE
`Underthe Paperwork Reduction Act of 1995, no personsare required to respondto a collection ofinformation unlessit displays a valid OMB centrol number.
`PROVISIONAL APPLICATION FOR PATENT COVER SHEET - Page 1 of 2
`This is a requestforfiling a PROVISIONAL APPLICATION FOR PATENT under 37 CFR 1.53(¢).
`Express Mail Label No.
`
`INVENTOR(S)
`Residence
`Given Name(first and middle[if any] )
`Family Name or Surname
`
`(City and either State or Foreign Country)
`
`|Youngsville, North Carolina
`Leon
`Neuteboom
`
`Sherry R.
`Whitt
`Raleigh, North Carolina
`John
`McElver
`[_Durham, North Carolina
`
`
`
`
`
`Paulik
`Jill Stevenson
`Mankin
`Scots L.
`Additional inventors are being named on the
`
`Cary, North Carolina
`| Raleigh, North Carolina
`separately numbered sheets attached hereto.
`
`METHODS AND COMPOSITIONS FOR ISOLATING, IDENTIFYING AND CHARACTERIZING MONOCOT
`PLASTIDIC ACCASE HERBICIDE TOLERANT MUTATIONSUSING A MODEL SYSTEM
`
`
`Direct ail correspondenceto:
`CORRESPONDENCE ADDRESS
`[x] The address corresponding to Customer Number:
`
`55392
`
`Firm or
`Individual Name
`
`
`
`
`.
`
`
`
`ENCLOSED APPLICATION PARTS(checkail that apply)
`
`[_ [Application Data Sheet. See 37 CFR1.76
`CD(s), Number of CDs
`
`98
`Drawing(s) Number of Sheets
`Other(specify)
`[x] Specification (e.g. description of the invention) Number ofPages
`78
`Fees Due: Filing Fee of $220 ($110 for small entity).
`If the specification and drawings exceed 100 sheets of paper, an application size fee is also
`due, which is $270 ($135 for small entity)
`for each additional 50 sheets orfraction thereof. See 35 U.S.C. 41(a)(1)(G) and 37 CFR 1.18(s).
`METHOD OF PAYMENTOF THE FILING FEE AND APPLICATION SIZE FEE FOR THIS PROVISIONAL APPLICATION FOR PATENT
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`|] Applicant claims small entity status. See 37 CFR 1.27.
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`|_| Acheck or moneyorder made payable to the Director ofthe United States Patent and Trademark Office
`is enclosed to coverthefiling fee and application size fee (if applicable).
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`USE ONLY FOR FILING A PROVISIONAL APPLICATION FOR PATENT
`
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`PROVISIONAL APPLICATION COVER SHEET
`Page 2 of 2
`
`PTO/SB/16 (12-08)
`Approved for use through 06/30/2010. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE
`Underthe Paperwork Reduction Act of 1995, no personsare required to respondto a collection ofinformation unless it displays a valid OMB contro! number.
`
`The inventlon was made by an agencyof the United States Government or undera coniract with an agency of the United States Government.
`[x] No.
`
`
`
` Yes, the narne of the U.S. Government agency and the Government contract numberare:
`
`WARNING:
`is cautioned to avoid submitting personal
`information in documents filed in a patent application that may
`Péetitioner/applicant
`coniribute to identity theft. Personal information such as social security numbers, bank account numbers, or credit card
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`or issuance of a patent. Furthermore, the record from an abandoned application may also be available to the public if the
`application is referenced in a published application or an issued patent (see 37 CFR 1.14). Checks and credit card
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`
`publicly available.
`
`SIGNATURE
`
`TYPED or PRINTED NAME
`
`Lawrence J. Carroll
`
`Date
`
`November 14, 2011
`
`REGISTRATION NO.
`(if appropriate)
`
`40,940
`
`TELEPHONE
`
`(202) 467-6900
`
`Docket Number:
`
`B248 1400.P1
`
`WCSR 7031096v1
`
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`Atty. Dkt. No. B248 1400.P1
`
`METHODS AND COMPOSITIONS FOR ISOLATING, IDENTIFYING AND
`CHARACTERIZING MONOCOT PLASTIDIC ACCASE HERBICIDE TOLERANT
`MUTATIONS USING A MODEL SYSTEM
`
`1.
`
`FIELD OF THE INVENTION
`
`(0001)
`
`The present invention relates compositions and methodsfor identifying and
`
`isolating herbicide tolerant mutations in plant acetyl-CoA carboxylases.
`
`2.
`
`BACKGROUND OF THE INVENTION
`
`[0002]
`
`Aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) herbicides are
`
`used post-emergence in dicot crops to control gramineous weeds. Because these herbicides
`
`effectively kill most monocotyledonous species at low concentrations, there is low toxicity to
`
`non-target organisms. Great potential exists for developing cereal varieties that can be
`
`treated post-emergence to control weedy grasses that escape other pre-emergent herbicides
`
`treatments (Somers, 1996). Furthermore, these herbicides have low persistence in soil and
`
`provide growers with increased flexibility for weed control and crop rotation. For example,
`
`red rice is the mast pervasive and expensive pest in U.S. rice production (USDA-ARSDale
`
`Bumpers National Rice Research Center 2006 Annual Report) and canserve as a host for rice
`diseases. CLEARFIELD®Riceis the premiertool for managingred rice in infested areas;
`however, gene flow between red rice and CLEARFIELD® Ricecan result in~170 Fl
`hybrids/ha (Shivrainet al., 2007). Thus, stewardship guidelines limit CLEARFIELD® Rice
`market penetration to two out of any four years on any given field. Therefore, the generation
`of cultivated rice with tolerance to different herbicides will provide farmers CLEARFIELD®
`
`with a rotation partner to help managered rice weed populations.
`
`[0003]
`
`FOPs and DIMstarget the enzyme Acetyl-CoA Carboxylase (EC 6.4.1.2), which
`
`catalyzes thefirst committed step in fatty acid (FA) biosynthesis. ACCaseis a biotinylated
`
`enzymethat converts acetyl-CoA to malonyl-CoA in a 2-step reversible reaction. The
`
`enzymefirst carboxylates the biotin group and then theintrinsic carboxytranferase activity
`
`transfers the carboxyl group from carboxybiotin to acetyl-CoA (Nikolau et al., 2003).
`
`ACCaseactivity is necessary in the plastid which is the primary site for FA biosynthesis for
`
`membrane biogenesis. ACCaseactivity is also present in the cytosol, where it is involved in
`
`the synthesis of very long chain FA and flavonoids (Chugh and Eudes, 2008). The
`
`multidomain, cytoplasmic monocot and dicot ACCases are not sensitive to the FOP and DIM
`
`herbicides. It is the plastidic form of ACCase that confers the selectivity to this class of
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`Atty. Dkt. No. B248 1400.P1
`
`herbicides. Dicot plastidic ACCasesare naturally insensitive while monocotplastidic
`ACCases are herbicide-sensitive. The plastidic ACCases are highly expressed in
`
`meristematic tissue to feed the high demand for membrane biogenesis in rapidly growing,
`
`young seedlings (Podkowinskiet al., 2003). This essential role for growth explains the
`
`effectiveness of targeting ACCase inhibition in post-emergent weeds.
`
`[0004]
`
`Since the inception of FOP and DIM usefor controlling weeds in world
`
`agriculture in the 1980s, there has been an emergence of tolerance amid various weed
`
`species. Amongthese, the most extensively studied are Alopecurus myosuroides (blackgrass)
`
`and Avenasterilis (wild oat). Comprehensive studies ofnatural blackgrass and wild oat
`
`mutants have revealed six residues within the carboxyltransferase domainofthe plastidic
`
`isoform that confer tolerance to FOPs and/or DIMs (Delyeet al., 2003; Delye et al., 2005;
`
`Liu et al., 2007) and these are [1781L, W2027C, I2041N, G2096A, D2078G and C2088R
`
`(designation according to standard blackgrass ACCase reference sequence). Interestingly,
`
`11781L and D2078G confer tolerance to both FOPs and DIMswhile the other four mutations
`
`confer tolerance only to FOPs, suggesting that the bindingsites of the two classes of
`
`herbicides is overlapping, yet distinct.
`
`(0005)
`
`Two approaches to develop DIM tolerant rice that have beentried include the
`
`following. In thefirst approach, previously identified mutations in natural blackgrass and
`
`wild oat are introduced by means of molecular biological techniques in rice plastidic
`
`ACCase. Theeffects of the mutationsare studied in rice after Agrobacterium-mediated
`
`transformation of the modified ACCase genes. Ultimately, the same mutations can be
`
`engineeredat the endogenouslocus through oligonucleotide gene targeting (Beetham etal.,
`
`1999).
`
`In the second approachrice callus is propagated in medium with gradually increasing
`
`DIM concentrations. This procedure can enrich the callus for cells in which plastidic
`
`ACCasehas mutated to become moretolerant to the herbicide. Plants can be regenerated
`
`from the callus whenasatisfactory tolerance level has been reached. However, both
`
`approaches are limited in the number of mutations that can be generated and/ortested.
`
`[0006]
`
`Yeast is an excellent model organism for screening andtesting large numbers of
`
`mutated rice ACCase genes for increased herbicide tolerance. However, yeast contains a
`
`single, endogenous ACCase gene (ACC1), which encodes a multidomain protein that is
`
`highly tolerant to herbicides. Haploid yeast in which ACCI is disrupted is not viable
`
`(Hassiacheret al., 1993). Joachimiak et al. (1997) introduced wheat cytoplasmic ACCase
`
`into a diploid strain heterozygous for ACC1. Standard tetrad analysis demonstrated that the
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`ACC] mutation was complemented by the plant ACCase. A similar experiment with the
`
`herbicide-sensitive wheat plastidic ACCase gene showedthat this gene was notable to take
`
`over the function of ACC/ (Nikolskayaet al., 1999). A series of chimeric constructs
`
`consisting ofthe N-terminus of wheat cytoplasmic ACCase and the C-terminus of wheat
`
`plastidic ACCase was tested for complementation and herbicide sensitivity. Wheat c60p40
`
`ACCase, which is comprised ofthe first 60% of cytoplasmic ACCase and last 40% of
`
`plastidic ACCase, complemented an ACCase deletion mutant, Aaccl, while showing the
`
`highest sensitivity towards haloxyfop, clodinafop, quizalafop, cethoxydim and sethoxydim of
`
`all constructs tested. The c60p40 chimeric ACCaseis a suitable target for mutagenesis,asall
`
`herbicide-conferring mutations known have been mappedto the last 40% ofplastidic
`
`ACCase. Yet, the Joachimiak approachis limited in its efficiency in that each new mutant
`
`plastid ACCase construct is separately introduced into yeast cells to create a new yeast
`
`knockout complement.
`
`[0007]
`
`DIMsand FOPs are important herbicides and there is a need for methods and
`
`compositions to isolate, identify and characterize herbicide tolerant ACCase variants. The
`
`methods and compositions described herein are suitable for isolating, identifying, and
`
`characterizing such herbicide tolerant ACCase variants. Citation or discussion of a reference
`
`herein shall not be construed as an admission that suchis prior art to the present invention.
`
`3.
`
`SUMMARY OF THE INVENTION
`
`[0008]
`
`In some embodiments, the present invention provides methods of producing an
`
`acetyl-CoA carboxylase (ACCase) enzymethatis tolerant to at least one herbicide.
`
`Typically, the methodsof the invention are high throughput methods. Such methods will
`
`typically comprise, providing an ACCase-deficient yeast that comprises a nucleic acid
`
`encoding a chimeric ACCase. Any nucleic acid molecule known in the art may be used for
`
`this purpose. Suitable examples include, but are not limited to, plasmids, for example single
`
`copy plasmids. The chimeric ACCase encoded by the nucleic acid will comprise two or more
`
`regions. In some embodiments, the nucleic acid will comprise an N-terminal region and a C-
`
`terminal region, wherein the C-terminal region comprises an herbicide sensitivity region
`
`(HSR). The ACCase-deficient yeast comprising the nucleic acid will be contacted with at
`
`least one mutagenic oligonucleotide underconditions that permit site-directed mutagenesis of
`
`at least one codonof the nucleic acid encoding the chimeric ACCase. The mutagenized yeast
`
`can then be grown thereby forming a library of mutagenized yeast colonies. The library of
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`mutagenized yeast colonies can be cultured in the presence ofat least one ACCase-inhibiting
`herbicide to form treated colonies; and at least one of said treated colonies isolated so as to
`
`identify at least one mutagenized yeast that grows in the presenceof the herbicide, wherein
`the mutagenized yeast that grows in the presence ofthe herbicide comprises a mutagenized
`
`ACCasethat has a toleranceto the herbicide that is greater than that exhibited by the chimeric
`
`ACCaseprior to mutagenesis. As indicated above, typically these methods are high
`
`throughput, for example, in an embodimentof the present method, one trained worked can
`
`generate and screen at least 500 different HSR-variant ACCases, each from its own cell
`
`colony, in 1 month, more preferablyat least or about 1000, 2000, or 4000, and preferably up
`
`to or about 5000, 8000, or 10000 per month; as compared to the Nikolskaya yeast method
`
`(tetrad dissection) in which one worker could accomplish 10 to less than about 50. In some
`
`embodiments invention, the present methods are capable of producingat least about 100, 200,
`
`300, 400, 500, 1,000, 2,500, 5,000, or 10,000 different HSR-variant ACCases per month per
`
`trained worker. Preferably methodsof the invention are capable of producing at least about
`
`500, 1,000, 2,500, 5,000, or 10,000 different HSR-variant ACCases per month pertrained
`
`worker. This assumesthat the trained workerin either case begins with all needed supplies
`
`of competent cells and vectors useful to transform those cells either with mutagenic oligos
`
`according to a present embodiment or with the ACCase variant according to the Nikolskaya
`
`method; and that no cell colonies are split to form multiplicates for assaying, after the clonal
`
`colonies are established.
`
`[8009]
`
`The present invention relates to methods of producing,isolating, identifying and
`
`characterizing herbicide tolerant ACCase variants. In one embodiment, the invention
`
`encompasses a method of screening for an acetyl-CoA carboxylase (ACCase) enzyme which
`
`is tolerant to at least one herbicide, comprising: a) providing an ACCase-deficient yeast with
`
`a chimeric ACCase, said chimeric ACCase comprising: at least two regions wherein said
`
`regions further comprise; i) an N-terminal region, said N-terminal region derived from yeast,
`
`fungi or monocot cytoplasmic ACCases, preferably a yeast ACCase;ii) a C-terminal region,
`
`said C-terminal region derived from monocotplastidic ACCases and comprising an HSR; and
`
`iti) said N-terminal region comprises about 50% to about 60% of the chimeric ACCase;b)
`
`isolating herbicide tolerant yeast cells after culturing in the presenceof at least one herbicide;
`
`and c) further comprising identifying the mutation(s) not present in chimeric ACCaseprior to
`
`culturing, which confers tolerance to at least one herbicide.
`
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`Atty. Dkt. No. B248 1400.P1
`
`In other embodiments, the present invention relates to a yeast cell tolerantto at
`[0010]
`least one herbicide whercin the cell is produced by: a) complementing an ACCase-deficient
`yeast with a chimeric ACCase, said chimeric ACCase comprisingat least two regions
`wherein said regions further comprise; i) an N-terminal region, said N-terminal region
`derived from yeast, fungi or monocot ACCases;ii) a C-terminal region, said C-terminal
`region derived from one or more monocot-species plastidic ACCases and comprising an
`HSR;andiii) said N-terminal region comprises about 50% to about 60% of the chimeric
`ACCase; and b)isolating herbicide tolerant yeast cells after culturing in the presenceofat
`
`least one herbicide.
`
`In yet other embodiments, the present invention relates to an isolated yeast cell
`[0011]
`comprising: a) no active genomic ACCase;b) a nucleic acid encoding a chimeric ACCase,
`said chimeric ACCase comprising at least two regions wherein said regions further comprise;
`i) an N-terminal region, said N-terminal region derived from yeast, fungi or monocot
`ACCases; ii) a C-terminal region, said C-terminal region derived from monocot-species
`plastidic ACCase and comprising an HSR;andiii) said N-terminal region comprises about
`50% to about 60% of the chimeric ACCase; and c) at least one oligonucleotide thatis
`
`mutagenic for the non-yeast ACCase.
`
`[0012]
`
`In yet other embodiments, the present invention relates to a mutant ACCase which
`
`is tolerant to at least on herbicide wherein the ACCaseis identified by: a) providing an
`
`ACCase-deficient yeast with a chimeric ACCase, said chimeric ACCase comprisingat least
`
`two regions wherein said regions comprise; i) an N-terminal region, said N-terminal region
`
`derived from yeast, fungi or monocot ACCases; il) a C-terminalregion, said C-terminal
`region derived from monocot-speciesplastidic ACCases and comprising an HSR;andiii)
`said N-terminal region comprises about 50% to about 60% ofthe chimeric ACCase;b)
`isolating herbicide tolerant yeast cells after culturing in the presenceofat least one herbicide;
`
`c)further comprising identifying the mutation(s) not present in chimeric ACCase priorto
`culturing, which confers tolerance to at least one herbicide; and d) recapitulating said
`mutation in a full-length monocotplastidic ACCase gene.
`
`[0013]
`
`In yet other embodiments, the present invention relates to an isolated DNA
`
`molecule encoding a chimeric ACCase whichis tolerant to at least one herbicide, said
`
`chimeric ACCase comprising at least two regions wherein said regions comprise; a) an N-
`
`terminal region, said N-terminal region derived from yeast, fungi or monocot cytoplasmic
`ACCases; b) a C-terminal region, said C-terminalregion derived from monocotplastidic
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`ACCases and comprising an HSR, wherein said C-terminal region further comprisesat least
`
`one mutation that confers tolerance to the herbicide ; and c) said N-terminal region comprises
`
`about 50% to about 60% of the chimeric ACCase.
`
`[0014]
`
`In yet other embodiments, the present invention relates to an isolated chimeric
`
`ACCase whichis tolerantto at least one herbicide, said chimeric ACCase comprising at least
`two regions wherein said regions comprise: a) an N-terminal region, said N-terminal region
`derived from yeast, fungi or monocot cytoplasmic ACCases; b) a C-terminal region, said C-
`
`terminal region derived from monocotplastidic ACCases and comprising an HSR; and c)
`
`said N-terminal region comprises about 50% to about 60% of the chimeric ACCase.
`
`[0015]
`
`In other embodiments, methods and compositions of the invention encompass
`
`ACCase-deficient yeast due to a mutation of the genomic yeast ACCase gene which include a
`
`single point mutation, multiple point mutations, a partial deletion, a partial knockout, a
`
`complete deletion and a complete knockout.
`
`[0016]
`
`In other embodiments, methods and compositions of the invention encompass an
`
`chimeric or mutant ACCase, wherein said N-terminal regionis derived from a yeast or fungi
`
`genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Candida,
`
`Ascomycetes Neurospora, Kluyveromyces, Picha, Cryptococcus, Chrysosporium, Yarrowia,
`
`Arxula, and Hansenula.
`
`[0017]
`
`In other embodiments, methods and compositions of the invention encompass a
`
`chimeric or mutant ACCase, wherein said N-terminal region or C-terminal region is derived
`
`from a monocot genus selected from the group consisting of Saccharum, Poa, Agrostis,
`
`Lolium, Festuca, Zoysia, Cynodon, Stenotaphrum, Paspalum, Eremochloa, Axonopus,
`Bouteloua, Arundinaria, Bambusa, Chusquea, Guadua, Shibataea, Erharta, Leersia,
`Microlaena, Oryza, Zizania, Triticeae, Aveneae, Hordeum, Lolium, Digitaria, Cyperus,
`
`Kyllinga, Erigeron, Hydrocotyle, Kummerowia, Euphorbia, and Viola, Zea, Sorghum,
`
`Pennisetum, Panicum, Setaria, Eleusine, Ananas, and Musa.
`
`[0018]
`
`Ina specific embodiments, methods and compositions of the invention encompass
`
`a chimeric ACCase, wherein said N-terminal region is derived from a cytoplasmic ACCase
`
`from an Oryza species.
`
`[6019]
`
`In yet other specific embodiments, in other embodiments, methods and
`
`compositions of the invention encompass a chimeric ACCase, wherein said C-terminal region
`
`is derived from a plastidic ACCase from an Oryza species.
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`[0020]
`
`In other embodiments, methods and compositions of the invention encompass at
`
`least one herbicide that is a aryloxyphenoxyproprionate (FOP) or cyclohexadione (DIM)
`
`herbicide. In other embodiments, methods and compositions of the invention encompass a
`
`FOP herbicide selected from the group consisting of haloxyfop, cyhalofop, quizalofop,
`
`diclofop, clodinafop, fluazifop, metamifop, propaquizafop, and fenoxyprop. In other
`
`embodiments, methods and compositionsof the invention encompass a DIM herbicide
`
`selected from the group consisting of alloxydim, butroxydim, clethodim, cycloxydim,
`
`tepraloxydim, sethoxydim, tralkoxydim, and profoxydim. In yet further embodiments,
`
`methods and compositions of the invention encompass at least one herbicide present at a
`
`concentration from about 0.02 uM to about 200 uM.
`
`(0021)
`
`In other embodiments, methods and compositions of the invention encompass
`
`mutagenesis of the chimeric ACCase. In further embodiments, methods and compositions of
`
`the invention éncompass mutagenesis induced by chemical agents, ultraviolet radiation, or a
`
`library of DNA oligos provided to the yeastcell.
`
`{0022}
`
`In other embodiments, methods and compositions of the invention encompass a
`
`chimeric ACCase were C-terminal region comprises about 50% to about 60% of the chimeric
`
`ACCase.
`
`[0023]
`
`In other embodiments, methods and compositions of the invention encompass
`
`yeast celis cultured at a temperature of about 23°C to about 30°C. In other embodiments,
`
`methods and compositions of the invention encompass yeastcells are cultured in liquid or on
`
`solid media.
`
`(0024)
`
`In other embodiments, methods and compositions ofthe invention encompass a
`
`chimeric or mutant ACCase wherein said C-terminal region comprises a sequence which
`
`encodes for at least one mutation selected from the group consisting of 11781L, W1999G,
`
`11781T, V2049F, V2075L, V2075I, D2078G, and V2098A.
`
`4. BRIEF DESCRIPTION OF THE FIGURES
`
`[6025]
`
`Forthe purpose of illustrating the invention, there are depicted in the drawings
`
`certain embodiments onthe invention. However, the invention is not limited to the precise
`
`arrangements and instrumentalities of the embodiments depicted in the drawings.
`
`[0026]
`
`Figure 1A provides a schematic view of the domain structure of a monocot
`
`plastidic ACCase.
`
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`[0027]
`
`Figure 1B provides an overview of the OsJACCc60p40 chimeric protein. Unique
`
`restriction sites introduced in the corresponding DNA sequence for subcloning are indicated.
`
`Amino acids for which substitutions have been found to confer herbicide tolerance are
`
`indicated by white rectangles.
`
`[0028
`
`Figure 2A provides an overview of plasmid RTP3240-1; Figure 2B provides an
`
`overview of plasmid RTP4108; and Figure 2C provides the DNA sequence of RTP4108
`
`(SEQ ID NO:13).
`
`[0029]
`
`Figure 3A provides an overview of plasmid RTP3378; and Figure 3B provides the
`
`DNA